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Dearborn, Kristina Ok-Hee.
The characterization of non-ionic surfactant vesicles :
b a release rate study for drug delivery
h [electronic resource] /
by Kristina Ok-Hee Dearborn.
[Tampa, Fla] :
University of South Florida,
ABSTRACT: Drug delivery methods for the treatment of brain tumor cells have been both inefficient and potentially dangerous for cancer patients. Drug delivery must be done in a controlled manner so that the effective amount of medication is delivered to the patient and ensure over-dosage does not cause adverse side reactions in the patient. The focus of this investigation is to design a drug delivery system that would allow for site-specific administration of the drug, protection of the drug from the surrounding environment, and controlled sustained release of the drug. We have proposed a model that incorporates a niosome, which is a non-ionic surfactant vesicle, within a biodegradable polymer hydrogel. The drug is encapsulated in the niosome, and the niosome is embedded within a three-dimensional hydrogel network. It is therefore critical that the release rate of the drug from the niosome be studied. This investigation provides information about the release rate and behavior of the drug within the niosome as it is placed in a semi-permeable membrane. The niosome and dye solution in the cellulose membrane are placed in contact with water or PBS. Intensity measurements are taken using fluorescence spectrometry, and the readings are converted to concentration and moles values. The release rates of the dye from of the niosome and across the membrane are studied as the concentration data is collected over time. The results indicate that most of the niosomes will release their dye within ten hours. The water will create instability in the niosomes, while the PBS solution will maintain the stability of the niosomes. The concentration that diffuses across the cellulose membrane will steadily increase and can be predicted well by a simple diffusion model. We hope to use the information provided in this study to continue to design a drug delivery method that will stabilize the niosomes and allow for the maximum control over the release rate of the drug.
Thesis (M.A.)--University of South Florida, 2006.
Includes bibliographical references.
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Adviser: Norma Alcantar, Ph.D.
Brain tumor treatment.
x Chemical Engineering
t USF Electronic Theses and Dissertations.
The Characterization of Non -Ionic Surfactant Vesicles: A Release Rate Study for Drug Delivery by Kristina Ok-Hee Dearborn A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department of Chemical Engineering College of Engineering University of South Florida Major Professor: Norma Alcantar, Ph.D. Julie Harmon, Ph.D. Peter Stroot, Ph.D. Ryan Toomey, Ph.D. Michael VanAuker, Ph.D. Date of Approval: April 7, 2006 Keywords: niosome, hydrogel, fluorescence spectrometry, cellophane membrane, brain tumor treatment Copyright 2006, Kristina Ok-Hee Dearborn
Dedication I dedicate the accomplishment of this achievement to my husband, Andy, for believing in me when I no longer could, for en couraging me when I had lost hope, for being patient with me when hope had returned, and for teachi ng me to take time out each day to look at the beau ty of life. Thank you for teaching me that life is more than just a GPA. I also dedicate this work to my moth er, Cathy, my childhood hero and now my adult role model and best friend. You have ta ught me how to work hard, even when all I wanted to do was give up. You have driven me to become the person I am today. And you have shown me that there is sti ll some good left in this world. I also dedicate this work to my sister, Kiera, for being able to do more than I could ever do and for influencing me in ways no one else has ever done.
Acknowledgments I would like to thank my major professor, Dr. Norma Alcantar, for her encouragement, guidance, and support in the completion of this thesis. This thesis could not be done without her patience and effort. I would like to thank my committee members, Dr. Michael VanAuker and Dr. Ryan Toomey, for their time and advice spent towards this thesis. I would also like to give a special thank you to Ms. Elizabeth Hood for her knowledge, time, and mentorship. I would also like to mention: The faculty and staff in the Department of Chemical Engineering for their continued support of my education. The USF Graduate Studies Delores Auzenne Fellowship for their support in my post-graduate studies. Dr. Jaroszeski for the use of his lab equipment. Kevin Young for his continued moral support and ability to laugh with me at the daily insanity of graduate school in all her glory. Dr. VanAukers lab group for their help with the niosome preparation.
i Table of Contents List of Tables iv List of Figures vi Abstract x Chapter One: Introduction 1 1.1. Brain Tumor Disease 1 1.2. The System 6 1.3. Significance of This Work 8 Chapter Two: The Components 12 2.1. Niosome 12 2.1.1. Self-Assembly of Niosomes 14 2.1.2. Nature of Encapsulated Drug 18 2.1.3. Preparation of Niosomes 23 2.1.4. Drug Delivery Applications 24 2.2. Hydrogels 25 2.2.1. Properties of Hydrogels 27 2.2.2. Chemical and Physical Hydrogels 29 2.2.3. Free Radical Polymerization 31 2.2.4. Ionic Crosslinking 33 2.2.5. Proposed Hydrogel 34 2.2.6. Chitosan 35 2.2.7. Chitosan Hydrogel Synthesis 36 2.2.8. Intermolecular Interactions 37 2.2.9. Biomedical Applications 38 2.3. Cellulose Membrane 38 2.4. 5(6)-Carboxyfluorescein 40 Chapter Three: Problem Presentation 42 3.1. Previous Work One 42 3.1.1. Gliadel Wafer 42 3.2. Previous Work Two 45 3.2.1. Liposomes in Hydrogels 45 3.3. Comparison to Smart-Packaging 48 3.4. Research Objectives 51 3.4.1. First Objective 51
ii 3.4.2. Second Objective 51 3.4.3. Third Objective 52 3.5. Research Aims 53 3.5.1. First Aim 53 3.5.2. Second Aim 53 3.5.3. Third Aim 54 Chapter Four: Experimental and Characterization Techniques 55 4.1. Concentration Characterization Technique 55 4.1.1. Fluorescence Spectroscopy 55 4.2. Calibration Curve Technique 63 Chapter Five: Experiment al Design and Procedure 68 5.1. Reagents 68 5.2. Equipment 70 5.3. Instruments 74 5.4. Experimental Procedure 75 5.4.1. Niosome Preparation 76 5.4.2. Membrane Measurements 78 5.4.3. Beaker Measurements 80 5.4.4. Density Calculation 81 5.4.5. Incorporation of Two Units 82 5.4.6. Elapsed Time for Experiment 83 5.4.7. Fluorescence Spectrometer 84 Chapter Six: Results 88 6.1. Calculations 89 6.1.1. Volume 89 6.1.2. Concentration 91 6.1.3. Moles in Cuvette 93 6.1.4. Moles in the System 96 6.1.5. Diffusion from Membrane to Medium 99 6.1.6. Diffusion/Release from Niosome to Membrane 104 6.1.7. Release Rate from Niosomes 111 Chapter Seven: Discussion 113 7.1. Moles of Dye in Membrane 113 7.2. Moles of Dye in the Medium 117 7.3. Experimental Concentration of Dye vs. Time 120 7.3.1. Niosomes 120 7.3.2. Diffusion from Membrane to Medium 127 7.4. Experimental vs. Theoreti cal Concentration of Dye 133 7.4.1. Membrane 1 33 7.4.2. Niosome 1 35 7.5. Experimental Flux 1 39
iii 7.5.1. Membrane 139 7.5.2. Niosome 141 7.6. Predicted Concentration of Dye Over Time 143 7.6.1. Membrane 143 7.6.2. Niosome 1 47 7.7. Release Rate of Dye from Niosomes 15 0 Chapter Eight: Conclusion 15 4 Chapter Nine: Future Work 1 58 9.1. Future Goals of Project 1 59 9.1.1. Future Goal One 1 59 9.1.2. Future Goal Two 1 59 9.1.3. Future Goal Three 1 60 9.1.4. Future Goal Four 1 61 9.2. Future Tasks for Project 16 2 9.2.1. Future Task One 16 2 9.2.2. Future Task Two 16 2 9.2.3. Future Task Three 16 3 9.2.4. Future Task Four 16 5 9.3. SFA and ATR-FTIR 16 6 9.3. Closing Remarks on Future Work 17 6 References 1 78 Appendices 19 4 Appendix A. Approximate Sizes of Quanta 19 5 Appendix B. Density (g/ml) Calculation for 4mM, 5mM, 14mM, and 5mM/PBS 19 6 Appendix C. Intensity Measurements 19 7 Appendix D. Material Safety Data Sheets 199 D.1. Phosphate Buffer Saline MSDS 199 D.2. Triton-X 100 20 4
iv List of Tables Table 1. The Effect of Alkyl Chai n Length on Encapsulation Capacity 21 Table 2. Preparation of Dilutions and Concentrations for Calibration Curve Measurements 65 Table 3. Primary Reagents Used 68 Table 4. Primary Equipment Used 70 Table 5. Instruments Used 74 Table 6. Initial and Final Volumes for 4mM, 5mM, 14mM, and 5mM/PBS 90 Table 7. Slope Obtained from Calibration Curve 91 Table 8. Initial and Fina l Concentrations (4mM) 92 Table 9. Initial and Final Concentrations for (5mM) 92 Table 10. Initial and Final Concentrations (14mM) 92 Table 11. Initial and Final Concentrations (5mM/PBS) 93 Table 12. Initial and Final Mo les in the Cuvette (4mM) 94 Table 13. Initial and Final Mo les in the Cuvette (5mM) 94 Table 14. Initial and Final Mo les in the Cuvette (14mM) 95 Table 15. Initial and Final Moles in the Cuvette for (5mM/PBS) 95 Table 16. Moles in Vesicles (4mM, 5mM, 14mM, and 5mM/PBS) 96 Table 17. Total Moles in the System (4mM) 98 Table 18. Total Moles in the System (5mM) 98
v Table 19. Total Moles in the System (14mM) 98 Table 20. Total Moles in the System (5mM/PBS) 98 Table 21. Flux and Diffusion Coeffici ent for Cellulose (4mM, 5mM, 14mM, and 5mM/PBS) 102 Table 22. Flux and Diffusion Coefficien t of the Niosomes (4mM, 5mM, 14mM, and 5mM/PBS) 109 Table 23. Various Types of Radiati on and Corresponding Sizes of Quanta 195 Table 24. Density Calculations (g/ml) 196 Table 25. Intensity Measurements for 4mM 197 Table 26. Intensity Measurements for 5mM 197 Table 27. Intensity Measurements for 14mM 197 Table 28. Intensity Measurements for 5mM/PBS 198
vi List of Figures Figure 1. Primary Brain and Centra l Nervous System (CNS) Tumors by Histology 1998-2002 (n=63,698) 2 Figure 2. Illustration of As trocyte, Oligodendrocyte, and Ependyma Glial Cells 3 Figure 3. Illustration of a Niosome 13 Figure 4. Factors that Influen ce the Properties of Niosomes 14 Figure 5. The Structure for Sorbitan M onoester Vesicle Forming Surfactants 15 Figure 6. The Interaction of Cholestero l and Sorbitan Monostearate in the Niosome Bilayer 17 Figure 7. The Effect of Surfactan t on the Properties of Niosomes 18 Figure 8. The Effect of the Encap sulated Drug on Niosome System 19 Figure 9. A Comparison of Sorbitan Monoe sters on Encapsulation Efficiency 22 Figure 10. Hydrogels in Nanopackaging 26 Figure 11. Differences Between a Chemi cal Hydrogel and a Physical Hydrogel 30 Figure 12. Differences Between a Semi -IPN Hydrogel and a Graft Hydrogel 32 Figure 13. Mechanism of Chitosan Cr osslinking with Glutaraldehyde 32 Figure 14. Ionic Crossl inking in Hydrogel 34 Figure 15. Chemical Structure of Chitosan 35 Figure 16. The Chemical Structure of Cellulose 39 Figure 17. Chemical Structure of 5(6)-Carboxyfluorescein 41 Figure 18. The Transition from Ground St ate to Higher Vibr ational Levels 57
vii Figure 19. General Schematic Diagram of a Fluorescence Spectrometer 59 Figure 20. Schematic of the LS-B Luminescence Spectrometer 61 Figure 21. Optical System of the LS-3B Fluorescence Spectrometer 63 Figure 22. Typical Calibration Curve Using 5mM CF Dye 66 Figure 23. Moles of Dye in Membrane vs. Time (4mM, 5mM, and 14mM) 114 Figure 24. Moles of Dye in Membrane vs. Time (5mM and 5mM/PBS) 116 Figure 25. Moles of Dye in Medium vs. Time (4mM, 5mM, and 14mM) 118 Figure 26. Moles of Dye in Medium vs. Time (5mM and 5mM/PBS) 119 Figure 27. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Niosome-4mM) 121 Figure 28. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Niosome-5mM) 123 Figure 29. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Niosome-14mM) 125 Figure 30. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Niosome-5mM/PBS) 126 Figure 31. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Membrane-4mM) 127 Figure 32. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Membrane-5mM) 129 Figure 33. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Membrane-14mM) 131 Figure 34. Experimental Concentrati on of Dye vs. Time Plotted on the Log-Scale (Membrane-5mM/PBS) 132 Figure 35. Experimental vs. Theoretical Concentration of Dye Using Fick's First Law of Diffusion (4mM, 5mM, and 14mM) 133 Figure 36. Experimental vs. Theoretical Concentration for 5mM of Dye in PBS 134
viii Figure 37. Experimental and Theoretical Concentration of Dye Using Fick's First Law of Diffusion (4mM) 135 Figure 38. Experimental and Theoretical Concentration of Dye Using Fick's First Law of Diffusion (5mM) 136 Figure 39. Experimental and Theoretical Concentration of Dye Using Fick's First Law of Diffusion (14mM) 137 Figure 40. Experimental and Theoretical Concentration of Dye Using Fick's First Law of Diffusion (5mM/PBS) 138 Figure 41. Flux vs. Time in Membrane (4mM, 5mM, and 14mM) 139 Figure 42. Flux vs. Time in Membrane (5mmM and 5mM/PBS) 140 Figure 43. Flux vs. Time in Niosome (4mM, 5mM, and 14mM) 141 Figure 44. Flux vs. Time in Niosome (5mM and 5mM/PBS) 142 Figure 45. Predicted Concentration of Dye Over Time for the Membrane System Plotted on the Log-Scale (4mM) 143 Figure 46. Predicted Concentration of Dye Over Time for the Membrane System Plotted on the Log-Scale (5mM) 144 Figure 47. Predicted Concentration of Dye Over Time for the Membrane System Plotted on the Log-Scale (14mM) 145 Figure 48. Predicted Concentration of Dye Over Time for the Membrane System Plotted on the Log-Scale (5mM/PBS) 146 Figure 49. Predicted Concentration Over Time for the Niosome System Plotted on the Log-Scale (4mM) 147 Figure 50. Predicted Concentration of Dye Over Time for the Niosome System Plotted on the Log-Scale (5mM) 148 Figure 51. Predicted Concentration of Dye Over Time for the Niosome System Plotted on the Log-Scale (14mM) 149 Figure 52. Predicted Concentration of Dye Over Time for the Niosome System Plotted on the Log-Scale (5mM/PBS) 150 Figure 53. Release Rate of Dye from Niosomes (4mM, 5mM, and 14mM) 151
ix Figure 54. Release Rate of Dye vs. Time (5mM and 5mM/PBS) 153 Figure 55. Basic SFA (Mark 4 Model) 169 Figure 56. The Path of Light Throu gh the Mica Surfaces and to the Spectrograph in an SFA 170 Figure 57. Schematic of a Michelson Interferometer 173 Figure 58. Scheme of an ATR 175 Figure 59. Schematic of the FTIR Spectrometer and ATR Attachment for the Measurement of Diffusion in a Polymer 175
x The Characterization of Non-Ionic Surfact ant Vesicles: A Release Rate Study for Drug Delivery Kristina Dearborn ABSTRACT Drug delivery methods for the treatment of brain tumor cells have been both inefficient and potentially dangerous for cancer patients. Drug delivery must be done in a controlled manner so that the effective amount of medication is delivered to the patient and ensure over-dosage does not cause adverse si de reactions in the patient. The focus of this investigation is to design a drug delive ry system that would allow for site-specific administration of the drug, protection of the drug from the surrounding environment, and controlled sustained release of the drug. We have proposed a model that incorporates a niosome, which is a non-ionic surfactant ve sicle, within a biodegradable polymer hydrogel. The drug is encapsulated in the ni osome, and the niosome is embedded within a three-dimensional hydrogel networ k. It is therefore critical that the release rate of the drug from the niosome be studied. This i nvestigation provides information about the release rate and behavior of the drug within the niosome as it is placed in a semipermeable membrane. The niosome and dye solution in the cellulose membrane are placed in contact with water or PBS. Intensity measurements are taken using fluorescence spectrometry, and the readings ar e converted to concentration and moles values. The release rates of the dye from of the niosome and across the membrane are
xi studied as the concentration data is collected over time. The results indicate that most of the niosomes will release their dye within ten hours. The water will create instability in the niosomes, while the PBS solution will main tain the stability of the niosomes. The concentration that diffuses across the cellulo se membrane will steadily increase and can be predicted well by a simple diffusion model. We hope to use the information provided in this study to continue to design a drug de livery method that will stabilize the niosomes and allow for the maximum control ov er the release rate of the drug.
1 Chapter One Introduction 1.1. Brain Tumor Disease Brain tumor disease refers to the dis ease that is caused by the presence of undesirable tissue or the abnormal formation of tissue in the brain. In 2000, it was estimated that approximately 40,900 people liv ing in the United States had developed a primary brain tumor, either malignant or benign . Remarkably, 3,140 of those patients were under the age of 20 years. In 2004, approximately 18,400 new patients were diagnosed with brain cancer and other nervous system tumors [1, 2]. In that same year, approximately 12,690 patients died of brain/ nervous system cancer . Of that population, approximately 75% of the people we re living with a benign brain tumor, 23% with a malignant brain tumor, and about 2% ar e uncertain of the tumor type . Brain tumors effect all ages, races, ethnic backgrounds, genders, and religions. However, it is reported that women have a higher incide nce rate (15 per 100,000) than men (14 per 100,000) . Brain tumor disease is the s econd leading cause of cancer death in men ages 20-39 years and the fifth leading cause of cancer death in women in that same age bracket . The three most common types of primary brain tumors are: astrocytomas (12%), meningiomas (27%), and glioblasto mas (23%) as shown in Figure 1 .
Figure 1. Primary Brain and Central Nervous System (CNS) Tumors by Histology 1998-2002 (n=63,698) Primary tumors originate in the brain . These tumor types differ in location in the brain in which the tumor is found and in the grade development of the tumor. Metastatic brain tumors are tumors th at originate from other tu mors in the body, commonly from breast cancer or lung cancer, and spread to ar eas in the brain. They have an incidence rate that is four times greater than primary tumors . In 1992-1999, the five-year survival rate of people diagnosed with malignant brain tumors was 33% . 2 Glioma tumors can often result in the mo st severe types of brain tumors. Glioma refers to any type of tumor that involves the glial cells, which are the supportive tissue in the brain that help to keep the brain neurons functioning properly . Approximately 70% of brain tumors are glioma tumors . The fiveyear survival rate for patients diagnosed with a glioma is less than three perc ent, and the risk increases as age increases . The recurrence of glioblastoma is be tween six and twelve months and between 18-
36 months for astrocytoma . Astrocyte glial cells lead to astrocytoma tumors, oligodendrocyte cells lead to oligodendrogliomas tumors, a nd ependymal ce lls lead to ependymomas tumors. The astrocyte cells are properly named so due to their long branch arms and star-like appearance . Figure 2 illustrates the location and shape of these cells . Figure 2. Illustration of Astrocyte, Oli godendrocyte, and Ependyma Glial Cells 3 Astrocytoma is a type of brain tumor that can range from a Grade I to a Grade IV tumor with Grade IV being the most severe. There are several types of as trocytoma tumors that include cerebella (located in the cerebellum) desmoplatics infantile (Grade I), pilocytic (Grade I), subependymal giant ce ll, diffuse astrocytoma (Grade II), anaplastic Grade III), and the astrocytomas Grade IV (glioblastoma multiforme) . The treatment for these tumors can range from surgery, radiation therapy, and chemotherapy. The chemotherapy drugs that are often used are Carmustine (BCNU), Procarbazine, and Temozolomide (Temodar). Oligodendroglioma is a type of brai n tumor that can range from a Grade II to a Grade IV tumor. The oligode ndroyte cells have short bran ch arms and have a fried egg like shape . This tumor is freque ntly located in the cerebral hemisphere and
4 about half are found in the frontal lobe . The treatment for this type of tumor is usually by surgically removing the tumor a nd possible radiation or chemotherapy to follow up. Ependymoma is a type of brain tumor th at can range from a Grade I to a Grade IV tumor. These tumors line the vent ricles of the brain and center part of the spinal cord . These tumors are relatively rare as about 2-3% of primary brain tumors, but comprise the most common type of brain tumor in children . The treatment for this type of brain tumor is surgery and radiation. The causes and risk factors for developing a primary brain tumor are still not well confirmed. Environmental and genetic fact ors have been identified that may be associated with the disease, but no one fact or can predict if someone is at risk of developing a brain tumor. It has been shown that exposure to ionizing radiation increases the risk of a brain tumor [1, 6]. Several othe r environmental factors that may be linked to brain tumor disease include, but are not lim ited to, exposure to vinyl chloride, diet, common infections and viruses, smoking, exposure to agricultural work, exposure to air pollution, and history of head trauma [1, 6]. It has been shown that only about 5-10% of brain tumors are caused by genetic disorders . In addition to the genetic syndromes associated with brain tumors researchers have identified several other genes in brain tumors that do not function properly. These genes include tumor suppressor genes (inhibit tumor growth), oncogenes (create tumor growth in an uncontrolled manner), growth factors (normal cell growth), cyclin-d ependent kinase inhib itors (normal growth cell cycle), DNA repair genes (control re pair of DNA), carcinogen metabolizing genes (break down toxic chemicals), and immune response genes (controls virus and infection
5 response) . Certain chromosomes have been identified and a ssociated with brain tumors including chromosomes 1, 10, 13, 17, 19, and 22 . The treatment for brain tumors incl udes surgery to remove the tumor, chemotherapy, and radiation therapy. These treatments can be used as the primary treatment alone for the tumor or in combina tion with the other two treatment methods. Surgery, of an average 5 cm diameter tumor, can increase survival time up to six months . Surgery can often be difficult with br ain tumors due to the lack of well-defined boundaries of the tumor and its location to crit ical areas in the brain . Chemotherapy refers to the use of certain drugs to kill existing tumor cells and prevent the growth of more cells . The drug works by inhibiti ng cell duplication or re plication. They may either be cell-cycle specific targeting drugs (Procarbazine, Temozolomide) or non cellcycle specific targeting drugs (BCNU) . Ch emotherapy drugs may be administered either by systemic delivery (injected into the bloodstream via an artery, muscle, or vein) or by local delivery (implanted on or ar ound the actual tumor) . The blood brain barrier is the protective layer that surrounds and protects the brain from foreign species. Systemic delivery can overcome the blood br ain barrier by using the drug Mannitol to break through this barrier. Local delivery can be delivered direc tly between meninges spaces (intrathecal) or into the cavity that the tumor left behind (intracavitary), brain tissue (interstitial), tumor by gravity (convec tion enhanced), tumor (intratumoral), or a ventricle (intraventricular) . The use of chemotherapy drugs can cause normal cells in the body to be exposed to the doses of medici ne given to treat th e tumor cells. Side effects are often experienced by the patients in response to the effect of the drug on their normal cells. Side effects of Temozolomide include nausea, vomiting, headache,
6 constipation, diarrhea, weakness, fatigue, se izures, bleeding, and a decrease in white blood cell count . Some patients may also develop a resistance to the chemotherapy drugs and the over-dosage that must be used to overcome this . Radiation therapy is also used in the treatment of brain tumors. Radiat ion uses an external beam of light to slow the growth or shrink tumors . Patie nts can only tolerate so much radiation, so other treatment methods are used in conjunction with this technique. Side effects due to radiation include nausea, fatigue, sexual side effects, hair loss, and changes in skin . Researchers are investigating new treatment therapies that include gene therapy, viral gene therapy, adenovirus vector s, adeno-associated virus v ectors, herpes simplex virus vectors, retroviral vectors, other viruses, hybrids, targeted viral vectors, and non-viral vectors . One particular gene therapy a pproach uses polymer microspheres to deliver adenovirus vectors to the brain tumor . The goal of this approach is to increase the effectiveness of the gene tr ansfer that is occurring a nd to decrease the amount of subsequent doses that mu st be given . 1.2. The System The system that this thesis proposes as a novel drug delivery technique consists of two components. The first component is a nonionic surfactant vesicle that is made up of a synthetic amphiphilic bilayer and cholesterol. This vesicle is commonly referred to as a niosome. Niosomes are composed of synt hetic amphiphiles that self assemble into bilayers, while liposomes are composed of natural amphiphilc lipids that self assemble into bilayers. Niosomes have been shown to be more chemically stable when used as a vesicle, easier to store, safer to handle, a nd less expensive than liposomes. The polar
7 core in niosomes can entrap hydrophili c solutions, while the non-polar shell can accommodate hydrophobic solutions. A dye that represents the drug to be delivered can be encapsulated into the vesicl e by thin film sonication. The dye that is used is 5(6)carboxyfluorescein. The second component of the system is the biodegradable polymer hydrogel. The hydrogel is a three dimensional polymer network that can form a gel-like solution based on ionic crosslinking. The main feature of hydrogels is their ability to swell in water but still resist dissolution. The polymer that is proposed for use in the hydrogel is chitosan with a -glycerophosphate polyol salt. Chitosan is derived from the natural polysaccharide chitin and is created by deacetlyation. Chitosan has shown to be biocompatible and biodegradable in the body. The advantage to usi ng a chitosan polymer is that it can be made to respond to ch anges in environmental conditions such as temperature and pH. The hydrogel will begin to gel at 37C and a pH of 6.8-7.2 . The pH of the brain is 7.32 [ 12] and the temperature of the brain ranges from 37.84+/1.03C to 38.39+/-0.33C, depending on the distan ce from the scaffold . These conditions suggest the chitosan hydrogel will begin to gel once implanted inside of the brain. The in vitro experiments will involve the use of a semi-permeable membrane that is designed specifically for dialysis expe riments. The semi-permeability of the membrane will prevent larger molecules from crossing the membrane and allow smaller molecules to cross. The system will be placed within this membrane and submerged in a bulk liquid solution. Two bulk liquid medi ums will be used: Milli-Q water and phosphate buffer solution (PBS). The niosomes will break or burst when they come into contact with the water due to the osmotic pr essure difference between the niosomes and
8 the water. Water in used so that the study of the effect of pH and ionic strength can be made in later studies. Water is also present in the body, so the behavior of the niosomes with water is very important. PBS is also st udied to observe the behavior of the niosomes in a buffer solution with constant pH of 7.4 and ionic strength of 281 mOsm . This thesis explored the behavior of the niosome solution alone in this environment. Future studies will be conducted incorporating th e niosomes inside the polymer hydrogel. The main goal of the system is to be used in drug delivery to brain tumor cells. After a malignant tumor is removed, malignant cells can still exist within the brain and the tumor cavity. These cells can reproduce an d cause a recurrence of the cancer. The proposed system would be inj ected into the tumo r cavity directly af ter surgery. The solution would gel once inside of the brain, and the opening to the brain would be closed and sealed. This eliminates contamination th at may occur due to transport and the use of other instrumentation. The stability of th e system would be known at the time of injection, thus providing better knowledge of when the drug will actu ally be released. The solution would take the shape of the cavity, reaching the entire surf ace of the cavity. The system aims at prolonging the time that the available drug is in it s therapeutic range. 1.3. Significance of This Work This thesis proposes a package within a package system that would allow for the maximum control over the release rate of the drug being delivered to brain tumor sites. The goal is to conve rt the basic insight of na nopackaging to design a more authentic device that would deliver drugs more accurately in order to reduce the cost of expensive drugs and the toxicity of the drug with other organs. The system proposed
9 involves a nanostructured drug delivery system that can control re lease rates from the nanoscopic to the macroscopic scales. The hydr ogel medium can either play a static role as a passive scaffold or an ac tive role as a stimuli-responsiv e network for the stability and release of the niosomes. The anticipated opportu nities with the system include: 1. Packaging of different type of vesicl es with respect to size, membrane composition, or loaded cargo to determin e the controlled release time-scales and chemical composition of the vesicles. 2. Packing of vesicles with gradient pr operties such as crosslink density and chemical composition to alter the releas e profiles for identical groups of the vesicles. 3. Packaging the vesicles so that they are sensitive to micro-environmental changes such as temperature and pH to allow for another control parameter. A fundamental aspect of this project is to study the re lation between the chemical composition, of both the niosome and hydrogel, to the resulting phys ical properties and its effect on the transport of molecules embedde d in the hydrogel. It is proposed to see if alterations on the nanoscopic sc ale can control for the release behavior of the system at the macroscopic level. The first control over the release rate of the drug is the release of the niosomes from the biodegradable hydrogel. The inte ractions between the properties of the niosomes and the properties of the hydrogel will provide insight on how to improve or modify the composition of either one separa tely. The hydrogel will also allow for a protective vehicle for the niosomes as the syst em is injected into the body. The hydrogel will protect the integrity of the niosome, thus, allowing the preservation of the drug inside
10 of it. This, in turn, will allow for the most e ffective delivery of the drug to the tumor site. The system will also allow for the control of when to release the niosomes and drug. This is important because re lease of the niosome and dr ug too early could result in medication being delivered to other parts of the body that do not require treatment. The hydrogel component of the system allows for the niosomes and drug to be delivered directly to the tumor site and control at what time the drug is released. This, in turn, reduces the side effects often experienced from toxicity to other parts of the body. The hydrogel is also a gel solution so it can be mo lded to any desired shape or size. This feature allows for custom designed delivery system s that will ensure for the entire area of the tumor cavity to be filled and exposed to medication. The other advantage to this system is that the variability of each tumor found in the brain. Some tumors are quite large, while other tumors can present themselves as very small. The size control allows for the determination of the optimal amount of drug needed and also decreases the toxicity problem mentioned earlier. The second control over the release rate of the drug is the release of the drug from the niosome. The niosome also provides protection to the drug from the outside environment, and prevents premature exposure to other organs in the body. The niosome can also provide protection of the drug from adversely interacting with the monomer from the hydrogel. It has been shown that th is interaction, in th e past, has caused the integrity of both the hydrogel and niosome to degrade. The concentr ation of drug within the niosome can be controlled so that the appropriate dose is being provided to the patient. Control can also be given to th e composition of the niosomes and various concentrations of them can be explored. The niosome component also allows for the
11 control of the size of the vesicl es. It has been shown that diffe rent size vesicles result in different release rates. This is another way in which we may gain greater control over the release rate of the drug. The significance of this system is that it will allow for the understanding of the release process of the niosome and polymer system. It will also allow for the understanding of the reaction process that is also occurring in the system. By understanding the release/reaction process of the system, the best conditions for the niosomes performance can be determined. F actors that account for the release behavior will be manipulated. This concept can be used for the further design of delivery systems that will increase efficiency and decrease toxicity to other parts of the body. The system that will be studied in this thesis will involve the measurement of fluorescence intensity readings that can be tr anslated into concentration values. The concentration gradient can be determined by comparing the concentration in the dialysis tubing with the concentrati on of the bulk solution in which it is submerged. The concentration gradient can be analyzed over several days to determine the behavior of the system. This analysis will allow for the determination of the behavior of the dye when the medium comes into contact with the vesicles. The concentration on each side of the membrane will be determined to see the amo unt of dye is being released over time. These concentrations are important in the de sign of this system because they provide information on the amount of drug that will actua lly be delivered to the site of infection and provide insight into how a lternations to the system can be made to improve this amount.
12 Chapter Two The Components 2.1. Niosome Since 1965, amphiphilic vesicles that are able to self-assemble have been used as vehicles for drug transport and drug delivery . The first of these vesicles was the liposome. A liposome is an amphiphilic, spherical membrane that consists of a phospholipid bilayer. The aque ous core is able to encapsu late hydrophilic drugs, while the hydrophobic membrane is able to transpor t non-polar solutions [ 15]. Liposome drug delivery systems have been reported to increase the stability of the encapsulated drug, promote the effectiveness of the drug, increase circulation time, and support the rate at which the body accepts the drug [16-23]. Howeve r, liposomes in drug delivery have their setbacks. Drug degradation by hydrolysis of the phospholipid molecule has been a problem in aqueous solutions . There have also been some stability problems of the liposome suspensions . It has been suggest ed that liposomes be prepared and stored in a vacuum or nitrogen environment to prevent phospholipid do not oxidation . Alternatives to liposome drug delivery have be en suggested. Niosomes, in particular, are an attractive alternative to liposomes in drug delivery. Niosomes were first used in the cosmetic industry by LOreal in 1972 . Niosomes consist of a non-ionic surfactant dissolved in an aqueous solv ent. Niosomes self-assemb le from non-ionic amphiphiles
and form spherical bilayers [ 25]. Energy in the form of heat or physical agitation is usually required in niosome synthesis . Similar to liposomes, the aqueous core is able to store hydrophilic molecules, while the non-polar membrane can accommodate hydrophobic solutions as seen in Figure 3 . Figure 3. Illustration of a Niosome Niosomes have many advantages over li posome for the use in drug delivery. For example, they are more chemically stable in solution, less expensiv e to make, easier to store . They also have a greater choice of non-ionic surfactants to choose, reduce the side effects in patients, prolong the rel ease of the drug, and perform transdermal procedures well . There are reports of niosomes used successfully in transdermal drug delivery, and the deliver y of anticancer, anti-tubercular, anti-leishmanial, antiinflammatory, hormonal, and oral drugs [ 26-33]. Niosomes consist of non-ionic surfactants that may derive from alkyl ethers alkyl esters, alkylamides, fatty acids, or amino acid compounds . There are severa l factors that affect the properties of niosomes. Figure 4 illustrates these factors and their relationship to one another . 13
Figure 4. Factors that Influen ce the Properties of Niosomes These factors may be used to formulate niosom es specific for a particular application and control the design of them. 2.1.1. Self-Assembly of Niosomes Structure of Surfactant : The structure of the non-ioni c surfactant affects the selfassembly of niosomes. Niosome formati on involves an amphiph ile and an aqueous solvent . Cholesterol is also needed in most cases to prevent the aggregation of the vesicles that occurs due to the repulsive steric or electrostatic interactions between molecules . An example of steric stab ilization is the addition of Solulan C24, a cholesteryl poly-24-oxyethylen e ether, in doxorubicin (DOX) sorbitan monostearate (Span 60) . An example of electrosta tic stabilization is th e addition of diacetyl phosphate in 5(6)-carboxyfluorescein encapsu lated Span 60 niosomes . Sorbitan monoester niosomes consists of alkyl esters. Figure 5 shows some common structures of these surfactants . 14
A = Sorbitan Monolaurate (Span 20), B = Sorbitan Monopalmitate (Span 40), C = Sorbitan Monostearate (Span 60) Figure 5. The Structure for Sorbitan Mo noester Vesicle Forming Surfactants The alkyl chain group of the surfactant commonly ranges from C 12 C 18 [34, 35]. The hydrophobic head group was investigated and designed to create new niosomes with unique properties . The hydrophilic lipophilic balance (HLB ) is a number that indicates the ability of any surfactant to form a vesicle . Sorbitan monostearate should have an HLB of about four to ei ght in order to be a good vesicle forming surfactant [34, 35]. Membrane Additives : The critical packing parame ter (CPP) was formulated by Israelachvili  and takes into account the geometric features of the amphiphilic monomer aggregation . It is a dimensionless number that predicts the ability of the amphiphile to form aggregates and is calculated by Equation 1 : ocaI CPP Eqn. 1 15
16 where = hydrocarbon chain volume, I c = critical hydrophobic chain length (the length above which the chain fluidity of th e hydrocarbon may no longer exist), and a o = area of hydrophilic head [25, 35]. Israel achvili  reports that a CPP value of 0.5 1.0 predicts that the amphiphile will form a vesicle. Additives must of ten be incorporated in the preparation of niosomes to st abilize the vesicles . C holesterol and diacetyl phosphate are the most common additive to niosome soluti ons to increase their stability . The bilayer of the niosome membrane may exist in th e gel state or the liqui d crystal state . The liquid state exists at a higher temperatur e than the gel state, and thus requires an increase in temperature . The increas e in energy increases the enthalpy and entropy of the system . The overall free ener gy of the system decrea ses [25, 35]. Van der Waals interactions, hydrophobic forces, hyd rogen bonding formation, and electrostatic interactions all play a role in the self-assembly of niosomes . The Effect of Cholesterol: The addition of cholesterol results in more stable niosomes. Cholesterol has s hown to eliminate the gel to liquid phase transition of niosomes  by affecting the chains in the bilayers when present in certain concentrations . The ultimate effect is that the niosomes will be less leaky, and thus more stable. Studies have shown that an e quimolar ratio of cholesterol to surfactant is optimal . Cholesterol can affect severa l biological membrane properties such as ion permeability, aggregation, elasticity, size, a nd shape [39-41]. Cholesterol also shows several mechanical properties such as cohe sion, stiffness of membranes, and membrane permeability to water . Figure 6 shows how the cholesterol and sorbitan monostearate may interact in the niosome bilayer .
Figure 6. The Interaction of Cholesterol a nd Sorbitan Monostearate in the Niosome Bilayer The small 3 -hydroxyl of the cholesterol head group is able to come parallel to the acyl chain of the sorbitan monostearate surfactan t ester group . A study has shown that a 29 mol% amount of cholesterol added to vesi cles resulted in a 50-100% increase in encapsulation efficiency . Surfactant and Lipid Levels: The surfactant/lipid level to make niosomes is usually about 10-30 mM [41, 44-52]. The properties of the niosome system can be changed by using different ratios of surfactan t to water during hydration of the niosomes . The surfactant/lipid level may also ha ve an effect on the encapsulated drug. An increase in surfactant/lipid level will incr ease how much drug will be encapsulated . Figure 7 illustrates how the selection of su rfactant may affect th e properties of the niosome system . 17
Figure 7. The Effect of Surfactant on the Properties of Niosomes Temperature: The temperature at which the niosome system is hydrated during the niosome preparation should greater than th e gel to liquid phase transition temperature of the system . 2.1.2. Nature of Encapsulated Drug 18 The nature of the encapsulated drug can a ffect the stability of the niosome. For example, Span 60 niosomes with diacetyl phosphate were loaded with carboxyfluorescein, and the resulting system formed a homogeneous dispersion . However, when the amphiphathic drug DOX was loaded into the same system, aggregation occurred . This is an i ndication that the CPP number must consider
hydrophobic and amphiphathic drugs th at could be incorporated into the niosomes . Figure 8 shows the effect the encapsulated drug may have on the niosome system . Figure 8. The Effect of the Encapsulated Drug on Niosome System The stability of liposomes has been evaluate d using dialysis met hods [54, 55]. Peer & Margalit  developed a novel procedure to entrap drugs in liposomes that involved the preparation and lyophilization of regular a nd surface-modified drug-free liposomes and drug encapsulation during liposome reconstituti on via rehydration of the drug in an aqueous solutions. It was found that the proper ties of the different drug determined the diffusion coefficient as evaluated using a dialysis membrane assay. Encapsulation Efficiency and Stability: The encapsulation efficiency of the niosomes is the result of the stability of the niosome dispersion, the loading of the 19
20 vesicles, and the intrinsic properties of the niosomes . The st ability of the niosome dispersion depends on the stability of the en capsulated drug, the retention the membrane has for the drug, the stability of the surfactant, and the structure of the vesicles . The loading of the drug and efficiency are based on the nature of the solute, the temperature of hydration, and the method used for loading [ 35]. The intrinsic properties that affect the encapsulation efficiency include vesicle size, cholesterol levels, and the composition of the membrane . The stability of the system is based not only on the aggregation of the vesicles but also on othe r factors such as osmotic beha vior and other size and shape change of the system . C holesterol has been demonstrated to enhance the retention of the solute, which is a measure of encapsula tion efficiency . The nature of the hydrophilic head group also increases the stabili zation of the system . The nature of the hydrophilic head group and the hydrophobic tail may be varied in order to increase the drug loading . Stable niosomes will also have constant particle size, constant levels of entrapped drug, and possess no memb rane precipitation . The encapsulated drug must be compatible with the components that make up the bilayer membrane . Smaller niosomes are less stable than large niosomes due to the high amount of energy needed to break them . Niosomes are considered thermodynamically stable and can exist in the metastable state that includes an excess of energy . Micelles are soluble surfactants and are not compatible with ni osomes; they will disrupt the system and a mixture of micelles and aggregates will form [41, 46, 47, 57, 58]. The length of the alkyl chain has an eff ect on encapsulation efficiency as shown in Table 1 .
Table 1. The Effect of Alkyl Chain Length on Encapsulation Capacity The table indicates that with longer the alkyl chain length, the encapsulation capacity increases. Sorbitan monoester niosomes with 5(6)-carboxyfluor escein and DOX have shown better encapsulation with C 18 alkyl chains than C 12 chains . Span 60 (C 18 ) and Span 80 (C 16 ) have shown excellent CF and DOX encapsulation efficiency and are the least leaky due to their high phase transiti on temperature . Figure 9 illustrates the encapsulation efficiency and size of CF loaded niosomes . 21
Figure 9. A Comparison of Sorbitan Monoes ters on Encapsulation Efficiency. The Solid Bars Represent the Size and the Shaded Bars Represent the Encapsulation Efficiency The stability of niosomes may be improved in other ways such as decreasing the air water interface , adding a polymer ized surfactant, including charged molecules , and the inclusion of hydrophobic drugs . Osmotic activity: The osmotic activity of niosome systems is observed in the dispersion of hypotonic mediums [2 5, 38, 45]. There is an observe d change in the size of the niosome during this occurrence. Toxicity: The toxicity of the niosome syst em was observed when there was an increase in the alkyl chain and gel to liquid transition. There was an observed decrease in the toxicity of the system and gel to liquid pha se transition when there was increase in the polyoxyethylene chain length . 22
23 2.1.3. Preparation of Niosomes The preparation of niosomes involves the self-assembly of th e vesicles through the addition of energy, which aids the pro cess. The most common way to prepare niosomes is through the hydration of a mixture of the surfactant at high temperatures . Size reduction is then performed to get the final dispersion . Centrifugation, gel filtration, or dialysis is then performed to separate the unentrapped dye from the encapsulated dye . Niosomes have been produced on the industr ial scale through a technique called Novasome . Hydration: The following hydration techniques are commonly used in laboratory settings. 1. Injecting the surfactant organic solution in to an aqueous solution of the drug and heating above the boiling point of the organic solvent . 2. Creating a surfactant film by evaporating the organic solvent in which the surfactant is dissolved. The film is hydrated with the drug solution [45, 62]. 3. Emulsifying the surfactant solution and aqueous solution of the drug in an oil in water emulsion. The niosomes are left in the dispersed aqueous phase after the organic solvent is evaporated . 4. Adding a heated aqueous phase to the surfact ant solution . Note: the use of an organic solvent is not required. 5. Using enzymatic activity in a mixed mice llar solution to form niosomes . 6. Homogenizing the surfactant mixture a nd bubbling nitrogen gas through it . Reduction of Niosome Size: Niosomes prepared by hydration are usually produced in the micron size range [24, 62]. Sub-micron sizes are used for intravenous
24 and transdermal delivery; sizes up to 10 m ar e used for intraperitoneal, intramuscular, and intracavity administration; and sizes greater than 10 m are used for drug delivery to the eye . The following are ways in wh ich the size of the niosome may be reduced, after hydration, which may play an important role in the function of the niosome. 1. Probe sonication to obtain 100-140 nm niosomes [45, 62]. 2. Use of 100 nm Nucleopore filters to obtain 140 nm niosomes . 3. Combination of sonication and filtrati on to obtain 200 nm niosomes . 4. Microfluidizer to obtain sub-50 nm niosomes . 5. High pressure homogenization to obtain 100 nm niosomes . Separation of Unentrapped Drug: A final step of removi ng an unentrapped drug is necessary because not all of the drug gets encapsulated. The met hods that have been described to separate the unentrapped dye from the encapsulated dye include: exhaustive dialysis [45, 66], gel filtration [34, 53], and centrifugation . 2.1.4. Drug Delivery Applications Niosomes have been used in a variety of drug delivery applications. They have been used intravenously as anti-infective agents, particularly for the treatment of leishmaniasis . They have also been used to deliver antica ncer drugs such as methotrexate (intravenously or orally ), DOX (cardiotoxic drug), and NDOX N(2hydroxypropylmethacrylamide) (tumors) . Niosome have also been used as antiinflammatory agents and in diagnostic imaging. Niosomes were first demonstrated in oral administration by Azmin in 1985 . It was reported that the delivery of drugs in niosomes resulted in higher levels of the drug in targeted areas when compared to the free
25 drug alone . Niosomes have also been used in transdermal drug delivery. The flexibility of their bilayers aids in the tran sdermal transport . Niosomes have also been used in ophthalmic drug delivery . Niosomes have been studied for use in drug delivery due to their attractive selfassembly and amphiphilic properties. There seems to be an endless number of different non-ionic surfactants that can be used, and the manipulation of several factors that make them ideal for any situation. The stability of niosomes has been explored and factors such as the nature of the su rfactant, the encapsulated drug us ed, and temperature all must be considered. In terms of encapsulated effi ciency, factors such as the stability of the niosome dispersion, the load ing of the drug, and intrin sic properties are all very important. The use of niosomes in drug delive ry applications is virtually unexplored. 2.2. Hydrogels Hydrogel networks have become in creasingly important for use in many biomedical and pharmaceutical applications. In particular, the use of biodegradable hydrogels coupled with drug-encapsulated niosomes has received attention for use in drug delivery. Hydrogels are crosslinked, thre e-dimensional polymer networks that swell in water but do not dissolve[ 69]. These hydrogels often unde rgo a volume change due to their swelling characteristics in response to surrounding stimuli such as temperature, pH, light, ionic strength, electric field, or even some chemicals [69, 70]. Hydrogels can be used in nanostructures and Figur e 10 summarizes these applications.
Figure 10. Hydrogels in Nanopackaging The most commonly used polymer in hydrogel networks is poly(N-isopropylacrylamide) (PNIPA), which has the ability to transition fr om a swelling characteristic to a collapsed characteristic at temperatures greater than 33C . Specifically, this means that PNIPA has a lower critical solution temp erature (LCST) of 33C. Below this temperature, it can dissolve in water. However, at temperatures above the LCST, the polymer precipitates out of solution due to the interruption of the hydrogen bonding and the hydrophobic interactions of the isopropyl groups . This alteration, based on 26
temperature and pH, allows for better control over the system and can be used for such applications as protecting drugs from degrad ation and for delivery of drugs to specific sites in the body . There have been quite a few number of research papers devoted to incorporating co-polymers in to the PNIPA network to improve the properties of the system. The use of co-polymers allows th e hydrogel network to take on advantageous properties of both polymers and improve upon th e function of the hydrogel as a whole. 2.2.1. Properties of Hydrogels Structural Parameters: There are three important structural parameters in biomedical hydrogels: the volume fraction of the polymer in the swollen state, v 2,s the number average molecular weight between cross-link units, cM and the correlation length, . The correlation length represents the distance between cross-links in the hydrogel network . The volume fraction of the polymer in the swollen state can be determined by the volume of the polymer and the volume of the swollen gel as shown in Equation 2: 1 ,2 Q V V vgel p s Eqn. 2 where V p = volume of the polymer, V gel = volume of the swollen gel, and Q = volume swelling ratio . The number average mo lecular weight between crosslink units is equal to the molecular weight of the repeating unit (M o ) divided by two times the degree of crosslinking (X)  as shown in Equation 3: X M Mo c2 Eqn. 3 27
28 Models for Network Prediction : Models that predict the formation of the network structure of hydrogels systems have been deve loped. There are cu rrently three models that are used with confidence and include ki netic models, statistical models, and Monte Carlo simulations . The kinetic models uses di fferential equations to predict the properties of the gel such as the final conve rsion of polymer to hydrogel and the average molecular weight . The component mass balance is represented by the differential equations . These models can predict aver age values for the prop erties of biomedical hydrogels and so statistical and Monte Carl o simulations have been developed that provide a more in depth analysis to the st ructure formation. They can determine the process of network formation, information on heterogeneities, and sol and gel regions . Swelling Properties: Polymer networks will swell when placed into contact with an aqueous solution because of the therm odynamic compatibility between the polymer chain and water . The swelling behavior of a polymer network is very important and can affect the proper ties of the network. Diffusive Characteristics : The diffusion of a drug or solute through a hydrogel network is very important in many biomedical applications. The diffusion coefficient of the solute can describe how a solute mol ecule will behave and takes into account the structure and size of the gel pore, the com position of the polymer, the amount of water present, and the behavior and size of the dr ug . The diffusion of the solute through the gel solvent is dependent on interactions between the drug, the gel polymer, and the solvent . The diffusion process may be slowed down from interactions between the drug and gel polymer . Large drugs will diffuse very differently than small, rigid
29 drugs . It is thus impor tant to consider the diffusive characteristics of the hydrogel system under consideration. Surface Properties : Hydrogels have been described as behaving similar to polymer brushes due to the various polymer chains that stem from the attacked end of the network . This property makes hydrogels adjust to different solvent environments . The frictional properties of hydrogels have been studied , and it was determined that hydrogels have a lower frictional coefficient than most solids, which may be due to the water solv ation of the hydrogel . 2.2.2. Chemical and Physical Hydrogels Hydrogels can be classified in two categ ories: chemical hydrogels or physical hydrogels. Chemical hydrogels, al so referred to as permane nt hydrogels, are gels that are formed from irreversible covalent links between different polymer chains [76-78]. Physical hydrogels, also referred to as rev ersible hydrogels, are gels that are formed from several different reversible links and phys ical interactions . They are held together by secondary forces such as ionic bonding, hydrogen bonding, and hydrophobic forces . Chemically crosslinked hydrogels can be made in several different ways: 1. crosslinking by radical polymerization; 2. crosslinking by ch emical reaction of complementary groups such as aldehydes, crosslinking by addition reactions, or crosslinking by condensation r eactions; 3. crosslinking by hi gh energy irradiation or; 4. crosslinking using enzymes [7 7]. Physically crosslinke d hydrogels can be made in several different ways as well: 1. crossli nking by ionic interactions; 2. crosslinking by crystallization in homopolymer systems and stereocomplex formation; 3. physically
crosslinked hydrogels from amphilphilic bl ock and graft copolymers; 4. hydrophobized polysaccharides; 5. crosslinking by hydroge n bonding or; 6. crosslinking by protein interactions . Figure 11 is an illustration of the difference between a chemical hydrogel and a physical hydrogel : Figure 11. Differences Between a Chemic al Hydrogel and a Physical Hydrogel Chemical hydrogels can be synthesized by th e following methods: 1. the polymer can crosslinked in the solid state or in solution with radiation, chemical crosslinkers, or multifunctional reactive compounds; 2. a monome r and crosslinker in solution can be copolymerized; 3. a monomer and a multi-func tional macromer can be copolymerized; 4. a monomer can be copolymerized within a different solid polymer to form an interpenetrating polymer network (IPN) or; 5. a hydrophobic monomer can be chemically converted to a hydrogel . Physical hydroge ls can be synthesized by the following methods: 1. a polymer solution can be warmed to form a ge l; 2. a polymer solution can be cooled to form a gel; 3. crosslink a polymer in an aqueous solution; 4. two different polymers in the same aqueous solution can form a hydrogen-bonded gel by lowering the 30
31 pH; 5. a polyanion and a polycation can be mixe d together to form a coacervate gel or; 6. a polyelectrolyte solution and a multivalent ion can gel together . 2.2.3. Free Radical Polymerization Hydrogels can be formed with or without the addition of a crosslinking agent. The crosslinking agent is very important in the formation of a hydrogel because it interconnects the polymeric chains and form s the 3-D structure of the gel . Crosslinkers are generally mu ch smaller in molecular weig ht than the monomer chains they connect, and the selection of a crosslinke r is critical for the properties that the gel will exhibit . Crosslinked chitosan hydrogels may be linked either covalently or ionically. The synthesis of both will be discussed in this paper. There are several different notable macromolecular structures for covalently crosslinked hydrogels : chitosan crosslinked with it self, hybrid polymer networ ks (HPN), semior fullinterpenetrating polymer networks (IPN)  block or grafted copolymers, or physical blends. The IPN and the grafted copolym ers are the most popular examples of chemically crosslinked hydrogels in the literature and will be discussed further. Physically crosslinked ionic hydrog els will be discussed later. The difference between a semi-IPN and a grafted chitosan hydrogel is that the semi-IPN hydrogel traps unreacted chitosan with in a polymer matrix, while the grafted chitosan hydrogel is functiona lized by grafting a nother polymer. Both hydrogels are desired because the hydrogel wi ll exhibit the characteristics of both polymers. Figure 12 is an illustration of a semi-IPN and a grafted chitosan hydrogel .
Figure 12. Differences Between a Semi -IPN Hydrogel and a Graft Hydrogel The polymerization of the hydrogel is usually done via a free radical polymerization or gamma-irradiation. UV light is a common initi ator of the polymerization. Covalent crosslinked chitosan requires multifunctional molecules as crosslinkers . Dialdehydes, such as glutaraldehyde, is one common crosslinker used in the polymerization of chitosan hydrogels. Figur e 13 illustrates the mechanism from which chitosan is able to crosslink with glut araldehyde to form a Schiffs base . Figure 13. Mechanism of Chitosan Crosslinking with Glutaraldehyde 32
33 The nitrogen of the amino group acts as a nucleophile and att acks the carbon of the aldehyde. A C=N bond, a Schiffs base, is formed when a water molecule is lost due to the displacement of the oxygen from the aldehyde . 2.2.4. Ionic Crosslinking Ionically crosslinked chitosan hydrogels contain reversible links. The chitosan polymer contains a positive charge and can react with ions or molecules that are negatively charged. These an ions or anionic mo lecules create ionic bridging between chitosan chains and form the hydrogel network [ 76]. This type of chitosan hydrogel is often preferred over the covalently cros slinked chitosan hydrogels, especially in biomedical applications, due to the toxicity that most crosslinkers posses. Ionic crosslinking requires chitosan in solution and at least one charged i onic crosslinker . The ionic crosslinker must have multivalent counter-ions . The crosslinking process is very simple and easy compared to the covalent crosslinking procedure, and a catalyst is not required for ionic crosslinking  Crosslinking can occur when the ionic crosslinker is added to chitosan; this can be done by dipping strips of chitosan into the crosslinker solution or by adding them together with a syringe . Figure 14 illustrates the interactions in an ionic crosslinking hydrogel network .
Figure 14. Ionic Crosslinking in Hydrogel Ionically crosslinked chitosan hydrogels can be used in a variety of biomedical and pharmaceutical applications such as controlled drug delivery, films, gel beads, and bone regeneration . These hydrogels are also able to swell in acidic and basic pH solutions; however, they are extremel y sensitive to pH conditions . 2.2.5. Proposed Hydrogel The literature suggests many different opt ions and choices for the number and type of hydrogels available. It is desire d to choose a physical hydrogel over a chemical hydrogel to eliminate the need for a toxic cr osslinking agent. Many chemical hydrogels also undergo free radical polymerization whic h often requires the use of UV light to initiate the process. It is desired to use a physical hydrogel that wi ll form from physical interactions and will not requi re the use of any external energy. Alginate hydrogels and chitosan hydrogel are of the most common physi cal hydrogels in the literature. The use of alginate hydrogels requires the presence of a divalent metal cation to form the gel [82, 83]. These hydrogels have also shown limitatio n in degradation  These properties are not attractive as the presen ce of a divalent metal cation could be challenging to find in 34
the body in enough concentration to induce gellat ion. Another disadvantage is that the hydrogel must be degradable in the body. Chitosan hydrogels have been used in biomedical applications and have shown to be both biodegradable and biocompatible. They can be synthesized to be temperature or pH sensitive and have shown to gel at body temperature. I have chosen to study chitosa n hydrogels because it has the potential to be a good hydrogel for our system. At this point, th e choice of chitosan is only theoretical in nature as in depth investigations of its proper ties will be studied in the future work of this project. 2.2.6. Chitosan Chitosan contains a hydroxyl group a nd an amino functional group from the deacetylation of chitin [70, 71, 79, 84, 85]. Fi gure 15 illustrates the chemical structure of chitosan . Figure 15. Chemical Structure of Chitosan 35 It is biocompatible, pH-dependent, cationic, and water soluble up to pH 6.2 . Chitosan can be dissolved in acidic solutions, thus protonating the amino group and creating a polycation [70, 85]. Chitosan behaves as a polycation at physiological pH  and is pH sensitive due to the many amino groups it contains [69, 70]. Chitosans reactivity makes it ideal for ch emical reactions, salt formation , and the formation of
36 intermolecular complexes with carboxylic a nd polycarboxylic acids . Chitosan is also an attractive polymer for use in drug de livery because it ha s shown to be highly biocompatible and biodegradable [70, 71, 76, 85], nontoxic [70, 71], abundant , cheap to produce , cell growth stimulator and protein absorber , affinity for cells , bioadhesive [71, 76], and hydrophili c . Chitosan has been used in such applications as topical ocular applications , implantati on , injection , drug delivery carriers [69-71] surg ical thread , wound healin g [69, 70, 86], anticoagulant , thickening and film forming , penetration enhancer , and is metabolized by enzymes in the body such as lysozyme  The molecular weight and degree of deacetylation govern the way that chitosan will behave . 2.2.7. Chitosan Hydrogel Synthesis One particular chitosan hydrogel uses the polyol counterionic monohead salt glycerophosphate (GP) as the io nic crosslinker. Chitosan/ -GP hydrogels can be prepared by preparing the chitosan solution by itself in deionized water at room temperature. The chitosan powder is added to the DI water and continued to stir and mix for one hour [11, 87, 88]. The solution is steri lized in an autoclave. A chilled solution of -GP is also prepared in deionized water. It is sterilized by filtration. The two solutions are chilled in an ice bath for about 15 minutes. The -GP solution is added drop wise to the chitosan while stirring. Th e entire solution is stirred for about ten more minutes [11, 87, 88]. The -GP lowers the temperature at which chitosan will begin to gel; it creates a shield around the chitosan molecules that allo ws them to retain their shape until large amounts of energy is added to break them ap art. Once the temperature is raised, the
37 carbon-carbon bonds will be disturbed. The gellation process occurs due to the following interactions: electrostatic attraction between the ammoni um group of the chitosan and the phosphate group of the glycerophosphate the hydrogen bonding between the chitosan chains, and the chitosan-chitosan hydrophobic interactions . Upon gellation, the neutralized chitosan amine groups will begin hydrogen bonding and hydrophobic interactions after the repulsive electrostatic forces are gone . 2.2.8. Intermolecular Interactions Temperature and pH Sensitivity As stated above, there are three interact ions that occur between chitosan and GP: electrostatic attraction between th e ammonium group of the chitosan and the phosphate group of the glycerophosphate, th e hydrogen bonding be tween the chitosan chains, and the chitosan-chitosan hydr ophobic interactions . The chitosan/ -GP will remain a liquid at low temperatures and a pH up to 6.2 . An increase in temperature will begin the gellation process. Chitosan ch ains are stable at low temperatures and weakened at high due to repulsive forces between the chains [11, 89]. It is suspected that the polyol part of the -GP prevents the gellation of ch itosan at low temperatures [11, 89]. The polyol most likely forms a shield of water around the chit osan molecules , thus increasing the transition temperature of th e solution [91, 92]. This, in turn, requires more energy, or temperature to break through th is barrier. Ruel-Gariepy  suggested that the addition of -GP would prevent the gel formation of the chitosan network at low temperatures and neutral pHs. They postula ted that when the temperature was raised, there would be an increase in chitosan-ch itosan hydrophobic interactions and gellation
38 would occur at this point. The chitosan would lose its water shields and the chitosan chains would be allowed to bond . The st ability of the chitosan at room temperature and the time of gellation increases as th e degree of deacetylat ion of the chitosan decreases . One study showed that an 84% deacetylation could remain stable stored for three months at 4C . 2.2.9. Biomedical Applications Hydrogel systems have been used in a variety of biomedical applications. Hydrogels have been used in applications wh ere a controlled release of a drug is needed. They have been used in systems where a target concentration of the drug must be maintained in the blood stream for extended peri ods of time . They can be used for drug diffusion, environmental sensitivity applications, and incorporation of functional groups to target specific sites . They ha ve been used in swelling-controlled release systems where controlled release is desired [ 72]. They are attractive for environmental response systems due to their response to cha nge in pH, temperature, ionic strength, and solvent composition . They have been used as blood-compatible hydrogels, in contact lenses, as artificial tendons, artificial kidneys, me mbranes for plasmapheresis, artificial liver, artificial skin, mammaplast y, vocal cord reconstruction, sexual organ reconstruction, ophthalmic applicati ons, and articular cartilage . 2.3. Cellulose Membrane A cellulose membrane is used to mimic the brain cavity in vitro in the experiments performed in this thesis. It is used in dialysis experiment in which the
behavior of the cell membrane is desired. Cellulose is a natural linear natural polysaccharide polymer that consists of (1 4) linked -D-glucopyranosyl unit . Figure 16 illustrates the chemical structure of cellulose . Figure 16. The Chemical Structure of Cellulose A typical cellulose polymer chain will contai n about 7,000 to 15,000 repeat units . Cellulose is the worlds most abundant natu ral resource and can be found in vegetable matter, plants, trees, cell walls, and cotton fibers . Cellulose is the main structural component of the cell wall of mo st plants . Cellulose was originally used in the paper-making industry and sparked the interest of chemists and biologists . Cellulose can be manufactured into cellulose derivates such as ethers an d esters or into regenerated materials such as fibers, films, me mbranes, sponges, and food casing . Cellulose derivatives have been used in the manufacturing of membranes . Microfiltration and ultrafiltration membranes have been made from cellulose acetate and reverse osmosis membranes have been manufact ured from cellulose triacetate . Cellulose acetate can be made from acetic a nhydride, acetic acid, and the catalyst sulfuric acid to produce a degree of substitution of about 2.4 . Regenerated cellulose has been a very attractive source for dialysis membrane due to its high hydrophilic nature 39
40 . The cellulose membrane is also resistant to dissolving in water, which makes it an ideal choice in dialysis anal ysis . Regenerated cellu lose membranes have a high degree of swelling due to their hydrophilic tendency and negative fixed charge created from the COOH group . The semi-permeab ility of the cellulose membrane allows for the transport of small molecules such as ions and low molecular weigh substances through the pores of the membrane . The molecular weight cut off (MWCO) of the membrane will prohibit larger molecules or high molecular weight macromolecules from diffusing . The control of molecules acro ss the regenerated cellulose membrane can be attributed to the cr ystalline nature of the cellulose as well as the hydrogen bonding that takes place between the hydroxyl groups of the molecule . The behavior of transport across a cellulose membrane is based on the permeability of the solute, the water, and vapour . The transport of molecules across the membrane can also be characterized by electrical parameters, when dealing with an electrolytic solution, and include conductivity, ion tran sport, and concentration of fixed charge in the membrane . The carboxylic acid group (-COOH) in the structure of cellulose makes the membrane weak to cation-exchange [101, 102]. 2.4. 5(6)-Carboxyfluorescein A fluorescent dye, 5(6)-carboxyfluorescein can be encapsulated in the aqueous core of niosomes. The dye can be an i ndication of how similar molecular weight molecules will behave under the same conditions. The advantage of using a fluorescent species is that fluorescent sp ectroscopy methods can be used to measure the intensity of the dye. The 5(6)-carboxyfluorescein dye that will be used in the experiments performed
in this paper is obtained from Biotium Inc. The chemical formula for it is C 21 H 12 O 7 The molecular weight of the dye is 376 g/mol. Fi gure 17 illustrates the chemical structure for 5(6)-carboxyfluor escein . Figure 17. Chemical Structure of 5(6)-Carboxyfluorescein The 5(6)-carboxyfluorescein molecule will form a trivalent anion when the pH is increased (physiologic and alkaline) due to the deprotonation of the hydroxyl groups . The carboxyl group will attacked at either the 5 or 6 carbon in the structure . 5(6)-Carboxyfluorescein is a ye llow orange solid that can be dissolved in water with a pH that is greater than 6 . It should be stored at a temp erature of 4C and have minimal exposure to light . When the dye is in solution, there should be specia l care taken to avoid extended periods of light exposure. At pH 9, the excita tion/emission wavelength ( ex / em ) of 5(6)-carboxyf luorescein is 492 nm/514 nm . 41
42 Chapter Three Problem Presentation The following chapter will discuss a current method that is used for the treatment of brain tumors and a controlled drug deliv ery method that uses liposomes in hydrogels that could be designed for brain tumor treat ment applications. The chapter will then compare these treatments to my proposed drug delivery system. I will then present the goals and aims of the project. 3.1. Previous Work One 3.1.1. Gliadel Wafer The challenges of drug deliv ery to brain tumor sites ha ve been investigated in several instances. There is a general unde rstanding that a contro lled delivery system would eliminate some of the complications that are often associated with brain tumors. The most significant of the c ontributions to this field of work would be the use of biodegradable polymer wafers. A chemotherapeutic drug is incorporated into a polymer and implanted in the cavity of a brain tumor af ter surgery. The wafers slowly release the drug over a two week time period as the po lymer slowly degrades [104, 105]. The Polymer-Brain Treatment Tumor Group was one particular group involved in some of the early studies of implanting these wafers in patie nts. Studies first began with patients who
43 required surgery and treatment after a tumor ha d reoccurred. Later studies conducted by various other research groups ha ve been studied on the effect of the wafers after a first surgery . The motivation for the use of this delivery system was the high recurrence rate of brain tumors, the limitations of drug delivery through the blood stream to the brain, and the risk of exposing other parts of the body to the drug . The average survival time for a patient diagnosed with a high-grade glioma, even after surgery and subsequent therapy, is less than one year . The idea to implant a biodegradable polymer offers sustained release to the targeted site and eliminates the need to cross the blood brain barrier . The wafers consisted of the polymer poly(carboxyphenoxypropane/sebaic acid) anhydride. This polyanhydride polymer is commonly called BIODEL and consists of a 20/80 ratio of poly-carboxyphenoxypropane and seba ic acid . Methyl chloride was used to dissolve the BIODEL and the dr ug. The hydrophobic environment of the polymer protects the drug from hydrolysis [ 108]. The chemotherapy drug that was used in the wafers was carmustine (BCNU). The resulting solution was sprayed into 1.4 cm (diameter) by 1.0 mm (thickness) microspheres and sterilized with gamma irradiation . The polymer was loaded with 50 g carmustine/mm 3 and resulted in wafers that contained 7.7 mg of the drug . There could be up to eight wafers placed in the tumor cavity so that the maximum patient dose was 62 mg . The Polymer Brain Tumor Treatment Group administered these wa fers to 222 patients in 27 at medical centers . The selection criterion wa s based on patients who needed to be reoperated on due to a recurrent malignant brain tumor . The results of this study showed that the six-month survival time in polymer disc treated pa tients was 50% greater
44 than those patients treated with a placebo pill . Fr om early studies, the wafer appeared to be easy to implant, safe from adverse side effect s, and clinically effective for treatment of high grade gliomas [104, 108, 109]. Recent studies suggest that the earlier optimism asso ciated with the Gliadel wafers was premature. Implantation has shown to cause extensive local edema and inflammation . The could lead to compli cations such as seizures, cerebral edema, tumor bed cyst formation, abnormal wound heal ing, and increase for infection [108, 112, 113]. Earlier studies report an infection rate of less than 5%, while recent studies suggest that the rate of infection was as high as 15%-23% [114, 115]. A study conducted at the University of Pennsylvania (Philadelphia) co mpared the risk of patients developing a surgical site infection (SSI) based on their use of Gliadel wafers. They found that 9 out of the 32 patients included in the study developed a surgical site infection . The 28% of patients who developed an infecti on after Gliadel wafer implantation is the highest percentage reported [ 111]. The infections that de veloped included one patient with cellulitis, one patient with subgaleal absc ess, four patients with bone flap ostetitis, two patients with epidural abs cesses, four patients with pa renchymal brain abscesses, and one patient with meningitis . The averag e time from the time of implantation to the time of infection was 23 days, and there were four patients who developed more than one infection . The study suggest that Gliade l wafers may contribute to the formation of these abscesses due to the prolonged exposure of drug to th e surrounding tissue, extended inflammation, and continued edema . Th e wafers are believed to contribute, along with other factors such as radiation th erapy and subsequent operations, to the development of infections [ 111]. Gliadel wafer were first approved for clinical use on
45 September 24 th 1996 by the US Food and Drug Administration . They were approved for use in the treatment of brain recurrent brain tumors. The FDA later approved the wafers for use in newly diagnosed patients with high grade gliomas on February 26, 2003 . The most recent study by We stphal (2003)  indicated that the average survival of a patient treated w ith Gliadel wafers was 13.9 months compared to 11.6 months for those patients receiving a placebo pill. 3.2. Previous Work Two 3.2.1. Liposomes in Hydrogels Liposomes alone have been investigated fo r use in drug delivery, particularly for chemotherapy applications. It has been shown that normal chemotherapy involves the administration of very toxic drugs to the brai n and that only about 1% of the drug will be delivered to the infected site . The remainder drug will cause toxicity to other organs of the body. Liposomes have been used to encapsulat e the drug to reduce toxicity. However, the reduced toxicity is actually due to the quick uptake of the liposomes by the immune system . Theref ore, treatment with liposomes resulted in reduced toxicity but also a reduced in therapeutic efficacy  However, the development of sterically stabile liposomes reduced the uptake of th e vesicles. This phe nomena is due to opsonization, which is the adso rption of proteins based on tagging foreign particles for the uptakes by the bodys immune system . Sterically stable liposomes are effective in brain tumor treatment due to the nature of the tumor tissue. The tumor tissue is comprised of poorly formed and leaky vascul ature . Small, stable vesicles are allowed to gather in these sites. Liposomes alone have show n promise for drug delivery;
46 however, other suggestions in drug deliver y have been made incorporating these characteristics of liposomes with other mechanisms. There have been several suggestions for th e use of in-situ forming systems for use in drug delivery [120-124]. Photoinitiation methods have been suggested for use in injured arteries , but re quires the use of a photoiniti ator and laser . Selfassembling amino acids gels have to be carefully selected due to their tendency to gel at room temperature , The idea of incor porating a water solubl e polymer in a watermiscible solvent and initia ting a precipitate polymer im plant [125, 126] poses the possibility of premature release of the drug due to the risk of partial precipitation . Thermosensitive systems have been suggested to eliminate the need for organic solvents and copolymerization components . The polymer must be carefully selected to be biodegradable and biocompatible. A formulatio n that involves the in -situ gel forming of a biodegradable and biocompatible polymer ha s been suggested that can be heated to about 45C and will gel when cooled to body temperature . However, this method is impractical because it invo lves the burdensome step of heating the solution. RuelGariepy  discussed one particular form ulation that addresse s this problem and involves the formation of a gel when the temp erature is raised to body temperature. He also decided to incorporate liposomes in thes e temperature sensitive hydrogels in order to sustain the release of hydrophi lic molecules. It was show n that drugs incorporated directly into the hydrogel system have a high release rate due to the presence of pores created by the high water cont ent of the hydrogel . Ruel-Gariepy  previously demonstrat ed that the release of small hydrophilic compounds incorporated into a chitosan--glycerophosphate occurs within 24 hours .
47 The concentration and the nature of the pol ymer did not improve on the release time. Therefore, Ruel-Gariepy  decided to load the hydrophilic com pounds into liposomes and incorporate them into the hydrogel netw ork. The researchers investigated what effects the liposomes would have on the gelli ng behavior of the hydrogel. It was shown that the liposomes decreased the sol-gel lag time and increased the strength of the gel . It was observed that high concentratio ns of liposomes disrupted the crosslinks of the polymer . The authors also studied th e viscoelasticity of the system, as well as the release kinetics of encapsulated dye . They found that the re lease rates of the dye was strongly dependent on the size of the li posome and the composition of them . For example, certain amounts of cholesterol were added to the liposomes to determine the effect it would have on the system. The au thors found that they were able to produce a controlled release rate over a two week time period . Murdan conducted research that uses niosomes and sorbitan monostearate/polysorbate 20 organogels. This work was primarily used to study these vesicle-in-water-in-oil (v/w/o) gels for the de livery of vaccines . The v/w/o gels are formed from an emulsion process at high temp eratures. The gel forms when the solution is cooled. The aqueous niosomes are dispersed throughout th e oil medium. The niosomes form mixed surfactant films at the interface of the water and oil. A thermosensitive semi-solid gel was form ed upon cooling and the niosomes selfassembled themselves into tubular and fibr illar aggregates [129, 130]. The organogel consisted or sorbitan monostearate and polys orbate 20 dissolved in an organic solvent such as hexadecane or isopropyl myristate. Th e results showed that the v/w/o gel showed strong properties after intramus cular administration . Th e w/o and v/w/o gels were
48 shown to improve the primary and secondary humoral immune responses to haemagglutinin antigen . 3.3. Comparison to Smart-Packaging The smart-packaging system is an impr oved design of the Gliadel wafer system for several reasons. Although the proposed system of this thesis seeks to be applied for drug delivery in a similar manner, the design addresses several factors that make these wafers undesirable. The pr oposed design improves upon the ove r exposure and toxicity problems that these wafers cause because the delivery of the drug from the wafer is done only in a semi-controlled fashion. Once the wa fers are implanted, they will degrade over a set period of time, depending on how many wafe rs are placed in the cavity. The smartpackaging system will allow us to have maxi mum control over the release of the drug and could be designed specifically for each indi vidual. The smart-packaging design allows for the controlled release of th e drug, thus, eliminating the ex posure of other organs in the body to unneeded medication. The drug delivery design also incorporates the use of a flexible, moldable hydrogel that will fill the shape of the entire cavity, thus, allowing for maximum contact area between the drug and th e tumor cells. The wafers possessed a predetermined shape, thus, reducing the contact surface area between the drug and the cavity . The smart-packag ing system has a similar en d-goal in mind; however, the design greatly increases the delivery of the drug to the tumor cavity while eliminating harmful side effects. The Gliadel wafers ha ve also been shown to increase survival by only a couple of months. The smart-packagin g design wishes to not only increase the survival of these patients, but would elim inate the risk of death due to the tumor
49 altogether. Any increase in survival time is an improvement, as the Gliadel wafers currently offer. The novel control system th at is being proposed will hopefully increase survival to a significant amount of time and eliminate the tumor completely. Liposomes have shown positive potenti al in drug delivery, especially for chemotherapy applications. However, the ster ic stability of the liposomes plays a major role in the efficacy of the system. In th e proposal, it is suggested to use a non-ionic surfactant vesicle which has shown better stability than liposomes. It is the intention that this advantage will improve the current systems available based on liposomes. The publication that discusses the use of liposomes and chitosan/-glycerophosphate hydrogels for the delivery of low molecular weight compounds such as carboxyfluorescein dye is simila r to our design; however, th ere are several differences and improvements in the smart-packaging desi gn . The author s found great success in their design. However, the smart-packag ing design is different in that synthetic amphiphilic surfactants, niosomes, will be used instead of liposomes. Niosomes have shown to be more chemically st able in the vesicle state. This could provide for a better release rate of the drug. Niosomes have also been shown to be easier to store and handle. This ultimately means that they will be more convenient and user-f riendly than liposomes for use in drug delivery applications. Niosomes are also less expensive than liposomes. This is advantageous because it means that we can design a better, improved system for less money. For the consumer, this means th at a life-saving procedure can be performed at a lower cost and be more effective, t hus, increasing the demand from the market if more people are able to afford it and have it improve their quality of life. The authors of the liposome/hydrogel system are also interest ed in observing the behavior of release
50 based primarily on changes in the niosome composition. For example, they altered the amount of cholesterol content, the size of the niosomes, and added an enzyme in some cases. The smart-packaging system will be interested in observing the behavior of the system when both or either the hydrogel or ni osome component is altered. The proposed plan is to determine how the composition of the hydrogel changes the release behavior and what is the best composition for intera ction between the niosome and hydrogel. The proposed system is interested in the niosom e and hydrogel as they work together and not as individual pieces. It is the intent to treat the niosome and hydrogel as separate but equal entities, but moreover, how they behave as one. The me ntioned article also did not have a specific aim in mind. They noted that their system could have the potential to be used in drug delivery. The advantage and improvement of the proposed smart-packaging design is that there is a specific market that it is targeting. The system will be able to be custom designed to be used in brain tumor surgeries. Although the desire to remain broad and multifunctional is also intended, th e proposed system suggests one particular application for the invention that will en able the testing of the design in specific in vivo samples. The work concerning the niosomes in organogels only slightly compares to the proposed smart-packaging design. The focus of the niosome/organogel was to use that system for the delivery of antigens. There are different considerations that must be addressed when comparing both systems. Th e main focus of the proposed system is a controlled delivery system of the drug, while the main focus of their system was to determine whether the niosome/organogel woul d actually delivery the antigen to the respective site. It is known that the niosome/hydrogel will make it to the desired site; it is
51 desired to focus on controlled delivery at the site. The niosome/organogel system was also prepared by heating the mixture and coolin g it to gel. This heating factor in their design process is impractical and neith er time nor cost effective. 3.4. Research Objectives 3.4.1. First Objective The first objective of this thes is is to study the behavior of the release of the 5(6)carboxyfluorescein from the niosomes. This is a critical, initial st ep towards the design of the smart-packaging system that include s the hydrogel because the behavior of the niosomes must first be understood. It is desired to observe how the dye will act when encapsulated in the niosomes and how the dye will behave once released from the niosomes. The release behavior will provide crucial information about the system such as the concentration, volume, and size of the vesi cles that will result in the most efficient design. The release behavior will also provide information about the interactions between the niosomes in the solution and the bulk solution that comes into contact with them. The majority of the dye initially rele ased and the behavior of the dye over time can be determined. Any equilibrium tendencies the high concentration of the niosomes solution has with the neutral concentration of the surrounding bulk fluid can also be observed. 3.4.2. Second Objective The second objective of this thesis is to run the system with in vitro experiments. It is desired to do this because this system is being designed to one day be used in brain
52 tumor patients. By creating an in vitro environment, a better understanding of how the system will behave under similar conditions can be seen. It is important to run the experiment in vitro because there are unforeseen problems that may occur during traditional bench top experiments that in vitro experiments will expose before in vivo experiments are performed. There are more specific conditions of the brain that will need to be addressed for future work; howev er, it is desired to create several in vitro conditions in this thesis that will provide informa tion on how the system will work when run at comparable situations of the brain environment. 3.4.3. Third Objective The third objective of this th esis is to look ahead to the future of the system. The objective is to one day create the perfect system for drug delivery to tumor patients by increasing survival time and lowering side eff ects. The information obtained from this thesis will provide insight as to the next step in the project. The purpose of this thesis is not to save the world overnight, but make a contribution to the understanding of the design of a delivery system that will one day be flawless. There are several different complex components of the smart-packaging system that are proposed and time must be spent on each of them to ensure that all fa ctors are considered. The information obtained here will provide a better perspective on what is essential for the future of the project and the possibility that what was once considered im portant may not be sign ificant after all.
53 3.5. Research Aims 3.5.1. First Aim The first aim of this thesis attempted to achieve the first objective of the thesis: to determine the release rates of the dye. This was done by setting up bench top experiments and observing the diffusion of the dye into a bulk fluid separated by a semipermeable membrane. The change in concen tration of the bulk fluid and the initial solution over several time intervals was determ ined. The use of fluorescent dye allowed for the use of fluorescence spectrometry to measure the intensity of the dye. These intensities were translated into concen tration measurements and the change in concentration provided information on the release of the dye from the niosomes. The experiments were run at different time inte rvals to determine the crucial release time behavior of the dye that should be noted. The moles of the dye were considered with the concentration of dye because the number of mo les is a direct indication of the amount of dye present. 3.5.2. Second Aim The second aim of this thesis attempted to address the second objective of this project: to run the experiments in vitro This was accomplished by setting certain environmental conditions that were similar to the conditions of the br ain. For example, a semi-permeable membrane was used to mimic the cell membrane of the body that will only allow certain molecules from passing th rough it. Regenerated cellulose has been used in previous dialysis experiments and will be used to observe th e behavior of the dye across it. The fluorescent dye that was chos en based on the molecula r weight of the drug
54 that will one day be loaded it its place. The molecular wei ght of the CF dye is about 376 g/mol, while the molecular weight of the drug that will be considered (Temozolomide) is about 194 g/mol. Different dosages of Temozolomide were found  and the concentration encapsulated in the niosomes was determined based on similar amounts. The niosomes were initially loaded with a 5mM concentration of CF dye. Different concentrations of the CF dye that were compar able to the loading of drug that are already used for treatment were considered. 3.5.3. Third Aim The third aim of this thesis attempted to accomplish the third objective of this thesis: to design, for the future, the perfect drug delivery system. This was accomplished by 1) gathering information on past systems that failed and addressing the downfalls of them; 2) obtaining information on potentia l components that have shown to be successful; and 3) using the information from th e experiments performed in this thesis to decide on what information was still needed and what information needed to be revised. The design of a perfect system will take tim e to develop, but by considering aspects of each of the components, crucial information can be obtained. It is the hope of this thesis to gain valuable information from the trials and tribulations of the experiments and to move forward in an efficient direction.
55 Chapter Four Experimental and Characterization Techniques 4.1. Concentration Characterization Technique The main interest of this thesis was to determine the change in concentration over an elapsed amount of time. This information would allow us to determine the behavior of the release of the 5(6)-carboxyf luorescein dye from the niosomes and the time it took to diffuse into the beaker. The use of fluor escence spectroscopy was used to obtain the concentration changes based on intensity reading from the dye. The following chapter discusses the method of fl uorescence spectroscopy and how it was used to determine concentration. 4.1.1. Fluorescence Spectroscopy Fluorescence spectroscopy is the class of spectroscopy in which absorbing energy from an external source excites samples and th e intensity of the ener gy is observed as the atoms transition back to the gr ound state . Luminescence is the general term that is used to describe all forms of light emission th at is not from an increase in temperature . The process involves the loss of energy from th e system, where energy is provided from an external source to make the emission continuous. Photoluminescence occurs when the emission of light is caused by the absorption of infrared, ultraviolet, or visible light [133, 134]. Chem iluminescence occurs when the emission of light is based
56 on a chemical reaction, and bioluminescence is from enzymes in living systems . Photoluminescence can either be categoriz ed as fluorescence of phosphorescence. Fluorescence involves the immediate re lease of absorbed energy, about 10 -8 second, while phosphorescence involves the delayed release of absorbed energy, about 10 -6 10 2 second . The absorption and emission of light is referred to as energy being absorbed in definite units cal led quanta . The quanta ar e usually referred to in terms of N (where N = 6.023 x 10 23 molecules) quanta, or an Einstein. Appendix A shows the approximate sizes of quanta for certain ra diation types. Fluorescence spectroscopy is only concerned with the ultraviolet and visi ble regions of the spectrum where absorption results in the excitati on of the outermost electrons . The absorbed light spectrum is similar to the excitation spectrum; however, the emission spectrum has a longer wavelength than the absorbed light . Th e intensity that is emitted is proportional to the concentration of dilute so lutions that allows for quantitative measurements . There are several advantages to fluorescence sp ectroscopy that include the sensitivity that is allowable in the pictogram range, the selectivity from two characteristic wavelengths, and the variety of methods available for sa mpling such as dilute, concentrated, and suspended solutions . The phenomena th at occur in fluorescence are a result of the electromagnetic nature of light, the mol ecular structure of the species, and the environment in which the speci es has been exposed . The electronic absorption spectrum represen ts graphically the intensity of light absorbed when electrons transition in a molecu le . This is a function of frequency or wavelength of the light . Strong abso rption bands result from regions of intensity where absorbed light is high, while weak absorption bands result from regions of
intensity where absorbed light is low [ 135]. Most molecules occupy the lowest vibrational level of the ground st ate when at room temperatur e . When the molecule absorbs light, it may be excited to the first state, S 1 or second state, S s vibrational levels. Figure 18 shows the transition that the mol ecule may follow when excited to higher vibrational states . Figure 18. The Transition fr om Ground State to Higher Vibrational Levels. The Return to the Ground State Produces Fluorescence 57
58 The molecule may be excited to any of the leve ls within each electronic state. It should be noted that Figure 18 does not account for any rotational levels that may be associated with the molecule due to the fact that such inclusion would result in numerous absorption bands that would make any individual absorption band indi stinguishable . The molecule has absorbed a high amount of energy to excite it to the higher levels of each electronic state and will even tually loose energy and fall to the lower levels of each excited state. Most molecules that occupy higher than the second excited el ectronic state will undergo internal conversion, causing them to fall from the lowest level of one excited state to the highest level of the next lowest level. All molecules will eventually loose enough energy to fall to the lowest level of the first electronic state. They will occupy this level for approximately 10 -8 seconds until they fall from th e excited state back to the ground state . During this transition, they will emit energy in the form of visible or ultraviolet fluorescence [134, 135]. The lifetime of fluorescent molecules is approximately of the order of 10 -8 seconds, but the actual fluorescence occurs in approximately the order of 10 -5 seconds . A fluorescence instrument consists of generally six components [134, 135]: 1) Light source to excite the sample. 2) Monochromator to separate the light of the source from the light of the sample. 3) Sample compartment to place the sample to be analyzed. 4) Second monochromator to separate the en ergy of excitation from the energy of the samples fluorescence.
5) Photodetector to interpre t the fluorescent light into an recordable electrical signal. 6) Readout system with an amplifier to obt ain the intensity of the fluorescent light that was emitted from the molecule. Slits are also present on both sides of the monochromator to unify the exciting and fluorescent light and to contro l the wavelength range of exci ting light from the sample and fluorescent light that the photodetector re ceives . Fluores cent spectrometers can either have filters or grat ings as monochromators to co ntrol the wavelengths of the species. Figure 19 represents a typical fluorescent sp ectrometer . Figure 19. General Schematic Diagram of a Fluorescence Spectrometer The excitation, or light, source should possess the following characteristics: be relatively intense, contain wavelengths in the ultraviolet to visible range, have continuum radiance, and be stable . Xenon sources may pr ovide a good continuum from 250 to 500 nm. 59
60 The disadvantage to a xenon s ource is that stability can only be obtained when an expensive highly regulated D.C. power supply is used . Devices must be used to control the amount wavelength that is allowed to excite the sample and control the wavelength that is emitted by the sample. Filter or monochromators may be used to fulfill this responsibility. Filters allow for a broader range of wavelengths to ex cite the sample and that are emitted by it . Monochromators allow a smaller range of wave length to pass. They usually consist of diffraction gratings and slit arrangements to control the wavelengths. The user is able to select both the excitation and emission wave length for each sample by changing the width of the slits independently . Gratings limit the wavelengths by using destructive and constructi ve principles of interferences of light . The slits focus the light onto the monochromators a nd are responsible for the wave length that will be read by the detector. Therefore, the width of the slits is crucial when it come to the resolution of the machine and the selectivity that wi ll be employed. Sample compartments are where the sample to be studied is placed. It usually consists of a cell that is painted black to minimize any stray exciting light and is c overed when in use to prevent outside light from entering . The compartment is us ually positioned so that the emitted light leaves at a 90 angle from the angle that light is excited to the cell. This minimizes any stray exciting light that may interfere with the reading. The cells are typically 1 cm 2 can be made of Pyrex glass, quartz, or fused silica . The detector consists of a photomultiplier. The photomultiplier is a phototube that is able to effect the emission of the photocathode to obtain a linear amplification of primary phot ocurrent within the tube . The photomultiplier produces an electrical current that can be amplified to levels
that can be measured, when exposed to li ght. The readout device is where the output from the detector is sent and displayed . They may be galvanometers, recorders, or oscilloscopes . The fluorescence spectrometer that is used in this procedure can measure the fluorescence in a continuous scan over the ra nge of wavelength of the machine or at specified points within a range . Figur e 20 illustrates the sc hematic of the LS-3B luminescence spectrometer that is used in the experiments in this thesis . Figure 20. Schematic of the LS -B Luminescence Spectrometer The instrument has two reflection grating monochromators and a photomultiplier detector. The output from the detector is proportional to the level of emission produced from the sample . The ex citation source is a pulse xenon flash tube. The ellipsoidal mirror collects the energy from the source, and the toroid reflects it onto the entrance slit 61
62 of the excitation monochromator. The excitation monochromator consists of and entrance slit 2, 1200 lines per mm grating, a s pherical mirror, and an exit slit 1 . The grating diffracts a narrow wa velength of energy that exits at slit 1 and is determined by the setting of the grating. A stepper motor controls the angle. The toroidal and plane mirrors direct the energy from slit 1 to the sample. Upon emission, the toroidal and plane mirrors direct the energy into the entrance slit to the emission monochromator. The emission monochromator is simila r to the excitation monochromator in that it consists of a entrance slit 1, a spherical mirror, a 960 line pe r mm grating, and a slit 2 that transmits the energy to the detector. The stepper mo tor controls the angl e of the grating and determines the narrow wavelength that is a llowed to be detected by the photomultiplier . Figure 21 illustrates the optical system of the LS-3B fluorescence spectrometer .
Figure 21. Optical System of the LS-3B Fluorescence Spectrometer The emission and excitation monochromators ca n be made independent or synchronized over their ranges or set to certa in points within their range [ 133]. The slits are 10 nm in width but can be adjusted to 5 nm (excitatio n) or up to 20 nm (emission). The excitation range is from 230 nm to 720 nm. The emission range is from 250 nm to 800 nm. 4.2. Calibration Curve Technique 63 The topic of interest in measuring the fluorescent intensity of solutions was to obtain the concentration from these readings, for the purpose of this thesis. The intensity measurements taken from the fluorescent spectrometer were interpreted into concentration values based on a calibration cu rve for intensity versus concentration that
was generated. The idea be hind the calibration curve wa s to prepare several known concentrations of a sample and measure the fluorescent intensity of those samples. A plot of concentration versus intensity was made. For dilute solutions (up to about 5x10 -7 M), the relationship between concentration an d intensity were dire ctly proportional and therefore create a linear curve in this region. In the case of the e xperiments performed in this thesis, 5(6)-carboxyfluorescein dye was the dilute solution that wa s used and studied. Samples of unknown concentrations of 5(6) -carboxyfluorescein were placed in the fluorescent spectrometer, and their intensities were taken. This was the crucial procedure of this thesis, because the diffusion of the dye from the niosomes and into the beaker created concentrations that were unknown and required calculat ed. Referring back to the concentration curve and finding the values gr aphically or analytically determined the concentration of these samples. A sample of dye that was encapsulated in the niosomes was provided to create the calibration curve. The dye was given as the concentration that was in the niosomes and required dilution. For example, if 5mM (5x10 -3 M) dye was encapsulated in the niosomes, the first diluted sample was made as 5x10 -5 M. If 10 ml of this new dilution was desired, Equations 4 and 5 were used. 2211VCVC Eqn. 4 PBSmlinMxofmlV mlMxVMx 9.91051.0 )10(*)105()(*1053 1 5 1 3 Eqn. 5 It was desired to create five samples of known concentrations: 1x10 M, 1x10 M, 2.5x10 M, 3.5x10 M, 5x10 M. In order to make these samples, five dilutions of the 5x10 -5 M sample had to be made. Those five dilutions were diluted further when 64
65 placed in the fluorescence spectrometer in a 1:1 00 ratio with PBS to yield the five desired concentrations. Table 2 illustrates the preparation of the dilutions and the concentrations that were obtained when 5mM was encapsulated in the niosomes. Table 2 was used to prepare these dilutions. Table 2. Preparation of Dilutions and Concentrations for Calibration Curve Measurements Dilution Volume of 5x10 -5 M CF dye (ml) Volume of PBS water (ml) New Dilution (M) New Concentration (M) 1 0.02 0.98 1x10 -6 1x10 -8 2 0.2 0.80 1x10 -5 1x10 -7 3 0.5 0.50 2.5x10 -5 2.5x10 -7 4 0.7 0.30 3.5x10 -5 3.5x10 -7 5 1 0 5x10 -5 5x10 -7 Intensity was plotted on the y-axis, while c oncentration was plotted on the x-axis. The calibration curve yielded an equation for the line in the form of y = mx + b, where b in this case was zero. The fluorescent intensity of the unknown sample was equal to y, and the concentration was equal to x. The unknown concentration of the sample wa simply be found by dividing the fluores cent intensity by th e slope of the calibration curve. Figure 22 illustrates a typical calibration curve. In this case, more than five known samples were made and tested.
CF Concentration versus Intensity Calibration Curve y = 3.3711E+08x R2 = 9.9655E-01 0 20 40 60 80 100 120 140 160 180 0.00E+001.00E-072.00E-073.00E-074.00E-075.00E-076.00E-07 Concentration (mol/L)Intensity Figure 22. Typical Calibration Curve Using 5mM CF Dye A new calibration curve was made each time a new batch of dye was encapsulated in the niosomes. For dye that did not come from a new batch of dye, testing one known concentration was made to verify that the intensity and concentration were consistent with the previous dye. The linear relationship between intensity and concentration wa s only observed for dilute concentrations. At high concentratio ns, the curve did not behave linearly, but rather parabolic. Therefore, in order to obtain the concentrations for unknown samples, their concentration had to lie in the linear range of the concentrat ion curve. If the concentration was too high, they were diluted so that they fell in the linear range. The linear range was observed to be concentrations up to about 5x10 -7 M, which corresponded 66
67 to an intensity of about 160. The linear relationship was been observed for intensity readings up to about 250. Therefore, an intensity of much greater than 250 required diluted. The corresponding calculations accounted for the dilution.
68 Chapter Five Experimental Design and Procedure The reagents, equipment, and instrument s used in the experiments are described in Table 3, Table 4, and Table 5, respectively. 5.1. Reagents Table 3. Primary Reagents Used. Gray Shaded Area Denotes Reagents That Were Used by Dr. VanAukers Lab Group to Prepare Niosomes Reagent Common Name Concentration Serial Number Manufacturer Lot Number Description Milli-Q water H 2 O pH ~ 7.1 N/A Millipore N/A Temperature: 22.6C; TOC: 5 ppb @ 25C; 18.2 M .cm Dulbeccos Phosphate Buffer Saline (Modified) without calcium, magnesium PBS 9.55 g/L; pH ~ 7.3 Catalog #: 1760426 MP Biomedical, Inc. 17604014 Storage 2C to 8C; 5-(6)Carboxyfluorescein C 21 H 12 O 7 MW 376 g/mol Cat # 51013 Biotium N/A Store at 4C. Protect from light, especially when in solution; pH ~ 7.3 Triton-X-100 Electrophoresis grade C 14 H 22 O; Ethoxylated p-tertoctylphenol 10% MW: 628 g/mol Cat # BP151500 Fisher Scientific 33078 Storage code gray G; Shake well before using
69 Table 3 (Continued) Chloroform HPLC grade CHCl 3 99+ % Chloroform 0.75 % Ethyl Alcohol FL-141296 Fisher Scientific 42013 C606-1; RQ Chloroform UN1888; UV cutoff 244 nm; Storage code blue B Dicetyl Phosphate (Dihexadecyl phosphate) C 32 H 67 O 4 P MW: 206.1534 g/mol D2631 Sigma Aldrich 112K1306 CAS #: 2197-63-9; Store at less than 0C; EC#218-5947; Sephadex G-50 N/A N/A G508050G Sigma Aldrich 085K1268 CAS #: 9048-71-9; Dont breathe dust, moisture sensitive; Dry bead diameter: 2080 m, Bed volume: 9-11 mL/g; SL05374 Sorbitan Monostearate C 24 H 46 O 6; Span 60 MW: 430.62 g/mol S-7010 Sigma Aldrich 023K1171 CAS #: 1338-41-6; EC # 215664-9; Store at room temperature Cholesterol (5Cholesten-3 -ol) C 27 H 46 O; Equivalent to USP/NF MW: 386.65 g/mol C-8503 Sigma Aldrich 032K0060 CAS #: 5788-5; Approx. 95% (GC); EC#200-3532
70 Table 3 (Continued) Phosphate Buffered Saline PBS pH 7.4 P-3813 Sigma Aldrich 025K8208 Avoid contact and inhalation; Target organs: heart; 10 packets; Dry powder dissolved in 1L deionized water will yield 0.01 M 5.2. Equipment Table 4. Primary Equipment Used. Gray Shade d Area Denotes Reagents That Were Used by Dr. VanAukers Lab Group to Prepare Niosomes Equipment Manufacturer Model Number Serial Number Range Description Autoclavable Nichiryo, Oxford Benchmate TM Micropipette 8885-500945 Oxford Worldwide Cat # 8885501945 N/A 100 ~ 1000 L Liquid volume: 100, 500, 1000; Accuracy: 1.0 %, .8 %, 0.7 %; Reproducibility: <0.5 %, <0.3 %, <0.2 %; Adjustable increment: 1.0 L; Accuracy: +/2.0 to +/8.0 L; Reproducibility CV: < 0.5 to < 0.3 Autoclavable Nichiryo, Oxford Benchmate Micropipette 8885-500937 Oxford Worldwide Cat # 8885501937 N/A 20 ~ 200 L Liquid volume: 20, 100, 200; Accuracy: 1.0 %, .8 %, 0.8 %; Reproducibility: <0.5 %, <0.3 %, <0.2 %; Adjustable increment: 1.0 L; Accuracy: +/0.6 to +/2.0 L; Reproducibility: < 1.0 to < 0.3
71 Table 4 (Continued) Autoclavable Nichiryo, Oxford Benchmate Micropipette 8885-500911 Oxford Worldwide Cat # 8885501911 N/A 2 ~ 20 L Liquid volume: 2, 10, 20; Accuracy: .0 %, 1.0 %, 1.0 %; Reproducibility: <3.0 %, <1.0 %, <0.4 %; Adjustable increment: 0.1 L; Accuracy: +/0.16 to +/0.4 L; Reproducibility: < 3.0 to < 0.5 Redi-Tip Fisher Scientific Cat # 211978A N/A 1011000 L General purpose tips; Fisherbrand; Nonsterile; Volume: 201-1000 L; Package: 10 racks of 100 each ART100 Tips (Aerosol Resistant Tips) Molecular Bioproducts Cat # 2065 Lot # 72101 101 L Prevent contamination even under the most demanding conditions; Nuclease, nucleic acid, and pyrogen free; Presterilized; certified pure 96 tips/10 tray/5 packs/case Disposable Serological Pipettes Fisher Scientific Cat # 1367610K N/A 25 ml in 2/10 ml Individually wrapped; 50 Pipettes; Fisher Brand; Sterile plugged; Polystyrene disposable serological pipettes with magnifier stripe Regenerated Cellulose Dialysis Membrane Fisher Scientific Cat # 21152-18 N/A Nominal MWCO 1200014000 Dalton Dialysis tubing; regenerated cellulose; Fisherbrand; Type T3 membranes; 20 m wall thickness; EOT#7933; Flat width: 25 mm, vol/cm: 1.98 ml, dry cylinder diameter: 15.9 mm
72 Table 4 (Continued) Spectrum Spectra/Por closures Spectrum Laboratories Cat # 142152 N/A N/A Red closures for cellulose membranes; Gripping width: 50 mm; Quantity: 10; Strong leak proof seal; Polyamide 6.6 from DuPont (Nylon); Maximum operating temperature: 90C; Minimum operating temperature: 0C; Sanitation: Ethylene oxide, gamma irradiation, ethanol, or formaldehyde; Nonautoclavable Elastic Bands Goody 71291 1712911W0 75 pieces Ouchless Goody hair bands Disposable Centrifuge Tubes Fisher Scientific Cat # 05539-1 N/A 15 ml (in 2-14.5 ml) Sterile; Fisher brand; Polypropylene; Plug seal cap; O.D x L: 17 x 119 mm; Max RCF: 6000xG; Graduated in 0.5ml subdivisions from 2 to 14.5 ml Fisher Isotemp Ceramic Top Stirring Hotplates Fisher Scientific Cat # 11600-49SH 312N3531 Stir: 60 1200 rpm Hot top: 30 550C; Laboratory Instruments (1000186); 120 volts, 7.5 amps, 60 Hz; Isotemp reflective white ceramic plate (7x7 in, 17.8x17.8 cm); Acid/alkali resistant; Load capacity: 40 lb. (18.1kg); heating surface area: 49 sq in (316cm2) Cimarec TM Digital Stirring Hot Plates Barnstead International Model SP131325 1313050139770 50 to 1200 rpm Phase 1; Amp: 8.9, 120 volts, 60 Hz; Aluminum top/ceramic top; Adjustable in 5 increments, from 5540C
73 Table 4 (Continued) Corning PC410 Stirrer Corning PC-410 04050569 60 to 1100 rpm 120 V, 60 Hz, 73 Watts; Top size: 5 x 7 in; Top style: pyroceram; Weight: 6.5 lbs; Dimensions (H x W x D): 4.4 x 5.8 x 9.6 in Corning PC353 Stirrer Corning PC-353 N/A 80 to 1000 rpm Top: 5 x 7 in; Ceramic top resistant to scratches, chemicals, and corrosion; Base: epoxy coated aluminum Vortex Genie 2 Fisher Scientific Cat # 12-812 2-148767 Scientific Industries; Model G-560; 0.5 amps, 120 volts, 60 Hz Parafilm M Fisher Scientific Cat # 13374-16 N/A 2in W x 250 ft L American National Can PM 992; 1 in diameter core; Flexible, moldable, self sealing, odorless, thermoplastic, moisture resistant, semi-transparent, practically colorless, waterproof Aluminum Foil Reynolds N/A N/A 200 ft 2 (66 2/3 yds x 12 in) 18.5 m 2 (60.9 m x 304 mm) Rotary Evaporator Buchi Rotavapor R200 N/A N/A Buchi-Vacuum Controller V-800; Heating Bath B-490 AKTA Prime Amersham Bioscience Hiload Superdex 30 Lot #: 216902 N/A N/A All of the water that was used in these expe riments was generated fr om a Millipore Water (Milli-Q) water station. The water was ultra purified and meet the requirements of resistance < 18 M .cm and organic contents < 5 ppb [ 136]. The purification process involveed three components: the Elix water system (Serial # 495000240926), the Automatic Sanitation Module (Serial # 495000240927), and the Milli-Q Synthesis A10
74 system (Serial # 495000240928). Tap water was passed through to a Progard pretreatment pack where particles and chlori ne were removed from the water . The water was pressurized and reverse osmosis was used to purify the water. The reverse osmosis water was then passed through to an electrodeionisation module where the organic and mineral content was brought down to an acceptable level . 5.3. Instruments Table 5. Instruments Used Instrument Manufacturer Model Number Serial Number Range Description AccuSeries II Electronic Toploading Balance Fisher Scientific Accu-2202 Cat # 13265-225 0 to 2200 g Readability: 0.01 g; Tarring; Repeatability: 0.01 g; Linearity: .02 g; Stabilization time 2 sec; Pan Size: 7x7 in (18 x 18 cm); Dimensions: 14 x 9 3/8 x 3 in; 115 Volts, 50/60 Hz; Single range Accumet 1003 pH Meter Fisher Scientific Accu met 1003 N/A -6 to 20 pH Resolution: 0.1, 0.01; Accuracy: 0.002; Buffer Values: 1.68,4.00,7.00,10.00,12.45; mV Range: -999 to 999; Temperature Range: 0C to 100C; Dimensions: 6.5 in x 2.88 in x 1.2 in; Weight: 9.3 ounces Accumet Gelfilled Polymer Body pH/ATC DoubleJunction Combination Electrode Fisher Scientific SN4175147P2 Cat # 13620-111 0 to 14 pH Temperature: -5C to 80C; Accuracy: <0.05pH at 25C; Efficiency: >95 at 25C; Sodium Ion Error: <0.05 pH 13 and 25C; Response Time: 30 seconds; Diameter: 3/8 in. (body) and 5/8 in. (cap)
75 Table 5 (Continued) Lab Fume Hood Safeaire Fisher Hamilton Part # 70864 54L PL822; Serial 110000 N/A Hood type: bench, prewired; Fumehood monitor: Phoenix Controls Corporation (part FHM610) LS-3B Fluorescence Spectrometer Perkin Elmer C659-0000 6999403 12 Volts, 60 Hz Monochromator = 488 nm Excitation Filter to 515 nm Emission Filter 5.4. Experimental Procedure The experimental procedure consists of a series of diffusion experiments. The experimental procedure requires several step s. The first step was to weigh out and measure the materials and reagents that were used in the experiment. This included the components for the membrane and the components for the medium. The next step was to incorporate the two components together. The experiments we re allowed to run for an elapsed time and the final samples were taken to the fluorescence spectrometer. From previous experience, the best design for in vitro experiments was determined. Two types of regenerated cellophane membranes were tested as a membrane. They were manufactured in shee ts of cellophane. The cellophane membrane was secured to the bottom of a tube, and the t ube was filled with a sample. Experimental design showed that the cellophane membrane caused a pressure differential and subsequently leakage from the membrane. The regenerated cellulose membrane was manufactured in spools of t ubing and was specifically used for dialysis experiment. The end closures also allowed for a reliable a nd secure seal of th e membrane. A 5(6)carboxyfluorescein dye was used because it has a similar molecular weight to Temozolomide, a chemotherapeutic drug, and is the dye of choice in Dr. VanAukers
76 laboratory. Temozolomide is the chemothe rapeutic drug that is currently used in vivo at Moffitt Cancer Center, where it is intended to test this drug in vitro and in vivo The concentrations encapsulated in the niosomes were 4mM, 5mM, and 14mM. These concentrations were based on similar amounts of Temozolomide used in experimental testing. Span 60 niosomes were prepared due to their great stability, which reduced leaking. TX-100 was the traditional surfacta nt that was used to break niosomes in fluorescence spectrometry. Time intervals we re used based on previous experiments. Several time intervals were originally made, and it was decided that intermediate points were needed for comprehensive data. Earlier experiments were performed at the original time intervals and later experiments were pe rformed at the modified time points. The experiment was performed at a room temperatur e. The pH of the Milli-Q water used was approximately 7.1 and the pH of the PBS used was 7.3-7.4. 5.4.1. Niosome Preparation The niosome solutions were prepared in the laboratory of Dr. VanAuker. The students who prepared the niosomes in the lab provided the following procedure . Niosome Films : The niosomes were prepared in a 1:1:0.105 molar ratio of surfactant : cholesterol : di cetyl phosphate (DCP). A 10x solution for 3 films of niosomes required 0.294 g of Span 60 surfact ants, 0.264 g cholesterol, and 0.039 g DCP. The components were then added to 9 ml of chloroform and swirled until all of the chemicals are dissolved. The flask was covere d to avoid evaporation of the chloroform. Too much mass would be aliquoted if exces sive evaporation occurs and additional chloroform could be added if necessary during the film-making process. Three
77 millimeters of the solution was added to 1 ml of chloroform in a 50 ml round bottom flask. The flask was attached to the Bu chi rotary evaporator (removing the large collection flask) and allowed to slowly rotate at a near flat angle. The solution was put into a rotary evaporator. Nitrogen gas was pumped into the flask at 3 L/min. The chloroform evaporated to reveal the film. The film was allowed to dry for at least 10 minutes after the last liquid is gone. The flask was then covered with Parafilm M and placed upside down in a dish in the fume hood where it was allowed to dry for 8-24 hours. Hydration of the Thin Films : The hydration of the thin films was done with 1X PBS solution. Span 60 has a gel phase tran sition temperature of 56-58C. The hydrating temperature must be 60C to make a stable ordered lamellar membrane . The rotary evaporator was filled with a deionized water bath and the temperature is set to 60C. Three ml of the hydrating solution was added to the think film flask. Three milliliters of the deionized water was added to a blank place -filling flask. The flask was secured with a clip on the wide side of the flask. The flasks were allowed to slowly spin in the water. After one hour, the flasks were taken off the ro tary evaporator and ag itated. They were covered with Parafilm M and mixed on a vortex mixer that removed the lipids evenly by moving around the outer surface. The flask was then returned to the evaporator. Hydration was continued for one more hour and sonicated for 60 minutes in a sonicator. The sonicator bath was filled three quarters of the way full with deionized water. The flask, covered with Parafilm M, was suspended in the water bath using a clamp stand. The bulk niosome solution was filtered and heated before it was GEC injected into the Akta Prime system. The plunger of a 10 ml disposab le syringe was removed. Two filter discs
78 (5/8) were put into the syringe and pushed to the bottom of the syringe. The syringe was held over a flask while 2.25-2.5 ml of the bul k solution was added to it. The plunger was pushed back into the syringe and forced the bulk solution through the filter. Another syringe was used to take about 2.0 ml of the bulk solution, and the tip of that syringe was wrapped in Parafilm M. The contents of that syringe were swirled in the heated water bath at 60C. 5.4.2. Membrane Measurements The first step in the experimental proce dure was to prepare the components of the membrane system. The procedure is discussed in terms of using water as the medium, but the procedure for using PBS instead is analogous; one would simply substitute the PBS information in for the water information. A five to seven centimeter strip of regenerated cellulose dialysis membrane was cut from the reel of membrane. A 250 ml beaker (Beaker A) was placed on the AccuSe ries II Electronic Toploading Balance Accu-2202 mass balance and zeroed. The membrane was placed in the zeroed beaker, and the mass of the membrane was taken. All mass measurements were taken in grams, g. Beaker A was later used in the meas urement of the entire membrane system and offered a contained environment for the co mponents of the membrane system to be recorded in. A solution of PBS was prepared by dissolving the entire container of Dulbeccos Phosphate Buffer Saline (Modified) without calcium, magnesium powder in one liter of Milli-Q and stirred. A 100 ml b eaker was filled with approximately 100 ml of the PBS solution. The membrane strip was placed in the PBS solution and allowed to soak for approximately ten minutes. Beaker A was zeroed again, and two Spectrum
Spectra/Por closures were placed in the beaker to be weighed. The beaker and closures were removed from the balance. The ba lance was zeroed again, and the two Goody elastic bands were placed directly on the bala nce to be measured. Due to the small mass of the elastic bands, they were placed dir ectly on the balance. This eliminated any disturbances that may have altered the reading of such a lightweight component. The membrane strip also had a small mass; howev er, the delicate nature of the membrane required that it be measured in the beaker. The closures did not require being measured directly on the balance and could be measured in the zeroed beaker because their mass was not greatly affected by disturbances from the zeroed beaker. Elastic bands were used to ensure proper closure of the membrane and eliminate the reduced tension of the closures that could result over time. The to tal mass of the dry membrane system could be calculated by adding the mass of the dry memb rane, the two dialysis-membrane closures, and the elastic bands. bands elastic closures membrane dry drysystem membranemm m m Eqn. 6 79 Beaker A, the two closures and elastic bands were placed aside while the components of the beaker are prepared. Please see section 5.4.2. After the beaker measurements were completed, the next step was to add the niosomes to the membrane. The soaked membrane was taken out of the PBS so lution with tweezers and placed onto approximately three sheets of Kimwipes paper tissue. Three more Kimwipes paper tissues were placed on top of the membrane and the excess moisture from the PBS solution was allowed to be absorbed. The membrane was gently opened by delicately rubbing the sides of the membrane back and fo rth. Once the ends of the membrane were opened, the tweezers were inserted into the membrane and expanded the remaining
length of the membrane. This was an essentia l step in the procedur e because otherwise, the bottom section of the membrane would have remained closed and the niosomes solution would have spilled out of the top wh en added. One dialysis-membrane closure was placed at one end of the membrane and securely shut. Over time, the closures could loose their firmness and leak due to an unsecured closure. The elastic bands were wrapped around the closures to ensure maximum secur ity. A Redi-Tip (101-1000 l) pipette tip was fitted to the Autocl avable Nichiryo Oxford Benchmate TM (~100-1000 l) micropipette. The micropipette was used to add 2 ml (2000 l) of the niosomes to the membrane. The tweezers were used to hold the membrane upright with one hand, while the other hand measured out the 2 ml. The ope n end was then closed with the remaining dialysis-membrane closure, and the remain ing elastic band was wrapped around the end of the closure. Beaker A was placed on th e mass balance once again and zeroed. The entire membrane unit was pla ced in the beaker and the mass was recorded. The weight of the niosome solution was determined by subtra cting this mass from the mass of the dry membrane, two dialysis-membrane cl osures, and elastic bands. drysystem membrane ttotalsystem membrane t niosomesm m mo o @ @ Eqn. 7 This information was used to determine the density of the niosomes solution and determined the final volume of the niosome solution in the membrane. 5.4.3. Beaker Measurements The next step in the experimental procedure was to prepare the components of the medium. A 250 ml beaker (Beaker B) wa s labeled Experiment N, t=x and was weighed on the Accu-2202 mass balance. The balance was zeroed and a stirring rod was 80
placed in the beaker. This mass was recorded The total weight of the empty beaker system could be calculated by adding these two measurements together. rodstirring BBea drysystem mediumrbeamm m ker ker Eqn. 8 A 500 ml beaker was filled with approximately 500 ml of the Milli-Q water. A 25 ml disposable serological pipette was used to fill th e Beaker B, with stirring rod, with 85 ml of Milli-Q water. The filled beaker was weighed again. The mass of the 85 ml of the Milli-Q water could be determined by subtr acting this mass from the mass of the empty beaker and stirring rod. drysystem bea ttotalsystem bea twaterm m mo oker @ ker @ Eqn. 9 This information was used to determine the density of the water in the Beaker B and then later to determine the final volume of the solution in the beaker. The pH of the medium was taken, based on a sample from the medium in the 500 ml beaker, using the Accumet 1003 pH Meter with Accumet Gel-filled Polymer Body pH/ATC Double-Junction Combination Electrode. 5.4.4. Density Calculation The density of the both the niosome solution in the membrane and the water medium solution in the Beaker B were calcul ated. The equation for density was equal to the mass of the solution divided by the volume of the solution. volume mass Eqn. 10 81
The density of the membrane was as follows: ml mniosomes t solution niosomeo2@ Eqn. 11 The density of the water in the beaker was as follows: ml mwater twatero85@ Eqn. 12 A list of the density values for the experiments performed can be found in Appendix B. 5.4.5. Incorporation of Two Units The next step in the experimental pro cedure was to incorporate the two units together: the membrane system and the beaker system. The membrane system was submerged into the 85ml of Milli-Q water in Beaker B, with the dialysis-membrane closures guiding the orientation of the membra ne to face downward. This was necessary to ensure that the entire area of the memb rane is submerged in the water. Parafilm M was placed over the entire system and placed on the mass balance. The total weight of the system was recorded and compared to the to tal weight of the system at the end of the experiment to determine if the mass remained constant throughout the elapsed time. The entire system was placed on th e Fisher Isotemp Ceramic Top Stirring Hotplate (1), the Cimarec TM Digital Stirring Hot Plate (3), the Corning PC-410 Sti rrer (1), or the Corning PC-353 Stirrer in the Lab Fume H ood Safeaire. The ro tation was placed on approximately 300-350 rpm. The system was covered with Reynolds aluminum foil to 82
prevent any exposure to light that could resu lt in bleaching of the dye. The system was allowed to run for the specified elapsed amount of time. 5.4.6. Elapsed Time for Experiment The experimental procedure was performed for each elapsed time desired. The original experiment was run at 0, 24, 72, and 144 hours. The times of 6 and 12 hours were then added. One experiment was performed to fill in intermediate data points to a previous experiment. It was discovered later that the concen tration that was thought to be equal to the original concentration was not. The experiment was taken as a separate experiment; only the times at 3, 6, 9, a nd 24 hours were obtained. In the latter experiment, it was determined that the times of 3, 6, 9, 12, 24, and 72 hours provided the most comprehensive data. At the end of each experiment, the foil was removed from the system and the stirring plate turned to zero. The total system was weighed to determine the consistency in weight. The Parafilm M was removed and Beaker A was placed on the mass balance and zeroed. The membrane unit was taken out of the beaker system with tweezers and as much excess liquid was shaken off. The membrane system was placed in the beaker on the balance and the final weight of the system was determined. The mass of the contents in the membrane (n ow consisting of both the original niosome solution and the water from the beaker) could be found. drysystem membrane ttotalsystem membrane t membranem m mf f @ @ Eqn. 13 The elastic bands and closures were remove d and the contents of the membrane were placed in a 15 ml disposable centrifuge tube and labeled Experiment N, t=x, MEMBRANE. The mass balance was zeroed and Beaker B was placed on it to be 83
84 weighed. The contents of the Beaker B were placed in a 15 ml disposable centrifuge tube and labeled Experiment N, t=x, BEAKER. The pH was taken of the remaining solution in Beaker B using the Accumet 1003 pH Meter with Accumet Gel-filled Polymer Body pH/ATC Double-Junc tion Combination Electrode. 5.4.7. Fluorescence Spectrometer The intensity of the 5(6)-carboxyfluores cein dye was taken using the LS-3B Luminescence Spectrometer. A small sample of the original niosome solution was used for the measurement. It is important to note that no volume was taken from the niosome solution already in the dialysis membrane. The sample was taken from the bulk niosome solution prepared in the plastic sample container. It was labeled, Experiment N, t=0, MEMBRANE. A sample of Mi lli-Q water was also taken from the bulk Milli-Q water in the 500 ml beaker, and no volume was removed from what was already in Beaker B. It was labeled, Experiment N, t=0, BEAKER. The LS-3B fluorescence spectrometer was turned on. The 5(6)-carboxyf luorescein dye excitation/emi ssion range was 492/514 nm. The excitation/emission wavelength was se t to 488/515 nm which was sufficient for detection of the dye. For the beaker sample, 1000 l of PBS solution was added to a quartz cuvette using the Autoclavable Nichiryo Oxford Benchmate Micropipette (~100-1000 l) micropipette with a Redi-Tip (101-1000 l) pipette tip. The cuvette was placed in the sample cell. There was a small black Q marked on the cuvette. The cuvette was placed in the fluorescence spectro meter with the Q facing the front right hand corner of the machine. The AUT O ZERO button was pushed and the PBS was taken as the zero. The PBS solution was discarded and 1000 l of the sample from the
85 beaker was added to cuvette using a 1000 l micropipette. The outsi de of the pipette tip was wiped with a Kimwipes to prevent any excess sample from being measured. The cuvette was placed in the sample cell. The INT button was pressed and the measurement was taken and recorded as I ntensity of the beaker @ t=0 before surfactant. This procedure was done until th ree consistent readings are taken. A surfactant didnot need to be added to the sa mple because there were no niosomes present in the initial milli-Q water in the beaker. The cuvette was cleaned each time between readings with water, paper towels, and Kimwipes The same procedure was performed for the sample from the beaker at each elapsed time except one step was added. A 10% solution of TX-100 was prepared by diluting 1 ml of TX-100 in 9 ml of Milli-Q water. The 10% TX-100 solution prepared was used in subsequent procedures. Ten microliters of TX-100 are added to the 1000 l of sample using the Autoclavable Nichiryo Oxford Benchmate Micropipette (~20-200 l) micropipette, with an ART100 Tips (101 l) pipette tip, and the outside of the pipette tip with Kimwipes are wiped. The Autoclavable Nichiryo Oxford Be nchmate Micropipette (~20-200 l) micropipette was able to read down to approximately 10 l. The solution was allowed to mix for five seconds on the Vortex Genie 2 and placed back in the sample cell. The intensity was allowed to stabilize, and the INT button was pressed. The r ecording was taken as Intensity of the beaker @ t=x after surfactant. The niosome readings were taken next. There are 990 l of PBS added to the cuvette using the 1000 l micropipette. The cuvette was placed in the fluorescence spectrometer, and the AUTO ZERO was presse d again. Ten microliters of the niosome sample were taken using the Autoclavable Nichiryo Oxford Benchmate Micropipette
86 (~2-20 l) micropipette with ART100 Tips (101 l) pipette tip and placed in the cuvette, wiping the outside of the pipette tip with Kimwipes The solution was mixed for five seconds on the Vortex Genie 2 and placed back in the cell. The reading was allowed to stabilize and the INT button was pressed. The intensity was recorded as Intensity of the membrane @ t=0 before surf actant. This reading represented the signal from the small amount of free dye in the soluti on as well as about 70% of the signal from the dye within the niosomes. There was a bout 30% quenching of the signal from the niosomes. Ten microliters of TX-100 are a dded to the cuvette using the Autoclavable Nichiryo Oxford Benchmat e Micropipette (~20-200 l) micropipette (20-200 l) micropipette and wiping the outside of the pi pette tip. The resulting solution was mixed for five seconds on the Vortex Genie 2, and the cuvette was again placed in the sample cell. The reading was allowed to stabilize and the intensity was recorded as Intensity of the membrane @ t=0 after surfactant. This reading represented the signal from supposedly all of the dye present. The TX -100 should have broken all of the niosomes and just free dye should be left. It repres ented everything that wa s present, inside and outside of the vesicles. The same proced ure was performed for the sample from the membrane at each elapsed time and recorded appropriately. It is important to note a slight variation in the procedure when high concentrations were to be tested. The linear relationship between intensity and concentration was only observed for dilute concentration, about 5.00x10 -7 M, which corresponded to around and intensity of 160. The linear relationship would continue until intensity readings of about 250. Higher concentrations would behave in a parabolic manner, reaching a high intensity and then lowering back down. Wh en high concentrations were observed, an
87 added step to the procedure was included to dilute the sample. The concentration was diluted by half by making a 1:1 ratio of the sa mple to PBS. If further dilutions were needed, the same technique was used, dilution by PBS in a 1:1 ratio. The procedure in the LS-3B fluorescence spectrometer was carried out in the same fashion as before by adding 10 l of the sample to 990 l of PBS. However, in the calculations to follow, the diluted sample was accounted for accordingly.
88 Chapter Six Results The diffusion of a niosome solution thr ough a cellulose membrane into a bulk solution was studied for several different concentrations. The experiments consisted of 4 mM dye in 0.01 M PBS diffused into water; 5.0 mM dye in 0.01 M PBS diffused into water; 14 mM dye in 0.01 M PBS diffused in to water; and 5 mM dye in 0.01 M PBS diffused into PBS. The 4mM ran for t = 0, 3, 6, 9, and 24 hours. The 5mM ran for t = 0, 6 12, 24, 72, and 144 hours. The 14mM ran for t = 0, 3, 6, 9, 12, 24, and 72 hours. The 5mM/PBS ran for t = 0, 24, 72, 144. The vari ation in time with the experiments was due to the fact that it took several tests to determ ine the best times to run the experiment at. However, the experiments that were not run at the formulated times behaved in a similar manner, and therefore, their times were recorded as usual. In some instances, interpolation was necessary to extend the beha vior to a greater time. There are several important calculations that needed to be made that included the concentration, moles, diffusion from the membrane to the medium, diffusion from the niosome to membrane, and release rate of dye. The following secti on describes these calculations and presents a comparison of the data.
6.1. Calculations 6.1.1. Volume The final volume of the contents in th e beaker containing the water/dye solution was calculated using the density calculated fo r each experiment for each time run. The final contents of the beaker are weighed. The weight of the water/dye solution can be determined by: f fttotalsystem bea drysystem bea tdyewaterm m m@ ker ker @/ Eqn. 14 The final volume of the water/dye solution can be determined from: o f ftbeainwater tdyewater tdyewaterm V@ker @/ @/ Eqn. 15 The density of water in the beaker at t=0 can be used to determine the volume of the water/dye solution at t f based on Equation 15 because the density of the dye was approximately close to the density of dye. Wh en the dye was in solution with the water, the density of the solution could be approximated by the density of water. The final volume of the contents in the membrane containing the noisome solution could be calculated in a similar manner. The final cont ents of the membrane were weighed. The weight of the noisome soluti on could be determined from: drysystem membrane ttotalsystem membrane tsolution niosomem m mf f @ @ Eqn. 16 89
The final volume of the noisome so lution could be determined from: o f ft niosome t solution niosome t solution niosomem V@ @ @ Eqn. 17 The density of the niosome solution at t=0 co uld be used to determine the volume of the niosome solution at t f based on Equation 17 because the density of the noisome solution would be approximately close to the density of the noisome solution at the end of the experiment. Table 6 represents the initial and final volumes of the 4mM, 5mM, 14mM, and 5mM/PBS. It is very clear that the vol ume in the beaker initially decreases at the beginning of the experiment, while the volume in the membrane initially increases. This indicated that initially the medium rushes in to the membrane. Volume after the first few hours fluctuates as the system dynamics are ch anging, but it is importa nt to observe that in all cases, the bulk solutions influxes into the medium. Table 6. Initial and Final Volumes for 4mM, 5mM, 14mM, and 5mM/PBS 4mM 5mM 14mM 5mM/PBS Time [hr] Beaker Final Volume [ml] Membrane Final Volume [ml] Beaker Final Volume [ml] Membrane Final Volume [ml] Beaker Final Volume [ml] Membrane Final Volume [ml] Beaker Final Volume [ml] Membrane Final Volume [ml] 0 85 2 85 2 85 2 85 2 3 84.02 2.78 83.83 2.95 6 83.88 2.91 84.04 2.71 83.64 3.11 9 84.05 2.65 83.90 2.83 12 83.18 2.67 83.69 3.01 24 83.71 3 84.12 2.51 83.80 2.81 84.34 2.445 72 84.24 2.47 83.31 3.25 84.455 2.365 144 84.39 2.27 84.265 2.485 90
6.1.2. Concentration The calculation of concentration was need ed to compare how the concentration changes over time and with varying concentrations of dye entrapped within the niosomes. The concentration was also dire ctly related to the number of moles in the system, which was also of interest. Concentration could be calculated initially, be fore the surfactant was added, and after the surfactant was added to obtain a total concentration. The concentrations were directly related to the intensity readings and were based on a concentration standard. The three consiste nt intensity readings for the fluorescence spectrometer were averaged. The calibration curve yielded an equation in the following form: bmxy Eqn. 18 )int() (*) ( ercept zeroion concentrat curvetheofslope Intensity The initial and final concentrations coul d therefore be determined by dividing the averaged intensity by the slope of the curve: curvetheofslope Intensity MC ] [ Eqn. 19 This concentration represented the amount of original sample in the PBS in the cuvette. Table 7 lists the slopes obtained from the calibration curves: Table 7. Slope Obtained from Calibration Curve Experiment 4mM 5mM 14mM 5mM/PBS Slope from calibration curve 3.3711E8 6.125E8 4.733E8 3.3711E8 91
92 It does not represent the actual concentration in either the beaker or the membrane. Further calculations were performed to scal e down this concentration to represent the concentrations that are present within the tw o systems. Table 8 Table 11 represents the initial (before TX-100 is added) and final (after TX-100 is added) concentrations of 4mM, 5mM, 14mM, and 5mM/PBS, respectively. It should be noted here that the concentration, and subsequently the number of moles, in th e beaker originally would be zero because there was just water at the beginning of the experiment. Table 8. Initial and Final Concentrations (4mM) Initial Concentration [mol/L] Final Concentration [mol/L] Time (hr) [Beaker] o [Membrane] o [Beaker] f [Membrane] f 0 0 2.140E-07 0 5.475E-07 3 1.517E-07 3.192E-07 1.535E-07 4.662E-07 6 1.616E-07 3.012E-07 1.497E-07 4.298E-07 9 2.319E-07 2.993E-07 2.297E-07 4.316E-07 24 3.612E-07 2.427E-07 3.257E-07 3.4604E-07 Table 9. Initial and Final Concentrations for (5mM) Initial Concentration [mol/L] Final Concentration [mol/L] Time (hr) [Beaker] o [Membrane] o [Beaker] f [Membrane] f 0 0 2.361E-07 0 6.559E-07 6 1.294E-07 2.597E-07 1.864E-07 5.645E-07 12 2.008E-07 2.674E-07 2.865E-07 5.335E-07 24 2.539E-07 2.822E-07 3.250E-07 5.497E-07 72 5.557E-07 2.305E-07 6.136E-07 3.767E-07 144 5.651E-07 3.816E-07 5.744E-07 4.513E-07 Table 10. Initial and Final Concentrations (14mM) Initial Concentration [mol/L] Final Concentration [mol/L] Time (hr) [Beaker] o [Membrane] o [Beaker] f [Membrane] f 0 0 6.439E-07 0 1.638E-06 3 5.226E-07 9.992E-07 5.157E-07 1.351E-06 6 5.182E-07 9.311E-07 4.899E-07 1.311E-06 9 8.820E-07 8.488E-07 9.156E-07 1.211E-06
Table 10 (Continued) 12 6.612E-07 8.020E-07 6.238E-07 1.153E-06 24 1.546E-06 6.932E-07 1.534E-06 1.062E-06 72 1.902E-06 4.955E-07 1.799E-06 9.009E-07 Table 11. Initial and Final Concentrations (5mM/PBS) Initial Concentration [mol/L] Final Concentration [mol/L] Time (hr) [Beaker] o [Membrane] o [Beaker] f [Membrane] f 0 0 1.689E-07 0 3.995E-07 24 6.318E-08 1.785E-07 6.575E-08 3.860E-07 72 5.717E-08 1.861E-07 5.489E-08 3.603E-07 144 6.100E-08 2.223E-07 4.263E-08 3.405E-07 6.1.3. Moles in Cuvette The moles in the cuvette could be calcul ated to determine how many moles were present in the sample that was measured in the cuvette. This was an important intermediate step in calculating the total mole s in the system. The moles in the cuvette had to be scaled up to account for the actual moles in eith er the beaker or membrane system. The moles in the cuvette represented the moles of the sample in the 1 ml (or 1.01 ml) sample in the cuvette. There was an initial (before TX-100 is added) and a final (after TX-100 is added) value for the moles in the cuvette. The initial moles in the cuvette for both the beaker and the membrane could be calculated by converting L to ml and was multiplyed by the total volume in the cuvette initially (1 ml): ml L ml L mol C moleso cuvetteininitial1* 1000 Eqn. 20 93
The final moles in the cuvette for both the be aker and membrane were calculated in a similar manner. However, the total volum e in the cuvette was now 1.01 ml since 10 l of TX-100 was added. The calcula tion for the final moles in th e cuvette for both the beaker and the membrane was as follows: ml L ml L mol C molesf cuvetteinfinal01.1* 1000 Eqn. 21 The initial and final moles in the cu vette are represented in Table 12 Table 15 for 4mM, 5mM, 14mM, and 5mM/PBS, respectively. Table 12. Initial and Final Moles in the Cuvette (4mM) Initial Moles in Cuvette [moles] Final Moles in Cuvette [moles] Time (hr) [Beaker] o [Membrane] o [Beaker] f [Membrane] f 0 0 2.140E-10 0 5.530E-10 3 1.517E-10 3.192E-10 1.550E-10 4.708E-10 6 1.616E-10 3.012E-10 1.512E-10 4.341E-10 9 2.319E-10 2.993E-10 2.320E-10 4.359E-10 24 3.612E-10 2.427E-10 3.289E-10 3.495E-10 Table 13. Initial and Final Moles in the Cuvette (5mM) Initial Moles in Cuvette [moles] Final Moles in Cuvette [moles] Time (hr) [Beaker] o [Membrane] o [Beaker] o [Membrane] o 0 0 2.361E-10 0.000E+00 6.624E-10 6 1.294E-10 2.597E-10 1.883E-10 5.701E-10 12 2.008E-10 2.674E-10 2.894E-10 5.388E-10 24 2.539E-10 2.822E-10 3.283E-10 5.552E-10 72 5.557E-10 2.305E-10 6.198E-10 3.805E-10 144 5.651E-10 3.816E-10 5.801E-10 4.558E-10 94
95 Table 14. Initial and Final Moles in the Cuvette (14mM) Initial Moles in Cuvette [moles] Final Moles in Cuvette [moles] Time (hr) [Beaker] o [Membrane] o [Beaker] o [Membrane] f 0 0 6.439E-10 0.000E+00 1.655E-09 3 5.226E-10 9.992E-10 5.208E-10 1.365E-09 6 5.182E-10 9.311E-10 4.948E-10 1.324E-09 9 8.820E-10 8.488E-10 9.247E-10 1.223E-09 12 6.612E-10 8.020E-10 6.301E-10 1.165E-09 24 1.546E-09 6.932E-10 1.549E-09 1.073E-09 72 1.902E-09 4.955E-10 1.817E-09 9.099E-10 Table 15. Initial and Final Moles in the Cuvette for (5mM/PBS) Initial Moles in Cuvette [moles] Final Moles in Cuvette [moles] Time (hr) [Beaker] o [Membrane] o [Beaker] o [Membrane] o 0 0 1.689E-10 0.000E+00 4.035E-10 24 6.318E-11 1.785E-10 6.640E-11 3.898E-10 72 5.717E-11 1.861E-10 5.544E-11 3.639E-10 144 6.100E-11 2.223E-10 4.305E-11 3.439E-10 It is important to note that the moles in the cuvette for the beaker only represented 1 ml of the sample. The moles in the cuvette for the membrane only represented 10 l of the sample. This information is important when the total moles of the beaker and membrane are desired. The moles in the vesicles were calculated by subtracting the initial moles in the cuvette from the final moles in the cuvette. The final moles represented the presumably total moles in the system afte r supposedly all of the niosomes had burst and there was just free dye left. The initial moles in the cuvette represented the signal from the free dye and about 30% of the reading of the dye within the niosomes. The difference between the initial and final moles represented the moles that were entrapped within in the niosomes. Table 16 shows the moles within the vesicl es for 4mM, 5mM, 14mM, and 5mM/PBS. The unusual observation was that the moles with in the vesicles were negative for some
96 certain times and experiments in the beaker. The behavior was due to the fact that the TX-100 is not sufficiently break ing all of the niosomes and the final signal should be much higher. Because it is not as high as it should be, negative numbers results. The negative numbers also resulted from the fact that there were no vesicles with dye in the beaker. The negative number was a result of an inaccurate mass balance as well as an indication that there were zero moles of dye in the vesicles in the beaker. Table 16. Moles in Vesicles (4mM, 5mM, 14mM, and 5mM/PBS) 4mM 5mM 14mM 5mM/PBS Time [hr] Beaker Moles in Vesicles Membrane Moles in Vesicles Beaker Moles in Vesicles Membrane Moles in Vesicles Beaker Moles in Vesicles Membrane Moles in Vesicles Beaker Moles in Vesicles Membran e Moles in Vesicles 0 0 3.390E-10 0 4.26362E-10 0 1.011E-09 0 2.345E-10 3 3.341E-12 1.516E-10 -1.745E-12 3.654E-10 6 -1.039E-11 1.330E-10 5.888E-11 3. 10442E-10 -2.341E-11 3.926E-10 9 1.203E-13 1.366E-10 4.268E-11 3.747E-10 12 8.854E-11 2.71410E-10 -3.109E-11 3.626E-10 24 -3.228E-11 1.068E-10 7.437E-11 2.72986E-10 3.507E-12 3.794E-10 3.218E-12 2.114E-10 72 6.408E-11 1.49971E-10 -8.540E11 4.144E-10 -1.735E-12 1.778E-10 144 1.504E-11 7.42231E-11 -1.795E-11 1.217E-10 6.1.4. Moles in the System The total moles in the system provided information of interest. The moles in the system represented the total number of mo les present in both the beaker and the membrane. It told us how much dye has diffused across the membrane and how much dye has been released from the vesicles. The total moles in the system were determined based off of the moles found in the cuvette. However, in order to calculate the total moles, the moles found in the cuvette were scaled up by multiplying by the volume that was found for each experiment and for each time run. It is important to note that 1 ml of
the beaker sample was measured and 10 l of the noisome solution was measured. Therefore the number of moles in the cuvette for the beaker was only based on 1 ml. The number needed to be multiplied by the volume of the system to determine the moles in that particular system. The total moles in the system were calculated for the free dye alone or for the total dye. The data for the total dye is shown below; however, the total free moles are discussed in Chapter Seven. The total moles in the beaker system were therefore be calculated by the following method: ftdyewater bea cuvetteinfinal system total beaV ml moles moles@/ ker)( ker* 1 Eqn. 22 The calculation for the number of moles to tal in the membrane was performed in a similar manner. The number of moles in the cuvette for the beaker was only based on 10 l. The conversion from l to ml needed to be made. The number then needed to be multiplied by the final volume of the membrane to determine the number of moles in that particular system. The total moles in the me mbrane system were therefore be calculated by the following method: ft solution niosome membrane cuvetteinfinal systemtotal membraneV ml l l moles moles@ )(* 1 1000 10 Eqn. 23 The total moles in the beaker and noisome sy stem were calculated and are represented in Table 17 Table 20 for 4mM, 5mM, 14mM, a nd 5mM/PBS, respectively. The gray shaded areas represent extrapolated points. 97
98 Table 17. Total Moles in the System (4mM) Total Moles in the System [moles] Time (hr) Beaker Membrane 0 0.000E+00 1.106E-07 3 1.302E-08 1.309E-07 6 1.268E-08 1.263E-07 9 1.950E-08 1.155E-07 24 2.753E-08 1.048E-07 72 5.213E-08 5.948E-08 Table 18. Total Moles in the System (5mM) Total Moles in the System [moles] Time (hr) Beaker Membrane 0 0.000E+00 1.325E-07 6 1.572E-08 1.716E-07 12 2.396E-08 1.439E-07 24 2.761E-08 1.393E-07 72 5.221E-08 9.398E-08 144 4.896E-08 1.035E-07 Table 19. Total Moles in the System (14mM) Total Moles in the System [moles] Time (hr) Beaker Membrane 0 0.000E+00 3.309E-07 3 4.366E-08 4.026E-07 6 4.138E-08 4.116E-07 9 7.758E-08 3.462E-07 12 5.273E-08 3.505E-07 24 1.298E-07 3.014E-07 72 1.514E-07 2.957E-07 Table 20. Total Moles in the System (5mM/PBS) Total Moles in the System [moles] Time (hr) Beaker Membrane 0 0.000E+00 1.614E-07 24 1.121E-08 1.906E-07 72 9.364E-09 1.721E-07 144 7.256E-09 1.709E-07
6.1.5. Diffusion from Membrane to Medium The analysis of the diffusion of the propos ed system was separated into two subsystems: the diffusion from inside the cellulose membrane to the medium (the term beaker will now be referred to in terms of the medium) and the diffusion from inside the niosome outside into the membrane area. The diffusion of the dye from the membrane to the medium was important to understand because it allowed for the determination of what the concentration was in the medium and how many moles were released to the surroundings. The diffusion also allowed for the determination of a relationship between the concentrations in the membrane with th e concentration in the medium. A model was developed to see if it would pr edict this information, or wh ere the model would begin to deviate from the experimental data. The diffusion through the membrane was also important because provided insight into the be havior of the system. Ficks First Law of Diffusion was used and described the flux of a chemical species through a membrane : cellulose final medium final membrane cellulose celluloseh C C DJ) (2) (1 Eqn. 24 where C 1 [=] mol/m 3 was the concentration in the membrane, C 2 [=] mol/m 3 was the concentration in the beaker, h [=] m wa s the thickness of the membrane, D [=] m 2 /hr was the diffusion coefficient of the dye, and J [=] mol/hr/m 2 was the flux of the dye across the membrane. The thickness of the cellulose me mbrane was 40 m. The concentration in the membrane was the total concentration in the membrane, not jus the free dye in the membrane, because it was of interest to se e what the maximum effect the concentration 99
in the membrane had on the flux across the membrane. Theoretically, if all of the niosomes were to break, the total concentratio n would affect the flux. In this case, only the free dye was moving across the membrane. It was decided it would be better to include the other dye in the nios omes than not to include them due to the fact that there was still some uncertainty of the actual fr ee dye at any given moment. The niosomes were constantly breaking and the dye was constantly diffusi ng across the membrane so it was very hard to determine the actual fr ee dye at any given moment. The diffusion coefficient can be determined from the following equation : )@ ( )@ (log*f otfinal membrane t tfinal membrane o cellulose celluloseC C t A D Eqn. 25 where A [=] m 2 was the area of the membrane, t [=] hours was the time of the experiment, C 0 [=] mol/m 3 was the initial concentration in the membrane, and C t [=] mol/m 3 was the concentration in the membrane at time, t. The length of our membrane was approximately 7 cm, which was equivalent to 0.07 m. The wi dth of our membrane was approximately equal to 25 mm, which was equivalent to 0.025 m. The area of the membrane is therefore: 20035.0025.0*07.0**2 m mmWL Acellulose Eqn. 26 The initial concentration in the membrane wa s constantly changing due to the breaking of the niosomes in the membrane Therefore, the quantity (C o /C t ) was determined by taking the initial concentration from the previous time over the concentration at the current time. Since the initial concentration was constantly changing, the previous concentration at time, t, will be the new initial concentra tion at our new time. Several values were 100
101 obtained for the diffusion coefficient based on the concentration and time. An average of the positive diffusion coefficients was taken to determine one diffusion coefficient for the system. Negative values were obtained for th e diffusion coefficient in some cases. This meant that the initial concentration from the previous time was not greater than that final concentration at time, t. This was very possible, as each different experiment ran at each different time could result in a variation in the actual concen tration present at each time. More niosomes may have been breaking from one set-up to the next that could account for this variation. The negative values were not used to calculate the average diffusion coefficient. The equation for the diffusi on coefficient assumed that either the concentration in the medium or the concentr ation in the membrane was kept relatively small to determine the diffusion coefficient for the system. It was assumed that the concentration in the medium was relatively small compared to the concentration in the membrane (1E-2 vs. 1E-4 mol/m 3 ). The concentration in the membrane, C 1 and C 0 /C t and the concentration in the medium, C 2 were scaled up based on the volume of the system. The concentration in the system wa s found by taking the intensity measurements and dividing by the slope of the calibration cu rve. However, this was the concentration based on the amount of sample was measured, ei ther 10 l for the membrane or 1 ml for the medium. In order to scale this concen tration up to account for the concentration in the volumes we actually have, the total moles in the system was used that were found for each. The total moles in the system were divided by the total volume in the system to obtain the final concen tration scaled up:
systemintotal systemintotal upscaledV moles C Eqn. 27 The values for D and J were obtained. The diffusion coefficients and fluxes for 4mM, 5mM, 14mM, and 5mM/PBS are shown in Table 21. Table 21. Flux and Diffusion Coefficient for Cellulose (4mM, 5mM, 14mM, and 5mM/PBS) 4mM 5mM 14mM 5mM/PBS Diffusion Coefficient m 2 /hr = 4.175E-5 Diffusion Coefficient m 2 /hr = 2.143E-5 Diffusion Coefficient m 2 /hr = 3.226E-5 Diffusion Coefficient m 2 /hr = 1.849E-6 Time [hr] Flux [mol/hr/m 2 ] Flux [mol/hr/m 2 ] Flux [mol/hr/m 2 ] Flux [mol/hr/m 2 ] 0 0.0577 0.0355 0.133 3.730E-3 3 0.0490 0.110 6 0.0451 0.0338 0.106 9 0.0453 0.098 12 0.0287 0.093 24 0.0361 0.0296 0.085 3.598E-3 72 0.0201 0.072 3.359E-3 144 0.0241 3.176E-3 It was also desired to obtain theoretical values of the concentration in the beaker based on a known concentration in the membrane. A re lationship between the concentration in the membrane and flux was determined by plotting the experimental values for the concentration in the membrane ve rsus the experimental values ju st calculated for the flux. The relationship was a linear relationship an d an equation for the line was easily obtained in the form of: bmxy Eqn. 28 where y = flux, m = slope, x = concentration in membrane, and b = y-intercept. A list of concentrations in the membrane was genera ted and plugged into the above equation to 102
obtain theoretical values for flux. The theoretical values for the concentration in the medium was obtained by rearra nging Equation 24 to solve for C 2 : cellulose cellulose cellulose final membrane final mediumD hJ C C *) (1) (2 Eqn. 29 The average diffusion coefficient was used in Equation 29 to obtain the theoretical values of C 2 A list of the theoretical values for fl ux and the concentrati on in the medium are not presented here but will be commented on in Chapter 7. It is also of interest to determine whether this model was able to predict the concentration in the medium (and perhaps the flux) over a cert ain period of time. To do this, Equation 25 was rea rranged to solve for C t : cellulose cellulose o fA tD tfinal membrane o tfinal membrane tC C* )@ ( )@ (10 Eqn. 30 The original C o was the total concentration at t = 0 hrs. Subsequent concentrations, C t were determined and acted as the new C 0 in the following time interval. These were predicted concentrations in the membrane. These predicted concentration were then plugged into Equation 28 to solve for the predicte d flux of the experiment at time, t. The predicted concentration in th e medium was determined using Equation. 29, plugging in the predicted values for C 1 and J. The predicted values are not presented here but will be commented on in Chapter 7. 103
104 6.1.6. Diffusion/Release from Niosome to Membrane The diffusion/release of the dye from within the niosome to the membrane was important to understand because it allowed for the determination of the behavior of the niosomes throughout the experiment. The concentration that was being released and available to diffuse through the membrane in to the surroundings was determined. This allowed for the determination of how the concentration being released was changing and the relationship between the c oncentration in the niosomes and the concentration being released. A model was developed to see how well that model predicted the experimental data. The actual system consisted of a certa in number of niosomes containing a specified concentration of dye (i.e. 5 mM) in a specif ic total volume with a certain amount of moles. Ficks First Law of Diffusion mode led the diffusion of a species of higher concentration into an area of lower concentratio n. However, in for this particular case, the dye was not diffusing across the niosome membrane appreciably. The real dye release mechanism was the breaking of the su rfactant membrane layer. It was proposed that the real system can be related to an analogous counterpart that included that total surface area of all of the niosomes in the real system, the concentration of the real system, and the membrane thickness of the real system. The new model consisted of one continuous theoretical membrane with a th ickness of the niosome membrane, the total surface area of all of the niosomes in the system, and was filled with dye at the concentration inside of the niosomes. It was assumed that the theoretical membrane thickness was equal to the thickness of the ni osome because the dye would never have to go through a thickness larger than that in th e real system. The diffusion out of the theoretical membrane would be the same as the rate of dye released due to the breaking
105 niosomes in the real system. The concentr ation in the theoretical membrane was the concentration in the real system that was still contained in the niosomes left in tact. The concentration in the theoretical membrane ch anged the same as the concentration in the real system changed due to dye being released from the breaking niosomes. The missing pieces were obtained to solv e Ficks First Law of Diffusion. The total volume of the niosomes in the membrane was determined so that the calculation of the total concentration in the niosome membrane at any given time was determined. (Please note that the niosome membrane now refers to the theoretical niosome membrane). In order to do so, the total number of niosomes present in the niosome membrane was calculated. For this, the dimens ions of a single niosome were taken. The diameter of Span 60 niosomes that were made for these experiment were determined by a particle sizer/counter to be about 800 nm, or 8x10^-7 m. The radius was therefore r = d/2 = 4x10^-7 m. The thickness of the niosome membrane has rarely been cited in the literature. The literature reports the thickne ss of a niosome membrane to be between 915 nm . The maximum thickness was used to determine what the maximum possible diffusion or release would be. The number of total moles that are within the niosome membrane was known. It was obtained from subtracting the initial moles of free dry in the cellulose membrane system from the tota l moles in the cellulose membrane system. The difference between the two will provide the number of moles that were not free, but were contained in the niosome membrane. To determine the concentration in the niosome membrane, the total volume of the niosomes in the niosome membrane were determined. The total volume [m 3 ] of the niosomes required the total number of niosomes in the system and the volume of one niosome:
total niosomes niosome total membrane niosomenV V *1 Eqn. 31 The volume of one niosome was determined by the following equation for the volume of a sphere: 3 2 219682.2)74( 3 4 3 4 mE mE r Vsphere Eqn. 32 The total number of niosomes was dete rmined from the following Equation 33: niosome membrane niosome total membrane niosome total niosomesV C moles n11 Eqn. 33 The total moles in the niosome membrane at each time were known, and the initial concentration in the niosome membrane was known based on the requested concentration of dye encapsulated in the niosomes, i.e. 5 mM. The number of niosomes was then calculated based on a known concentration of dye originally encapsulated in the niosomes. The actual original concentration was determined by dividing the total number of moles in the niosome membrane by the to tal volume of the niosome in the membrane Equation 31. The total volume can be determined from Equation 31. The concentration [mol/m 3 ] within the niosome membrane at each time was determined from the following equation: membrane niosomeintotal membrane niosomeintotal membrane niosomeV moles C Eqn. 34 106
The total moles represent the total number of moles in the niosomes membrane was determined by the difference between total moles in the membrane and free moles in the membrane. Ficks Law was used: niosome initial membrane membrane niosome niosome niosomeh C C DJ) (2) (1 Eqn. 35 where C 1 [=] mol/m 3 was the concentration in the niosome membrane, C 2 [=] mol/m 3 was the concentration released into the cellulose membrane, h [=] m was the thickness of the niosome membrane, D [=] m 2 /hr was the diffusion coefficient of the dye across the niosome membrane, and J [=] mol/hr/m 2 was the flux of the dye across the niosome membrane. The diffusion coefficient was determined using: )@ ( )@ (log*f ot membrane niosomet t membrane niosomeo niosomes all niosomeC C t A D Eqn. 36 where A [=] m 2 was the total surface area of the a ll of the niosomes, t [=] hours was the time of the experiment, C 0 [=] mol/m 3 was the initial concentration in the niosome membrane, and C t [=] mol/m 3 was the concentration released into the cellulose membrane at time, t. The total surface area of all of the niosomes was used because the system is modeled with one theoretical membrane, but in the real system that is of interest was the dye that was bursting from all of the niosomes so total surface area was used. The initial concentration in the niosome membrane was constantly changing due to the breaking of the niosomes. Therefore, the quantity (C o /C t ) was determined by taking the initial concentration from the previous time over the concentration at the current time. Since the initial concentration was constantly changi ng, the previous concentration at time, t, 107
108 was the new initial concentration at the ne w time. Several values for the diffusion coefficient were obtained based on the conc entration and time. The average of the positive diffusion coefficients was taken to determine the one diffusion coefficient for the system. Negative values of the diffusion coeffi cient were obtained in some cases. This meant that the initial concentration from the previous time was not greater than that final concentration at time, t. This was very possi ble as each different experiment run at each different time could have resulted in a vari ation in the actual concentration present at each time. More niosomes may have been breaking from one set-up to the next that could account for this variation. The negative values were simply not used to calculate our diffusion coefficient because a negative valu e did not fit the Ficks Law model. The equation for the diffusion coefficient assumed th at either the concentration in the niosome membrane or the concentration in the released into the cellulose membrane was kept relatively small to determine the diffusion coefficient for the system. It was assumed that the concentration released into the cellulose membrane was relatively small compared to the concentration in the niosome membrane. The initial concentration in the cellulose membrane, or the free dye in the membrane, ha d to be scaled up in a similar fashion as was done for the final concentra tions in the medium and membra ne in Section 6.1.5. The initial concentration was used as the concentration in the cellulose membrane in Ficks First Law. The concentration in the system was originally found by taking the intensity measurements and dividing by the slope of the calibration curve. However, this was the concentration based on the amount of sample wa s measured, 10 l. In order to scale this concentration up to account for the concentratio n in the actual volume, the total moles in the system had to be found and the divided by the actual volume:
total membrane cellinitial membrane system initial membraneV ml l l moles moles 1 1000 10 Eqn. 37 The new concentration was determined by dividing the total number of moles by the volume of the system: total membrane system initial membrane upscaledV moles C Eqn. 38 The values for D and J were be obtained. The diffusion coefficients and fluxes for 4mM, 5mM, 14mM, and 5mM/PBS are shown in Table 22. Table 22. Flux and Diffusion Coefficient of the Niosomes (4mM, 5mM, 14mM, and 5mM/PBS) 4mM 5mM 14mM 5mM/PBS Diffusion Coefficient m 2 /hr = 8.152E-24 Diffusion Coefficient m 2 /hr = 1.815E-24 Diffusion Coefficient m 2 /hr = 7.683E-24 Diffusion Coefficient m 2 /hr = 1.803E-25 Time [hr] Flux [mol/hr/m 2 ] Flux [mol/hr/m 2 ] Flux [mol/hr/m 2 ] Flux [mol/hr/m 2 ] 0 2.149E-15 0.023E-16 7.137E-15 5.990E-17 3 9.488E-16 2.541E-15 6 8.309E-16 4.375E-16 2.738E-15 9 8.544E-16 2.615E-15 12 3.820E-16 2.531E-15 24 6.671E-16 3.858E-16 2.657E-15 5.395E-17 72 2.101E-16 2.915E-15 4.534E-17 144 1.007E-16 3.091E-17 The theoretical values of the concentration released into the cellulose membrane wee desired based on a known con centration in the niosome membrane. A relationship between the concentration in the niosome me mbrane and flux was determined by plotting the experimental values for the concentrati on in the niosome versus the experimental values just calculated for the flux. The relationship was a linear relationship and an equation for the line was easily be obtained from Equation 28 where y = flux, m = slope, 109
x = concentration in niosome membrane, and b = y-intercept. A list of concentrations in the niosome membrane was generated and plugged into the above equation to obtain theoretical values for flux. The theoretical va lues for the concentration released into the cellulose membrane was obtained by: niosome niosome niosome membrane niosome initial membraneD hJ C C *) (1) (2 Eqn. 39 The average diffusion coefficient was used in Equation 37 to obtain the theoretical values of C 2 A list of the theoretical values for fl ux and the concentrati on in the medium are not presented here but will be commented on in Chapter 7. It is also of interest to determine whether this model was able to predict the concentration released into the cellulose membrane (and perhaps the flux) over a certain period of time. Equation 40 was used: niosomealloftotal niosome o fA tD t membrane niosomeo t membrane niosometC C* @ ( )@ (10 Eqn. 40 The original C o was the total concentration at t = 0 hrs. Subsequent concentrations, C t were determined and acted as the new C 0 in the following time interval. These are the predicted concentrations in the niosome memb rane. These predicted concentrations were then be plugged into Equation 28 to solve for the predicted flux of the experiment at time, t. The predicted concentration in the me mbrane was determined using Equation 37, plugging in the predicted values for C 1 and J. The predicted valu es are not presented here but will be commented on in Chapter 7. 110
6.1.7. Release Rate from Niosomes Section 6.1.5 and 6.1.6 described calcula tions in which the total system was separated into two separate systems to determine if Ficks First Law of Diffusion modeled and predicted the concentrations in the experiments well. However, the release rate of the niosomes could be determined based on a total mole balance of the system. The rate of change of the free dye in the me mbrane (not entrapped in the niosomes) was determined from the rate of change in concentration over time: C l k S t CVgen Eqn. 41 where represented the free dye in the cellulose membrane, S gen represented the burst/leak rate of the dye from the niosomes l represented thickness of the membrane, k represented the diffusion coefficient, and C represented the concentration difference across the cellulose membrane. The collective third term in the equation represented the diffusion across the cellulose membrane. The ra te of change of dye in the medium was determined based on the third term for the diffusion across the membrane: C l k t CV Eqn. 42 where represented the dye outside in the medium The rate of change of dye entrapped in the niosomes was represented by S gen being the burst/leak rate: genS t CV Eqn. 43 111
where represented the dye entrapped in the ni osomes. The combination of Equation 41, Equation 42, and Equation 43 yi elded the governing equation: 0 t CV t CV t CV Eqn. 44 The rate of release of dye from the niosomes was therefore determined based on data that was known for the free dye in the cellulose membrane ( ) and the dye in the medium ( ). The values are not presented here but will be commented on in Chapter 7. 112
113 Chapter Seven Discussion The results from the experiments provi ded a myriad of information open for discussion. The discussion section will comme nt on the behavior of the system as it relates to the activity of the moles in the syst em, the concentration in the system, the flux, and the release rate of the niosomes. 7.1. Moles of Dye in the Membrane The behavior of the free moles of dye in the membrane was of particular interest. Figure 23 depicts the behavior of the free mo les of dye in the membrane over time for 4mM, 5mM, and 14mM.
Figure 23. Moles of Dye in Membrane vs. Time (4mM, 5mM, and 14mM) The number of free moles dye in the membrane initially increased and reached a peak very early on during the experiment. The mo les then gradually decreased steadily over the 72 hours time period. This indicated that initially an influx of water was entering from the beaker across into the membrane. The water was creating an osmotic pressure gradient and causes the niosomes to burst ope n, and released any encapsulated dye that was within. This occured during the first ten hours of the experiment, in some cases sooner than this time. The p eak in moles indicated that th e initial influx of water was responsible for the large majority of the nios omes to burst. The moles in the membrane eventually decreased as the bursting decreased due to the fact that there are less intact niosomes present. The moles in the membra ne decreased as the dye diffused from the membrane into the water medium. The similar behavior of the moles in the membrane of 114
115 all three experiments indicates that the con centration of the dye encapsulated in the niosomes does not affect the overall behavior of the system. The affect that the concentration did have was the point at wh ich each system has its major decrease in moles. For 4mM and 5mM, there was a gra dual decrease in moles after the first 12 hours, but a greater slope was observed from 24 hours to 72 hour s. The dashed line for 4mM at 72 hours indicates an extrapolated point. The 14mM had a greater slope from 12 hours to 24 hours and a more constant decreas e from 24 hours to 72 hou rs. Overall, the behavior of the three systems is similar. The co ncentration was dir ectly proportional to the number of moles present; how ever, the behavior of the system was not dependent on the concentration of the dye encapsulated. This means that one can encapsulate of a variety of concentrations of drugs in our niosomes, and would see a peak during approximately the first six hours when most of the bursting is occu rring, followed by a gradual decrease in drug in the membrane as the drug is released across the membrane starting at about 12 hours. This data indicates the general behavior of the system as well as supports the consistency of the behavior of the system with different concentrations. The data indicates that a higher initial concen tration of dye encapsulated in the niosomes will ultimately result in a higher amount of mo les present in the system, but the general behavior is not affected by the concentration. The free moles of dye in the membrane have also been compared for 5mM and 5mM/PBS. Figure 24 illustrates th e behavior of the moles over time.
Figure 24. Moles of Dye in Membran e vs. Time (5mM and 5mM/PBS) It is important to note that these experime nts were both carried out over 144 hours, but is illustrated only for 72 hours. The moles in 5mM increased to a maximum at about 24 hours and the gradually decreased until about 72 hours. After 72 hours, the moles began to increase again. The moles initially increased due to the breaking of the niosomes in the membrane and then gradua lly decreased as moles of dye are diffusing across the cellulose membrane. The importa nt point to note here is the comparison of 5mM/PBS. The moles in 5mM/PBS did not do a sudden in crease at the beginning of the experiment, but more of a gradual increase over time. Th is indicates that the free moles in the PBS experiment did not increase and that PBS stab ilized the breaking of the niosomes better than the water. The niosomes were released gradually over time due to natural instability 116
117 of the niosomes, but it clear that the water me dium is the solution that creates instability and causes the niosome to release dye. 7.2. Moles of Dye in the Medium The behavior of the moles in the medium was also of importance when analyzing the system. The moles in the medium were an indication of how many moles actually diffuse across the cellulose membrane. This was important because it provide information on the number of moles, or amount of drug that will cross the membrane and be exposed to the surroundings. Figure 25 depicts the behavior of the moles in the medium over time for 4mM, 5mM, and 14mM. The plot shows that the behavior of the moles in the membrane over time is a clear increase in the number of moles over time. The 4mM and 5mM both began with relati vely similar concen trations of dye encapsulated in the niosomes, and their beha vior of moles in the medium followed a similar pattern. There was a steady gradual in crease in the number of moles that diffused into the medium. The dashed line for the 4mM at 7 hours indicates an extrapolated point. During the first six hours, the behavior of the moles in the medium was somewhat variable; however, the overall behavior was a constant increase. This was in agreement with the data of the moles in the membrane. Most of the niosomes broke during the first ten hours at which time, as indicated in Figure 25, there was a steady increase in moles in the medium as the dye from those broken ni osomes diffused across the membrane.
Figure 25. Moles of Dye in Medium vs. Time (4mM, 5mM, and 14mM) The moles in the membrane began to steadily decrease at about 12 hours, while the moles in the medium began to steadily increase at 12 hours. The 14mM initially had a higher con centration of dye encapsulated in the niosomes. The behavior of the moles in th e medium was somewhat variable during the first ten hours when the burstin g of the niosomes reaction was dominant. This variability could be due to the system trying to reach e quilibrium while more ni osomes are bursting, keeping the concentration variable until about ten hours when the moles do not fluctuate as dramatically. Once the majority of th e niosomes have broken, there was a sharp increase in the moles that diffused into the medium followed by a steady increase in the moles after about 24 hours. After the major ity of niosomes burst at approximately ten hours, there was a sharp decrease in the moles in the membrane and a sharp increase in 118
the number of moles in the medium. This was due to the systems attempt to balance the concentration gradient and reach equilibrium After about 24 hours, the attainment of equilibrium reached an approximate steady beha vior. The overall behavior of the three systems was similar and indicates the stability of the niosomes with varying concentrations. The absolute values of moles in the system will of course be different, and the intermediate path of the system ma y vary, but the behavior of the system was more or less consistent regardless of the concentration of dye encapsulated. The moles in the medium of 5mM and 5mM/PBS are shown in Figure 26. Figure 26. Moles of Dye in Medium vs. Time (5mM and 5mM/PBS) This plot clearly indicates that the water in 5mM forced the niosomes to burst and the released dye was diffusing across the cellulose membrane. The steady increase in moles in the medium indicates that dye from the membrane was diffusing out, and this can only 119
120 happen if dye was begin released by the niosomes. In contra st, the moles in the medium for 5mM/PBS indicates that the there was only a small rise in concentration at the beginning. This was when the dye released naturally from the niosomes diffused across the membrane. The fact that the moles in the medium did not increase over time indicates that the there was minimal release of dye from the niosomes in the membrane. This indicates that the medium of PBS did not induce the niosomes to break. It would therefore be recommended that if one desired the niosomes to be forced to break, water would be the preferred medium over PBS. 7.3. Experimental Concentration of Dye vs. Time 7.3.1. Niosomes The concentration in the niosome and the concentration released were plotted on the same graph versus time for 4mM in Figur e 27 from 0-24 hours. The concentration for all plots in this section are viewed in the log scale so th at a clear comparison can be made.
Figure 27. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Niosome-4mM) 121 The plot indicates that concentration in the niosomes was sharply de creasing in the first three hours. This means that there was an init ial burst and release of niosomes at the very beginning of the experiment. During this tim e, the concentration released into the cellulose membrane was increasing as indicat ed by that initial release. There was a period in which the concentration in the niosome membrane was practically constant from three hours to nine hours. During this time of nios omes not constant concentration, the concentration released decreased as that dye was diffusing across into the medium. After about nine hours, the conc entration in the niosomes d ecreased again. The release rate of the niosomes was appr oximately constant over this time as indicated in Section 7.7. This decrease in concentration c ould be explained by several reasons. The concentration may have begun to decrease as more stable niosomes began to break over
122 this time. The larger, more unstable niosomes burst first, and the smaller, more stable niosomes broke after time. The decrease in concentration could be misleading since there are two separate sub-systems that have been m odeled. The decrease could really just be a constant concentration but did not recognize th e other diffusion that was occurring in the cellulose membrane sub-system. The decreas e in concentration could also possibly be due to a concentration differential between the concentration in the niosome and the concentration outside of the niosomes. When this differential is too low, the niosomes may not burst until the differential increas es and promotes bursting again. The concentration released continued to decrease as the dye continued to diffuse into the water medium. The concentration in the niosome and the concentration released were plotted on the same graph versus time for 5mM in Figure 28 from 0-144 hours.
Figure 28. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Niosome-5mM) 123 This plot indicates that the concentration in the niosomes decreased over the entire time range. There was an initial decrease in concentration as water initially bursts the vesicles, at which time the concentration released into the cellulose membrane increased as the dye moves into the membrane. Like with the 4mM, there was a time interval in which the niosomes appeared to not be releasing dye as indicated by a steady line from 12 hours to 24 hours. The concentration then decreased. This again can be explained by several reasons. The concentration may begin to decrease as more stable niosomes begin to break over this time. The larger, more unsta ble niosomes burst first, and the smaller, more stable niosomes will broke after time. The decrease in concentration could be misleading since the system was separated into two sub-systems. The decrease could really just be a constant concentration but did not recognize the ot her diffusion that was
124 occurring in the cellulose membrane sub-sy stem. Another possible reason could be the initial burst of dye created an environmen t outside of the niosomes that was too concentrated, and the niosomes did not wa nt to release any more dye until this concentration decreased. It is noted again that the concentration released into the membrane greatly decreases over this time of no release from the niosomes as the dye is diffusing across the cellulose membrane to try to decrease that concentration the niosomes detect outside. It is also importa nt to note that this time of niosomes not breaking occurs later in the time interval a nd for a longer amount of time that of the lower concentration, 4mM. This could be due to the fact that more stable niosomes will burst later during the experiment at the higher concentration. This difference could also be due to the fact that the higher concentra tion release provided a higher concentration outside of the niosomes and the rate thr ough the membrane was approximately the same, so it took a little longer to diffuse that higher concentra tion in the water medium. The niosomes then began to release dye consta ntly from 24 hours to 72 hours, while the concentration released in the membrane gradua lly increased over this time as the dye was released and was now steadily diffusing into th e medium. At 72 hours the slope of rate of the niosomes releasing dye changed; it be came slower. The majority of niosomes broke at the beginning of the experiment, but at 74 hours there were even less niosomes to break and so the rate of breaking may have decreased again. This shift in niosomes that broke occurred at 72 hour s and remained constant ther eafter. The concentration released into the membrane followed the behavi or of the niosome rel eased change by also increasing at a slower rate.
The concentration in the niosome and the concentration released are plotted on the same graph versus time for 14 mM in Figure 29 from 0-72 hours. Figure 29. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Niosome-14mM) 125 This plot indicates a similar behavior as th e 4mM and 5mM. There was an initial release of dye from the niosomes at the beginning of the experiment, as s hown by a decrease in concentration in the niosomes and an increase in concentration in released dye to the membrane. This was again followed by a pe riod of constant concentration in the niosomes which was explained by the several possibilities disc ussed earlier. The higher concentration, again, resulted in a longer time of this constant be havior from 12-72 hours. This again, could be due to the fact that for higher concentrations, the niosomes mostly all broke at the beginning and that the smaller, more stable niosomes took even longer to break. It may be that there were no more niosomes to break. It could also mean that it
took longer for the concentration differentia l to reach a point again that promotes breaking. The concentration outside still decreased over tim e as it was continuously release into the membrane. Concentration wa s the driving force behind the breaking and release of the niosomes in th is experiment as well, and indicates the independence of original concentration encapsulated on this phenomena. The concentration in the niosome and the concentration released are plotted on the same graph versus time for 5mM/PBS in Figure 30 from 0-144 hours: Figure 30. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Niosome-5mM/PBS) This plot indicates that PB S was not inducing a release of dye from the niosomes as indicated by the constant decrease in nios ome concentration and the constant increase in released concentration. There was no indicat ion of an initial burst of niosomes or any 126
significant change in how the dye was released from them. In the experiment, the only dye that was being released from the niosom es was the dye released over time due to natural instabilities in the niosomes structure. This supports the theory that osmotic interactions of water caused the niosomes to break, while PBS did not have an effect on the release. 7.3.2. Diffusion from Membrane to Medium The concentration in the membrane and the concentration in the medium are plotted for 4mM in Figure 31. Figure 31. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Membrane-4mM) The data indicates that as the niosomes burst, the dye from them was being released into the tubing, but then diffused out across the cellulose membrane. The concentration in the 127
128 membrane decreased as the moles of dye di ffused out into the beaker and as water entered into the membrane and created a dilution of the existing dye in the membrane. The dye in the medium was increasing gradually as the dye diffused out into it. There was a significant increase in the concentra tion in the medium at the beginning of the experiment as the concentration gradient was much higher at the beginning. Over time, the concentration gradient deceased and both si des changed in concentration steadily. It was important to observe that the concentrati on in the membrane and the concentration in the medium complimented each other throughout the experiment. This meant that as one concentration increased, the other one decreased It is important to note that 4mM was only run for a maximum of 24 hours. Extrapolation was not perf ormed for this plot because these plots are strictly experimental measurements only. Predicted values were determined and are discussed later. The concentration in the membrane and the concentration in the medium were plotted for 5mM in Figure 32.
Figure 32. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Membrane-5mM) 129 It is clear that 5mM behaves similarly to 4mM during the first 72 hours. The niosomes are initially breaking, releasing dye, and diffusing across the membrane. The concentration in the membrane decreased as the niosomes diffused out and the water diffused inward, while the concentration in th e medium increased as the dye diffused out into it. There was a significant increase in the concentration in the medium at the beginning of the experiment as the concen tration gradient was much higher at the beginning. Over time, the concentration gr adient deceased and both sides changed in concentration steadily. Afte r 72 hours, the concentration in the membrane increased, while the concentration in the medium decreased It was here that the moles of free dye in the tubing (5.693x10 -8 moles) was very close to the moles of dye in the beaker (5.221x10 -8 moles). Although the data indicates that the moles in the tubing was slightly
130 greater than the moles in the medium, based on the difference between these values at different times, it was safe to say that moles in the beaker could very well be greater at t=72 hours than the moles in the membrane. If the system sees more moles in the medium than in the membrane, the equilibrium shifted and the moles in the medium began to flow back into the membrane to create equi librium. After a certain amount of time, the moles in the membrane will again be greater than the moles in the beaker and expected diffusion will occur. The reason for the shift in equilibrium at this time in the experiment may be due to the fact that the niosomes after 72 hours de creased their rate of release, as indicated in Figure 28. The fr ee dye in the membrane was not increasing as steadily as it was in the past, so the moles in the medium at this point would be greater than in the membrane. Once the system adjusts to the new slightly slower release of dye from the niosomes, the system will again recognize that the concentration in the membrane was higher than in the medium and the dye will again diffuse into the medium until equilibrium is reached. The concentration in the membrane and the concentration in the medium are plotted for 14mM in Figure 33.
Figure 33. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Membrane-14mM) This plot indicates the same behavior as the 4mM and 5mM over 72 hours. The concentration in the membrane was slowly decreasing over time while the concentration in the water medium was slowly increasing over time as the dye diffuses across the cellulose membrane. The dye was diffusing st eadily from 12-72 hours as this is the time that the niosomes were not breaking and waiti ng for the concentration in the membrane to decrease. There was a significant increase in the concentration in the medium at the beginning of the experiment as the concen tration gradient was much higher at the beginning. Over time, the concentration gr adient deceased and both sides changed in concentration steadily. 131 The concentration in the membrane and the concentration in the medium were plotted for 5mM/PBS in Figure 34.
Figure 34. Experimental Concentration of Dye vs. Time Plotted on the Log-Scale (Membrane-5mM/PBS) This plot indicates that the concentration in the membrane decreased slightly as the dye from the niosomes was slowly released ove r time. There was no dip in concentration anywhere, which indicates that there was no sudden increase in dye from the niosomes. The concentration almost appeared constant, or at least was changing very slightly over the long time interval. This supports the fact that PBS was not inducing breaking of the niosomes. The concentration in the PBS me dium appeared to be decreasing over time; however, it was suspected that the sensitivity at such low concentration resulted in fluctuations in the measurements. It was be lieved that the concentration in the medium increased where there concentration in the me mbrane decreased and that there was error in the measurements of the experimental data. 132
7.4. Experimental vs. Theoretical Concentration of Dye 7.4.1. Membrane The theoretical concentration in the memb rane and medium were calculated using Ficks First Law of Diffusion as a model. The experimental concentrations were plotted as stray data points on the same plot. Th e results for the 4mM, 5mM, and 14mM were plotted together in Figure 35. Figure 35. Experimental vs. Theoretical Co ncentration of Dye Using Fick's First Law of Diffusion (4mM, 5mM, and 14mM) The plot indicates that the theoretical model of Ficks First Law predicted the experimental data well. The r 2 value for 4mM was 0.9338, for 5mM was 0.934, and for 14mM was 0.8675. The average di ffusion coefficients were 4.17x10 -5 m 2 /hr, 2.14x10 -5 133
m 2 /hr, and 3.23x10 -5 m 2 /hr, respectively for the three e xperiments. An average diffusion coefficient was found to be 2.98x10 -5 .46x10 -6 m 2 /hr. Figure 35 is plotted using the individual diffusion coefficien ts; a plot using the average diffusion coefficient yields a similar behavior. Ficks First Law of Diffusi on predicted that as the concentration in the membrane decreased, the concentration in th e medium increased in a constant manner. This was in agreement with the experimental data. The data indicates that the change in concentration in the membrane and the cha nge in concentration in the medium would always be the same regardless of the initial concentration. Figure 36 shows the theoretical and e xperimental concentrations for the 5mM/PBS. Figure 36. Experimental vs. Theoretical Concentration for 5mM of Dye in PBS 134
This plot indicates that the theoretical mode l was not able to fit the experimental data well due to the inaccurate m easurements in data that suggest a decrease in the concentration in the medium. It was most lik ely that the data point s were not indicative of what was actually occurring, and so the theoretical model appeared to be a bad fit. If the data points were measured with bett er confidence, it is predicted that the concentration in the medium would increase, and the theoretical model would fit it well. 7.4.2. Niosome The experimental and theoretical concentr ation in the niosome and concentration released into the membrane for 4mM were plotted in Figure 37. Figure 37. Experimental and Theoretical Co ncentration of Dye Using Fick's First Law of Diffusion (4mM) 135
This plot indicates that the theoretical model f its this plot moderately well. The error in the theoretical model was that is did not rec ognize the point in the experiment when the niosomes were not breaking. The concentratio n in the niosome was c onstant at that point and Ficks Law did not account fo r that delay predicts consta nt release. The two data points that were practically on top of one another indica ted the point at which the niosomes were not breaking and was where th e theoretical model failed. Figure 38 illustrates the experimental and theoretical concentrations for 5mM. Figure 38. Experimental and Theoretical Co ncentration of Dye Using Fick's First Law of Diffusion (5mM) 136 The conclusion is the same. The theoretical model predicted moderate ly well, but did not predict the constant concentration in the nios ome when they were not breaking. The two data points with the same concentration in niosome were the point at which the niosome was not releasing dye. Ficks First Law did not model this occurrence well. The
behavior was repeated when the experimental and theoretical concentrations were plotted for the 14mM in Figure 39. Figure 39. Experimental and Theoretical Co ncentration of Dye Using Fick's First Law of Diffusion (14mM) The pattern was repeated in this plot that showed that Ficks model did not model well for the system when the niosomes were not br eaking. It was very obvious in this plot because very early on in the 14mM experiment did the niosomes stop breaking. For the remainder of the experiment, they mostly did not want to break (or had mostly broken) as indicated by the practically c onstant concentration in the niosome of the experimental points. Figure 40 illustrates the experimental and theoretical concentration for the 5mM/PBS experiment: 137
Figure 40. Experimental and Theoretical Co ncentration of Dye Using Fick's First Law of Diffusion (5mM/PBS) y = -0.0022x + 0.0276 R2 = 0.9608 The plot indicates that Ficks First Law of Diffusion did mode l this system very well. The r 2 value for the experimental data was 0.9608. The system was modeled well because the release of dye from the niosome was slow and gradual. Since the PBS was not causing the niosomes to burst, there wa s not a sudden change in concentration followed by a period of niosomes not bursting. In this experiment, dye was gradually being released from the niosome over an extended amount of time, and it was indicated that Ficks Law was a good model for this system. 138
7.5. Experimental Flux 7.5.1. Membrane Ficks First Law of Diffusion was not ab le to model the concentration in the medium well. This was due to the dynamics of the system that were not accounted for in the equation. Ficks Law can be used with the experimental data to obtain the actual flux at each time. The fluxes for 4mM, 5mM, and 14mM are shown in Figure 41. Figure 41. Flux vs. Time in Me mbrane (4mM, 5mM, and 14mM) The plot indicates that for all experiments the flux decreased as the concentration gradient between the membrane and medium deceased. At the beginning of the experiment the niosomes first released a larg e amount of their dye. The concentration in the membrane was high in the beginning of the experiment. As time went on, the dye 139
diffused across the cellulose membrane the c oncentration between th e two sides evened out. Although there were some niosomes still bursting over time, the concentration gradient was more even now and continued to decrease as dye went into the medium and increased the concentration there. Figure 42 illustrates the flux for the 5mM and 5mM/PBS. Figure 42. Flux vs. Time in Me mbrane (5mmM and 5mM/PBS) The plot indicates that the flux of 5mM/PBS wa s practically constant which indicates that the niosomes were not dramatically br eaking and releasing dye, causing a major concentration gradient. The flux was influe nced by the concentration gradient and was constant/steady if there was not a major change in concentration. The PBS was not breaking the niosomes and therefore, the c oncentration gradient in the membrane and medium was always steady. The 5mM behaved as previously described: decreased as 140
the concentration gradient decreased and increased as the concentration in the membrane increased at 72 hours due to the shift in ni osomes breaking and the momentarily higher number of moles in the medium than membrane when the system was readjusting to this change. 7.5.2. Niosome The fluxes of dye from the niosome are shown in Figure 43 for 4mM, 5mM, and 14mM. Figure 43. Flux vs. Time in Ni osome (4mM, 5mM, and 14mM) Figure 43 indicates the flux was initially very high as the concentration gradient was very high. The flux decreased over time as the c oncentration gradient deceased. The steep slope at the beginning indicates that this was the time when the niosomes initially burst 141
due to their contact with water. The flux re mained constant because the niosomes were either not breaking or were releasing dye very slowly at a constant rate. The flux of 5mM and 5mM/PBS is shown in Figure 44. Figure 44. Flux vs. Time in Niosome (5mM and 5mM/PBS) This plot indicates that the release from the niosomes in the 5mM/PBS was relatively constant. There were not significant change s in the flux which meant that the dye from the niosomes was only being release over time and was not induced by the PBS. The flux was relatively constant because the difference inside and outside of the niosome (gradient) remained the same throughout the experiment. The low flux meant that there was not dye being released. The flux for the 5mM dye started high and decrease over time as the concentration gradient decreased. There was no indication that the niosomes 142
were bursting or had the need to burst. The flux was gradual and was only being released constantly as the stability of the niosomes weakened over time. 7.6. Predicted Concentration of Dye Over Time 7.6.1. Membrane The concentration in the membrane and the c oncentration in the medium were predicted for different times. All the plots were gra phed on a log-scale to illustrate better the behavior of the system. Figure 45 shows the results for 4mM. Figure 45. Predicted Concentration of Dy e Over Time for the Membrane System Plotted on the Log-Scale (4mM) 143
Figure 45 indicates that the concentrations were predicted well at the beginning of the experiment. It was clear that the predicted times agreed at the beginning of the experiment with the experimental data. Figure 46 illustrates the same plot for the 5mM. Figure 46. Predicted Concentration of Dy e Over Time for the Membrane System Plotted on the Log-Scale (5mM) 144 Figure 46 indicates Ficks Firs t Law predicted the concentration well at the beginning of the experiment. After 72 hours, there was th e shift in breaking niosomes and therefore a shift in the normal behavior of the concentr ation in the membrane. The predicted plot wanted to keep decreasing, even after 72 hours, but experimental data indicated that it did not decrease there but rather increased and then continued to decrease until it reached equilibrium with the medium. The niosomes we re breaking at a lower rate and thus a less concentration change occurred as indicated by the more steady concentration data in the membrane as opposed to the sharp decline in the predicted line. There was perhaps a
different model that could predict this behavior; however, this is for the future. Figure 47 illustrates the predicted concentration in the membrane and concentration in the medium for the 14mM. Figure 47. Predicted Concentration of Dy e Over Time for the Membrane System Plotted on the Log-Scale (14mM) 145 Figure 47 indicates that Ficks First Law pred icted the concentration moderately well at the beginning of the experiment for the 14mM. The data points at the beginning were slightly different than the predicted values (error bars were plotted to illustrate the discrepancy) due to the difference in the av erage diffusion coefficient used to calculate the predicted values and the actual diffusion coefficients used to calculate the experimental values. The experimental diffusion coefficients varied slightly from one time-run to the next. The predicted concentr ations used an average diffusion coefficient in their calculations which explains for the slight variation. The 4mM and 5mM
diffusion coefficient per experiment must ha ve been relatively close to the average diffusion coefficient that went into calcu lating the predicted values because those experimental points were very close to the predicted points. The theoretical concentrations deviated at 72 hours, which indicates that the concentration in the membrane was not decreasing as fast as the predicted values were. The concentration decreased only slightly because it was through this time interval that the niosomes were breaking at a very slow rate. Future work may be spent on finding a model that would predict this behavior. Figure 48 illustrates the predicted concentration in the membrane and concentration in th e medium for 5mM/PBS. Figure 48. Predicted Concentration of Dy e Over Time for the Membrane System Plotted on the Log-Scale (5mM/PBS) 146 Figure 48 indicates that the predicted concentration fitted well to the experimental concentrations over the entire time interval This would be correct because the PBS
system was somewhat stable, meaning that th ere were no bursting niosomes or changes in bursting niosomes. The concentration in the membrane steadily deceased over time, while the concentration in the medium steadily increased over time as the dye from the niosome was slowly being released over time. The plot supports the fact that PBS did not affect the release of the niosome and a simple system such as this can be predicted well with Ficks First Law for many different times. 7.6.2. Niosome The predicted concentration of dye in the niosome and concentration of dye released into the membrane were plotted for 4mM in Figure 49. Figure 49. Predicted Concentration Over Time for the Niosome System Plotted on the Log-Scale (4mM) 147
The plot indicates that the concentrations can be predicted moderately well at the beginning of the experiment. Error points we re plotted and indica ted the error in the average diffusion coefficient that was used to calculate the predicted concentration and the actual diffusion coefficient obtained for each time-run experiment. The model did not indicate the point at which the niosomes we re not breaking as seen as the relatively constant behavior of the experimental data points. Figure 50 illustrates the predicted concentrations for 5mM. Figure 50. Predicted Concentration of Dy e Over Time for the Niosome System Plotted on the Log-Scale (5mM) 148 The plot shows a moderately well fit at th e beginning of the experiment, but did not predict well the point at which the niosomes are not breaking. That point was indicated by the data points that were constant over a sp ecific period of time. The rate of breaking was constant and slower and so the concentrat ion in the niosomes slowly decreased over
time; Ficks Law predicted a steadier, steepe r decline. A better model is needed that describes this behavior. Erro r in the diffusion coefficient wa s indicated by th e error data points. Figure 51 illustrates the same graph for 14mM. Figure 51. Predicted Concentration of Dy e Over Time for the Niosome System Plotted on the Log-Scale (14mM) 149 The plot follows the same pattern as the 4m M and 5mM. The predicted concentrations did not fit the experimental data well wher e the niosomes were not breaking. The predicted curve assumed that the release had a relatively constant beha vior, but in reality, it did not. Error points were plotted i ndicating the error in the average diffusion coefficient used to calculate the predicted line and the actual diffusion coefficient used to calculate the experimental concentrations at each time-run experiment. Again, a better model is needed to predict the dynamics of the system. Figure 52 illustrates the same plot for the 5mM/PBS.
Figure 52. Predicted Concentration of Dy e Over Time for the Niosome System Plotted on the Log-Scale (5mM/PBS) The plot indicates that the predicted model fits the experimental data very well. Ficks First Law worked well with the PBS system because dye was being slowly diffused from the niosome over a long period of time, but there was no sudden release of dye that caused the system to behavior differently. The PBS system resulted in a stable system in which the niosomes were not breaking, but dye was slowly being released through natural causes. 7.7. Release Rate of Dye from Niosomes The release rate of dye from the niosomes was important to study because it provided information on how the dye will beha ve over time. The maximum release rate was determined and adjusted to control for the desired time and amount of released drug. 150
The dye was present in one of three areas: entrapped in the niosomes, free dye in the cellulose membrane, or free dye in the medium The release rate of the niosomes was determined from a total mole balance on the system in which the free dye in the cellulose membrane and the free dye in the medium was calculated based on measured data. Figure 53 illustrates the release rate of dye from the niosomes for the 4mM, 5mM, and 14mM. Figure 53. Release Rate of Dye from Niosomes (4mM, 5mM, and 14mM) The release rate was plotted on a scale that was normalized by the initial concentration. The data for the 4mM, 5mM, and 14mM had been normalized so that a clear comparison could be made between the three experiment s. The plot indicates that the 4mM and 14mM data points are extremely close to one another. The 5mM also followed a similar behavior, although a measurement at three hours was not made for this experiment. The 151
152 data indicates that most of the dye from th e niosomes was released during the first ten hours of the experiment, in all th ree cases. It appeared that af ter the initial release of the dye from the niosomes, there was little or no change in the rate of release from the niosomes. It is important to note, however, that experimental measurements indicate that there are still niosomes that are in tact at th e end of the experiment that have not released dye. This was based on the fact that there is a change in concentration before and after the TX-100 detergent was added, which indicated that there was still a small amount of dye in the niosomes that were not broken by the influence of the osmotic pressure difference in the niosomes and water. The si ze of the niosomes may play an important role in the study of this system. Larger niosomes were less stable than smaller niosomes and were the majority of niosomes that initia lly burst. The smaller, more stable niosomes may survive the length of the experiment and were why we saw a change in concentration even at the end. The data afte r the initial release of dye was still somewhat unclear. The experimental data dipped into the negative and back into the positive for some points. Error bars were plotted and an average behavior trend was illustrated. The concentration data indicates that the rate of release should be zer o for a period of time and then will be constant. Ev en though the rate of release wa s small, it was constant and would cause a change in the concentration as presented earlier in the concentration data. The release rate for the 5mM in water so lutions and 5mM/PBS is plotted in Figure 54.
Figure 54. Release Rate of Dye vs. Time (5mM and 5mM/PBS) It is indicated that the rel ease from the niosomes in the PBS was very much lower than the release of the dye from the niosomes in the water solutions. This means that the water induced breaking of the niosomes, while the PBS stabilized the niosomes. The niosomes will break in a similar aqueous e nvironment, while in a buffer solution will remain moderately stable. 153
154 Chapter Eight Conclusion There are several conclusions that can be made based on the experiments performed and the results obtained in this thesis: 1. It is clear that the PBS retain the stability of the niosomes. 2. The water does break the niosomes very fast initially. 3. The system was able to be characterized. 4. Diffusion data is all that is needed to characterize the system. 5. The dye from the membrane diffuses into the medium. 6. A hydrogel membrane that behaves similar to PBS is needed to stabilize the release of dye from the niosomes. The experiments performed in this thesis pr ovide important information on the behavior of the system. In these experiments, th e behavior of dye, encapsulated in non-ionic surfactant vesicles, through a ce llulose membrane into a beaker of medium was studied. The initial concentration of the dye encapsula ted in the niosomes was varied from 4mM, 5mM, and 14mM. The concentration was kept constant and the medium in which it was diffused into was changed. These experime nts led to several conclusions about the system. It is very clear that PBS retains th e stability of the niosomes. The PBS does not have any affect on the breaking of the ni osomes as seen by the constant flux, concentration in the niosome did not signif icantly decrease over time, and the moles or
155 concentration in the medium did not increa se significantly over time. The water does break the niosomes very fast initially as i ndicated by the high increa se in moles at the beginning of the experiment, the initial decr ease in concentration in niosomes, and the significant increase in concentration in the me dium. The release rate of the niosomes also indicates that most of the dye entrapped in the niosomes is released during the first ten hours of the experiment. Information on what is exactly happening in the system based on the data is kn own. Water initially rushes in and breaks the niosomes. The concentration in the niosomes decrease, th e moles in the membrane increase but the concentration will decrease as dye leaves the membrane and water comes in, and the concentration and moles in the medium increase. There is a point in the experiment in which the concentration in the niosome is cons tant and then slowly decreases. This can be explained by several reasons : the data does not give a tr ue perspective of what is occurring due to the independent systems taken, the smaller, more stable niosomes will break later in time, or the concentration di fferential in the nios ome and membrane may promote release. This point of no release is delayed in time and occurs for a longer period of time from the 4mM, 5mM, and 14mM. This could be due to the stability of smaller niosomes in higher concentrations or the fact that higher concentrations take longer for the concentration differential to decrease enough for breaking to occur again. The dye from the membrane is still diffusing into the medium during this point of niosomes not breaking because the concentra tion in the medium is still increasing. Ficks First Law of Diffusion predicts well for the membrane system because even though there are changes in the rate of the niosomes breaking, the diffusion between the membrane and medium is not effected by th at. Although there are fluctuations in the
156 gradient due to the niosomes, the system remains relatively simple and will follow Ficks First Law. Ficks First Law does not pred ict the niosome diffusi on/breaking well because it does not account for the point of no niosom es breaking/release of dye. A different model will be needed. The model therefor e does not predict time well for the niosome system because it does not recognize this shift. The objectives to the project changed as more information was made available and different failures resulted in other successes. The first objective of this project was to study the behavior of the release of the 5(6) -carboxyfluorescein from the niosomes. This was successfully accomplished by running the experiments and calculating moles, concentration, and flux to study the behavior of the system as a whole. The system was separated into two sub-systems and the behavior of each was obtained. A diffusion model was able to predict the behavior of one of the sub-systems and found that another model was needed to fit the other sub-system. The concentration in the niosomes was changed and the type of medium in which the niosomes were diffused was changed in order to observe the behavior of the system under these different conditions. The second objective of this project was to run the system with in vitro experiments. This goal was also accomplished. It is desi red to run the entire system in vitro in the future with the hydrogel, but this project just focused on the niosomes alone. In these experiments, as many parameters as possible were set to in vitro conditions such as the membrane, the dye size, the dye concentration, and the pH of the medium. The future will allow for more accurately run the experiment in vitro by setting other parameters such as temperature. The third objective of this proj ect was to look ahead to the future of the system. This goal was accomplished by inves tigating the different types of hydrogels and
157 proposing a hydrogel that has the potential to be compatible w ith the system. The future of this project was determined as we f ound out more information about the niosome system and were able to apply this information to the future design of the smartpackaging system. For example, the high conc entrations in the niosomes will result in high concentrations outside of the niosomes for a longer period of time. It would perhaps be suggested to use a smaller concentration so that there is not all of this released dye floating around for several hours that could possible cause exposure and toxicity to other organs. Water should also be present to induce the bursting of the niosomes; otherwise, it will take a very long time for the dye to be re leased naturally. Suggested future work has also be included and is presented in Chapter Nine. The smart-packing system has the potential to be a novel approach to drug delivery. The idea to have two control mech anisms is very attractive when over-exposure and increased toxicity in the body is a major problem. This project has begun the process for designing this system, and the results can provide us with valuable information as to the behavior of our vesicles. The future of th e project will lie in incorporating the second control mechanism and optimizing the properties of both. The investigation of one of the fundamental components has been performed and there is optimism about the success of the system.
158 Chapter Nine Future Work The information presented in this thesis can provide valuable insight into the direction that this pr oject will go towards. The st udy of the release rate of 5(6)carboxyfluorescein dye from niosomes in a dialysis membrane allows the understanding of the behavior of a drug in a stand alone nios ome. However, this is just the beginning of our smart-packaging system. The future of th e system is still yet to be seen, and there are still pieces of the puzzle th at must be scrutinized and in corporated into the whole. This chapter focuses on the future of the project by presenting the anticipated studies to be conducted. Several future goals of the proj ect will be presented, as well as tasks that must be performed to achieve those goals. Specific attention will be spent on describing the Surface Force Apparatus, as this will be a significant component in future studies. Attention will also be devoted to the Attenuated Total Reflection Fourier Transform Infrared technique. It is important to note that by the nature of research there is a good chance that these anticipated studies may be a ltered or a new course may be needed. As far as a PhD project is concerned, it is the hope that many of these analyses will be conducted; however, they should not be strictly interpreted at this time.
159 9.1. Future Goals of Project 9.1.1. Future Goal One The first goal for the future work of th is project is to c ontinue to study the characteristics of the behavior of the dye within the niosom es alone. Although this may seem tedious and redundant, it is important to understand this behavi or as conditions in the system change. The system was studied as the concentration of the dye within the niosomes was changed, as well as with chan ging the bulk solution to PBS; however, it is important to study the behavior of the release rate of the dye from the niosomes as environmental factors changes as well. Th e incorporation of th e niosomes into the hydrogel network is eagerly antic ipated, but it is desired to build a strong foundation on the understanding of the niosome component before a second component is added to our system. Currently, the proposed system studied in this thesis is moderately simple consisting of just the niosomes; however, when the system will be built upon by adding more complications, it will be advantage ous to have a solid understanding of the niosomes system alone. 9.1.2. Future Goal Two The second goal for the future work of this project is to develop the hydrogel membrane that will be used in the system. This is crucial for the future of this project because the hydrogel composes the second element of the smart-packaging system. The hydrogel must be developed in such a way that its prop erties compliment the behavior of the niosome component but do not greatly interfere with it. The two components must be designed in such a way that neither one stands alone, but either one
160 does not prohibit the unique char acteristics of the other. The hydrogel is very important in the system because it is the second control mechanism for drug delivery. A hydrogel system will be designed that will be biocom patible, biodegradable, pH and temperature sensitive, effective in controlling the release of the drug, and be alterable for optimization. Once the hydrogel of choice is decided upon, the next goal in conjunction with the hydrogel is to incorporate the niosomes into the network. It is desired to confirm that the two units fit together, meaning th at based on the theoretic al and experimental information obtained for the two units alone, they are actually compatible with one another and will release the drug in a controlled manner. It is important to show that these two components actually work together and their properties can be furthered studied. 9.1.3. Future Goal Three The achievement of incorporating the ni osome and hydrogel together to form a drug release system (see Goal Two) will lead to the third goal. The third goal for the future of this project is to now study the interactions between the niosome and hydrogel in detail. Now that the system is established and know that it is going to work, the interaction between the two will be studied. Th is information is impor tant because it will allow for the understanding of what propertie s govern the behavior of the system, what properties may be altered to improve th e system, and what the optimal operating conditions of the system are. It is importa nt to understand the structure of the hydrogel network and its mechanical properties such as its swelling behavior, the stiffness, the cross-link density (if applicable ), the thickne ss, its elasticity, and its dynamic response.
161 In conjunction with the mentioned propertie s, the normal forces between the hydrogel may also be of interest. The preparation conditions of the niosomes and hydrogels, the chemical composition of the niosomes and hydrogels, and the tribological behavior of the system could also be studied. These are ju st to name a few properties that could be chosen to be studied. The studies for the future work of this project are not limited to just these parameters and will not necessarily incl ude an analysis on all of the ones mentioned above. Other properties that could be of interest include the adhesion behavior of the hydrogel and the niosomes within the hydrogel Chemical reactions occurring in the system may also be of interest for the future work. It is desired to show that for a particular hydrogel with a particular cros slink density and embedded with a specific composite niosome, this is what will happen. 9.1.4. Future Goal Four The fourth goal for the future work of th is project is to design a drug delivery system that will be able to be tested in vivo in an animal rabbit model. It is desired to optimize the control of the release of the system so that the actual outcomes in a biological model can be studies. The long-rang e goal of the project is to design a system that may be used in human beings for the tr eatment of brain cancer. The goal to test the system in an animal rabbit model first so that it will give a better understanding of how the in vitro studies actually compare to the systems behavior in vivo experiments. This goal is optimistically lofty, considering all of the work that needs to be done to get to this point, so it is safe to say that this goal will remain in the far distant future but will remain important in the back of our minds as to re mind us what we are striving to attain.
162 9.2. Future Tasks for Project 9.2.1. Future Task One The first task for the future of this projec t will attempt to address the first future goal of the project: to continue to study the ch aracteristics of the niosomes alone. It is desired to observe the behavior of the re lease of the dye from the niosomes as environmental conditions are changed such as temperature and pH. The goal would be to ultimately inject the system into the huma n brain, and by altering sensitive conditions such as the temperature and pH, obtain a bette r idea of how the system will behavior once implanted in our target system. Understanding how the niosomes and dye behave under certain temperatures and pHs will allow fo r the determination of what conditions will work and what conditions will not work. Th e determination of how extreme temperature or pH changes drastically alter the release of the dye, the integrity of the niosomes, and the optimal operating range is feasible. Base concentrations can be changed to work with and hold that concentration constant while we change the other conditions. It is also possible to change the temper ature and pH for different c oncentrations if needed. 9.2.2. Future Task Two The second task for the future of this project will attempt to address the second future goal of the project: to develop a hydrogel system that will be used in the system. A literature search has already been conduc ted on several different types of polymer hydrogels that are available. The hydrogel system that is believed to have the most potential for use in biomedical applications was presented in this thesis: the chitosan/ glycerophosphate hydrogel. The first observati on would be the under workings of this
163 hydrogel in the lab such as method of synthesi s, the ease of synthesis, the temperature and pH sensitivity, and other properties that will help for the determination if this is, in fact, an acceptable hydrogel to us e. Once that is determined the incorporation of the niosomes into the hydrogel will be done. Ba sed on the literature, the chitosan hydrogel will begin to gel via physical alterations and the niosomes will be entrapped within the three dimensional network. Since a crosslinki ng agent is not needed for the formation of the chitosan hydrogel, the crosslinking density is something that may not be easily controlled for. The chitosan will form its own cross-linking density based on ionic crosslinking (see Section 2.2.4). However, other properties of the hydrogel can be studied such as the effect the chitosan deacetylation has on the release rate of the drug and the composition of the chitosan and -glycerophosphate. Thes e are all suggestions that may be desired to observe. However, it is important to note that they may not be necessary or required to study based on the literature and ot her concrete data that has been previously generated. 9.2.3. Future Task Three The third task for the future of this proj ect will attempt to address the third goal of this project: to study the interactions betw een the niosomes and th e hydrogel. The main approach that will be taken will make use of the Surface Force A pparatus, or commonly called the SFA. The SFA is an instrument that is used to measure the forces between two surfaces and can provide information on the su rface interactions, structural information about the two samples, and other interfacial pr operties . A detailed description of the SFA will be provided in Section 9.3. Th ere are three main properties that influence
164 the performance of the compone nts of a hydrogel: their mechanic al strength, their surface friction, and their response kinetics . The SFA will allow for application and measurement of the shear and normal forces between two surfaces as a function of the distance of separation between the two to the 1 The normal and shear forces will depend strongly on the structure of the hydrogel. The SFA will allow for the determination of the force profiles of the empty and niosome-embedded hydrogels. It will help to determine how the normal and adhesive forces are affected by solvent conditions, crosslink density (if applicab le), thickness, and the ionic or hydrophobic constituents in the hydrogel. It can provide information on the friction coefficients and dissipation effects between the hydrogel an d niosome and how they will affect the properties of the network. The SFA can also provide insight into effect that surface chemistry, roughness, and confinement have on the swelling behavior of the gel. The effect of changing certain ambient conditions such as temperature, pH, and solvent ionic strength have on the response of the hydr ogel can also be observed . The SFA will allow for the determination of the structure of our hydrogel network and how it behaves under certain conditions. It will allow for the determination of the surface forces of the hydrogel and compare that with the surface forces of the hydrogel and the niosomes. The cushi oning and stiffness of our hydr ogel can be determined and compared with the cushioning and stiffness of the hydrogel with the niosomes. The SFA will use a friction device and a bimorph slider An optical multiple beam interferometry (MBI) technique will provide data on the phys ical characteristics of the hydrogel network such as their thickness, thei r elastic modulus, and their normal and lateral interaction forces. The amount of force required to compress the hydrogel can be determined which
165 will provide insight into the swelling and elastic ity of the system. Recovery times of the hydrogels can be found which will provide information on the dynamic response of the system. The SFA is also a good tool for meas uring adhesion forces. The SFA will be a powerful tool in the study of our smart-p ackaging system. The information obtained from the SFA will allow us to determine the behavior of the system and thus, dictate the alteration and design of the final system. An Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) technique wi ll also be used to determine the surface reactions of our system. It will allow us to obtain a surface analysis between the empty hydrogel and the hydrogel with the niosomes. The ATRFTIR data will depend on how much force was used to compress the syst em and will be based off of the SFA measurements. The SFA can determine the surface forces of the system, and the ATRFTIR can provide information on the surface interactions of the sample. For each surface force associated with each altered system, a different set of surface interactions will be obtained. Both techniques will be discussed in Section 9.3. 9.2.4. Future Task Four The fourth task for the future of this pr oject will attempt to address the fourth goal of this project: to test the smart-packaging system in vivo experiments using animal rabbit models. This will be achiev ed by taking all of the data collected, and presenting the optimal system to the surgeons at the Moffitt Cancer Center and Research Institute. It is interest to test the system in animal models so the effects it will have once exposed to actual physiological conditions can be determin ed. It is impossible to mimic the entire environment of the body in vitro, so in vivo experiments will allow for the determination
166 of how well the system will stand up in the real world. The survival times of the animal models and any adverse side effects they may experience will be observed. Based on the information obtained from the in vivo experiments, the system will be able to be fine tuned so that it could possibly one da y be used in actual human beings. 9.3. SFA and ATR-FTIR The surface force apparatus, as discussed earlier, is an instrument that can be used to study the surface interactions of polymer sy stems, biological systems, and molecular systems in various environments. The simplest way to measure the adhesion between two solid surfaces is to measure the deflection of a spring that one surface is suspended from and observe the force required to separa te the two bodies . In short, the SFA will measure the attractive and repulsive intermolecular forces between two layers supported on a solid (or soft) substrate as a function of surface separation. The surfaces can be brought into contact with one anot her or can be separated by using highly controlled micrometers and piezoelectric disp lacement transducers. The deflection of a spring supporting one of the surfaces is used to determine the forces between them. A detailed description of the SFA will be presented here. Direct force measurements are often very straightforward, however, the problem with surface force measurements comes when there are very weak forces at very small surface separations that must be controlled a nd measured within 0.1 nm . The first direct measurements were attractive van der Waals forces made by Derjaguin and coworkers [145, 146] between a convex lens and a flat glass surf ace vacuum . The simplest way to measure the force as a functi on of surface separation is to move the base
of a force-measuring spring by a set amount, D o and observing if the spring deflects by D s if there is indeed a force between the tw o surfaces . The surface separation will change by D, and the three distance changes ca n be related by the following equation : DDDos Eqn. 45 and the force differential between the initia l and final separation can be represented by : ssDKF Eqn. 46 where K s is the stiffness of the force-measuring spring (spring constant). The measurement of the forces between two flat surfaces is extremely hard due to the rigorous effort to perfectly align the surfaces for reli able angstrom measurements . It is much easier to measure the forces between two curved surfaces such as two spheres, a sphere and a flat surface, or two crossed cylinders . The formula for the general force law is: D DW DF )( ) ( Eqn. 47 The Derjaguin approximation is used to rela te the force, F(D), measured between two curved surfaces with the energy per unit ar ea, E(D), between two flat surfaces at a distance, D, apart from each other . Th e surface forces depend on the geometry of the surfaces in contact, and for two spheres in contact with one a nother, the Derjaguin approximation would take the following form : )( 2)(21 21DE RR RR DF Eqn. 48 167
where R 1 and R 2 are the radii of the spheres in contact with one another. For the case of two cylinders, the Derjaguin approximation becomes : )sin( )( 2 )(21 DWRR DF Eqn. 49 where D is much smaller than R 1 or R 2 For the case where R 1 = R 2 the formula for the Derjaguin approximation would be as follows : r DF DE2 )( )( Eqn. 50 The Derjaguin approximation can be applied to ay force law including attractive, repulsive, or oscillatory for ces . The only requirement is that the range of interaction, or separation between the two su rfaces, is much less that the radii of the spheres . The Derjaguin approximation can be used to derive the interaction energy for two planar surfaces if the easier to meas ure forces between two curved surfaces are known. The SFA was developed to measure the interactions betwee n dynamic and static surfaces in vapors or liquids with precision do wn to the Angstrom level . Previous techniques developed by Tabor and Winterton (1969)  and Israelachvili and Tabor (1972, 1973) [148, 149] measured the van der Waals forces between smooth mica in air or in a vacuum. The technique s were in agreement with the Lifshitz theory of van der Waals forces as small as 1.5 nm separati ons . The techniques were improved upon to include measurements in liquids. The SFA th at we will be using is one such apparatus. The SFA can directly measure the force be tween two surfaces in controlled vapor or immersed in liquids [150, 151] Figure 55 illustrates a basic SFA. 168
Figure 55. Basic SFA (Mark 4 Model) The distance resolution is about 0.1 nm and has a force sensitivity of about 10 -8 N. Israelachvili (1992)  provide s a detail description of ho w the SFA works. The SFA consists of two curved mica surfaces that are molecularly smooth with a radius of about 1 cm and a thickness of about 2 m. The mica sheets are transparent, which allow a beam of light to pass thr ough the mica and onto the sample. The interaction forces are measured by force-measuring springs. The mica surfaces are oriented in a cross cylinder manner, which would be similar to contacting a sphere and a flat su rface or contacting two spheres. An optical technique is used to measure the distance of separation between the two surfaces from microns to molecular le vels. The optical technique uses multiple beam interference fringes, or Fringes of Equal Chromatic Order (FECO) to determine this distance. The mica surfaces are coated with a layer of pure silver and glued silver side 169
down onto the silica discs. A light is passed up through the two surfaces, where the beam is forced onto the beam of a grating spectrometer. Figure 56 illustrates this procedure. Figure 56. The Path of Light Through the Mi ca Surfaces and to the Spectrograph in an SFA 170 The FECO fringes can provide information on the distance betw een the two surfaces (usually to better than 0.1 nm), the exact shape of the two surfaces, and the refractive index of the material between them base d on the fringes positions and shapes. The shapes of the fringes also allow for the dete rmination of the radii of the surfaces and any deformation in the surfaces that may occur du e to the interaction . The multiple beam interference fringes allow for the su rface separation to be measured to 1 . A three-stage mechanism is used to control the distance between the surfaces. A coarse control can control for positioning within 1 m, a medium control can position distances within 1 nm, and a piezoelectric crystal tube can position distances to 0.1 nm by vertically expanding or contracting by about 1 nm per volt applied axially across its
171 cylindrical wall . The force measurem ents can be made by the expansion or contraction of the piezoelectric crystal by a sp ecific quantity. The measurements are then able to be taken optically by observing how much the two surfaces have moved. The force difference between the initial and fina l positions can be determined by multiplying the difference in how much the two surfaces have moved by the stiffness of the spring. A full force law can be determined for any distance and for both re pulsive and attractive forces. A single-cantilever or a double-cantilever fi xed-stiffness spring can be used as the force measuring spring, and the stiffness of the spring can be adjusted and changed during the experiment by a factor of 1000 . The force between any other curved surfaces can be scaled by R once the force as a function of D is determined for the two su rfaces. The Derjaguin approximation can be used to relate the interfacial (or adhesi on) energy E per unit ar ea between two flat surfaces. The SFA has been used to measure the basic interactions, between surfaces in both aqueous and nonaqueous solutions, and includes van der Waals forces, electrostatic double-layer forces, oscillatory forces, repulsive hydration forces, attractive hydrophobic forces, steric interactions in polymer systems, capillary forces, and adhesion forces . The use of the SFA has been extended to measurements of dynamic interactions and time-dependent effects [152-154], shear and fric tion forces , and the fusion of lipid bilayers . The SFA has been used to study the forces between a mica surfaces coated with lipid bilayers . The forces between two bilayers ab sorbed onto the mica surface have shown to correlate well to larger vesi cles without undulation forces . The SFA can be used to measure the magnitude, range, and origin of the forces that control for
172 vesicle stability . For our system, th e SFA will allow us to determine how much force is needed to compress the hydrogel and what the strength of the hydrogel is. We will be able to compare the forces in the hydrogel alone with the forces that are present when the niosomes are added to the network. A certain force will be associated with each manipulated hydrogel (i.e. crosslinki ng density if applicable, composition of components, amount of components, etc.), and we will be able to determine which system will work best for our drug delivery. We will also be able to gain other important information from the SFA about our system and ultimately be able to gather this knowledge and present an optimized system to Moffitt. We are also interested in using ATR-FTIR techniques to study the surface interactions of our sample. Ot her analytical surface techniques that have previously been used include X-ray and UV photoelectron spectroscopy, electron energy loss spectroscopy, Auger electr on spectroscopy, low-energy electron diffraction, field emission microscopy, field ionization sp ectroscopy, scanning tunneling spectroscopy, and atomic force microscopy . The prob lem with these techniques is that they require the use of an ultra-high vacuum whic h can change the surfac e characteristics of the sample . Other techniques have show similar setbacks. ATR-FTIR is a technique that has shown to not alter the surface interactions of the sample to be studied . ATR-FTIR is a specific form of a F ourier Transform techniqu e. A FT technique applies the principles of infrared spectrosc opy (IR) which is to pass an infrared light through a sample and observing what part of the light is absorbed by the sample at a particular frequency . FTIR has improved most aspects of traditional IR
spectroscopy through the use of an interf erometer. Figure 57 illustrates a typical Michelson interferometer . Figure 57. Schematic of a Michelson Interferometer The interferometer consists of two mirrors that are perpendicular to one another: one mirror that is able to move and one mirror that is stationary. The mirrors are bisected by a beam splitting mirror, or a semi-reflecting mirror. The beam splitter splits the beam from the source. One beam is sent to the fixed mirror where it is reflected and sent back to the beam splitter. The two beams interfere on recombination. The beam is controlled by the movable mirror and leaves the interferometer to go to the detector (interferogram). The detector receives a signal from the intens ity of the combined b eams as a function of the position of the mirror. The intensity is converted into a spectrum by a Fourier transform function. There are se veral steps that must be take n in order to obtain an IR transmission spectrum. A interferogram mu st be obtained for both a beam with and without a sample and be translated from inte rferograms into spectra of the source with and without sample absorption . 173
174 ATR-FTIR has been successf ul in measuring the diffusion in polymers  based on its robust, noninvasive, in situ technique. ATR-FT IR is a reliable method for short-time data that is easy to use and co mparably inexpensive against other techniques such as NMR spectroscopy. ATR-FTIR is unique from other techniques in that it is able to distinguish between chemical species by providing molecular-molecular differences between solute and polymer through the abso rption of light at different wavelengths . Shifts in the IR spectrum can al so allow the determination of chemical interactions between the solute and the polymer. ATR involve s the passing of a beam of light through a dense material where a rarer medium reflects the light at the interface . The attenuated terms derives from the process of the rare medium absorbing light at the interface [ 160]. For ATR, a special ATR crystal is placed in optical contact with a sample . The ATR crystal has a high refractive index. The spectrometer uses mirrors to focus an IR beam onto the beveled (inclined) edge of ATR element where it is reflected and absorbed through the crystal and another set of mirrors sends the signal to a detector. ATR is based on that fact that some radiation will penetrate some of the sample (evanescent wave) even though most internal reflection occurs at the sample-crystal interface. The distance of pe netration depends on the wavele ngth of the incident light . The sample will inte ract with the evanescent wave and the sample will absorb radiation . This radiat ion resembles the transmission spectrum for the sample. The interface is where chemical bond s from the existence of solute molecules in the polymer (or change in the polymer due to interactio ns with solute molecules) are measured through the evanescent wave . Figure 58 i llustrates the simple ATR process 
while Figure 59 illustrates a FTIR sp ectrometer and ATR attachment for the measurement of diffusion in polymers . Figure 58. Scheme of an ATR Figure 59. Schematic of the FTIR Spect rometer and ATR Attachment for the Measurement of Diffusion in a Polymer 175 The ATR spectrum will depend on several fa ctors including the a ngle of the incoming radiation, the wavelength of th e radiation, and the sample and ATR crystals refractive indexes . ATR is a fast technique to obtain IR spectrum th at will not harm the sample There is usually no sample prepar ation required; however, attention must be spent on the sample to ATR crystal optical contact. The FTIR-ATR can be used for a
176 variety of pharmaceutical applications in cluding characterizi ng the structure and properties of biological systems, studying noninvasive drug release, measuring drug release from polymer systems, investigating drug penetration into artificial and biological membranes, and characterizing interactions between drugs and synthetic macromolecules . 9.3. Closing Remarks on Future Work The future work of this project is very promising for the development and investigation of the smart-packaging system. The scope of this project has just begun to uncover the properties of the system, and the future work of the project will begin to build on this information. The future will also see the incor poration of the hydrogel polymer, the essential second component to the system. The interactions, compatibility, and behavior of the system will be explor ed with such tools as the Surface Force Apparatus and the Attenuated Total Reflectio n Fourier Transform Infrared technique. The SFA will allow us to comp are the surface forces of our empty hydrogel with that of our hydrogel with the niosomes embedded within its network. The surface force information will allow us to determine proper ties such as elastic ity, swelling behavior, dynamic response times, and other properties that will ultimately go into the final design of the system. This information is important because it will allow us to know the exact the behavior the system under certain conditi ons so that we may present our data to Moffitt for future investigations. The AT R-FTIR will also be a powerful tool in obtaining information about the surface interactions of the system. We will be able to determine the surface characteristics when a part icular force is applied to the system. We
177 will be able to determine any changes in the surface interactions between the niosome and hydrogel while maintaining the integrity of the system. The information obtained from these studies will allow use to present our data to surgeons at the Moffitt Research and Cancer Institute. We will be able to tell them how the sy stem will behave and hopefully run the system in vivo to determine the behavior in an actual biological model. The ultimate goal of the system is to be admi nistered intracavitary to brain tumor patients to increase survival time and decrease adverse side effects. The information and analyses proposed in this thesis and fu ture work section all aspire towards the unde rstanding and improvement of our system to attain this goal.
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Appendix A. Approximate Sizes of Quanta Table 23. Various Types of Radiation and Corresponding Sizes of Quanta  195
196 Appendix B. Density (g/ml) Calculatio n for 4mM, 5mM, 14mM, and 5mM/PBS Table 24. Density Calculations (g/ml) 4mM 5mM 14mM 5mM/PBS Time Beaker g/ml Membrane g/ml Beaker g/ml Membrane g/ml Beaker g/ml Membrane g/ml Beaker g/ml Membrane g/ml 3 0.980 1.02 0.985 6 0.981 1.00 0.980 1.04 0.986 1.055 9 0.981 1.03 0.986 1.015 12 0.987 1.03 0.990 1.01 0.986 1.02 24 0.986 1.05 0.986 1.015 0.987 1.015 0.993 1.023 72 0.980 1.015 0.986 1.02 0.993 1.025 144 0.990 1.065 1.005 0.990 1.03
197 Appendix C. Intensity Measurements Table 25. Intensity Measurements for 4mM Time (hr) Beaker (before TX) 1 ml from beaker Beaker (after TX) 1 ml sample + 10 l TX Membrane (before) 990 PBS + 10 l sample Membrane (before) 990 PBS + 10 l sample + 10 l TX 0 0 0 0 0 0 0 132.9 130. 0 130.4 337.5 333.5 335.1 3 92.60 93.68 92.39 94.87 93.58 93.54 194.9 194.8 196.9 286.0 282.5 288.1 6 99.55 99.20 98.16 91.80 91.71 91.55 184.4 184.3 184.7 264.4 261.2 264.2 9 142.9 142.2 141.0 141.2 139.8 141.1 183.4 183.5 183.1 265.2 264.9 263.0 24 236.9 235.5 236.3 225.1 224.5 223.8 136.7 164.3 163.0 238.6 236.1 236.1 Table 26. Intensity Measurements for 5mM Time (hr) Beaker (before TX) 1 ml from beaker Beaker (after TX) 1 ml sample + 10 l TX Membrane (before) 990 PBS + 10 l sample Membrane (before) 990 PBS + 10 l sample + 10 l TX 0 0 0 0 0 0 0 79.56 78. 36 80.82 219.8 221.2 222.3 6 43.17 43.91 43.80 62.29 63.17 63.08 88.26 86.76 87.63 189.5 189.2 192.2 12 67.54 67.18 68.40 96.77 95.99 97.01 90.76 89.61 90.04 182.5 179.1 177.9 24 85.15 85.96 85.66 109.6 110.2 108.9 95.67 95.46 94.25 190.2 183.5 182.2 72 187.4 187.5 187.1 206.1 205.8 208.7 77.69 77.55 77.90 127.8 126.1 127.1 144 189.1 191.8 190.6 190.9 198.1 191.9 115.0 115.5 115.4 125.7 151.9 151.8 Table 27. Intensity Measurements for 14mM Time (hr) Beaker (before TX) 1 ml from beaker Beaker (after TX) 1 ml sample + 10 l TX Membrane (before) 990 PBS + 10 l sample Membrane (before) 990 PBS + 10 l sample + 10 l TX 0 0 0 0 0 0 0 74.72 76. 23 77.62 191.9 194.5 195.1 3 124.5 124.7 121.8 121.9 121.9 122.3 117.7 118.0 119.0 159.7 160.4 159.5 6 123.1 123.9 120.9 116.0 114.7 117.1 110.0 109.3 111.2 155.8 154.8 154.6 9 209.3 208.3 208.6 215.5 216.7 217.8 100.2 100.1 101.0 144.6 143.0 142.4 12 155.1 157.9 156.4 146.8 148.7 147.4 94.81 94.90 94.98 137.1 135.7 136.5 24 182.0 183.2 183.5 179.9 182.1 182.5 81.37 82.31 82.40 125.2 125.1 126.7 72 225.8 223.2 226.2 212.0 213.4 213.1 58.3 58.0 59.6 106.0 105.3 108.5 It is important to note that these are the intensity reading for 14mM; however, the actual intensity readings are not indicative of the ab solute intensity as it is related to 4mM, 5mM, and 5mM/PBS. The samples in 14mM required dilution due to their high concentrations. The sample concentrations for the membrane were cut in half twice. For
198Appendix C. (Continued) t=0, 3, 6, 9, and 12 hours, the sample concentr ations for the beaker were cut in half. For t=24 and 72 hours, the sample concentrations for the beaker were cut in half twice. Table 28. Intensity Measurements for 5mM/PBS Time (hr) Beaker (before TX) 1 ml from beaker Beaker (after TX) 1 ml sample + 10 l TX Membrane (before) 990 PBS + 10 l sam ple Membrane (before) 990 PBS + 10 l sam ple + 10 l TX 0 0 0 0 0 0 0 113.2 114 9 113.6 269.0 269.4 269.6 24 42.54 42.53 42.73 44.18 44.49 44.31 120.1 120.2 120.7 259.7 259.0 262.0 72 38.84 39.13 37.67 37.15 37.18 36.69 125.5 125.1 125.9 243.7 241.3 243.8 144 41.46 40.89 41.03 39.15 38.43 38.64 149.8 150.7 149.1 2323.2 227.6 229.0
199 Appendix D. Material Safety Data Sheets D.1. Phosphate Buffer Saline MSDS MATERIAL SAFETY DATA SHEET Date Printed: 02/15/2006 Date Updated: 01/27/2006 Version 1.5 Section 1 Product and Company Information Product Name PHOSPHATE BUFF ERED SALINE, PH 7.4, TRUMEASURE CHEMICAL Product Number P3813 Brand SIGMA Company Sigma-Aldrich Street Address 3050 Spruce Street City, State, Zip, Country SAINT LOUIS MO 63103 US Technical Phone: 800-325-5832 Emergency Phone: 314-776-6555 Fax: 800-325-5052 Section 2 Composition/Information on Ingredient Substance Name CAS # SARA 313 PHOSPHATE BUFFERED SALINE (SOLID) None No Ingredient Name CAS # Percent SARA 313 SODIUM CHLORIDE 7647-14-5 83.8 No DI-SODIUM HYDROGEN PHOSPHA TE ANHYDROUS 7558-79-4 12 No POTASSIUM PHOSPHATE, MONOBASIC 7778-77-0 2 No POTASSIUM CHLORIDE 7447-40-7 2 No Section 3 Hazards Identification EMERGENCY OVERVIEW Caution: Avoid contact and inhala tion. Target organ(s): Heart. HMIS RATING HEALTH: 1* FLAMMABILITY: 0 REACTIVITY: 0 NFPA RATING HEALTH: 1 FLAMMABILITY: 0 REACTIVITY: 0 *additional chronic hazards present. For additional information on toxi city, please refer to Section 11. Section 4 First Aid Measures ORAL EXPOSURE If swallowed, wash out mouth with water provided person is
200 Appendix D. (Continued) conscious. Call a physician. INHALATION EXPOSURE If inhaled, remove to fresh air. If breathing becomes difficult, call a physician. DERMAL EXPOSURE In case of contact, immediately wash skin with soap and copious amounts of water. EYE EXPOSURE In case of contact with eyes, flush with copious amounts of water for at least 15 minutes. Assure adequate flushing by separating the eyelids with fingers. Call a physician. Section 5 Fire Fighting Measures FLASH POINT N/A AUTOIGNITION TEMP N/A FLAMMABILITY N/A EXTINGUISHING MEDIA Suitable: Noncombustible. Use extinguishing media appropriate to surrounding fire conditions. FIREFIGHTING Protective Equipment: Wear self-contained breathing apparatus and protective clothing to preven t contact with skin and eyes. Specific Hazard(s): Emits toxi c fumes under fire conditions. Section 6 Accidental Release Measures PROCEDURE(S) OF PERSONAL PRECAUTION(S) Exercise appropriate precautions to minimize direct contact with skin or eyes and prevent inhalation of dust. METHODS FOR CLEANING UP Sweep up, place in a bag and hold for waste disposal. Avoid raising dust. Ventilate area and wash spill site after material pickup is complete. Section 7 Handling and Storage HANDLING User Exposure: Avoid inhalation. Avoid contact with eyes, skin, and clothing. Avoid prolonged or repeated exposure. STORAGE Suitable: Keep tightly closed. Section 8 Exposure Controls / PPE ENGINEERING CONTROLS Safety shower and eye bath. Mechanical exhaust required. PERSONAL PROTECTIVE EQUIPMENT
201 Appendix D. (Continued) Respiratory: Use respirators and components tested and approved under appropriate government standards such as NIOSH (US) or CEN (EU). Respiratory protection is not required. Where protection from nuisance levels of dusts are desired, use type N95 (US) or type P1 (EN 143) dust masks. Hand: Protective gloves. SIGMA P3813 www.sigma-aldrich.com Page 2 Eye: Chemical safety goggles. GENERAL HYGIENE MEASURES Wash thoroughly after handling. Section 9 Physical/Chemical Properties Appearance Physical State: Solid Property Value At Temperature or Pressure pH N/A BP/BP Range N/A MP/MP Range N/A Freezing Point N/A Vapor Pressure N/A Vapor Density N/A Saturated Vapor Conc. N/A SG/Density N/A Bulk Density N/A Odor Threshold N/A Volatile% N/A VOC Content N/A Water Content N/A Solvent Content N/A Evaporation Rate N/A Viscosity N/A Surface Tension N/A Partition Coefficient N/A Decomposition Temp. N/A Flash Point N/A Explosion Limits N/A Flammability N/A Autoignition Temp N/A Refractive Index N/A Optical Rotation N/A Miscellaneous Data N/A Solubility N/A N/A = not available Section 10 Stability and Reactivity STABILITY
202 Appendix D. (Continued) Stable: Stable. Materials to Avoid: Strong oxi dizing agents, Strong acids. HAZARDOUS DECOMPOSITION PRODUCTS Hazardous Decomposition Products: Nature of decomposition products not known. HAZARDOUS POLYMERIZATION Hazardous Polymerization: Will not occur Section 11 Toxicological Information ROUTE OF EXPOSURE Skin Contact: May cause skin irritation. Skin Absorption: May be harmfu l if absorbed through the skin. Eye Contact: May cause eye irritation. Inhalation: Material may be irri tating to mucous membranes and upper respiratory tract. Ma y be harmful if inhaled. SIGMA P3813 www.sigma-aldrich.com Page 3 Ingestion: May be harmful if swallowed. TARGET ORGAN(S) OR SYSTEM(S) Heart. SIGNS AND SYMPTOMS OF EXPOSURE Ingestion of large amounts causes vomiting and diarrhea. Dehydration and congestion may occur in internal organs. Hypertonic salt solutions can produc e inflammatory reactions in the gastrointestinal tract. Section 12 Ecological Information No data available. Section 13 Disposal Considerations APPROPRIATE METHOD OF DISPOSAL OF SUBSTANCE OR PREPARATION Contact a licensed professional wast e disposal service to dispose of this material. Dissolve or mix the material with a combustible solvent and burn in a chemical incinerator equipped with an afterburner and scrubber. Observe all federal, st ate, and local environmental regulations. Section 14 Transport Information DOT Proper Shipping Name: None Non-Hazardous for Transport: This substance is considered to be non-hazardous for transport. IATA Non-Hazardous for Air Transport: Non-hazardous for air transport. Section 15 Regulatory Information EU ADDITIONAL CLASSIFICATION S: 22-24/25
203 Appendix D. (Continued) Safety Statements: Do not breathe dust. Avoid contact with skin and eyes. US CLASSIFICATION AND LABEL TEXT US Statements: Caution: Avoid contact and inhalation. Target organ(s): Heart. UNITED STATES REGULATORY INFORMATION SARA LISTED: No CANADA REGULATORY INFORMATION WHMIS Classification: This pr oduct has been classified in accordance with the hazard criteri a of the CPR, and the MSDS contains all the information required by the CPR. DSL: No NDSL: No Section 16 Other Information DISCLAIMER For R&D use only. Not for drug, household or other uses. WARRANTY SIGMA P3813 www.sigma-aldrich.com Page 4 The above information is believed to be correct but does not purport to be all inclusive and sh all be used only as a guide. The information in this document is based on the present state of our knowledge and is applicable to the product with regard to appropriate safety precautions It does not represent any guarantee of the properties of th e product. Sigma-Aldrich Inc., shall not be held liable for any damage resulting from handling or from contact with the above produc t. See reverse side of invoice or packing slip for additional terms and conditions of sale. Copyright 2006 Sigma-Aldrich Co. Li cense granted to make unlimited paper copies for internal use only. SIGMA P3813 www.sigma-aldrich.com Page 5
204 Appendix D. (Continued) D.2. Triton-X 100 Material Safety Data Sheet Triton X-100 ACC# 24515 Section 1 Chemical Product and Company Identification MSDS Name: Triton X-100 Catalog Numbers: BP151-100, BP151-500, NC9269787, XXBP15120LI Synonyms: Polyethylene glycol p-tert-octylph enyl ether; Poly(oxy-1,2-ethanediyl), alpha-[4-(1,1,3,3-tetramethylbutyl)phenyl ]-omega-hydroxy-; Poly(oxyethylene) p-tertoctylphenyl ether. Company Identification: Fisher Scientific 1 Reagent Lane Fair Lawn, NJ 07410 For information, call: 201-796-7100 Emergency Number: 201-796-7100 For CHEMTREC assistance, call: 800-424-9300 For International CHEMTREC assistance, call: 703-527-3887 Section 2 Composition, Information on Ingredients CAS# Chemical Name Percent EINECS/ELINCS 9002-93-1 Ethoxylated p-tert-oct ylphenol 100 unlisted Section 3 Hazards Identification EMERGENCY OVERVIEW Appearance: clear to s lightly hazy liquid. Warning! May cause allergic skin reaction. May cause eye and skin irritation. May cause respiratory and digestive tract irritation. May be harmful if swallowed. The toxicological
205 Appendix D. (Continued) properties of this material have not been fully investigated. Target Organs: Eyes. Potential Health Effects Eye: Causes severe eye irritation. Causes redne ss and pain. Risk of serious damage to eyes. Skin: May cause skin irritation. May cause skin sensitization, an allergic reaction, which becomes evident upon re-exposure to this material. Ingestion: May cause gastrointestinal irritation with nausea, vomiting and diarrhea. May be harmful if swallowed. Inhalation: May cause respiratory tract irritation. The toxicological properties of this substance have not been fully investigated. Chronic: Repeated exposure may cause sensitization dermatitis. Section 4 First Aid Measures Eyes: Immediately flush eyes with plenty of water for at least 15 minutes, occasionally lifting the upper and lower eyelids. Get medical aid. Skin : Immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. Get medical ai d if irritation develops or persists. Wash clothing before reuse. Ingestion: Do not induce vomiting. If victim is c onscious and alert, give 2-4 cupfuls of milk or water. Get medical aid. Wash mouth out with water. Inhalation: Remove from exposure and move to fres h air immediately. If not breathing, give artificial respira tion. If breathing is difficult, gi ve oxygen. Get medical aid if cough or other symptoms appear. Notes to Physician: Treat symptomatically and supportively. Section 5 Fire Fighting Measures General Information: As in any fire, wear a self-c ontained breathing apparatus in pressure-demand, MSHA/NIOSH (approved or equivalent), and full protective gear. During a fire, irritating a nd highly toxic gases may be generated by thermal decomposition or combustion. Vapors may be heav ier than air. They can spread along the ground and collect in low or c onfined areas. Runoff from fire control or dilution water may cause pollution. Extinguishing Media: Water or foam may cause frothing. Use water spray, dry chemical,
206 Appendix D. (Continued) carbon dioxide, or chemical foam. Flash Point: 288 deg C ( 550.40 deg F) Auto ignition Temperature: Not available. Explosion Limits, Lower:Not available. Upper: Not available. NFPA Rating: (estimated) Health: 2; Fl ammability: 1; Instability: 0 Section 6 Accidental Release Measures General Information: Use proper personal protective equipm ent as indicated in Section 8. Spills/Leaks: Absorb spill with inert material (e.g vermiculite, sand or earth), then place in suitable container. Avoid runoff into storm sewers and ditches which lead to waterways. Clean up spills immediately, observing precautions in the Protective Equipment section. Provide ventilation. Section 7 Handling and Storage Handling: Wash thoroughly after handling. Avoid br eathing dust, vapor, mist, or gas. Avoid contact with eyes, skin, and clothi ng. Keep container tightly closed. Avoid ingestion and inhalation. Use w ith adequate ventilation. Storage: Do not store in direct s unlight. Store in a tightly cl osed container. Store in a cool, dry, well-ventilated area away from incompatible substances. Section 8 Exposure Contro ls, Personal Protection Engineering Controls: Facilities storing or utilizing th is material should be equipped with an eyewash facility and a safety shower. Us e adequate ventilation to keep airborne concentrations low. Exposure Limit s Chemical Name ACGIH NIOSH OSHA Final PELs Ethoxylated p-tertoctylphenol none listed none listed none listed
207 Appendix D. (Continued) OSHA Vacated PELs: Ethoxylated p-tert-octylphenol: No OSHA Vacated PELs are listed for this chemical. Personal Protective Equipmen t Eyes: Wear chemical splash goggles. Skin: Wear appropriate protective glove s to prevent skin exposure. Clothing: Wear appropriate protective clot hing to prevent skin exposure. Respirators: Follow the OSHA respirator regulations found in 29 CFR 1910.134 or European Standard EN 149. Use a NIOS H/MSHA or European Standard EN 149 approved respirator if exposure limits are exceed ed or if irritation or other symptoms are experienced. Section 9 Physical an d Chemical Properties Physical State: Liquid Appearance: clear to slightly hazy Odor: aromatic odor pH: 6-8 Vapor Pressure: < 1 mm Hg @ 20 Vapor Density: 7.11 (Air=1) Evaporation Rate:Negligible. Viscosity: 240 cP at 25 deg C Boiling Point: 270 deg C Freezing/Melting Point:7 deg C Decomposition Temperature:Not available. Solubility: Soluble in water. Specific Gravity/Density: 1.082 Molecular Formula:C14H22O.(C2H4O)n Molecular Weight:206.1534 Section 10 Stability and Reactivity Chemical Stability: Stable under norm al temperatures and pressures. Conditions to Avoid: Light, exposure to ai r, exposure to moist air or water. Incompatibilities with Other Materials: Oxid izing agents, strong reducing agents, strong acids.
208 Appendix D. (Continued) Hazardous Decomposition Products: Ca rbon monoxide, carbon dioxide. Hazardous Polymerization: Will not occur. Section 11 Toxicological Information RTECS#: CAS# 9002-93-1: MD0907700; YM0616666; YM0683332 LD50/LC50: CAS# 9002-93-1: Draize test, rabbit, eye: 10 uL/24H Moderate; Draize test, rabb it, skin: 500 uL/24H Mild; Oral, rat: LD50 = 1800 mg/kg; Oral, rat: LD50 = 3800 mg/kg; Oral, rat: LD50 = 1900 mg/kg; Carcinogenicity: CAS# 9002-93-1: Not listed by ACGI H, IARC, NTP, or CA Prop 65. Epidemiology: No information found. Teratogenicity: No information found. Reproductive Effects: No information found. Mutagenicity: No data available. Neurotoxicity: No information found. Other Studies: Section 12 Ecological Information Ecotoxicity: No data available. Fish-toxici ty: Bluegill TL(96H): Dynamic Bioassay: >10 mg/l Static Bioassay: 12 mg/l Environmental: No information available. Physical: No information available. Other: No information available. Section 13 Disposal Considerations
209 Appendix D. (Continued) Chemical waste generators must determine whether a discarded chemical is classified as a hazardous waste. US EPA guidelines for the cl assification determination are listed in 40 CFR Parts 261.3. Additionally, waste generators must consult state and local hazardous waste regulations to ensure comple te and accurate classification. RCRA P-Series: None listed. RCRA U-Series: None listed. Section 14 Transport Information US DOT Canada TDG Shipping Name: Not regulated as a hazardous material No information available. Hazard Class: UN Number: Packing Group: Section 15 Regulatory Information US FEDERAL TSCA CAS# 9002-93-1 is list ed on the TSCA inventory. Health & Safety Reporting List None of the chemicals are on the Health & Safety Reporting List. Chemical Test Rules None of the chemicals in this product are under a Chemical Test Rule. Section 12b None of the chemicals are listed under TSCA Section 12b. TSCA Significant New Use Rule None of the chemicals in this material have a SNUR under TSCA. CERCLA Hazardous Substances and corresponding RQs None of the chemicals in this material have an RQ. SARA Section 302 Extremel y Hazardous Substances None of the chemicals in this product have a TPQ.
210 Appendix D. (Continued) SARA Codes CAS # 900293-1: immediate. Section 313 No chemicals ar e reportable under Section 313. Clean Air Act: This material does not contain any hazardous air pollutants. This material does not contain any Class 1 Ozone depletors. This material does not contain any Class 2 Ozone depletors. Clean Water Act: None of the chemicals in this produc t are listed as Hazardous Substances under the CWA. None of the chemicals in this product are listed as Priority Pollutants under the CWA. None of the chemicals in this product are listed as Toxic Pollutants under the CWA. OSHA: None of the chemicals in this pro duct are considered high ly hazardous by OSHA. STATE CAS# 9002-93-1 is not present on st ate lists from CA, PA, MN, MA, FL, or NJ. California Prop 65 California No Significant Risk Level: None of the chemicals in this product are listed. European/International Regulations European Labeling in Accordance with EC Directives Hazard Symbols: XN Risk Phrases: R 22 Harmful if swallowed. R 41 Risk of serious damage to eyes. Safety Phrases: S 26 In case of contact with ey es, rinse immediately with plenty of water and seek medical advice. S 39 Wear eye/face protection. WGK (Water Danger/Protection) CAS# 9002-93-1: 1 Canada DSL/NDSL CAS# 9002-93-1 is li sted on Canada's DSL List. Canada WHMIS This product has a WHMIS classification of D2B. This product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all of the information required by those
211 Appendix D. (Continued) regulations. Canadian Ingredient Disclosure List CAS# 9002-93-1 is listed on the Canadian Ingredient Disclosure List. Section 16 Additional Information MSDS Creation Date: 6/25/1999 Revision #3 Date: 10/13/2003 The information above is believed to be accurate and re presents the best information currently available to us. However, we ma ke no warranty of merchantability or any other warranty, express or implied, with respect to such information, and we assume no liability resulting from its use. Users should make their own investigations to determine the suitability of the informa tion for their particular purposes. In no event shall Fisher be liable for any claims, losses, or damages of any third party or for lost profits or any special, indirect, incidental, consequentia l or exemplary damages, howsoever arising, even if Fisher has been advised of the possibility of such damages.