POLYMERIC HYDROGEL BASED INSULIN DELIVERY SYSTEM

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POLYMERIC HYDROGEL BASED INSULIN

DELIVERY SYSTEM

by

AMIT KUMAR

Centre for Biomedical Engineering

Submitted

In fulfillment of the requirements of degree of

Doctor of Philosophy

to the

INDIAN INSTITUTE OF TECHNOLOGY, DELHI

April, 2007

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CERTIFICATE

This is to certify that the thesis entitled "Polymeric Hydrogel Based Insulin Delivery System" being submitted by Mr. Amit Kumar to the Indian Institute of Technology, Delhi for the award of the degree of Doctor of Philosophy, in Biomedical Engineering is a record of the original bonafide research work carried out by him. Mr. Amit Kumar has worked under my guidance and supervision and has fulfilled the requirement for the submission of this thesis.

The results presented in this thesis are original and has not been submitted in partial or full, to any other university or institute for the award of any degree or diploma.

(Dr.Harpal Singh)

Professor

Centre for Biomedical Engineering Indian Institute of Technology, Delhi Hauz Khas, New Delhi-110016

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DEDICATED

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Acknowledgement

I wish to eArress my deep sense 0-Fatima to Dr. 'aural Singh, Professor, Centre of

Biomedical Engineering (CBME) for his invaluable guidance, encouragement and

constructive criticism during this work It would not be possible for me to bring this

work in the present form without their support. Working with them has been a rich

experience and shall always be a pleasant memory. I am also grateful to faculty

members of the CBME for their constant help.

I am thankful to Irufian Institute of technology Delhi for providing me necessary

facilities and financial aid to carry out the research.

I sincerely thank Dr. S.S.Lahiri, Sc.Officer-F, Institute of Npckar Medicine and

Meted Sciences (INMAS), DSO for his invaluable discussion during animal and

studies. I am indebted to him for his help in providing me laboratory facilities

and assistance in Experimental Anima! Facility, INMAS. I am thanicru to the entire

research staff and research scholar in his laboratory for constant help.

I am deep% grateful- to Dr. .91semBhatanagar, Sc-E, Department of Nuclear 91(edicine,

Institute of Nyclear Medicine and Aftled Sciences (INMAS), DSO for his guidance

and concrete discussion during the Gamma imaging studies. I would like to thanks to

all staff and research scholar in his laboratory for their invaluable help.

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It is also true that this work has been impossible without the cooperation and support

of my seniors and friends. In naming them, I am likely to miss many of those who

contributed in different ways. I would fike to thankRachna Jain, Chant Tyagi, Lomas

Tomar, Geeta Singh, Supriya, and Andendu for helping me throughout the work 914

**special' thanks to all Laboratory and Office Staff of Center of Biomedical* Engineering**

for their timely help.

My special thank to my parents my brother .91mcf Tyagi, and all my family members

for their love, support and unabated faith in me, which kept me going through this

work In the end, I have a special word of praise and appreciation for my wife

Prtyanka Tyagi, for her cooperation and encouragement, without whom the completion

of this dissertation was impossible.

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ABSTRACT

The application of synthetic polymers to medical problems has increased substantially in the last twenty years. Recently, the study of application of controlled release formulations containing the active substances has grown enormously. In the preparation of these systems, biologically active substances are immobilized in a carrier, generally a polymeric material. These polymeric materials must have the right balance of hydrophilicity/ hydrophobicity and the right charge character and be biocompatible. In the oral delivery of insulin, formidable barriers of digestion and absorption is essential to take in account to improve bioavilibility of insulin by the oral route so that it can attain the appropriate concentration in the body and cause the required hypoglycemic effect. One way to achieve this is to entrap insulin in a polymeric carrier which will protect the insulin from the degradation as long as it stays in the stomach. But upon reaching the small intestine, polymeric carrier becomes active in alkaline medium and releases the biologically active insulin. Many researchers have tried to immobilize or entrap insulin into various pH sensitive polymeric matrices to make the oral insulin delivery possible. A number of different strategies have been explored to improve the oral bioavailability of insulin, like use of penetration enhancers, enteric coatings, protease inhibitors and combinational strategies. The present work is devoted towards the development of a pH sensitive polymeric formulation with high insulin loading efficiency, sustained and efficient delivery of insulin in gastro intestinal tract and development of a polymeric hydrogel based implant for insulin delivery.

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An oral insulin delivery system based on copolymers of poly(ethylene glycol) dimethacrylate and methacrylic acid was developed and its functional activity was tested in non-obese diabetic rats. Poly(ethylene glycol) dimethacrylates (PEGDMA) were synthesized by the esterification reaction of different molecular weight poly(ethylene glycol) with methacrylic acid (MAA) in presence of acid catalyst. Their degrees of acrylation were found to be in the range of 93 to 95% using proton nuclear magnetic resonance (NMR) spectroscopy. PEG dimethacrylates of molecular weight ranging from 400 to 4000 and methacrylic acid were further copolymerized by suspension polymerization to obtain pH sensitive hydrogel microparticles. FTIR spectra of MAA had characteristic absorption peaks at 1635 cm-1 for carbonyl group and 1697 cm -1 for vinylic groups and a band from 3000 to 3450 cm-1 for OH group of carboxylic group. FTIR spectra of PEGDMA showed peaks at 1635 cm -1 for carbonyl group and 1697 cm -1 for vinylic groups was only observed upto 1000da molecular weight of PEGDMA. PEGDMA with molecular weight 2000da and 4000da showed a broader peak at 1639 cm 1 was observed due to merging of carbonyl and vinylic peaks. While in case of poly(PEGDMA-MAA) microparticles, FTIR spectra irrespective of PEGDMA molecular weight showed intense absorption peak of carbonyl group at 1695 cm-1 due to carboxylic acid groups of PEGDMA and MAA. Surface morphology of various microparticles were analyzed using SEM, which showed the spherical shape at pH 2.5 but microparticles coalesce with each other and appeared like a continuous film at pH 7.4. Surface morphology of various microparticles were also analyzed using AFM, which again confirmed the spherical shape of micropaticles at pH 2.5 and continuous film at pH 7.4. The diameters of poly(PEGDMA-MAA) microparticles increased with

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the increasing molecular weight of the poly(ethylene glycol) dimethacrylates and was found to be in the range of .4 to 2.7 p.m at pH 7.4 and 5.2 to 25.3 urn at pH 2.5. Insulin was loaded into the hydrogel microparticles by partitioning from concentrated insulin solution. It was found that poly(PEGDMA4000:MAA) microparticles showed the maximum loading efficiency (82%) while the lowest (43%) loading efficiency was observed in case of poly(PEGDMA400:MAA) copolymeric microparticles. In-vitro release studies of insulin-loaded microparticles were performed by simulating the condition of gastrointestinal tract, which showed the minimal insulin leakage (18-25%) at acidic pH (2.5) and significantly higher release at basic pH (7.4). Animal studies were carried out to investigate the abilities of the insulin loaded hydrogel microparticles to influence the blood glucose levels of the diabetic rats. In studies with diabetic rats, the blood glucose level reduced in animals that received the insulin loaded hydrogel microparticles and the effect lasted for 8-10 hrs. It was also observed, two capsules per day of poly(PEGDMA4000:MAA) hydrogel microparticles containing 80 I.U./kg of insulin dose were sufficient to control the blood glucose level of fed diabetic rats between 100-300mg/d1. In studies with diabetic rabbits, effect of oral administration of poly(PEGDMA4000-MAA) microparticles loaded with 50 IU/kg insulin dose to over night fasted diabetic rabbits reduced the blood glucose level by 78% within first 4 hrs of the treatment and maintained the same for next 2.5 hrs and then started rising slowly and approached the control value. The effect of insulin-loaded microparticles lasted for at least 10 hrs after the oral administration. Pharmacoscintigraphy studies of technetium-99m labeled insulin loaded in poly(PEGDMA4000-MAA) microparticles were carried out on diabetic rabbits which showed the sustained release of insulin from the 99m

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labeled insulin loaded poly(PEGDMA4000-MAA). LDO of the poly(PEGDMA4000- MAA) microparticles in rats is found to be above 4gm/kg body weight. Repetitive dose toxicity of poly(PEGDMA4000-MAA) microparticles was also carried out in rats and histopathological section of liver, stomach, small intestine and large intestine of rat fed with poly(PEGDMA4000-MAA) microparticles upto six month showed no toxicity. The present work was also devoted towards the development of poly(HEMA) based hydrogel device containing variable amount of water as porogen for implantable delivery of insulin and evaluation of their physical and insulin release properties. Poly(HEMA-co-EGDMA) hydrogel devices were prepared by bulk polymerization in a specially designed cylindrical mold of polypropylene. It was observed in the preliminary studies that hydrogel devices synthesized using AIBN as free radical initiator were transparent, bubble free and having good mechanical properties but difficult to remove from polypropylene mould. While hydrogel devices synthesized using APS/TEMED as free radical initiator was easily removed (within 2-3 minutes) from the PP mold probably due to incomplete polymerization and presence of water (used for initiator solution) which acts as a plasticizer for poly(HEMA), various amount of EGDMA was taken as crosslinker and different amount of water was also added in the reaction mixture as a pore generating solvent. These hydrogel devices were post cured at 80°C for 2 hrs. FTIR spectrum of poly(HEMA) shows the absorption bands associated with -C=0 stretching of carboxylic acid group (-COOH) at 1714 cm-1, C-O-C stretching vibration at 1152 cm -1 and appearance of broad band at 3500-3800 cm -1 corresponding to —OH stretching vibration of poly(HEMA) hydrogel. Hydrogel devices synthesized using 1% of EGDMA and 7% of water (used to dissolve initiators) showed the highest swelling percentage

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(50%), while the lowest swelling percentage (8.7%) was observed in hydrogel synthesized with 10% of EGDMA. Surface morphology studies of poly(HEMA) device was carried out using AFM. Device synthesized using 27% of water showed —800nm pores which were unevenly distributed on the hydrogel surface while device containing 47% of water showed 2500-5000nm pores which were unevenly distributed on the hydrogel surface. The hydrogel device containing 67 % of water showed the pores on the surface with pore size in the range of 3500-9000 nm. In-vitro release studies of

thallium"' (as a model compound) were carried out using hydrogel devices synthesized using 1% of EGDMA as crosslinker and it was observed that approximately 50% of activity release from the hydrogel device with in first 30 minutes, while the 90% of

thallium201 was released from the device with in next 45 minutes. Poly(HEMA) hydrogel device synthesized using 20% of water showed approximately 18% of insulin release with in first 30 minutes, only 50% of insulin release was observed in 60 minutes. Rest of the insulin takes longer time (165 minutes) to release. Poly(HEMA) hydrogel device synthesized using 20% of water filled with insulin loaded poly(PEGDMA4000-MAA) microparticles showed only 18% of insulin release with in 2days, only 50% of insulin release was observed in 5 days. Hypoglycemic effect of poly(HEMA) implantable device containing 120 I.U. of insulin loaded in poly(PEGDMA4000-MAA) microparticles showed 60% reduction in blood glucose level within first 24 hrs (Ist day), while highest reduction (70%) in blood glucose level was observed on 3 day after implantation. Animals maintain the 70% reduction in blood glucose level upto 4 days, while 5th day 40% reduction in blood glucose level was observed and then blood glucose level approach the control value by 6th day.

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1.4 Insulin for the Treatment of Diabetes 14 1.4.1 Nasal Delivery of Insulin 15 1.4.2 Transdermal Delivery of Insulin 16 1.4.3 Implantable Delivery of Insulin 18 1.4.4 Limitations of Current Insulin Therapy 18 1.4.5 Oral Delivery of Insulin 20 1.4.5.1 Physical Barrier in Oral Insulin Delivery 20 1.4.5.2 Enzymatic Barrier of Oral Insulin Delivery 20 1.4.5.3 Manufacturing Stability Barrier of Insulin 21 1.5 Objective of the Work 28

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Chapter II- Synthesis of Monomers and Polymers 44 2.1 Introduction 44 2.1.1 Polymeric Hydrogels 44 2.1.2 Classification of Hydrogels 45 2.1.2.1 Non-Ionic Hydrogels 46 2.1.2.2 Ionic Hydrogels 46 2.1.2.3 Polyampholytic hydrogels 46

Part A Synthesis of PEGDMA and its Copolymers

2.2 Experimental 48

2.2.1 Methods 48

2.2.1.1 Synthesis of PEG dimethacrylate 48 2.2.1.2 Synthesis of Poly(PEGDMA- MAA) Microparticles 49 2.2.1.3 Synthesis of poly(SMA) Copolymer 50 2.3 Results and Discussion 51 2.3.1 Synthesis of PEG dimethacrylate 51 2.3.2 Synthesis of Poly(PEGDMA- MAA) Microparticles 51 2.3.3 Synthesis of SMA Copolymer 51

Part B Synthesis of Poly(2-hydroxyethyl methacrylate) based Hydrogel Device

2.4 Experimental 53

2.4.1 Methods 53

2.4.1.1 Polymerization of poly(2-Hydroxyethyl Methacrylate) 53 2.5 Results and Discussion 54

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2.5.1 Synthesis of Polymeric Hydrogel 54

References 59

Chapter III- Characterization of Monomers and Polymers 61

3.1 Introduction 61

Part A Characterizations of PEG dimethacrylates and Their Copolymers

3.2 Experimental 63

3.2.1 Methods 64

3.2.1.1 Characterization of PEG dimethacrylates 64 3.2.1.1.1 Nuclear Magnetic Resonance Spectroscopy 64 3.2.1.1.2 Fourier Transform Infrared Spectroscopy 64

3.2.1.2 Characterization Of Poly(PEGDMA-MAA) Particles 65 3.2.1.2.1 Scanning Electron Microscopy 65 3.2.1.2.2 Fourier Transform Infrared Spectroscopy 65

3.2.1.2.3 Particle Size Analysis 65 3.2.1.2.4 Swelling Studies 66 3.2.1.2.5 Thermo Gravimetric Analysis 66 3.2.1.2.6 Atomic Force Microscopy 67 3.2.1.3 Characterization of Poly(Styrene-Maleic Anhydride) 67 3.2.1.3.1 Physical Properties of SMA Copolymer 67 3.2.1.3.2 Fourier Transform Infrared Spectroscopy 67 3.2.1.3.3 'H-NMR Spectroscopy 68 3.2.1.3.4 CHN Analysis 68 3.2.1.3.5 Thermal Characterization 68

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3.2.1.3.6 Acid Value Determination of SMA Copolymer 69 3.3 Results and Discussion 70 3.3.1 Characterization of PEG Dimethacrylates 70 3.3.1.1 Nuclear Magnetic Resonance Spectroscopy 70 3.3.1.2 Fourier Transform Infrared Spectroscopy 72 3.3.2 Characterization of Poly(PEGDMA-MAA) Particles 75 3.3.2.1 Scanning Electron Microscopy 75 3.3.2.2 Fourier Transform Infrared Spectroscopy 76 3.3.2.3 Particle Size Analysis 84 3.3.2.4 Swelling Studies 86 3.3.2.5 Thermo Gravimetric Analysis 87 3.3.2.6 Atomic Force Microscopy 88 3.3.3 Characterization of Poly(Styrene-Maleic Anhydride) 89 3.3.3.1 Physical Properties SMA Copolymer 89 3.3.3.2 Fourier Transform Infrared Spectroscopy 89 3.3.3.3 1H-NMR Spectroscopy 93 3.2.3.4 CHN Analysis 94 3.3.3.5 Thermal Characterization 95 3.3.3.6 Acid Value Determination 96

Part B Characterization of P(HEMA) Based Hydrogel Devices

3.4 Experimental 97

3.4.1 Fourier Transform Infrared Spectroscopy 97

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3.4.4 Swelling Studies 98 3.4.5 Atomic Force Microscopy 99 3.5 Results and Discussion 99 3.5.1 Fourier Transform Infrared Spectroscopy 99

3.5.2 Differential Scanning Calorimetry 100 3.5.3 Thermo Gravimetric Analysis 103 3.5.4 Swelling Studies 104 3.5.5 Atomic Force Microscopy 104

References 110

Chapter-IV Loading and In-Vitro Release Studies 111

4.1 Introduction 111

Part A: Loading and In-Vitro Release of Drugs from Poly(PEGDMA-MAA) Microparticles

4.2 Experimental 114

4.2.1 Methods 115

4.2.1.1 BSA Loading in Copolymeric Particles 115 4.2.1.2 In Vitro Release Studies of BSA 116 4.2.1.3 Preparation and Loading of 8-Hydroxyquinoline Hydrochloride in

Copolymeric Microparticles 116 4.2.1.4 In Vitro Release Studies of 8-HQ-Hcl 117 4.2.1.5 Insulin Loading of Microparticles 118 4.2.1.6 In-Vitro Insulin Release Studies 119

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4.2.1.7 SMA Coating of Insulin Loaded Microparticles 120 4.2.1.8 In Vitro Insulin Release from SMA Coated Microparticles 120 4.3 Results and Discussion 121 4.3.1 BSA Loading in Copolymeric Particles 121 4.3.1.1 In Vitro Release Studies of BSA 122 4.3.2 Preparation and Loading of 8-Hydroxyquinoline Hydrochloride in

Copolymeric Microparticles 126 4.3.2.1 In Vitro Release Studies of 8-HQ-Hcl 127 4.3.3 Insulin Loading of Microparticles 131 4.3.3.1 In Vitro Insulin Release 132 4.3.3.2 In Vitro Insulin Release from SMA Copolymer Coated

Insulin Loaded Microparticles 138

Part B: In-Vitro Release Of Studies From Poly(HEMA) Based Implantable Devices

4.4.1 Methods 139

4.4.1.1 In-Vitro Release of Thallium201 from poly(HEMA)

Hydrogel Device 139

4.4.1.2 Loading and In-Vitro Release of Insulin from

poly(HEMA) Hydrogel Device 139 4.4.1.3 Loading and In-Vitro Release of Insulin Loaded

Microparticles from Hydrogel Device 140 4.4.1.4 Antimicrobial Studies 140 4.5 Results and Discussion 142

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4.5.1 In-Vitro Release of Thallium20' from Hydrogel Device 143 4.5.2 Loading and In-Vitro Release of Insulin from Hydrogel

Device 144

4.5.3 Loading and In-Vitro Release of Insulin Loaded

Microparticles from Hydrogel Device 144 4.5.4 Antimicrobial Studies 144

References 147

Chapter-V In-Vivo Insulin Release and Toxicity Studies 149

5.1 Introduction 149

Part A : In-Vivo Insulin Release from Poly(PEGDMA-MAA) Microparticles and their Toxicity

5.2.1 Methods 153

5.2.1.1 In-Vivo Insulin Release Studies on Rats 153 5.2.1.1.1 Effect of Oral Microparticles on Fasted-Diabetic Rats 154 5.2.1.1.2 Effect of Oral Microparticles Coated with SMA

Copolymer on Fasted-Diabetic Rats 154 5.2.1.1.3 Effect of Oral Microparticles on Fed-Diabetic Rats 155 5.2.1.2 In-Vivo Insulin Release Studies on Rabbits 155 5.2.1.2.1 Effect of Oral Microparticles on Fasted-Diabetic Rabbits 156 5.2.1.2.2 Effect of Oral Microparticles on Fed-Diabetic Rabbits 156 5.2.1.3 Pharmacoscintigraphy for Evaluation of Microparticles 157

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5.2.1.3.1 Radiolabel ling of insulin by 99mTc-pertechnetate as

Radionuclide 157

5.2.1.3.2 99'Tc-insulin Loading of Poly(PEGDMA4000 158 -MAA) Microparticles

5.2.1.3.3 Gamma Camera Imaging of 99mtc-Insulin Loaded

Microparticles on Diabetic Rabbits 158 5.2.1.4 Toxicity Studies 159 5.2.1.4.1 Conventional Acute Toxicity (LD50) Test 159 5.2.1.4.2 Repetitive Dose Toxicity 159 5.2.1.4.3 Histopathological Testing of Poly(PEGDMA4000

-MAA) Microparticles 160 5.2.1.4.4 Preparation of Tissue Samples for Microtomy 160 5.2.1.4.5 Microtomy and Staining of Tissue Samples 161 5.3 Results and Discussion 162 5.3.1 Animal Studies on Rats 162 5.3.1.1 Effect of Oral Microparticles on Fasted-Diabetic Rats 162 5.3.1.2 Effect of SMA Coated Insulin Loaded Microparticles

on Fasted-Diabetic Rats 163 5.3.1.3 Effect of Oral Microparticles on Fed Diabetic Rats 164 5.3.2 Animal Studies on Rabbits 165 5.3.2.1 Effect of Oral Microparticles on Fasted-Diabetic Rabbits 167 5.3.3 Pharmacoscintigraphy for Evaluation of Microparticles 168 5.3.3.1 Selection of 99mtc-Pertechnetate as Radionuclide for

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Radiolabeling 168 5.3.3.2 99'Tc-Insulin Loading of Poly(PEGDMA4000

-MAA) Microparticles 169 5.3.3.3 Gamma Camera Imaging of 99nitc-Insulin Loaded Microparticles

on Diabetic Rabbits 169 5.3.4 Toxicity Studies 180 5.3.4.1 Conventional Acute Toxicity (LD50) Test 180 5.3.4.2 Repetitive Dose Toxicity 180 5.3.4.2.1 Histopathological Testing of Poly(PEGDMA4000-MAA)

Microparticles 180

Part B : In-Vivo Insulin Release Of Insulin From Poly(HEMA) Based Implantable Device

5.4.1 In-Vivo Insulin Release Studies of Poly(HEMA)

Implantable Device on Rabbits 188 5.4.1.1 In-Vivo Insulin Release Studies from Insulin

Loaded Poly(PEGDMA4000-MAA) Microparticles

Loaded in Poly(HEMA) Implantable Device 188 5.4.1.2 In-Vivo Insulin Release Studies from Poly(HEMA) Implantable

Device 188

5.5 Results and Discussion 189 5.5.1 In-Vivo Insulin Release Studies of Poly(HEMA)

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References

Implantable Device on Rabbits 189 5.5.1.1 In-Vivo Insulin Release Studies from Insulin

Loaded Poly(PEGDMA4000-MAA) Microparticles

Loaded in Poly(HEMA) Implantable Device 190 5.5.1.2 In-Vivo Insulin Release Studies from Poly(HEMA)

Implantable Device 190 191

Chapter-V Summary and Scope of Future Work 192

6.1 Summary 192

POLYMERIC HYDROGEL BASED INSULIN DELIVERY SYSTEM