A study on the ph temperature sensitive biodegradable hydrogels for controlled protein and drug delivery

List of Tables Table 2-1 Molecular weight and polydispersity of PCL-PEG-PCL triblock and PAE-PCL-PEG-PCL-PAE pentablock copolymers ···30 Table 4-1 Molecular weight and polydispersity of

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A Study on the pH/Temperature-Sensitive

Biodegradable Hydrogels for Controlled Protein and

Drug Delivery

Dai Phu Huynh

The Graduate School Sungkyunkwan University Department of Polymer Science and Engineering

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Drug Delivery

Dai Phu Huynh

The Graduate School Sungkyunkwan University Department of Polymer Science and Engineering

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Drug Delivery

Dai Phu Huynh

A Dissertation Submitted to the Department of Polymer Science & Engineering and the Graduate School of Sungkyungkwan University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

[May 2007]

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CONTENTS

List of Tables vi

List of Schemes .vii

List of Figures viii

Chapter 1 General Introduction 1

1.1 Scop of this study 1

1.2 Background 3

1.2.1 Stimuli-sensitive copolymer hydrogels 3

1.2.1.1 Temperature-sensitive block copolymer hydrogels 3

1.2.1.2 pH and temperature-sensitive block copolymer hydrogels 5

1.2.2 Controlled drug/protein delivery .8

1.2.2.1 Controlled drug/protein delivery 8

1.2.2.2 Drug/protein release mechanisms .10

1.3 Aims and outlines of this study 13

References .15

Chapter 2 A New pH/Temperature-Sensitive Block Copolymer Hydrogels Based on Poly(β-amino ester) 19

2.1 Introduction 19

2.2 Experimental 22

2.2.1 Materials 22

2.2.2 Synthesis of pH/temperature-sensitive PAE-PCL-PEG-PCL-PAE petablock copolymer hydrogel 22

2.2.2.1 Synthesis of temperature-sensitive PCL-PEG-PCL triblock copolymer 22

2.2.2.2 Synthesis of acrylated PCL-PEG-PCL triblock copolymers 23

2.2.2.3 Synthesis of pH/temperature-sensitive PAE-PCL-PEG-PCL-PAE pentablock copolymers 23

2.2.3 Characterization 25

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2.2.3.1 H-NMR analysis 25

2.2.3.2 GPC analysis 25

2.2.3.3 pH determination .25

2.2.3.4 Sol-gel phase transition measurement 26

2.2.3.5 Cytotoxicity evaluation 26

2.2.3.6 Storage stability 26

2.3 Results and discussions 28

2.3.1 Synthesis and characterization of block copolymers 28

2.3.2 Sol-gel phase transition diagram of triblock and pentablock copolymers 31

2.3.3 pH change of the pentablock copolymers with varying temperature 34

2.3.4 Control of Sol-gel phase transition diagram of copolymers 36

2.3.5 Cytotoxicity evaluation 43

2.3.6 Degradability evaluation 44

2.3.7 Storage stability evaluation 45

2.4 Conclusions 47

References .48

Chapter 3 Controlled Protein Release of Poly(β-amino ester) based Block Copolymer Hydrogels 51

3.1 Introduction 51

3.2 Experimental .54

3.2.1 Materials 54

3.2.2 Synthesis of pH/temperature-sensitive block copolymer hydrogel 54

3.2.3 Characterization of copolymer 54

3.2.4 Protein loading process .54

3.2.5 Degradability evaluation of complex gel in vitro 55

3.2.6 Degradability evaluation of complex gel in vivo 55

3.2.7 Insulin releasing in vitro 56

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3.2.8 Study insulin release in vivo using female Sprague Dawley (SD) rats 57

3.2.9 Controlled insulin release in vivo using female Sprague-Dawley (SD) rats 58

3.2.10 Controlled insulin release using diabetic fat rats (DFR) 58

3.3.1 Synthesis and characterization of copolymers 62

3.3.2 Change of sol-gel transition by insulin loading 63

3.3.3 Degradability evaluation of block copolymers and complex gel 63

3.3.4 Degradability evaluation of complex gel in vivo 64

3.3.5 Insulin loading and release mechanism 65

3.3.6 Insulin release in vivo 71

3.3.7 Insulin release in vivo using female Sprague Dawley (SD) rats 73

3.3.8 Controlled insulin release on DFR rats 74

3.3.9 hGH loading and release in vitro 78

References .82

Chapter 4 Biodegradation Rate Control of Poly(β-amino ester) based Block Copolymer Hydrogels 85

4.1 Introduction 85

4.2 Experimental 87

4.2.1 Materials 87

4.2.2 Synthesis of pH/temperature-sensitive block copolymer hydrogel 87

4.2.2.1 Synthesis of temperature-sensitive PCLA-PEG-PCLA triblock copolymers 87

4.2.2.2 Syhthesis of acrylated PCLA-PEG-PCLAs .88

4.2.2.3 Synthesis of pH/temperature-sensitive pentablock copolymers 88

4.2.3.1 1H-NMR and GPC analyses 90

4.2.3.2 pH determination 90

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4.2.3.4 Cytotoxicity evaluation 90

4.2.4 Insulin loading process 91

4.2.6 Degradability evaluation of the complex gel in vivo 92

4.2.7 Insulin release in vitro 92

4.2.8 Storage stability 92

4.3.1 Synthesis characterization of block copolymers 93

4.3.2 pH change of pentablock copolymers with varying temperature 97

4.3.3 Sol-sel phase transition diagrams 99

4.3.4 Cytotoxicity evaluation 105

4.3.5 Change of sol-gel transition by insulin loading 106

4.3.7 Degradatbility of the complex gel in vivo 109

4.3.8 Insulin release in vitro 109

4.4 Conclusions .114

References .115

Chapter 5 Biodegradation Rate Control of Sulfamethazine Oligomer-based Block Copolymer Hydrogels and theirs Controlled PTX delivery 117

5.1 Introduction .117

5.2 Experimental 120

5.2.1 Materials 120

5.2.2 Synthesis of OSM-based pH-sensitive block copolymer hydrogels 120

5.2.2.1 Synthesis of sulfamethazine oligomer 120

5.2.2.2 Synthesis of temperature-sensitive PCGA-PEG-PCGA triblock copolymers 121

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5.2.2.3 Synthesis of pH/temperature-sensitive OSM-PCGA-PEG-PCGA-OSM pentablock

copolymers 121

5.2.3.1 1H-NMR analysis 124

5.2.3.2 GPC analysis 124

5.2.3.4 Degradability evaluation 125

5.2.4 Drug loading and release in vitro .125

5.2.5 PTX assay by HPLC 125

5.3.1 Synthesis and characterization 126

5.3.2 Sol-gel phase transition diagrams 130

5.3.3 Sol-gel phase transition in vitro 138

5.3.5 Drug loading and release in vitro 141

5.4 Conclusions 144

References .145

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List of Tables

Table 2-1 Molecular weight and polydispersity of PCL-PEG-PCL triblock and

PAE-PCL-PEG-PCL-PAE pentablock copolymers ···30

Table 4-1 Molecular weight and polydispersity of PCLA-PEG-PCLA triblock and

PAE-PCLA-PEG-PCLA-PAE (CL/LA ~ 2/1) pentablock copolymers ···97

Table 5-1 Molecular weight of sulfamethazine oligomer ···127

Table 5-2 Molecular weight and polydispersity of PCGA-PEG-PCGA triblock and

OSM-PCGA-PEG-PCGA-OSM pentablock copolymers ···129

Table 5-3 Sol-gel phase transitions of OSM-PCGA-PEG-PCGA-OSM copolymer solutions

(concentration 20 wt%) obtain during injection testing to the environment at temperature 37 °C, and pH 8.0 and 7.4 ···138

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List of Schemes

Scheme 2-1 Synthesis of pH/temperature-sensitive PAE-PCL-PEG-PCL-PAE block copolymers, a)

Synthesis of PCL-PEG-PCL triblock copolymer, b) Synthesis of acrylated triblock copolymer,

c) Synthesis of PAE-PCL-PEG-PCL-PAE pentablock copolymers ···24

Scheme 4-1 Synthesis of pH/temperature-sensitive PAE-PCLA-PEG-PCLA-PAE block copolymers, a)

Synthesis of PCLA-PEG-PCLA triblock copolymers, b) Synthesis of acrylated triblock copolymer, c) Synthesis of PAE-PCLA-PEG-PCLA-PAE pentablock copolymer ···89

Scheme 5-1 Synthesis of pH/temperature-sensitive OSM-PCGA-PEG-PCGA-OSM, a) Synthesis of

PCGA-PEG-PCGA triblock copolymer, b) Synthesis of Oligosulfamethazine (OSM), c) Synthesis of OSM-PCGA-PEG-PCGA-OSM pentablock copolymer ···123

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List of Figures

Figure 1-1 Sol-gel phase transition of B-A-B temperature-sensitive triblock copolymer hydrogel ··· 4

Figure 1-2 Schematic diagram of the sol-gel mechanism of the pH and temperature sensitive block copolymer solution Reproduced from Ref [40] ··· 7

Figure 1-3 Schematic phase diagrams of block copolymers in buffer solution Reproduced from Ref [40] a) PCLA-PEG-PCLA, b) OSM-PCLA-PEG-PCLA-OSM ··· 8

Figure 1-4 The profile controlled release of drug/protein (discontinuous line: drug/protein release profile by traditional methods; continuous line: drug/protein release profile by sustained release from biopolymer hydrogels) ··· 9

Figure 1-5 The general sol-gel phase diagram of the PAE-PCL-PEG-PCL-PAE ···12

Figure 1-6 The general sol-gel phase diagram of the OSM-PCGA-PEG-PCGA-OSM ···13

Figure 1-7 General mechanism of controlled drug/protein release from these hydrogel ···13

Figure 2-1 1H-NMR spectrums of copolymers at composition: PEG 1650, PCL/PEG 1.8/1; PAE 1.26K, a) acrylated PCL-PEG-PCL, b) PAE-PCL-PEG-PCL-PAE ···29

Figure 2-2 GPC traces of PCL-PEG-PCL and PAE-PCL-PEG-PCL-PAE ···30

Figure 2-3 Sol-gel transition phase diagrams, a) Sol-gel transition of pentablock copolymer solution at pH 7.4 with various temperature and concentration, b) Sol-gel transition of triblock and pentablock copolymer solutions (20 wt%) with various temperature and pH ···32

Figure 2-4 The sol-gel reversible phenomena of PAE-PCL-PEG-PCL-PAE ···33

Figure 2-5 The sol-gel transition of PAE-PCL-PEG-PCL-PAE in vivo test ···33

Figure 2-6 pH change of PAE-PCL-PEG-PCL-PAE (PCL/PEG~1.5/1; PEA~1.25, 20 wt%) with various temperature, a) Change of pH depends on temperature, b) Engineered and real sol-gel transition phase diagram ···35

Figure 2-7 Sol-gel phase transition diagrams of PAE-PCL-PEG-PCL-PAE (PCL/PEG ~1.5/1, and PAE~1.25-1.3k) block copolymer solutions with different PEG molecular weight, a) Various copolymer concentration at pH 7.4, b) Various pH at copolymer concentration 20 wt% ···37

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Figure 2-8 Sol-gel phase transition diagrams of PAE-PCL-PEG-PCL-PAE (PEG 1500, and

PAE~1.25-1.3k) block copolymer solutions with different PCL/PEG ratio, a) Various copolymer

concentration at pH 7.4, b) Various pH at copolymer concentration 20 wt% ···38

Figure 2-9 Sol-gel phase transition diagrams of PAE-PCL-PEG-PCL-PAE (PEG 1650, and PAE~1.25k) block copolymer solutions with different PCL/PEG ratio, a) Various copolymer concentration at pH 7.4, b) Various pH at copolymer concentration 20 wt% ···40

Figure 2-10 Sol-gel phase transition diagrams of PAE-PCL-PEG-PCL-PAE (PEG 1500, and PCL/PEG ~ 1.8) block copolymer solutions with different PAE molecular weight ···42

Figure 2-11 Sol-gel phase transition diagrams of PAE-PCL-PEG-PCL-PAE (PEG 1650, PCL/PAE ~ 1.8/1, and PAE~1.25k) block copolymer solutions with different concentration···42

Figure 2-12 Cytotoxicity of PAE-PCL-PEG-PCL-PAE (PCL/PAE ~ 1.5/1, and PAE~1.25-1.3k) at various PEG molecular weight ···43

Figure 2-13 Change of copolymer molecular weights with time in vitro ···44

Figure 2-14 Change of molecular weight with time at various pH conditions ···45

Figure 2-15 Molecular weight change with time of PAE-PCL-PEG-PCL-PAEs (PEG 1.5k; PCL/PEG~1.8/1; PAE~1.25k) at different conditions ···46

Figure 3-1 Protein loading process.···55

Figure 3-2 Degradability of complex gel in vivo ···56

Figure 3-3 Protein release process in vitro ···57

Figure 3-4 Insulin release and assay in vivo ···58

Figure 3-5 DFR rats induced and treatment process ···60

Figure 3-7 Sol-gel phase transition diagram of PAE-PCL-PEG-PCL-PAE (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k) solution at 25 wt% ···62

Figure 3-8 Sol-gel phase transition diagrams of complex gel with different insulin formulation Pentablock (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k) copolymer solution (20%) ···63

Figure 3-9 Change of molecular weight with time in vitro ···64

Figure 3-10 Change of gel intergrity of PAE-PCL-PEG-PCL-PAE insulin complex gel with time in vivo (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k) 5 mg/ml insulin in copolymer solution (25%) ···65

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Figure 3-11 Mechanism of insulin loading and release, a) The polymer solution is sol state at 10 °C and pH

7.0 with the ionic complex between insulin and PAE-PCL-PEG-PCL-PAE, b) The gel formed

by insulin free PAE-PCL-PEG-PCL-PAE after injection to human body (37 °C and pH 7.4), c) Insulin release from gel by polymer degradation and Fickian diffusion [27] ···66

Figure 3-12 Insulin release in vitro (5 mg.ml-1 insulin in copolymers solutions 20 wt%) with different

sampling method, error bars represent the standard deviation (n = 4), a) Cumulative release of

insulin (%), b) Concentration of insulin in serum (mg/ml) ···68

Figure 3-13 Insulin release in vitro (5 mg.ml-1 insulin in copolymers solutions) with different copolymer

concentration, error bars represent the standard deviation (n = 4), a) Cumulative release of

insulin (%), b) Concentration of insulin in serum (mg/ml) ···69

Figure 3-14 Insulin release from triblock and complex gel in vitro (5 mg.ml-1 insulin in copolymer solutions

20 wt%) Error bars represent the standard deviation (n = 4), sampling method 1, a) Cumulative release of insulin (%), b) Insulin concentration in serum (mg.ml-1) ···71

Figure 3-15 Insulin release in vivo Insulin-only injected group, 200 µl insulin solution 0.25 mg.ml-1(in PBS

buffer (pH 7.4)) is administered by intraperitoneal injection In a insulin-loaded hydrogel group, the complexation insulin (5 mg.ml-1 in triblock and pentablock copolymer solutions 25 wt%) at

pH 7.0 and 10 °C is subcutaneously injected into the back side (200 µl per Female SD rat),

Error bars represent the standard deviation (n = 5) ···72

Figure 3-16 Controlled insulin concentration in plasma of blood in vivo on SD rat, error bars represent the

standard deviation (n = 5) ···74

Figure 3-17 Blood glucose concentration with time on STZ-induced diabetic rats for 5 days and insulin

releasing-induced for 19 days Error bars represent the standard deviation (n = 5) (the insulin formulations are 0 mg.ml-1 in the control group (only pentablock gel), 1 mg.ml-1 in group 1, 5 mg.ml-1 in group 2, and 10 mg.ml-1 in group 3) ···75

Figure 3-18 Insulin concentration in blood plasma with time on STZ-induced diabetic rats during insulin

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Figure 3-19 Body weight change with time on STZ-induced diabetic rats for 5 days and insulin

Figure 4-1 1H-NMR spectra of block copolymers, a) PCLA, b) Acrylated

PCLA-PEG-PCLA, c) PAE-PCLA-PEG-PCLA-PAE ···95

Figure 4-2 GPC traces of PCLA-PEG-PCLA and PAE-PCLA-PEG-PCLA-PAEs at PEG 1.5k,

PCLA/PEG=2.5/1 with various β-amino ester block length ···96

Figure 4-3 pH change of PAE-PCLA-PEG-PCLA-PAE (PCLA/PEG~2.0/1; PEA~1.3k, 20 wt%) with

various temperature, a) Change of pH depend on temperature, b) Engineering and Real sol-gel

transition phase diagram ···99

Figure 4-4 Sol-gel phase transition diagrams of PAE-PCLA-PEG-PCLA-PAE (PCLA/PEG ~2.0/1, and

PAE~1.3k) block copolymer solutions with different molecular weight of PEG, a) Various

copolymer concentration at pH 7.4, b) Various pH at copolymer concentration 20 wt% ···100

Figure 4-5 Sol-gel phase transition diagrams of PAE-PCLA-PEG-PCLA-PAE (PEG 1500, and

PAE~1.3k) block copolymer solutions with different PCLA/PEG ratio, a) Various copolymer

PCLA/PEG ~ 2.5/1) block copolymer solutions with different PAE molecular weight ···104

Figure 4-8 Sol-gel phase transition diagrams of PAE-PCLA-PEG-PCLA-PAE (PEG 1500, PCLA/PAE

~ 2.5/1, and PAE~1.3k) block copolymer solutions with different concentrations ···104

Figure 4-9 Cytotoxicity evaluation of PAE-PCLA-PEG-PCLA-PAE (PCLA/PAE ~ 2.0/1, and

PAE~1.3k) ···105

Figure 4-10 Sol-gel phase transition diagrams of complex mixture with different insulin formulation

Pentablock (PEG 1500; PCLA/PEG~2.5/1; PAE~1.3k) copolymer solution 20 wt% ···106

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Figure 4-11 Change of molecular weight of block copolymers and complex gel during the degradation in

vitro (PEG 1500; PCLA/PEG~2.5/1; PAE~1.3k, copolymer solution 20 wt%) ···107

Figure 4-12 Change of molecular weight of copolymers by the degradation in vitro (Group triblock, pentablock and complex gel 1: PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k Group triblock, pentablock and complex gel 1: PEG 1.65k; PCL/PEG~1.8;PEA~1.25k; 5 mg/ml insulin in copolymer solution 20 wt%), a) PAE-PCL-PEG-PCL-PAE and PAE-PCLA-PEG-PCLA-PAE, b) Complex gel of PAE-PCL-PEG-PCL-PAE and PAE-PCLA-PEG-PCLA-PAE ···108

Figure 4-13 Change of gel integrity of PAE-PCLA-PEG-PCLA-PAE insulin complex gel with time in vivo (PEG 1500; PCLA/PEG~2.5/1; PAE~1.3k), 5 mg/ml insulin in copolymer solution (20 wt%)···109

Figure 4-14 Cumulative release of insulin with time from complex gel of PAE-PCLA-PEG-PCLA-PAE (PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k) in vitro Error bars represent the standard deviation (n = 4), a) 5 mg of insulin/ml copolymer (20 wt%) with different sampling method, b) At the same sampling method but different insulin formulation ···111

Figure 4-15 Cumulative release of insulin with time from complex gels of PAE-PCL-PEG-PCL-PAE and PAE-PCLA-PEG-PCLA-PAE Experiment in vitro, error bars represent the standard deviation (n = 4) Complex gel 1: 5 mg/ml insulin loaded in copolymer (20 wt%) of PAE-PCLA-PEG-PCLA-PAE (PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k); complex gel 2: 5 mg/ml insulin loaded in copolymer (20 wt%) of PAE-PCL-PEG-PCL-PAE (PEG 1.65k; PCL/PEG~1.8;PEA~1.25k); Sampling method 1 ···112

Figure 4-16 Molecular weight change with time of PAE-PCLA-PEG-PCLA-PAEs (PEG 1.5k; PCLA/PEG~2.5/1; PAE~1.3k) under different conditions ···113

Figure 5-1 1H-NMR spectra of sulfamethazine, SMM, OSM, a) Sulfamethazine and SMM, b) SMM and OSM ···127

Figure 5-2 1H-NMR spectrum of PCGA-PEG-PCGA triblock copolymer ···128

Figure 5-3 1H-NMR spectrum of OSM-PCGA-PEG-PCGA-OSM pentablock copolymer ···128

Figure 5-4 GPC traces of triblock and pentablock copolymer ···130

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Figure 5-5 Sol-gel phase transition diagram of pH/temperature-sensitive hydrogel ···131

Figure 5-6 Sol-gel phase transition diagrams of OSM-PCGA-PEG-PCGA-OSM (C-2.1, C-2) block

copolymer solutions with different molecular weight of sulfamethazine oligomers (C-2.1, C-2; PEG 1750; PEG/PCGA= 1/2.15), a) Various copolymer concentration at pH 7.4, b) Various

pH at copolymer concentration 10 wt% ···133

Figure 5-7 Sol-gel phase transition diagrams of OSM-PCGA-PEG-PCGA-OSM (A-1, B-1, C-2.1, D-1)

block copolymer solutions with different molecular weight PEG (OSM=806), a) Various

copolymer concentration at pH 7.4, b) Various pH at copolymer concentration 10 wt% ···135

Figure 5-8 Sol-gel phase transition diagrams of OSM-PCGA-PEG-PCGA-OSM block copolymer

solutions with different PEG/PCGA ratio, a) Various copolymer concentration at pH 7.4, b)

Various pH at copolymer concentration 10wt% ···136

Figure 5-9 Sol-gel phase transition diagrams of OSM-PCGA-PEG-PCGA-OSM block copolymer

solutions with different PEG/PCGA ratio at copolymer concentration 20 wt% ···137

Figure 5-10 Change of molecular weight with time at 37oC and pH 7.4, a) PCGA-PEG-PCGA and

OSM-PCGA-PEG-PCGA-OSM, b) OSM-PCGA-PEG-PCGA-OSM and

OSM-PCLA-PEG-PCLA-OSM ···140

Figure 5-11 Cumulative release of PTX in vitro, a) PTX release from OSM-PCGA-PEG-PCGA-OSM

(C-4; PEG 1750, PCGA/PEG: 2.67/1, OSM 904) matrix, b) PTX release from

OSM-PCLA-PEG-PCLA-OSM (PEG 1750, PCLA/PEG: 1.89/1, OSM 1040) matrix [33] ···142

Figure 5-12 Change of molecular weight of OSM-PCGA-PEG-PCGA-OSM (C-4; PEG1750,

PEG/PCLA :1/2.67; OSM 904), which was kept as solution (20 wt%) at 5 °C, 0 °C and pH 8.0 over specified time period ···143

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Chapter 1

General Introduction

1.1 Scope of this study

In past few years, the number of persons, whose were died by many kinds of dangerous diseases such as flu epidemic, HIV-AIDS, cancer, hepatitis, and especial diabetes, increased very fast Those diseases have become the most commonly dangerous epidemic proportion in the world, and the prevalence of the diseases were expected to increase day by day [1] Because of the standard treatment methods, which were used to treatment for patients such as parenteral injection, oral, and drug transfusion through vein, show the disadvantages in the controlled release of drug/protein because of the enzymatic dehydration in the gastric fluid, and the limitation of the therapeutic concentration range of the drug/protein during treatment Such that, the controlled drug/protein delivery systems by using novel carrier materials for revolutionizing the treatment of the diseases became one of the most challenger achievements in medical science field [2] Many systems have been studied since 1980s [3-7] for controlled drug/protein delivery For examples, microspheres made of poly(methacrylic acid)-poly(ethylene glycol) [8] were studied and used as a nasal drug delivery system to delivers insulin for treatment the diabetes disease In fabrication of new oral delivery system for treatment the diabetes, insulin was protected from the enzymatic dehydration in the gastric fluid by poly(methacrylic acid) grafted with poly(ethylene glycol) [9] and soybean phosphatidylcholine [10], and the release of this peptide happened in small intestine In addition, Morimoto’s group studied the nasal drug delivery system base on microspheres of aminated gelatin [11] Insulin was loaded into this material and then absorbed to human body by the mucosal membrane Chiou [12] presented another systemic delivery, which delivered insulin through the eyes, base on an enhancing agent such

as polyoxyethylene ethers of fatty acids and bile salts and acids such as cholic acid, deoxycholic acid, etc

One of the most interesting carrier materials for drug/protein delivery, which were studying and developing

in recent years, is polymer hydrogels Among the hydrogels, the in-situ forming hydrogels, which exist as liquid aqueous solutions (or sol state) before administration but turn into gel immediately after administration, have attracted considerable interests for their potential applications in site-specific drug controlled-delivery systems

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[13] pH-sensitive hydrogels based on N-isopropylacrylamide (NIPA)/N-aminopropylmethacrylamide linked with N, N'-methylenebis(acrylamide) [14], poly(2-hydroxyethyl acrylate), acrylate, or methacrylate [15-18] were investigated for drug loading and release as subcutaneous transplantation delivery system in recent years Although drugs loading in the matrix are easier, those are non-degradable polymer Biodegradable hydrogels have been interested as an attractive insulin formulation because they have many advantages such as biocompatibility and high responsibility for specific factors The important advantage of the drug release from the hydrogels is that could be controlled by several elements, for instance pore size of hydrogels, the hydrophobicity, the presence of some specific function that make a particular interaction between the matrix and drug, and the controlling of the degradation of the biodegradable hydrogels Thermo-reversible biodegradable hydrogel made of poly(ethylene oxide)-b-poly(L-lactide-co-glycolide) (PEO-P(LLA/GA)), poly(ethylene oxide)-b-poly((D,L-lactide-co-glycolide) (PEO-P(DLLA/GA)) [18-20], and PEG-grafted chitosan [21] have been developed for polymeric drug carriers, implantation, and other medical devices over the past few years However, these hydrogels have several unresolved drawbacks, which limit their application in injectable drug delivery systems When temperature-sensitive hydrogels are hypodermic injected into the body with a syringe, they tend to change into a gel as the needle is warmed by the body temperature This change makes it difficult to inject them into the body Also, after injection, the hydrogels tend to undergo rapid degradation of the block copolymer, which consequently produce the acidic monomers such as lactic acid or glycolic acid Since the low-pH environment of the hydrogel caused by the acidic monomer is known to be deleterious to some proteins and nucleic acids, the pH change, which occurs within these biodegradable hydrogels, is an important consideration [22] Furthermore, even these hydrogels were limited to protein loading to either mechanical absorption by hydrophobic-hydrophobic interactions or sequestering the drug inside the voids in the hydrogel matrix This limits the loading capacity of the gel, the applications of the gel, and also the ability to control the release profile At the heart of the matter is the ability to use a functional group which could make a strong reversible linkage with the drug/protein to allow for a predictable and customizable loading and release

cross-In this study, the challenger of molecular design of novel pH and temperature-sensitive hydrogels is polymer and drug (special for ionic drug) should compatibility We focused on the synthesis, characterization and evaluation of the materials As such, drug and polymer could form a complex gel after injected to human body,

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and drug loading into this gel and releasing are controlled easily Then the application for drug and protein delivery systems was also investigated

1.2 Background

1.2.1 Stimuli-sensitive copolymer hydrogels

The term of stimuli-sensitive copolymers is that they can change their structure by themselves in respond to environmental stimuli The various stimuli-sensitivity that respond to pH [23], temperature [24-26], electric field [27,28], and other stimuli have been studied experimentally and theoretically [29,30] Hydrogels are interpenetrating polymeric networks, which are composed of chemical or physical interaction to form hydrophilic three-dimension structure The hydrophilic networks matrix structure of this material allow it absorb large quantities of water while remaining insoluble in aqueous solutions due to chemical or physical crosslinking of individual polymer chains Hydrogels are excellent candidates for loading drug, proteins, and DNA due to their chemical or physical interactions between biomacromolecules and the carrier materials, which exhibit many unique physicochemical properties that make them advantageous for biomedical applications including drug delivery [31]

The stimuli-sensitive polymer hydrogels, especially the thermo-reversible gels and pH-reversible gels have been developed for polymeric drug carriers, implantation, and other medical devices over the past few years [13,32,33]

1.2.1.1 Temperature-sensitive block copolymer hydrogels

Among the developed stimuli-sensitive materials, polymers showing a sol-to-gel transition with changing temperature have been proposed for use as injectable drug delivery systems because the sol-gel phase transition

of these materials can be easily controlled both in vitro and in vivo The hydrophobic interpenetrating crosslinks

forming, which makes the aqueous copolymer solutions change from sol to gel state, are simply influenced of temperature changing

One of the typical thermoreversible hydrogels is triblock copolymer BAB, which composes of hydrophilic block at the center and hydrophobic blocks at the ends of copolymers structure The hydrophobic block plays a

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role as a temperature-sensitive moiety, its hydrophobicity is directly proportional to the increase of temperature

As a result, the soluble-insoluble property of the triblock copolymer shows the displayment, which depending

on the change of temperature The mechanism of sol-gel phase transition of this copolymer is shown in figure

1-1

Figure 1-1 Sol-gel phase transition of B-A-B temperature-sensitive triblock copolymer hydrogel

At low temperature, PLGA plays as a weak hydrophobic block, which increases the solubility of the copolymer Because of this, most of copolymer molecules were dissolved in aqueous solution, the other molecules form the micelle structure with the hydrophobic block of PLGA in the core and hydrophilic of PEG locate at the shell Consequently, the copolymer solution stays at a sol state When the temperature increases, PLGA becomes more hydrophobic because of the responding to temperature of this block Therefore, most of copolymer molecules form micelles At a high concentration of copolymer solutions, many bridges between micelles are formed by hydrophobic interaction between PLGA blocks This structure absorbs a large amount

of water and exhibit both liquid-like and solid-like behaviors As a result, the aqueous copolymer solution changes from sol state to gel state However, PLGA becomes too hydrophobic when the temperature is increased to very high level, which makes the solubility of copolymer decrease In addition, the hydrophobic interaction, which leads to the bridges micelles, is too great Consequently, the gel structure aggregates and

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changes to sedimentation state because of the effect of the above factors, and then the water is effectively liberated as the result of the greater enthalpy of H2O The temperature, where copolymer solutions change from so-to-gel state, is called lower critical solution temperature (LCST), whereas the upper critical solution temperature (UCST) is used to describe the temperature where the systems undergo the opposite transition

The examples of this type of temperature-sensitive hydrogel were poly(ethylene glycolide) (PEO-P(LLA/GA)), poly(ethylene oxide)-b-poly((D,L-lactide-co-glycolide) (PEO-P(DLLA/GA)) [19,33,34], and PEG-grafted chitosan [35]

oxide)-b-poly(L-lactide-co-The other type of temperature-sensitive triblock copolymer hydrogels based on the ABA-type of poly(ethylene glycol)-poly(L-lactide)-poly(ethylene glycol) triblock copolymer (PEG-PLLA-PEG) [36] was studied and developed by Kim and coworkers However, the sol-gel phase transition diagrams of PEG-PLLA-PEG triblock and PEG/polyester diblock copolymers exhibited only two area-phases, the gel state at low temperature and the sol state at high temperature Therefore, the sol-gel phase transition of this type of copolymer such as Pluronic systems just showed UCST behavior and did not show the LCST This property limits the usage of some drugs/proteins because of the drug denature during loading at high temperature

1.2.1.2 pH and temperature- sensitive block copolymer hydrogels

Temperature-sensitive block copolymer hydrogels showed many disadvantages in drug/protein delivery as discussed above Many researchers developed new materials, which can resolve these problems, in the desire of increasing drug/protein capacity, and controllable biomolecules releasing to design the new drug/protein delivery system The common method, which has been used in recent years, is study and develop copolymer hydrogels which have more than one stimulus-sensitivity besides the temperature-sensitivity pH respond stimulus is usually used to modify with temperature-sensitive hydrogels to archite the new drug/protein delivery devices The sol-gel phase transition of products is predicted that respond to both pH and temperature

Anderson [37] and Determa [38] developed a series of pH/temperature-sensitive copolymer hydrogels type ABCBA based on Pluronic (F127) Here, BCB is temperature-sensitive triblock copolymer made of F127, and

A is an outer cationic block pH-sensitive moiety The pentablock copolymers were synthesized by combination reaction between F127 and pH-sensitive moieties made of PDEAEMA and PDMAEMA [37,38] The

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PDEAEMA-Pluronic-PDEAEMA and PDMAEMA-Pluronic-PDMAEMA aqueous solutions exhibited a closed-loop sol-gel-sol transition, which depend on the change in both pH and temperature However, the disintegration of PEG/polyesters block copolymer usually showe a rapid degradation, which can not do the drug sustain release in some cases In addition, the generated acidic monomer byproducts, which are formed by copolymers degration, such as lactic acid and glycolic acid cause the significant pH decrease in the hydrogels Its become poisonous and pernicious to tissue

Sulfamethazine oligomers (OSM) [39] showed the ionized/non-ionized transition because of narrow pH change around 7.4 OSM play a role of hydrophilic block at the ionized state, but it change to hydrophobic block at non-ionized state as a result of the crystallizing Based on the depence of solubility of OSM on pH, Lee and co-workers used OSM to conjugate to ends of poly(ε-CL-co-LA)-PEG-poly(ε-CL-co-LA) (PCLA-PEG-PCLA) to create a pH/temperature- sensitive pentablock copolymers (OSM-PCLA-PEG-PCLA-OSM) [40,41] Figure 1-2 shows the schematically illustrated of interconnected-micelle association sol-gel transition mechanism of the pentablock copolymer aqueous solutions At 15 °C and pH 8.0 (the “D” state), the block copolymer solution exists as a sol state with low viscosity due to the less hydrophobicity of the PCLA and ionized OSM block When the temperature increases from 15 °C to 37 °C (the copolymer solution changes from stage D to B), although PCLA becomes more hydrophobic responding to temperature increase, but OSM

is still a hydrophilic block as a result of the ionization at high pH Consequently, PCLA-OSM block is not enough hydrophobic to form the complete interactions between micelles So the viscosity of copolymer solution increases if compare with the stage D, but it exists in a sol state When the copolymer solution changes from stage D to C, OSM is de-ionized because of the decrease of pH from 8.0 to 7.4 Then the non-ionized sulfonamide side groups in OSM block have a tendency to crystallize The viscosity of the block copolymer solution increases caused by the weakly hydrophobic interaction between the deionized OSM blocks, but the solution still exists as a sol state in each state At 37 °C and pH 7.4 (the “A” state), the PCLA block and non-ionized OSM induce the strong hydrophobic associations between the PCLA-OSM blocks Consequently, many bridges between micelles are formed by hydrophobic interaction between PCGA-OSM blocks Therefore, the OSM-PCLA-PEG-PCLA-OSM block copolymer solution undergoes “the micellar-interconnecting gelation”

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Figure 1-2 Schematic diagram of the sol-gel mechanism of the pH and temperature-sensitive block

copolymer solution Reproduced from Ref [40]

Figure 1-3 shows the comparison of sol-gel phase transition diagram of temperature-sensitive hydrogel and pH/temperature-sensitive hydrogel As can be seen in this figure, the sol-gel phase transition of triblock copolymer hydrogel of PCLA-PEG-PCLA just depends on temperature, whereas OSM-PCLA-PEG-PCLA-OSM shows the respond to both pH and temperature in the sol-gel transition [40]

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Figure 1-3 Phase diagram of block copolymers in buffer solution Reproduced from Ref [40] a)

PCLA-PEG-PCLA, b) OSM-PCLA-PEG-PCLA-OSM

1.2.2 Controlled drug/protein delivery

1.2.2.1 Controlled drug/protein delivery

In past few years, controlled drug/protein release to treatment for dangerous diseases such as epidemic, AIDS, cancer, hepatitis, especial diabetes became one of the most challenging achievements in medical science fields [2] There is a large unmet therapeutic need for better drug delivery of drug/proteins Although many of proteins/drugs have demonstrated great success in treating patients, they have been limited in their application

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HIV-because of the concerns about side effects resulted from the manner in which the drugs are released within the body In addition, the standard controlled release methods such as parenteral injection, oral, and drug transfusion through vein, etc show the burst of drug/protein’s concentration, which become a poison because it is higher than the therapeutic range Furthermore, the reduced drug/protein profile to under therapeutic range during the releasing by those methods will require the new dose of drug/protein The approach of delivering native protein drugs is the most promising because it permits full release of active drugs It is expected that the reduced side-effect profile will allow for increased dosage of some proteins, thus extending their efficacy and potential applications [42] (Figure 1-4)

Figure 1-4 The profile controlled release of drug/protein (discontinuous line: drug/protein release profile by traditional methods; continuous line: drug/protein release profile by sustained release from

biopolymer hydrogels)

A successful drug delivery device relies not only on intelligent network design but also on accurate a priori mathematical model of drug release profiles An ordered polymer network composed of macromolecular with well-understood chemistries yields hydrogels with well-defined physicochemical properties and reproducible drug-release profiles Biodegradable polymeric systems, especially biodegradable hydrogels [43-47] have been

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interested as an attractive insulin formulation because they have many advantages such as biocompatibility, high responsibility for specific factors The important advantage of the drug release from the hydrogels is that could

be controlled by several elements, for instance: pore size of hydrogels, the hydrophobicity, the presence of some specific function that make a particular interaction between the matrix and drug, and the controlling of the degradation of the biodegradable hydrogels

1.2.2.2 Drug/protein release mechanisms

As discussed above, hydrogels have many advance properties that make them useful in drug delivery applications The cumulative release of drug/protein from hydrogel devices is a function of time Furthermore, most of the release models are based on the rate-limiting step for controlled release and are therefore categorized

Swelling-controlled release occurs when diffusion of drug/protein is faster than hydrogel swelling Therefore, there is a transfer of molecules between the interface of rubbery and glassy phases of swollen hydrogel as a moving boundary [50] For example, the hydrogel tablets of hydroxypropyl methylcellulose from Dow Chemical Company is the device from which allow small molecule drugs release [51]

Chemically-controlled release is used to modeling the release of molecule, which determined by reactions occurring within a delivery matrix The most ordinary reactions that occur within hydrogel delivery systems are disintegration of polymer chains via hydrolytic or enzymatic degradation or reversible or irreversible reactions occurring between the polymer network and releasable drug The type of chemically-controlled release is influenced by the chemical reaction occurring during drug release Generally, the liberation of drug/protein occur through the surface-erosion or bulk-degradation of the polymer hydrogels [49,52]

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1.3 Aims and outlines of this study

The aims of this study are as follows:

The first is to develop a new temperature/pH-sensitive block copolymer hydrogels to apply controlled drug and protein delivery systems These hydrogels consist of pH-sensitive block and the temperature-sensitive block to form pH/temperature-sensitive block copolymers These copolymers are responsive to both temperature and pH, and its sol-gel transition behavior was evaluated upon pH and temperature The sol-gel transition of these materials is investigated to summarize the influence of the factors on sol-gel transition properties of copolymers such as the molecular weight of hydrophilic block copolymer of PEG, the ratio of hydrophobic/hydrophilic block length, PAE/OSM molecular weight, and the concentration of copolymers In addition, the cytotoxicity of type 1 copolymer hydrogels is also studied by direct contact method to check the biocompatibility property

The second is to examine the formation of ionic complex between hydrogels and drug and protein after injection into the body The positive charge of ionized PAE block was used to encapsulation many specific drug such as protein PAE as the ionization stage has positive charges, and it can form the complexes with negatively charges on the drug/protein by forming the ionic linkages As a result, the drug can be loaded into these copolymer matrixes by the link to form complexes The cationic nature of the polymer was found to endow the gel with unique properties for drug loading through charge complexation with proteins as well as a sustained release profile by decomplexation through continuous matrix degradation

The third is to examine the mechanism of controlled drug and protein release inside body The release of

protein-insulin from the type 1 hydrogels is studied both in vitro and in vivo on Female Sprague-Dawley (SD)

rats to identify the drug/protein loading and releasing mechanism Moreover, the relationship between the controlled release of insulin from the matrix of these materials and the controlled degradation is also investigated

In controlling the release of insulin and diabetes treatment studies, the accommodation of the characteristics into insulin delivery systems resulted in the sustained zero-order drug release with insulin concentration in plasma on Female Sprague-Dawley (SD) rats and continuous blood glucose normalization in streptozotocin-induced diabetic rats Furthermore, the release of hydrophilic protein such as hGH from PAE-PCL-PEG-PCL-PAE is

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also surveyed in vitro To study the drug release mechanism from the type 2 hydrogel, the cumulative release of hydrophobic drug paclitaxel (PTX), a anticancer agent, from OSM-PCGA-PEG-PCGA-OSM is investigated

in vitro and compared with that from OSM-PCLA-PEG-PCLA-OSM to survey the effect of the desintegration

on the release

The outlines of this study are as follows:

In this study, we report two types of tailored polymeric materials of which aqueous solutions undergo to-gel transition by pH change as well as by temperature change It could be used as subcutaneous injection drug delivery systems

sol-1 Type 1

Lee [53] used poly (β-amino ester) (PAE) as a pH-sensitive block to design biodegradable polymers In addition, ionized pH-responsive moiety PAE has positive charges; it could constitute a complexation with protein or anionic drugs by ionic union The new concept is using PAE as a functional group with two assignments, the first is a pH-sensitive block and the second is that it can make powerful linkages with protein or anionic drugs, to become a pH/temperature-sensitive injectable pentablock copolymer hydrogels The material relies not only on intelligent network, which well responds to both temperature and pH, but also on the great drug/protein capacity In these studies, PAE is used to conjugated to temperature-sensitive triblock copolymer hydrogels of poly(ethylene glycol)(PEG) – poly(ε-caprolactone(CL)) (PCL-PEG-PCL) and poly(ethylene glycol) – poly(ε-caprolactone-co D, L-lactide) (PCLA-PEG-PCLA) to form pentablock copolymer Triblock copolymers are synthesized by the ring-opening polymerization from ε-caprolactone/D,L-lactide and polyethylene glycol (PEG) Then these triblock copolymers are acrylated by acryloyl chloride before conjugated with 1, 4-butandiol diacrylate, and 4, 4’- trimethylene dipiperidine by additional polymerization to form pH/temperature-sensitive pentablock copolymers of (PAE-PCL-PEG-PCL-PAE)/ (PAE-PCLA-PEG-PCLA-PAE) The physicochemical properties of triblock and penta-block copolymers are characterized by 1H-NMR spectroscopy and gel permeation spectroscopy Sol-gel phase transition behaviors of aqueous solutions

of PAE-PCL-PEG-PCL-PAE block copolymers are investigated The general sol (flow)-gel (no flow) phase diagram of the PAE-PCL-PEG-PCL-PAE copolymer in buffer solution (it is recorded using the inverting test method) was shown in Figure 1-5

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Figure 1-5 The general sol-gel phase diagram of the PAE-PCL-PEG-PCL-PAE

Type 2

The second type of pH/temperature-sensitive hydrogel (OSM-PCLA-PEG-PCLA-OSM) consiting of poly(CL-co-LA)-PEG-and pH-sensitive moiety of oligo-sulfamethazine(OSM) was synthesized and investigated by Shim and co-worker[40,41] However, the degradation period of this polymer was found to be too long, it made some disadvantages on application for drug/protein delivery device Thus, in the present study, the disintegrative property of the hydrogel is modified by replacing of PCLA with poly(glycolide-co-ε-caprolactone) (PCGA) block copolymer, which is faster degraded by hydrolytic reaction under influence of methyl group in their structure The general sol (flow)-gel (no flow) phase diagram of the OSM-PCGA-PEG-PCGA-OSM [54] copolymer in buffer solution (it was recorded using the inverting test method) was shown in Figure 1-6

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Figure 1-6 The general sol-gel phase diagram of the OSM-PCGA-PEG-PCGA-OSM

The mechanisms of controlled drug/protein release from these hydrogels are chemically and controlled by the degradation as shown in Figure 1-7

diffusion-Figure 1-7 General mechanism of controlled drug/protein release from these hydrogel

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weakness, difficult to load drugs and cells and then crosslink in vitro as a prefabricated matrix The other

disadvantage is the clogging problem: when a temperature-sensitive hydrogels are injected into the body via a syringe, it tends to form a gel as the needle is warmed by the body temperature, leading to the difficultly during injection pH-sensitive hydrogels based on acrylic copolymers [16], carboxymethyl chitosan-based polyampholyte [17], and copolymers of (N,N-dimethylacrylamide, acrylic acid, N-tert-butylacrylamide, and N-methacryloylglycine (p-nitrophenyl ester) [18] were studied as a biomaterials Those polymers have some problems in application in drug release by subcutaneous injection method

Thermo- and pH-sensitive hydrogels by using N-isopropylacrylamide aminopropylmethacrylamide cross-linked with N, N'-methylenebis(acrylamide) [19], poly(2-hydroxyethyl acrylate), acrylate, or methacrylate [20-23] were investigated as matrices for drug release in recent years

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(NIPA)/N-Although drugs loading in the matrix are easier, those are non-degradable polymer

Shim et al coupled the sulfamethazine oligomer (OSM) as a pH-sensitive moiety with PCLA-PEG-PCLA

as a temperature-sensitive block copolymers to form a pH/temperature-sensitive block copolymer [24] This block copolymer solution showed a reversible sol-gel transition by a small pH change in the range of pH 7.4-8.0 and also by the temperature change in the region of body temperature Although these materials have some advances in applications in drug/protein delivery system such as controllability of Sol-gel phase transition diagram by adjusting the ratio of hydrophobic/hydrophilic chain lengths, PEG molecular weight, the concentration of the copolymer in solution, and the molecular weight of the pH-sensitive component There is

no clogging problem during injection into body The releasing of drug (PTX) from these gel matrixes could be controlled by the degradation of the copolymer However, there are some disadvantages during using the materials as a drug/protein delivery system in several cases The pH-sensitive moiety, OSM, is non-degradable polymer, and the degradation of these materials was just the decomposition of PCGA, PCLA or PEG, which made some question of degradation controlling of the materials Furthermore, it is just used as a delivery system for some appropriate drugs/proteins such as hydrophobic drugs Hydrophilic drug or protein can not be carried

by the hydrophobic core, which forms by unionized OSM and PCLA/PCGA block of PCGA-OSM and OSM-PCGA-PEG-PCGA-OSM Even these hydrogels were limited to protein loading to either mechanical absorption by hydrophobic-hydrophobic interactions or sequestering the drug inside the voids

OSM-PCGA-PEG-in the hydrogel matrix This limits the loadOSM-PCGA-PEG-ing capacity of the gel, the applications of the gel, and also the ability

to control the release profile At the heart of the matter is the ability to use a functional group which could make

a strong reversible linkage with the drug/protein to allow for a predictable and customizable loading and release

Among numerous interests have been focused on the stimuli-sensitive polymeric hydrogels, with displacement of sol-gel transition behavior, as promising candidates as chemical and/or protein drug carriers to materialize sustained drug delivery systems Although promising results have been achieved with chemical drug delivery systems, inadequate drug loading, releasing, and stability issues remain critical impedimenta for sustained protein delivery systems To address these obstacles, a new type of pH- and temperature-sensitive polymeric hydrogel system was constructed by introducing pH-sensitive blocks on the amphiphilic poly(ethylene glycol) - poly(ε-caprolactone) triblock copolymers (PCL-PEG-PCL) The resulting pentablock

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copolymers represented unique sol-gel transitions under the physiological conditions (pH and temperature) Furthermore, the cationic nature of the pH-sensitive block was found to endow the gel with unique properties for drug loading through charge complexation with proteins as well as a sustained release profile by decomplexation through continuous matrix degredation

Poly (β-amino ester) (PAE) is known as a pH-responsive and biodegradable polymer Langer [25-31], Ferruti [32] and Lee [33] used poly (β-amino ester) as a pH-sensitive block to design biodegradable polymers The toxicity of poly (β-amino ester) was investigated [26], and the biodegradable polymers based on the pH-sensitive block were used for self-assembly, gene delivery [27, 28], paclitaxel delivery [29], promoting-cellular uptake of heparin and cancer cell death [30], nonviral genetic vaccines [31], and tissue engineering [32] In addition, ionized pH-responsive moiety PAE has positive charges; it could constitute a complexation with protein or anionic drugs by ionic union The new concept is using PAE as a functional group with two assignments, the first is a pH-sensitive block and the second is that it can make powerful linkages with protein or anionic drugs, to become a pH/temperature-sensitive injectable hydrogel

In this chapter, we develop the new material with the main aim of controlling the Sol-gel phase transition diagram of copolymer solution PAE- PCL-PEG-PCL-PAE copolymers were synthesized with various of PEG molecular weight, pH-sensitive block length and ratio of hydrophobic/hydrophilic block (PCL/PEG) The sol-gel transition of these materials was investigated to summarize the influence of the above factors on sol-gel transition properties of the novel pH/temperature-sensitive cationic hydrogel In addition, the cytotoxicity, and

the stored condition of the copolymer in vitro were studied

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2.2 Experimental

2.2.1 Materials

Poly(ethylene glycol)s (PEG) were purchased from Sigma-Aldrich (St Louis, MO) (Mn = 1500 and 1650) and ID Biochem, Inc (Seoul, Korea) (Mn =1750) The PEGs were recrystallized in n-hexane and dried in vacuum for 3 days prior to use ε -Caprolactone (CL), and phosphate buffered saline (PBS) were obtained from Sigma Chemical Co Tween 80, and cremophor EL were purchased from Aldrich Chemical Co Stannous octoate [Sn(Oct)2] was obtained from Sigma Chemical Co and was dried for 24 h under vacuum at ambient temperature prior to use Acryloyl chloride, triethylamine (TEA), 1, 4-butandiol diacrylate (BDA), and 4, 4’-trimethylene dipiperidin (TMDP) were purchased from Aldrich Chemical Co 2,3-bis(2-methoxy-4-nitro-5-susfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, dulbecco’s modified Eagle’s medium, fetal bovine, glucose, L-glutamine, sodium pyruvate, sodium bicarbonate using for cytotoxicity were purchased from Aldrich Chemical Co HCl 37 %, sodium chloride, chloroform (CDCl3), dichloromethane (DCM), and diethyl ether were all obtained from Samchun All other reagents were of analytical grade and used without further purification

2.2.2 Synthesis of pH/ temperature-sensitive PAE-PCL-PEG-PCL-PAE

pentablock copolymer hydrogels

2.2.2.1 Synthesis of temperature-sensitive PCL-PEG-PCL triblock copolymer

PCL-PEG-PCL triblock copolymer was synthesized by ring opening polymerization of ε-caprolactone (CL) initiated by hydroxyl groups at the end of PEG in the presence of stannous octoate as a catalyst Several PEGs with different molecular weights (1500, 1650, and 1750) were used to synthesize the triblock copolymers The compositions and molecular weights of triblock copolymers to control the balance of hydrophobic/hydrophilic of PEG/PCL were adjusted by the feed ratios of PEG and PCL The synthesis process of triblock copolymer, which have PCL/PEG = 1.8/1(weight ratio); PEG1650 can be described as below: 4 g PEG (1650) and 0.04 g stannous octoate in a dried two-neck round-bottom flask are dried at 110 °C for 2 h under vacuum After cooling to 60 °C, 7.6 g CL is added under a dry nitrogen atmosphere The reaction mixture is dried for 1 h under vacuum at 60 °C and was raised slowly to 130 °C The ring opening reaction is

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carried out for 18 h The reactant is cooled to room temperature before being dissolved in chloroform, and then the solution is precipitated in excess diethyl ether The precipitated product is dried under vacuum at room temperature for 48 h The overall yield of this triblock copolymer is over 85 % after drying

2.2.2.2 Synthesis of acrylated PCL-PEG-PCL triblock copolymers

Acrylate groups are conjugated to triblock copolymer by coupling reaction between hydroxyl groups at the end of PCL-PEG-PCL and acryloyl chloride in the presence of tri-ethylamine as a catalyst The reactants can be calculated based on molar ratio of triblock compolymer, acryloyl chloride and catalyst Every triblock was acrylated with the same reaction ratio:

Triblock/acryloyl chloride/TEA = 1/3.2/2 (molar ratio) (equation 2-1)

Based on equation 1-1, the quantity of the reactants, which used for acrylation the triblock have the composition PEG 1650, PCL/PEG ~1.8, are 4 g of PCL-PEG-PCL , 0.24 ml of triethylamine and 0.23 ml of acryloyl chloride (96%) The processing to acrylate this triblock can be described as below PCL-PEG-PCL in

a dried two-neck round-bottom flask is dried for 2 h under vacuum at 80 °C and then the triblock is dissolved in anhydrous chloroform at ambient temperature under a dry nitrogen atmosphere to get a solution at 20 wt% After that, TEA and acryloyl chloride are added at 10 °C The acrylation reaction is carried out for 48 h under a dry nitrogen atmosphere A rotary evaporator at ambient temperature is used to remove the chloroform solvent

of the reactant, and then the dry reactant is purified by excess diethyl ether The precipitated product is dried under vacuum at room temperature for 48 h

2.2.2.3 Synthesis of pH/temperature-sensitive PAE-PCL-PEG-PCL-PAE pentablock

copolymers

The pentablock copolymer is synthesized by additional polymerization between the vinyl groups at the end

of the acrylated triblock and 1, 4-butandiol diacrylate (BDA) with vivacious hydrogen in amine groups of 4, trimethylene dipiperidine (TMDP) The molar ratio of BDA/TMDP has to keep at 1/1 The molecular weight

4’-of pH/temperature-sensitive moiety PAEs, which are introduced into acrylated triblock by additional polymerization, can be controlled by the quantity of BDA and TMDP

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