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

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

i


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

i


A Study on the pH/Temperature-Sensitive
Biodegradable Hydrogels for Controlled Protein and
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]

ii



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-PCLtriblock copolymer ................................................22
2.2.2.2 Synthesis of acrylated PCL-PEG-PCLtriblock 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 1H-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 Results and discussions ...............................................................................................................................................................62
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
3.4 Results and discussions ...............................................................................................................................................................81
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-PCLAtriblock 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 Characterization ..................................................................................................................................................................90
4.2.3.1 1H-NMR and GPC analyses ..................................................................................................................................90
4.2.3.2 pH determination ......................................................................................................................................................90

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4.2.3.3 Sol-gel phase transition measurement ................................................................................................................90
4.2.3.4 Cytotoxicity evaluation .............................................................................................................................................90
4.2.4 Insulin loading process ......................................................................................................................................................91
4.2.5 Degradability evaluation ...................................................................................................................................................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 Results and discussions ...............................................................................................................................................................93
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
4.3.6 Degradability evaluation .................................................................................................................................................106

4.3.7 Degradatbility of the complex gel in vivo ..................................................................................................................109
4.3.8 Insulin release in vitro .......................................................................................................................................................109
4.3.9 Storage stability ..................................................................................................................................................................112
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-PCGAtriblock 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 Characterization ...............................................................................................................................................................124
5.2.3.1 1H-NMR analysis....................................................................................................................................................124
5.2.3.2 GPC analysis .............................................................................................................................................................124
5.2.3.3 Sol-gel phase transition measurement ............................................................................................................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 Results and discussions .............................................................................................................................................................126
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
5.3.4 Degradability evaluation .................................................................................................................................................139
5.3.5 Drug loading and release in vitro ..................................................................................................................................141
5.3.6 Storage stability ..................................................................................................................................................................143
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-PCLPAE pentablock copolymers ··················································································································30

Table 4-1

Molecular weight and polydispersity of PCLA-PEG-PCLA triblock and PAE-PCLA-PEGPCLA-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-PEGPCGA-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

vi


List of Schemes
Scheme 2-1

Synthesis of pH/temperature-sensitive PAE-PCL-PEG-PCL-PAE block copolymers, a)
Synthesis of PCL-PEG-PCLtriblock 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

vii

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

1

H-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-PCLand 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

viii


Figure 2-8

Sol-gel phase transition diagrams of PAE-PCL-PEG-PCL-PAE (PEG 1500, and PAE~1.251.3k) 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

ix


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
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) ··················································································77

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Figure 3-19 Body weight change with time on STZ-induced diabetic rats for 5 days and insulin releasinginduced 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) ·····································································································78
Figure 4-1

1

H-NMR spectra of block copolymers, a) PCLA-PEG-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
concentration at pH 7.4, b) Various pH at copolymer concentration 20 wt% ······························102

Figure 4-6

Sol-gel phase transition diagrams of PAE-PCLA-PEG-PCLA-PAE (PEG 1750, and
PAE~1.3k) block copolymer solutions with different PCLA/PEG ratio, a) Various copolymer
concentration at pH 7.4, b) Various pH at copolymer concentration 20 wt% ······························103

Figure 4-7

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

xi


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-PCLAPAE, 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-PEGPCLA-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

1

H-NMR spectra of sulfamethazine, SMM, OSM, a) Sulfamethazine and SMM, b) SMM and

OSM. ······················································································································································127

Figure 5-2

1

Figure 5-3

1

Figure 5-4

GPC traces of triblock and pentablock copolymer ··········································································130

H-NMR spectrum of PCGA-PEG-PCGAtriblock copolymer ··················································128
H-NMR spectrum of OSM-PCGA-PEG-PCGA-OSM pentablock copolymer ····················128

xii


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/PCGAratio 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 OSMPCGA-PEG-PCGA-OSM, b) OSM-PCGA-PEG-PCGA-OSM and OSM-PCLA-PEGPCLA-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-PCLAPEG-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

xiii


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


[13]. pH-sensitive hydrogels based on N-isopropylacrylamide (NIPA)/N-aminopropylmethacrylamide crosslinked with N, N'-methylenebis(acrylamide) [14], poly(2-hydroxyethyl acrylate), acrylate, or methacrylate [1518] 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.
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,

2


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

3


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 11.

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

4


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 oxide)-b-poly(L-lactide-coglycolide) (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].
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-PLLAPEG 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
ABCBAbased on Pluronic (F127). Here, BCB is temperature-sensitive triblock copolymer made of F127, and
Ais 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

5


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-PEGPCLA) 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 PCLAbecomes 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 nonionized 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 micellarinterconnecting gelation”

6


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-PEGPCLA-OSM shows the respond to both pH and temperature in the sol-gel transition [40].

7


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, HIVAIDS, 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

8