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7/27/2019 Polymer Sinh Hoc Va y Sinh 1
http://slidepdf.com/reader/full/polymer-sinh-hoc-va-y-sinh-1 1/99
Sungkyunkwan University 1
Novel pH and Temperature Sensitive
Biodegradable Block Copolymer Hydrogels
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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Sungkyunkwan University 2
1. Molecular design of novel temperature- and pH-sensitivebiodegradable block coplolymer hydrogels- Anionic hydrogels
- Cationic hydrogels
2. Application for DDS- In vitro test
- In vivo test
Objectives
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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1. Synthesis of OSM-PCGA-PEG-PCGA-OSM.2. Sol-gel transition-Degradation
3. In vitro
Part I Anionic hydrogels: OSM-PCGA-PEG-PCGA-OSM
Part II Cationic hydrogels: PAE-PCL-PEG-PCL-PAE
1. Synthesis of PAE-PCL-PEG-PCL-PAE
2. Sol-gel transition3. Degradation-In vitro drug/protein delivery test
4. Controlled release of insulin in vivo on Female Sprague-Dawley (SD) rats.
5. Treatment the Diabolical disease in vivo on Diabetic Fat Rats (DFR).
Contents
Part III Controlled the degradation of cationic hydrogels:PAE-PCLA-PEG-PCLA-PAE
1. Synthesis of PAE-PCLA-PEG-PCLA-PAE
2. Sol-gel transition
3. Degradation-In vitro drug/protein delivery test
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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Sungkyunkwan University 4
Minute change in
stimuli
Temperature
Electric FieldLight
Stress
pH
Ion species
Ionic strength
Solvent
composition
Biomolecules
Large change inresponse
(Transition)
Conformation
Solubility
Shape
Swelling
Permeation
Background: Stimuli- Sensitive Polymer
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Temperature-sensitive hydrogels : Pluronic
PPO
core
Hydrophilic PEO
Hydrophobic PPO
Concentration
Temperature
PEO shell
5~7.5nm3.5~15nm
15~35nm
- Cancer therapy- Protein delivery
- Gene delivery
- Temperature-sensitive hydrogel
- Non-biodegradable (PPO)
Pluronic F-127 ( PEO99-PPO65-PEO99)
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Cloud Point
10 15 20 25 30 35 40 45
0
20
40
60
80
100
120
Ge l So l
Concentration(wt% in solution)
T e m p ( o C )
Temperature-sensitive hydrogels: ReGel
ReGel ( PEG-PLGA-PEG)
- Cancer therapy
- Protein delivery
- Gene delivery
- Biodegradable- Temperature-sensitive only
Clogging during injection
- Degradable product Low pH
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Clogging : Gelation occurs inside the needle during injection bytemperature change due to the body heat transfer
Gelation inside the body after injection
Gelation inside
the needle during
injection by
temperature
change
Clogging during injection
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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pH
Temp.
Sol Gel
Hydrophilic block hydrophobic block
A ---- --B----- C--- --B-- --- A
Anti-clogging: Introducing pH-sensitive moiety
pH sensitive blockpH sensitive block
Temperature sensitive block
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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A ---- --B----- C--- --B-- --- A pH sensitive block pH sensitive block
Acid moiety : pKa(7.8-7.4)
- Acid group
(-COOH, -SH, - SONH-)
- Anionic charge
- Complex with cationic drug
Basic moiety : pKb (6.2-7.5)
- Backbone amine group
- Pendent amine group
(Tertiary & secondary amine)
- Cationic charge
- Complex with anionic drug
- Screening work (also FDA list)
Poly(amino acid) derivertives
Poly(amido amine) derivertives
Temperature sensitive block
Prof. Yoo Han Bae Group
Prof. Doo Sung Lee Group
Screening works
Screening works of pH sensitive moiety
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Anionic & Cationic pH/Temp-sensitive hydrogels
A ---- --B----- C--- --B-- --- A pH sensitive block pH sensitive block
Type 1: Anionic hydrogel Type 2 : Cationic hydrogel
Ref.) Shim, W. S.; Kim, S. W.; Lee, D. S. Biomacromolecu les 2006, 7 (6), 1935
pH
T e m p e r a t u r e ( o C )
7.4
37
Sol
Gel
Sol (Sedimentat ion )
Human body
cond i t ion
pH
T
e m p e r a t u r e ( o C )
7.4
37
Sol
Gel
Sol (Sedimentat ion )
Human body
cond i t ion
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Anionic pH/temperature sensitive hydrogels OSM-PCGA-PEG-PCGA-OSM
pH
T e m p e r
a t u r e ( o C )
7.4
37
SolGel
Sol (Sedimentat ion )
Human body cond i t ion
Part I
1. Synthesis of OSM-PCGA-PEG-PCGA-OSM.
2. Sol-gel transition-Degradation
3. In vitro
Part I Anionic hydrogels: OSM-PCGA-PEG-PCGA-OSM
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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Sulfamethazine-dimethyl acrylamide copolymer
pH
5 6 7 8 9 10
% T
0
20
40
60
80
100
120 0.5 wt %
1 2
sulfamethazine 20%
turbidity of polymer solution
CH2 C CH2
C O
NH
CH3
S OO
CH
C O
NH3C CH3
N N
H3C CH3
x y
NH
pKa=7.4
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ionizationdeionization
As pH sensitive moiety is ionized, polymer solution will be a sol state.
pKa
A-B-A
pH-sensitive block pH-sensitive block
Temperature sensitive block
pH sensitive block (moiety)
CH2 C
C O
NH
CH3
S
O
ON
N
H3C
H3C
x
HN
S
H2C
H2C COO
H2
CC
CO
HN
CH3
S
O
O N
N
CH3
CH3x
HN
SH2
CH2
COOC
Ionization / deionization of pH-sensitive moiety
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Synthesis of OSM-PCGA-PEG-PCGA-OSM
HO PEG OH + O
O
+O
O
O
O
ε-caprolactone GA
Sn(Oct)2 130oC
H2
C
H2
C O Cn
OH2
C O C
H2
C
H2
C
OH2
C
H2
C
H2
C Ox
Hy
CO
H2
COC
H2
C
H2
CO
H2
C
H2
C
H2
CO xH Oy
PEG
PCGA-PEG-PCGA
PCGA-PEG-PCGA triblock copolymer
7/27/2019 Polymer Sinh Hoc Va y Sinh 1
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[Sulfamethazine monomer]
+
[Methacryloyl Chloride]
CCH3
C
CH2
O
Cl
NH2
S OO
N
N N
CH3CH3
H
CCH3 C NH SO
O
N
N
N
CH3
CH3
CH2
HO
C
CH3
C
NH
S OO
N
N N
CH3CH3
CH2
H
n
O
[Sulfamethazine monomer]
+ C
O
HO CH2 CH2 SH
[3-Mercapto propionic acid]
NH
SO O
NH
N N
CH3CH3
C
CH2 S CH2 CH2 COOHC
CH3
H
O
n
[Oligo-sulfamethazine or
Sulfamethazine oligomer ]
Transfer agent
[Sulfamethazine]
Synthesis of OSM-PCGA-PEG-PCGA-OSM
Oligo-sulfamethazine (OSM)
OSM
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DCC, 4-DMAP
Biodegradable & Temperature-sensitive pH -sensitivepH -sensitive
NH
SO O
NH
N N
CH3CH3
C
CH2 S CH2 CH2 COOHC
CH3
H
O
n
PCGA-PEG-PCGA
OSM C
O
CH2CH2 C
O
O PCGA PEG PCGA O C
O
CH2CH2 C
O
OSM
Synthesis of OSM-PCGA-PEG-PCGA-OSM
OSM-PCGA-PEG-PCGA-OSM pentablock copolymer
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Characterization of OSM-PCGA-PEG-PCGA-OSM
PEG
CLGA
1H-NMR of PCGA-PEG-PCGA triblock copolymer
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Characterization of OSM-PCGA-PEG-PCGA-OSM
Sulfamethazine
1H-NMR of Sulfamethazine & Sulfamethazine monomer(SM)
Sulfamethazinemonomer(SM)
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OSM
1H-NMR of Sulfamethazine monomer(SM) and OSM
Characterization of OSM-PCGA-PEG-PCGA-OSM
SM
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200 400 600 800 1000
-0.02
0.00
0.02
0.04
0.06
0.08
R I
R e s p o n s e S i g n a
l
Time (min)
PCGA-PEG-PCGAOSM-PCGA-PEG-PCGA-OSM
GPC (elution solvent:THF)
GPC of OSM-PCGA-PEG-PCGA-OSM
Characterization of OSM-PCGA-PEG-PCGA-OSM
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OSM-PCGA-PEG-PCGA-OSMPEG
Mab
PEG/PCGA
(wt/wt)a
CL/GA
(mol/mol)
OSM
Mnc
Mw /Mnc
806-1091-1000-1091-806 (A-1) 1000 1/2.18 2.34/1 806 1.30
806-1590-1500-1590-806 (B-1) 1500 1/2.12 2.35/1 806 1.34
806-2474-2000-2474-806 (D-1) 2000 1/2.47 2.37/1 806 1.50
806-1881-1750-1881-806 (C-2.1) 1750 1/2.15 2.32/1 806 1.35
904-1461-1750-1461-904 (C-1) 1750 1/1.67 2.26/1 904 1.35
904-1881-1750-1881-904 (C-2) 1750 1/2.15 2.32/1 904 1.35
904-2117.5-1750-2117.5-904 (C-3) 1750 1/2.42 2.26/1 904 1.36
904-2336.2-1750-1336.2-904 (C-4) 1750 1/2.67 2.29/1 904 1.36
904-2494-1750-2494-904 (C-5) 1750 1/2.85 2.28/1 904 1.35
a PCGA-PEG-CLGA number-average molecular weights were calculated from 1H-NMR.b Provided by Aldrich.c Measured by GPC
Characterization of OSM-PCGA-PEG-PCGA-OSM
Mol. wt & MWD
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Sol – gel phase transition
Effece of PEG mol.wt
::
:
:(PEG/PCGA=1/2.18); PEG.1000; OSM.806
(PEG/PCGA=1/2.12); PEG.1500; OSM.806
(PEG/PCGA=1/2.15); PEG.1750; OSM.806
(PEG/PCGA=1/2.47); PEG.2000; OSM.806
Concentration (wt%)
(pH 7.4)
T e m p e r a t u r e ( o C )
Gel
Sol
Sol (Sedimen tation)
0 5 10 15 20 25
0
10
20
30
40
50
60
T
e m p e r a t u r e ( o C )
pH
(C: 10 wt%)
Gel
Sol (Sedimentation )
So l
7.0 7.2 7.4 7.6 7.8 8.00
10
20
30
40
50
60
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Effect of OSM mol.wt
:(PEG/PCGA=1/2.15); PEG.1750; OSM.806
: (PEG/PCGA=1/2.15); PEG.1750; OSM.904
T
e m p e r a t u r e ( o C )
Concentration (wt%)
(pH 7.4)
Gel
Sol
Sol (Sedimentation )
0 5 10 15 20 25
0
10
20
30
40
50
60
T e m p e r a t u r e ( o C
)
pH
(C: 10 wt%)
Gel
Sol (Sedim entation)
Sol
0
10
20
30
40
50
60
7.0 7.2 7.4 7.6 7.8 8.0 8.2
Sol – gel phase transition
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Effect of PEG/PCGA mol ratio ( PEG 1750, OSM.904)
(PEG/PCGA=1/2.15)
(PEG/PCGA=1/2.67):
:
: (PEG/PCGA=1/2.85)
:
: (PEG/PCGA=1/1.67)
(PEG/PCGA=1/2.42)
Gel
So l
Sol (Sedimentat ion )
0 5 10 15 20 25
0
10
20
30
40
50
60
T e m p e r a t u r e ( o C )
Concentration (wt%)
(pH 7.4)
Gel
Sol (Sedimentat ion )
So l
7.0 7.2 7.4 7.6 7.8 8.0 8.20
10
20
30
40
50
60
T e
m p e r a t u r e ( o C )
pH
(C: 10 wt%)
Sol – gel phase transition
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(PEG/PCGA=1/2.15)
(PEG/PCGA=1/2.67):
:
:
:
: (PEG/PCGA=1/1.67)
(PEG/PCGA=1/2.42)
Effect of PEG/PCGA and Concentration ( PEG 1750, OSM.904)
(PEG/PCGA=1/2.85)
Gel
Sol (Sedimentat ion )
Sol
0
10
20
30
40
50
60
7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6
T e m p e r a t u r e ( o C )
pH
(C: 20 wt%)
Gel
Sol (Sedimentat ion )
So l
7.0 7.2 7.4 7.6 7.8 8.0 8.20
10
20
30
40
50
60
T e m
p e r a t u r e ( o C )
pH
(C: 10 wt%)
Sol – gel phase transition
Ti i ti f l l t i ft i j ti
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Time variation of sol-gel transion after injection
Time remaining 5min 1 day 5 days 10 days 20 days 30 days
A-1pH 8.0 S S S S S S
pH 7.4 S S S S S S
B-1pH 8.0 S S S S S S
pH 7.4 G G G G G S
D-1pH 8.0 S S S S S S
pH 7.4 G G G S S S
C-2.1pH 8.0 S S S S S S
pH 7.4 G G G G G G
C-1pH 8.0 S S S S S S
pH 7.4 G G G G G G
C-2pH 8.0 S S S S S S
pH 7.4 G G G G G G
C-3
pH 8.0 G G S S S S
pH 7.4 G G G G G G
C-4pH 8.0 G G G S S S
pH 7.4 G G G G G G
C-5pH 8.0 G G G S S S
pH 7.4 G G G G G G
S; sol, G; gel state
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Sol-gel transition after injection at pH 8.0 & 37oC
Material: OSM-PCGA-PEG-PCGA-OSM; (PEG/PCGA=1/2.15); PEG.1750; OSM.904
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Material: OSM-PCGA-PEG-PCGA-OSM; (PEG/PCGA=1/2.15); PEG.1750; OSM.904
Sol-gel transition after injection at pH 7.4 & 37oC
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Time (day)0 10 20 30 40
oecuarweg
p
0
2000
4000
6000
8000
10000OSM-PCGA-PEG-PCGA-OSM (904-2118-1750-2118-904)
PCGA-PEG-PCGA (2118-1750-2118)
Time (day)0 10 20 30 40
oecuarweg
p
2000
4000
6000
8000
10000
OSM-PCGA-PEG-PCGA-OSM (904-2118-1750-2118-904)
OSM-PCLA-PEG-PCLA-OSM (1114-1820-1750-1820-1114)
Time variation of degradation rate
Controlled degradation of Anionic hydrogel at 37 oC and pH 7.4
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Time variation of degradation rate: Storage stability
M
o l e c u l a r w e i g h t ( M p )
0 50 100 150 200
2000
4000
6000
8000
Time (day)
Keeping at 5oC as a solution stage pH 8.0
Keeping at 0oC as a solution stage pH 8.0
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PTX loading and releasing
OSM-PCGA-PEG-PCGA-OSM Dissolving in
PBS buffer
at 3 oC Adjusting
pH to 8.0
Adjusting
pH to 7.4 Sampling Adding fresh
serum at 37oC Releasing
Loading and releasing experiment
PTX loading 2 days
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Sol-gel of OSM-PCGA-PEG-PCGA-OSM contents drug
7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6
10
20
30
40
50
60
T e m p e r a t u r e ( o C )
pH
Sol (Sedimentat ion )
Gel
So l
Polymer solution: 20wt% in PBS buffer (PEG 1750; PCGA/PEG= 2.67; OSM 904)
0 mg PTX/ml polymer solution (0 Wt% drug in matrix)10 mg PTX/ml polymer solution (5 Wt% drug in matrix)
20 mg PTX/ml polymer solution (10 Wt% drug in matrix)
40 mg PTX/ml polymer solution (20 Wt% drug in matrix)
Sol-gel region shift by PTX loading
Drug loading
Influence
New
Moleculer design
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Time (day)0 10 20 30
ComulativeReleaseofPTX(%
)
0
20
40
60
80
100
PTX loading and releasing in vitro
OSM-PCGA-PEG-PCGA-OSM
(PEG 1750; PCGA/PEG= 2.67; OSM 904)
Time(day)
0 4 8 12 16 20 24 28 32 36
CumulativeReleaseofPTX(
)
0
20
40
60
80
100
2.5 mg PTX/ml of polymer solution (20wt%)
5 mg PTX/ml of polymer solution (20wt%)
10 mg PTX/ml of polymer solution (20wt%)
OSM-PCLA-PEG-PCLA-OSM
(PEG 1750; PCGA/PEG= 2.67; OSM 904)
Controlled the release by the degradation
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Ionic complex mechanism of PTX loading and releasing
Mechanism of anionic protein loading and release.
Advantages
• Good absorption.
•
Ease to control drug level in the bloodbetween the desired maximum and
minimum for an extended period of time
• Protein and matrix are a complete gel:
good mechanical property.
• Ease to apply by injection.
• Useful for cationic protein drug
Protein loading at pH 8.0, 15 oC Gel formation at pH 7.4, 37 oC Sustained Release
(Degradation & Diffusion)
+++
+
+
++
++
+
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• OSM-PCGA-PEG-PCGA-OSM penta-block copolymers of varying PEG molecular
weight and PCGA/PEG ratios were synthesized.
•The gel phase regions of OSM-PCGA-PEG-PCGA-OSM can be controlled;
- PEG molecular weight
- PCGA/PEG ratio and the concentration
- OSM molecular weight
• Temperature & pH dependence of OSM-PCGA-PEG-PCGA-OSM pentablock
copolymer ;
- Gel at pH 7.4 & 37℃.
- Sol at pH 8.0 & 37℃
• The degradation rate of OSM-PCGA-PEG-PCGA-OSM is notably faster than OSM-
PCLA-PEG-PCLA-OSM, indicating its potential for drug delivery.
• This polymer can be kept in aqueous solution at 0℃ for more than 7 months without
degradation.
• More than 90% of the PTX drug was released from this matrix after 20-22 days. The
results of this investigation demonstrate the potential usefulness of this
pH/temperature sensitive block copolymer for application in drug delivery systems.
Part I Summary
Part II
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Cationic pH/temperature sensitive hydrogel PAE-PCL-PEG-PCL-PAE
pH
T e m
p e r a t u r e ( o C )
7.4
37
Sol
Gel
Sol (Sediment at ion)
Human body
cond i t ion
Part II
Part IICationic hydrogels: PAE-PCL-PEG-PCL-PAE
1. Synthesis of PAE-PCL-PEG-PCL-PAE
2. Sol-gel transition
3. Degradation-In vitro drug/protein delivery test
4. Controlled release of insulin in vivo on Female Sprague-Dawley (SD) rats.
5. Treatment the Diabolical disease in vivo on Diabetic Fat Rats (DFR).
A i t bl k l
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β-Aminoester block copolymer
pKb= 6.50 - 6.75
H2
CC
H2
H2
CN
H2
CCH2
H2
CCH2
H2
CCH2
CCH2
H2
C
O
O C
H2
CCH2
O
O *N*
n
0.1N NaOH aqueous solution(ml)
0 2 4 6 8 10 12 14
pH
1
2
3
4
5
6
7
8
9
10
11
12
I i ti / d i i ti f H iti i t
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ionization deionization
As pH sensitive moiety is ionized, polymer solution will be a sol state.
pKb
Ionization / deionization of pH-sensitive moiety
NO N O
O O
n
B-C-BNO N O
O O
n
pH-sensitive block pH-sensitive blockTemperature
sensitive block
pH sensitive block (moiety)
Synthesis of PAE PCL PEG PCL PAE
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Synthesis of PAE-PCL-PEG-PCL-PAE
PCL-PEG-PCL
PCL-PEG-PCL triblock polymer
ε-caprolactone
Sn(Oct)2 130oC
PEG
+ O
O
HO
H2
C
H2
C O Hn
H2
C
H2
C O
n
C
H2
C
H2
C
OH2
C
H2
C
H2
C O H
x
OC
H2
C
H2
C
O
H2
C
H2
C
H2
COH
x
Cl C
O
CH
CH2
+
Acrylation in
Chloroform
10oCHO PCL PEG PCL OH
O PCL PEG PCL O C CH
CH2
O
C
O
CH
H2C
Acryloyl chlorideTriblock copolymer
PCL-PEG-PCL - Diacrylated
Synthesis of PAE PCL PEG PCL PAE
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PAE-PCL-PEG-PCL-PAE
NHHN+
OO
O
O
+
50oC
4,4’ trimethylene dipiperidine 1,4 – Butane diol diacrylate
PAE-PCL-PEG-PCL-PAE
O PCL PEG PCL O C CH
CH2
O
CHCH2C
O
DCM
Synthesis of PAE-PCL-PEG-PCL-PAE
NNO
OO
O
O
O
N NO
OO
OOn PCL PEG PCL O
n
Synthesis of PAE PCL PEG PCL PAE
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Triblock Acrylation Amino ester reaction
in DCM
VaporizingDCM
Dissolving
in THF
Vaporizing
THFPrecipitation in
ethyl ether Vacuum drying
Final product
(powder-type)
Filtering
Processing of synthesis of PAE-PCL-PEG-PCL-PAE
Synthesis of PAE-PCL-PEG-PCL-PAE
Characterization of PAE PCL PEG PCL PAE
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Characterization of PAE-PCL-PEG-PCL-PAE
G3 G1
6.0 5.0
ppm 2.03.04.05.06.0
A, A’
CB,F
D E
G2
O
H2
C
H2
C O
H2
C
H2
C O
H2
C
H2
C O
n-2
C
H2
C
O
H2
C
H2
C
H2
C
H2
C O C
O
CH
C
H
Hy
A A A'A'B B C D G1 G2
G3
D'E F
PEG
CL
Acrylate
Acrylate
1H-NMR of PCL-PEG-PCL acrylated
Characterization of PAE-PCL-PEG-PCL-PAE
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Characterization of PAE-PCL-PEG-PCL-PAE
ppm 2.03.04.05.06.0
PEG
CL
A, EB C
GF
D
NNO
O
O
O
O
O
n PCL PEG PCL PAE
G G E E DAAADBCHH
Aminoester
1H-NMR of PAE-PCL-PEG-PCL-PAE
Characterization of PAE-PCL-PEG-PCL-PAE
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Characterization of PAE-PCL-PEG-PCL-PAE
Elution time
RIresponses
ignal
PCL-PEG-PCL
PAE-PCL-PEG-PCL-PAE
PEG 1.65k; PEG/PCL=1.8
GPC of triblock and pentablock copolymer
Characterization of PAE-PCL-PEG-PCL-PAE
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Characterization of PAE-PCL-PEG-PCL-PAE
PCL-PEG-PCL(Mn a)
PEG/PCL(wt ratio)
PEG(Mn)
PAE-PCL-PEG-PCL-PAE b PAE-PCL-PEG-PCL-PAE c D
984-1500-984 1/1.3 1500 2000-984-1500-984-2000 1285-984-1500-984-1285 1.43
1110-1500-1110 1/1.5 1500 2000-1110-1500-1110-2000 1301-1110-1500-1110-1301 1.46
1364-1500-1364 1/1.8 1500 1000-1364-1500-1364-1000 762-1364-1500-1364-762 1.35
1364-1500-1364 1/1.8 1500 2000-1364-1500-1364-2000 1225-1364-1500-1364-1225 1.45
1364-1500-1364 1/1.8 1500 3000-1364-1500-1364-3000 1925-1364-1500-1364-1925 1.52
1364-1500-1364 1/1.8 1500 4000-1364-1500-1364-4000 2345-1364-1500-1364-2345 1.58
1104-1650-1104 1/1.3 1650 2000-1104-1650-1104-2000 1249-1104-1650-1104-1249 1.42
1262-1650-1262 1/1.5 1650 2000-1262-1650-1262-2000 1287-1262-1650-1262-1287 1.43
1584-1650-1584 1/1.8 1650 2000-1584-1650-1584-2000 1258-1584-1650-1584-1258 1.41
1310-1750-1310 1/1.5 1750 2000-1310-1750-1310-2000 1254-1310-1750-1310-1254 1.43
a Number-average molecular weight calculated from 1H-NMR
b Number-average molecular weight (ratio of reaction)
c Number-average molecular weight calculated from GPC
pH of PAE PCL PEG PCL PAE
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Temperature (oC)
0 10 20 30 40 50 60 70
pH
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
pH depending on temperature
PAE-PCL-PEG-PCL-PAE (PCL/PEG~1.5/1; PEA~1.25) 20 wt% copolymer solution
Sol-gel transition
pH of PAE-PCL-PEG-PCL-PAE
PEG 1.5k
PEG 1.75k PEG 1.65k
pH
5.5 6.0 6.5 7.0 7.5
Tem
perature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sediment at ion)
PEG 1.5k
Solid: Real diagram
Dash: Engineering diagram
PEG 1.65k PEG 1.75k
Phase diagram of PAE PCLA PEG PCLA PAE
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Concentration (wt%) (pH 7.4)
5 10 15 20 25 30
Temperature(oC)
0
10
20
30
40
50
60
Effect of PEG mol. wt
Phase diagram of PAE-PCLA-PEG-PCLA-PAE
PAE-PCL-PEG-PCL-PAE (PCL/PEG~1.5/1; PEA~1.25k)
pH (20wt%)
5.5 6.0 6.5 7.0 7.5
Temperature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sediment at ion)
Sol
Gel
Sol (Sediment at ion)
PEG 1.5k
PEG 1.75k
PEG 1.65k
Phase diagram of PAE PCL PEG PCL PAE
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Effect of PCL/PEG ratios (PEG 1500, PAE~1.25k)
Phase diagram of PAE-PCL-PEG-PCL-PAE
Concentration (wt%) (pH 7.4)5 10 15 20 25 30
Temperature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sediment at ion)
pH (20wt%)
5.5 6.0 6.5 7.0 7.5
Te
mperature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sedimentat ion )
PCL/PEG ~1.3/1
PCL/PEG ~1.5/1
PCL/PEG ~1.8/1
PAE-PCL-PEG-PCL-PAE (PEG 1.5k; PEA~1.25k)
Phase diagram of PAE PCL PEG PCL PAE
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Effect of PCL/PEG ratios (PEG 1650, PAE~1.25k)
Phase diagram of PAE-PCL-PEG-PCL-PAE
PCL/PEG ~1.3/1
PCL/PEG ~1.5/1
PCL/PEG ~1.8/1
pH (20wt%)
5.5 6.0 6.5 7.0 7.5
Tem
perature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sediment at ion)
Concentration (wt%) (pH 7.4)
5 10 15 20 25 30
Tem
perature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sedimentat ion )
PAE-PCL-PEG-PCL-PAE (PEG 1.65k; PEA~1.25k)
Phase diagram of PAE PCL PEG PCL PAE
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pH (20wt%)
5.5 6.0 6.5 7.0 7.5
Te
mperature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sedimentat ion )
PAE-PCL-PEG-PCL-PAE (PEG1.5k; PCL/PEG~1.8)
Effect of PAE mol. wt
Phase diagram of PAE-PCL-PEG-PCL-PAE
β-amino ester 1.25kβ-amino ester 0.76k
β-amino ester 1.95kβ-amino ester 2.35k
β-amino ester 0k
Phase diagram of PAE PCL PEG PCL PAE
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PAE-PCL-PEG-PCL-PAE (PEG1.65k; PCL/PEG~1.8)
Effect of copolymer concentration (PEG 1650)
Phase diagram of PAE-PCL-PEG-PCL-PAE
pH
5.5 6.0 6.5 7.0 7.5
Temperature(oC)
0
10
20
30
40
50
60
Sol
Gel
Sol (Sedimentat ion ) Sol state at
pH 6.4, 10 °C
Gel state at
pH 7.4, 37 °C
Tem. & pH
So-Gel phenomena
20 wt%
25 wt%
30 wt%
Phase diagram of PAE PCL PEG PCL PAE
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At pH 6.6; 0oC and pH 7.4; 37oC
Phase diagram of PAE-PCL-PEG-PCL-PAE
PAE-PCL-PEG-PCL-PAE (PEG1.65k; PCL/PEG~1.8; PAE~1.25k)
C t t i it f PAE PCL PEG PCL PAE i i t t
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*Cell line: NIH 3T3 (fibroblast). *Growth medium: DMEM
(90% Dulbecco’s modified
Eagle’s medium, 10% fetal
calf serum, penicillin 100
units/mL, streptomycin
100 µg/mL).* XTT assay
(XTT :2,3-bis(2-methoxy-
4-nitro-5-susfophenyl)-
2H-tetrazolium-5-carbox
anilide)
* 96-well plates, incubator.Microplate reader.
Cytotoxicity of PAE-PCL-PEG-PCL-PAE in vivo test
PAE-PCL-PEG-PCL-PAE (PCL/PEG~1.5; PAE~1.25k)
0
20
40
60
80
100
120
0
20
40
6080
100120
PEIPEG1500
PEG1650PEG1750
R e l a t i v e c e l l v i a b i l i t y ( % )
P o l y m
e r C o n
c . ( µ g / m
l )
Time variation of degradation rate
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Time variation of degradation rate
Copolymers
as powder state
Dissolving in
PBS buffer at
2 oC
Adjusting
pH to 3.0-4.0 Insulin loading
12 hours Adjusting
pH to 7.4
1. Copolymers solutions were prepared by dissolving in PBS buffer (contain 2-4 v % of HCl 37 %) at 2℃
for 2 days.
2. pH of these solutions are adjusted by NaOH 5 M and HCl 5 M at 2℃ to 3.0-4.0.
3. Insulin was loaded in to the solution by mixing at 2℃ for 12 hours.
4. pH of the complex mixture then are adjusted to 7.4 to do in vitro release experiments, or 7.0 to do in vivo
experiments.
Processing of insulin loading
Materials
Triblock or Pentablock (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k)
Drug/protein loading
Mixture
Time variation of degradation rate
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Time variation of degradation rate
Degradation in vitro/vivo
Sampling Adding fresh
serum at 37oC Free drying Dissolving
in THF
GPC
In vitro
In vivo
Subcutaneous
injection
Surgery
Mixture
Mixture
Time variation of degradation rate
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pH (20wt% copolymer)5.5 6.0 6.5 7.0 7.5
Temperature(oC)
0
10
20
30
40
50
60
0 mg/ml insulin5 mg/ml insulin
10 mg/ml insulin
Sol (Sedimen tat ion)
Gel
so l
Time variation of degradation rate
Time (days)
0 10 20 30 40 50
M
olarcularweight(M
p)
0
2000
4000
6000
8000
PCL-PEG-PCLPAE-PCL-PEG-PCL-PAE
Complex Gel (5 mg/ml of Insulin in coplymer solution)
Triblock and Pentablock (PEG1.65k; PCL/PEG~1.8/1; PAE~1.25k)
copolymer solution (20%)
In vitro
Sol-gel transition with difference
Insulin loadedDegradation
Time variation of degradation rate
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Time variation of degradation rate
In vivo PAE-PCL-PEG-PCL-PAE (PEG1.65k; PCL/PEG~1.8/1; PAE~1.25k)
5 mg/ml insulin in copolymer solution (20%)
1 day 1 week 2 week
5 week 7 week
Insulin releasing in vitro
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Insulin releasing in vitro
Step 1
Step 2 Step 3
Sampling
method 1
Sampling
method 2
1. 0.5 ml of the complex mixture at pH 7.4 is placed in a 6 ml vial
2. The sample vials were incubate at 37 oC for 30 min, at 37 oC is added to the vial samples.
3. Sampling the insulin release to serum by two methods:
• Method 1: The amount of beginning fresh serum was 3 ml. At a given time, 1.5 ml of the serum in vials (releasi
ng sample) was extracted from the vial samples, and 1.5 ml of fresh serum was supplemented.
• Method 1: The amount of beginning fresh serum was 6 ml. At a given time, the releasing sample was 3 mle, and
fresh serum supplemented was 3 ml.
Mixture
Insulin releasing in vitro
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Insulin releasing in vitro
Time (days)0 10 20 30 40
Cum
ulativereleaseofinsulin(%)
0
20
40
60
80
100
PCL-PEG-PCL gel. Sampling method 1
Complex gel. Sampling method. 1Complex gel. Sampling method. 2
Triblock or Pentablock (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k)
5 mg/ml insulin in copolymer solution (20%)
Time (days)
0 10 20 30 40Insulinconcentrationinserum
(mg.ml-1)
0.0
0.2
0.4
0.6
0.8
1.0
PCL-PEG-PCL gel. Sampling method 1
Complex gel. Sampling method. 1
Complex gel. Sampling method. 2
Insulin releasing in vitro
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Insulin releasing in vitro
PAE-PCL-PEG-PCL-PAE (PEG 1.65k; PCL/PEG~1.8; PAE~1.25k)
5 mg/ml insulin in copolymer solution
Time (days)0 10 20 30 40
Cum
ulativereleaseofi
nsulin(%)
0
20
40
60
80
100
Complex gel 20wt%. Sampling method. 1
Complex gel 30wt%. Sampling method. 1
Time (days)0 10 20 30 40In
sulinc
oncentrationinserum
(mg.ml-1)
0.0
0.2
0.4
0.6
0.8
Complex gel 20wt%. Sampling method. 1
Complex gel 30wt%. Sampling method. 1
Mechanism of anionic protein loading and release
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PEG hydrophilic
Amino ester ionized
Amino ester de-ionized
hydrophobic
PCL hydrophobic
Insulin
Negative charge on Insulin
B
C
A. Copolymer solution and insulin
at 10oC and pH 5.0B. Complex gel of copolymer and
insulin at 37oC and pH 7.4
C. Insulin releasing depend on the
degradation of copolymer at 37oC
and pH 7.4
PAE-PCL-PEG-PCL-PAE at
low pH and low temperature
PAE-PCL-PEG-PCL-PAE at high
pH and high temperature
A
Mechanism of anionic protein loading and release
Insulin releasing in vitro
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Insulin releasing in vitro
Insulin releasing in vivo on SD rats
Tail cutting bloodSampling
Mixture pH 7.0and 10 oC
Subcutaneousinjection (200 µl/rat)
Insulin solution
0.25mg/ml
Injection
200 µl/rat
Tail cutting blood
Sampling
Centrifuging toget Serum
Centrifuging to
get Serum
Insulin essay
Insulin essay
Insulin releasing in vivo
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Insulin releasing in vivo
Insulin releasing in vivo on SD rats
Time (days)0 5 10 15 20 25
Insulininplasmaofblood(mU.l-1)
0
1000
2000
3000
4000
Insulin only
Insulin - PCL-PEG-PCL gel
Complex gel
Time (hours)0 10 20 30 40 50
Insulininplasmaofblood(m
U.l-1)
0
1000
2000
3000
4000
PAE-PCL-PEG-PCL-PAE (PEG 1.65k; PCL/PEG~1.8; PAE~1.25k)
5 mg/ml insulin in copolymer solution
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Treatment the Diabolical disease on DFR
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Treatment the Diabolical disease on DFR
DFR rats induced
STZ
solution
Intraperitioneal
injection to SD rats
Tail cutting blood sampling
DFR , Glucose essay
Complex mixture
pH 7.0 and 10 oC
Subcutaneous
injection
DFR induced from SD rats
1. Streptozotocin (STZ) injected: 60
mg.kg-1.
Treatment the Diabolical disease
1. PAE-PCL-PEG-PCL-PAE (PEG
1.65k; PCLA/PEG~1.8/1; PAE~
1,25k)
2. Copolymers solutionconcentration: 30 wt%.
3. Insulin in the complex mixture 1-
10 mg.ml-1
4. Complex mixture injected: 200μl.
5. Blood sampling from the rat tailvein.
Treatment the Diabetical disease on DFR
Centrifuging to
get Serum
Insulin essay
Tail cutting blood sampling
DFR, Glucose essay
Treatment the Diabolical disease on DFR
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Glucose concentration in blood of diabetic ratwith insulin-hydrogel complex
Time (days)-5 0 5 10 15 20G
lucoseconcentrationinblood(mg/dL)
0
100
200
300
400
500
600
700
Control
1 mg insulin/ml polymer solution (30 wt%)5 mg insulin/ml polymer solution (30 wt%)
10 mg insulin/ml polymer solution (30 wt%)
Complex gel injected
STZ injected
Treatment the Diabolical disease on DFR
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Body weight of diabetic ratwith insulin-hydrogel complex
Time (days)-5 0 5 10 15 20
o
yweg
tg
100
150
200
250
300
350
Control
1 mg insulin/ml polymer solution (30 wt%)5 mg insulin/ml polymer solution (30 wt%)
10 mg insulin/ml polymer solution (30 wt%)
Complex gel Injected
STZ injected
Treatment the Diabolical disease on DFR
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Insulin concentrationin blood of Diabetic rat
Time (days)0 5 10 15 20
In
sulininplasma
ofblood(mU/l)
0
50
100
150
200
250
Control
1 mg insulin/ml polymer solution (30 wt%)
5 mg insulin/ml polymer solution (30wt%)
10 mg insulin/ml polymer solution (30 wt%)
Complex gel Injected
Human growth hormone (Hgh) releasing in vitro
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pH
5.5 6.0 6.5 7.0 7.5
Temperature(oC)
0
10
20
30
40
50
600 mg HGH
10 mg HGH
20 mg HGH
g ( g ) g
Time (days)0 2 4 6 8 10 12 14 16 18
Cumula
tiveReleaseofHGH
(%)
0
20
40
60
80
100
Sol (Sedimentat ion )
Gel
Sol
Triblock and Pentablock (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k)
copolymer solution (20%)
Sol-gel transition with difference
Hgh loadedHgh releasing
Keeping condition of PAE-PCL-PEG-PCL-PAE
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p g
• Degradation of copolymer during keeping time
Time (day)
0 20 40 60 80 100 120 140 160 180
Molecularweight(Mp)
2000
3000
4000
5000
6000
7000
8000
PAE-PCL-PEG-PCL-PAE (PEG 1.5k; PCL/PEG~1.8/1; PEA~1.25)
keeping at 0oC as a solution stage pH 6.6
keeping at 0oC as a powder stage
Part II Conclusion
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• PAE-PCL-PEG-PCL-PAE penta-block copolymers of varying PEG molecular weight,
PCL/PEG ratios, and PAE molecular weight were successfully synthesized.
• The gel phase regions of PAE-PCL-PEG-PCL-PAE can be controlled;
- PEG molecular weight
- PCL/PEG ratio and the concentration
- PAE molecular weight
• Temperature & pH dependence of PAE-PCL-PEG-PCL-PAE pentablock copolymer ;
- Gel at pH 7.4 & 37℃
- Sol at pH 6.4 & 4℃
• PAE plays as a duo-functional group:
- pH sensitive moiety.
- Encapsulation anionic drug/protein by forming ionic complex unit.
• The dominant factor of anionic drug/protein release is the degradation of PAE block
copolymer.
Part II Conclusion
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• The releasing of anionic drug/protein can be controlled by:
- The drug/protein formulations.
- The copolymer concentration.
• This polymer can be kept as a powder stage at 0℃ for more than 7 months without
degradation.
PAE-PCL-PEG-PCL-PAE is a novel pH/temperature sensitive hydrogen. It could
be applied for a subcutaneous injectable drug/protein delivery system
Part III
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Cationic pH/temperature sensitive hydrogen PAE-PCLA-PEG-PCLA-PAE
Part III Controlled the degradation of cationic hydrogels:
PAE-PCLA-PEG-PCLA-PAE1. Synthesis of PAE-PCLA-PEG-PCLA-PAE
2. Sol-gel transition
3. Degradation-In vitro drug/protein delivery test
pH
T e m p
e r a t u r e ( o C )
7.4
37
Sol
Gel
Sol (Sediment at ion)
Human body cond i t ion
Synthesis of PAE-PCLA-PEG-PCLA-PAE
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ε-caprolactone D,L-Lactide
Sn(Oct)2 130oC
PCLA-PEG-PCLA
PEG
+ O
O
+ O
O
O
O
C H 3
H 3 C
HO
H2
C
H2
C O Hn
H2
C
H2
C O C
n
OHC O C
H2
C
H2
C
CH3
OH2
C
H2
C
H2
C O
x
H
y
C
O
C
H
OC
H2
C
H2
C
CH3
O
H2
C
H2
C
H2
CO x
H O
y
PCLA-PEG-PCLA triblock polymer
Synthesis of PAE-PCLA-PEG-PCLA-PAE
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PCLA-PEG-PCLA –diacrylate
Cl C
O
CH
CH2
+
Acrylation in
Chloroform
PAE-PCLA-PEG-PCLA-PAE
NHHN+
OO
O
O
+ 50oC
10oC
4,4’ trimethylene dipiperidine 1,4 – Butane diol diacrylate
PAE-PCLA-PEG-PCLA-PAE
HO PCLA PEG PCLA OH O PCLA PEG PCLA O C CH
CH2
O
CHCH2C
O
O PCLA PEG PCLA O C CH
CH2
O
CHCH2C
O
NNO
OO
O
O
O
N NO
O O
OO
n PCLA PEG PCLA On
DCM
Acryloyl chlorideTriblock copolymer
Characterization of PAE-PCLA-PEG-PCLA-PAE
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O
H2
C
H2
C O
H2
C
H2
C O
H2
C
H2
C O C CH
n-2
O
CH3
O C
H2
C
O
H2
C
H2
C
H2
C
H2
C O C
O
CH
C
H
Hx y
A A A'A'B B C DE F1 F2
F3
2.003.004.005.006.00ppm(f1)
CLA, A’B, D
E a
5.006.00
E
E
F1 F2 F3
a
b
Acrylated
PEG
LA C
CL
Characterization of PAE-PCLA-PEG-PCLA-PAE
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5.010.0ppm
NN
OO
O
O
O
O
nm PCLA PEG PCLA PAE
PEG
LA
CLCL
A A A
A
B B
B
C C
C
D
E, F
E EF FGG
CLG
HH
H
I
I I
CL
Characterization of PAE-PCLA-PEG-PCLA-PAE
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Elution time
RI
ResponseSignal
Triblock (Controlled)
Pentablock-PAE ~1.3K
Pentablock-PAE ~2.0K
Pentablock-PAE ~2.5K
PEG 1.5k; PEG/PCLA=2.5
Characterization of PAE-PCLA-PEG-PCLA-PAE
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PCLA-PEG-PCLA
( Mn a )
PEG/PCLA
(wt ratio)
PEG
(Mn)PAE-PCLA-PEG-PCLA-PAE b PAE-PCLA-PEG-PCLA-PAE c D
1530-1500-1530 1/2.04 1500 2000-1530-1500-1530-2000 1335-1530-1500-1530-1335 1.40
1636-1500-1636 1/2.2 1500 2000-1636-1500-1636-2000 1383-1636-1500-1636-1383 1.43
1885-1500-1885 1/2.5 1500 2000-1885-1500-1885-2000 1340-1885-1500-1885-1340 1.44
1885-1500-1885 1/2.5 1500 1000-1885-1500-1885-1000 820-1885-1500-1885-820 1.32
1885-1500-1885 1/2.5 1500 2500-1885-1500-1885-2500 1764-1885-1500-1885-1764 1.45
1885-1500-1885 1/2.5 1500 3000-1885-1500-1885-3000 2018-1885-1500-1885-2018 1.48
1885-1500-1885 1/2.5 1500 4000-1885-1500-1885-4000 2566-1885-1500-1885-2566 1.52
1400-1750-1400 1/1.6 1750 2000-1405-1750-1405-2000 1351-1405-1750-1405-1351 1.38
1583-1750-1583 1/1.8 1750 2000-1583-1750-1583-2000 1310-1583-1750-1583-1310 1.44
1/1.8 1750 3000-1583-1750-1583-3000 1998-1583-1750-1583-1998 1.48
1726-1750-1726 1/2.0 1750 2000-1726-1750-1726-2000 1297-1726-1750-1726-1297 1.40
2050-2000-2050 1/2.5 2000 2000-2050-2000-2050-2000 1299-2050-2000-2050-1299 1.42
a Number-average molecular weight calculated from 1H-NMR
b Number-average molecular weight (ratio of reaction)
c Number-average molecular weight calculated from GPC
Sol-gel transition of PAE-PCLA-PEG-PCLA-PAE
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Temperature (oC)
0 10 20 30 40 50 60 70
pH
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
PEG 1.75k PEG 1.5k
pH depending on temperature
PAE-PCLA-PEG-PCLA-PAE (PCLA/PEG~2.0/1; PEA~1.3k)
Sol-gel transition
PEG 1.75k
PEG 1.5k
pH
6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8
Te
mperature(oC)
0
10
20
30
40
50
60
Eng. phase diagram PEG1500
Real phase diagram PEG1500
Eng. phase diagram PEG1750
Real phase diagram PEG1750
So l
Gel
Sol (Sediment at ion)
Phase diagram of PAE-PCLA-PEG-PCLA-PAE
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PAE-PCLA-PEG-PCLA-PAE (PCLA/PEG~2.0/1; PEA~1.3k)
Concentration (wt% at pH 7.4)
5 10 15 20 25 30 35
Tem
perature(oC)
0
10
20
30
40
50
60
PEG 1500
PEG 1750
PEG 2000
So l
Gel
Sol (Sediment at ion)
pH (20wt%)6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6
Te
mperature(oC)
0
10
20
30
40
50
60
PEG 1500
PEG 1750
PEG 2000
Sol
Gel
Sol (Sedimentat ion )
PEG 1.75k PEG 1.5k
PEG 2.00k
Effect of PEG mol. wt
Phase diagram of PAE-PCLA-PEG-PCLA-PAE
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Concentration (wt% at pH 7.4)5 10 15 20 25 30 35
Temperature(oC)
0
10
20
30
40
50
60
PCLA/PEG = 1.6
PCLA/PEG = 1.8
PCLA/PEG = 2.0
So l
Gel
Sol (Sedimentat ion )
PCLA/PEG ~1.8/1 PCLA/PEG ~1.6/1
PCLA/PEG ~2.0/1
PAE-PCLA-PEG-PCLA-PAE (PEG1.75k; PEA~1.3k)
Effect of PCLA/PEG ratios, PEG 1750
pH (20 wt%)
6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6
Temperature(oC)
0
10
20
30
40
50
60
PCLA/PEG = 1.6
PCLA/PEG = 1.8
PCLA/PEG = 2.0
So l
Gel
Sol (Sedimentat ion )
Phase diagram of PAE-PCLA-PEG-PCLA-PAE
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Concentration (wt% at pH 7.4)
0 5 10 15 20 25 30 35
T
emperature(oC)
0
10
20
30
40
50
60
PCLA/PEG = 2.0/1
PCLA/PEG = 2.2/1
PCLA/PEG = 2.5/1
So l
Gel
Sol (Sedimentat ion )
PAE-PCLA-PEG-PCLA-PAE (PEG1.5k; PEA~1.3k)
Effect of PCLA/PEG ratios, PEG 1500
pH (20 wt%)
6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6
T
emperature(oC)
0
10
20
30
40
50
60
PCLA/PEG = 2.0/1
PCLA/PEG = 2.2/1
PCLA/PEG = 2.5/1
So l
Gel
Sol (Sedimentat ion )
Phase diagram of PAE-PCLA-PEG-PCLA-PAE
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pH ( 20 wt%)
5.5 6.0 6.5 7.0 7.5
Te
mperature(oC)
0
10
20
30
40
50
60
Sol
Sol (Sedimentat ion )
Gel
PAE-PCLA-PEG-PCLA-PAE (PEG1.5k; PCLA/PEG~2.5/1)
Effect of PAE mol.wt
β-amino ester 0.8k
β-amino ester 1.3k
β-amino ester 2.0k
β-amino ester 2.55k
β-amino ester 0k
Phase diagram of PAE-PCLA-PEG-PCLA-PAE
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Effect of copolymer concentration (PEG 1500)
pH5.5 6.0 6.5 7.0 7.5
T
emperature(o C
)
0
10
20
30
40
50
60
20%
25%
30%
PAE-PCLA-PEG-PCLA-PAE (PEG1.5k; PCLA/PEG~2.5/1; PAE~1.3k)
Sol (Sedimentat ion )
Sol
Gel
Sol-gel transition of PAE-PCLA-PEG-PCLA-PAE
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PAE-PCLA-PEG-PCLA-PAE (PEG1.5k; PCLA/PEG~2.5/1; PAE~1.3k)
At pH 6.6; 0oC and pH 7.4; 37oC
Cytotoxicity of PAE-PCLA-PEG-PCLA-PAE in vivo test
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0
20
40
60
80
100
120
0
20
40
6080
100120
PEIPEG 1750
R e l a t i v e c e l l v i a b i l i t
y ( % )
P o l y m
e r C o n
c . ( µ g
/ m l )
*Cell line: NIH 3T3 (fibroblast).
*Growth medium: DMEM
(90% Dulbecco’s modified
Eagle’s medium, 10% fetal
calf serum, penicillin 100
units/mL, streptomycin
100 µg/mL).
* XTT assay
(XTT :2,3-bis(2-methoxy-
4-nitro-5-susfophenyl)-
2H-tetrazolium-5-carbox
anilide)
* 96-well plates, incubator.Microplate reader.
PAE-PCLA-PEG-PCLA-PAE (PEG1.75k;PCLA/PEG~2.0/1;PEA~1.3k)
Time variation of degradation rate
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Copolymers
as powder state
Dissolving in
PBS buffer at
2 oC
Adjusting
pH to 3.0-4.0 Insulin loading
12 hours Adjusting
pH to 7.4
1. Copolymers solutions were prepared by dissolving in PBS buffer (contain 2-4 v % of HCl 37 %) at 2℃
for 2 days.
2. pH of these solutions are adjusted by NaOH 5 M and HCl 5 M at 2℃ to 3.0-4.0.
3. Insulin was loaded in to the solution by mixing at 2℃ for 12 hours.
4. pH of the complex mixture then are adjusted to 7.4 to do in vitro release experiments, or 7.0 to do in vivo
experiments.
Processing of insulin loading
Materials
Triblock or Pentablock (PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k)
Drug/protein loading
Mixture
Time variation of degradation rate
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Degradation in vitro/vivo
Sampling Adding fresh
serum at 37oC Free drying Dissolving
in THF
GPC
In vitro
In vivo
Subcutaneous
injection
Surgery
Mixture
Mixture
Time variation of degradation rate
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In vitro
Time (days)
0 10 20 30 40 50
Molec
ularweight(Mp)
0
2000
4000
6000
8000
PCLA-PEG-PCLA
PAE-PCLA-PEG-PCLA-PAE
Complex Gel (5 mg/ml of Insulin in coplymer solution)
Degradation of PAE-PCLA-PEG-PCLA-PAE
(PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k) copolymer solution (20%)
pH (20 wt% copolymer)
6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6
Tem
perature(oC)
0
10
20
30
40
50
60
0 mg/ml insulin
5 mg/ml insulin
10 mg/ml insulin
Sol (Sedimentat ion )
Gel
Sol
Sol-gel transition with difference
Insulin loadedDegradation
Controlled of the degradation
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Time (days)0 10 20 30 40 50
Molecularweight(Mp)
2000
3000
4000
5000
6000
7000
8000
Pentablock gel 1
Complex Gel 1
Pentablock gel 2
Pentablock gel 2
Time (days)0 10 20 30 40 50
Molecularweight
(Mp)
3000
3500
4000
4500
5000
5500 Triblock gel 1
Triblock gel 2
In vitro
Group 2: PAE-PCL-PEG-PCL-PAE
(PEG 1.65k; PCL/PEG~1.8;PEA~1.25k)
Triblock gel:PCL-PEG-PCL
pentablock gel: PAE-PCL-PEG-PCL-PAE
Complex gel: 5mg/ml insulin in pentablock
solution
Group 1: PAE-PCLA-PEG-PCLA-PAE
(PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k)
Triblock gel:PCLA-PEG-PCLA
pentablock gel: PAE-PCLA-PEG-PCLA-PAE
Complex gel: 5mg/ml insulin in pentablock
solution
Time variation of degradation rate
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In vivo
1 day 1 week 2 week
3 week 5 week
PAE-PCLA-PEG-PCLA-PAE (PEG1.5k;PCLA/PEG~2.5/1; PEA~1.3k)
Insulin releasing in vitro
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Step 1
Step 2 Step 3
Sampling
method 1
Samplingmethod 2
1. 0.5 ml of the complex mixture at pH 7.4 is placed in a 6 ml vial
2. The sample vials were incubate at 37 oC for 30 min, at 37 oC is added to the vial samples.
3. Sampling the insulin release to serum by two methods:
• Method 1: The amount of beginning fresh serum was 3 ml. At a given time, 1.5 ml of the serum in vials (releasi
ng sample) was extracted from the vial samples, and 1.5 ml of fresh serum was supplemented.
• Method 1: The amount of beginning fresh serum was 6 ml. At a given time, the releasing sample was 3 mle, and
fresh serum supplemented was 3 ml.
Mixture
Insulin releasing in vitro
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PAE-PCLA-PEG-PCLA-PAE (PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k)
Time (days)
0 10 20 30 40
Cumulativ
eReleaseofInsulin(%)
0
20
40
60
80
100
Sampling method 1
Sampling method 2
5 mg/ml Insulin in complex gel (20wt% copolymer)
Insulin release in vitro
Sampling method 1 ( 20 wt% copolymer)
Time (days)0 10 20 30 40
Cumulativ
eReleaseofInsul
in(%)
0
20
40
60
80
100
5 mg/ml insulin in complex gel
10 mg/ml insulin in complex gel
Insulin loading and releasing in vitro
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Insulin release in vitro- Controlled the release by the degradation
5 mg/ml Insulin in complex gel (20wt%)- sampling method 1
Time (days)0 10 20 30 40
CumulativeR
eleaseofInsulin(%)
0
20
40
60
80
100
Complex gel 1
Complex gel 2
Group 2: PAE-PCL-PEG-PCL-PAE
(PEG 1.65k; PCL/PEG~1.8;PEA~1.25k)
Triblock gel:PCL-PEG-PCL
pentablock gel: PAE-PCL-PEG-PCL-PAE
Complex gel: 5mg/ml insulin in pentablock
solution
Group 1: PAE-PCLA-PEG-PCLA-PAE
(PEG 1.5k; PCLA/PEG~2.5;PEA~1.3k)
Triblock gel:PCLA-PEG-PCLA
pentablock gel: PAE-PCLA-PEG-PCLA-PAE
Complex gel: 5mg/ml insulin in pentablock
solution
Storage stability
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Time (day)0 20 40 60 80 100 120 140 160 180
Mo
lecularweight(M
p)
4000
5000
6000
7000
8000
9000
PAE-PCLA-PEG-PCLA-PAE, (PCLA/PEG~2.5/1; PEG= 1750) PAE~ 1300
Keeping at 0oC as a solution stage pH 6.6
Keeping at 0oC as a powder stage
Part III Summary
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• PAE-PCLA-PEG-PCLA-PAE penta-block copolymers of varying PEG molecular weight,
PCLA/PEG ratios, and PAE molecular weight were successfully synthesized.
• The gel phase regions of PAE-PCLA-PEG-PCLA-PAE can be controlled
- PEG molecular weight
- PCLA/PEG ratio and the concentration
- PAE molecular weight
• Temperature & pH dependence of PAE-PCLA-PEG-PCLA-PAE pentablock copolymer ;
- Gel at pH 7.4 & 37℃
- Sol at pH 6.4 & 4℃
• The releasing of anionic drug/protein can be controlled by:
- The drug/protein formulations.
- The copolymer concentration.
• The dominant factor of anionic drug/protein release is the degradation of PAE block
copolymer.
• This polymer can be kept as a powder stage at 0℃ for more than 7 months without
degradation.
Conclusion
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• Novel pH/temperature sensitive hydrogel based on acidic and basic pH
sensitive moiety were successfully synthesized.
• The gel phase regions of these new hydrogel can be controlled by1. PEG molecular weight
2. Ratio of hydrophobic/hydrophilic and the concentration
3. The molecular weight of pH sensitive moiety block.
• Temperature & pH dependence of PAE-PCLA-PEG-PCLA-PAE pentablock
copolymer ;1. Gel at pH 7.4 & 37℃
2. Sol at different human condition.
• The degradation of these hydrogel can be controlled by the biocompartibility
property of each block polymer
• These new pH sensitive moieties play as a duo-functional block:
1. pH Sensitive moiety
2. Encapsulation ion drug/protein by established the ionic complex unit
between protein and these block copolymer.
Conclusion
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• The dominant factor of drug/protein release is the degradation of hydro gels.
• The releasing of drug/protein can be controlled by:
1. The drug/protein formulations.
2. Copolymer concentration.
3. The degradation ration of hydrogel.
• These materials can be stored as a powder staga at 0OC for more than 6
months without degradation.
These novel materials could be applied for a subcutaneous
injectable drug/protein delivery system