Polymer Sinh Hoc Va y Sinh 1

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




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




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




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




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)




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


http://slidepdf.com/reader/full/polymer-sinh-hoc-va-y-sinh-1 7/99Sungkyunkwan University 7

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



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



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


Prof. Yoo Han Bae Group

Prof. Doo Sung Lee Group

Screening works

Screening works of pH sensitive moiety



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


Human body

cond i t ion



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


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



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



ionizationdeionization

As pH sensitive moiety is ionized, polymer solution will be a sol state.

pKa

A-B-A

pH-sensitive block pH-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



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



[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]


Oligo-sulfamethazine (OSM)

OSM



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


OSM-PCGA-PEG-PCGA-OSM pentablock copolymer



Characterization of OSM-PCGA-PEG-PCGA-OSM

PEG

CLGA

1H-NMR of PCGA-PEG-PCGA triblock copolymer





Sulfamethazine

1H-NMR of Sulfamethazine & Sulfamethazine monomer(SM)

Sulfamethazinemonomer(SM)




OSM

1H-NMR of Sulfamethazine monomer(SM) and OSM


SM




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





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


Mol. wt & MWD




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)


Gel

Sol

Sol (Sedimen tation)

0 5 10 15 20 25

0

10

20

30

40

50

60

T


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




Effect of OSM mol.wt

:(PEG/PCGA=1/2.15); PEG.1750; OSM.806

: (PEG/PCGA=1/2.15); PEG.1750; OSM.904

T


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





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


0 5 10 15 20 25

0

10

20

30

40

50

60


Concentration (wt%)

(pH 7.4)

Gel


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%)





(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

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


pH

(C: 20 wt%)

Gel


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%)


Ti i ti f l l t i ft i j ti




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




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




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




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




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





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




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


pH


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




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




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)

+++

+

+

++

++

+




• 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




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



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




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




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




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





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


NNO

OO

O

O

O

N NO

OO

OOn PCL PEG PCL O

n





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


Characterization of PAE PCL PEG PCL PAE




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






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






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






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




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


PEG 1.5k

Solid: Real diagram

Dash: Engineering diagram

PEG 1.65k PEG 1.75k

Phase diagram of PAE PCLA PEG PCLA PAE




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

Gel


PEG 1.5k

PEG 1.75k

PEG 1.65k

Phase diagram of PAE PCL PEG PCL PAE




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


pH (20wt%)

5.5 6.0 6.5 7.0 7.5

Te

mperature(oC)

0

10

20

30

40

50

60

Sol

Gel


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)





Effect of PCL/PEG ratios (PEG 1650, PAE~1.25k)


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


Concentration (wt%) (pH 7.4)

5 10 15 20 25 30

Tem

perature(oC)

0

10

20

30

40

50

60

Sol

Gel


PAE-PCL-PEG-PCL-PAE (PEG 1.65k; PEA~1.25k)





pH (20wt%)

5.5 6.0 6.5 7.0 7.5

Te

mperature(oC)

0

10

20

30

40

50

60

Sol

Gel


PAE-PCL-PEG-PCL-PAE (PEG1.5k; PCL/PEG~1.8)

Effect of PAE mol. wt


β-amino ester 1.25kβ-amino ester 0.76k

β-amino ester 1.95kβ-amino ester 2.35k

β-amino ester 0k





PAE-PCL-PEG-PCL-PAE (PEG1.65k; PCL/PEG~1.8)

Effect of copolymer concentration (PEG 1650)


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%





At pH 6.6; 0oC and pH 7.4; 37oC


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




*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 )






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






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





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 (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






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





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






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






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


Time (days)0 10 20 30 40In

sulinc

oncentrationinserum

(mg.ml-1)

0.0

0.2

0.4

0.6

0.8



Mechanism of anionic protein loading and release




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 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




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



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





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





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%)


Complex gel Injected

STZ injected





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


5 mg insulin/ml polymer solution (30wt%)


Complex gel Injected

Human growth hormone (Hgh) releasing in vitro




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


Gel

Sol

Triblock and Pentablock (PEG 1.65k; PCL/PEG~1.8/1; PAE~1.25k)

copolymer solution (20%)


Hgh loadedHgh releasing

Keeping condition of PAE-PCL-PEG-PCL-PAE




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




• 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;


- 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




• 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




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



pH

T e m p

e r a t u r e ( o C )

7.4

37

Sol

Gel


Human body cond i t ion

Synthesis of PAE-PCLA-PEG-PCLA-PAE




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




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


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




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





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





Elution time

RI

ResponseSignal

Triblock (Controlled)

Pentablock-PAE ~1.3K



PEG 1.5k; PEG/PCLA=2.5





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




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






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


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


PEG 1.75k PEG 1.5k

PEG 2.00k

Effect of PEG mol. wt





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


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






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


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






pH ( 20 wt%)

5.5 6.0 6.5 7.0 7.5

Te

mperature(oC)

0

10

20

30

40

50

60

Sol


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





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

Gel

Sol-gel transition of PAE-PCLA-PEG-PCLA-PAE




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




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)





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





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





In vitro

Time (days)

0 10 20 30 40 50

Molec

ularweight(Mp)

0

2000

4000

6000

8000

PCLA-PEG-PCLA


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


Gel

Sol


Insulin loadedDegradation

Controlled of the degradation




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


solution





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)





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





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




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


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


solution

Storage stability




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 powder stage

Part III Summary




• 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


- 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




• 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



• 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

Polymer Sinh Hoc Va y Sinh 1

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