Nanostructured Macromonomer Based Hydrogels Designed for Biomedical Applications

P.J. Lutz

Institut Charles Sadron, CNRS, UPR22, F-67083 Strasbourg Phone: 0033388414074, Facsimile: 0033388414099, e-mail: lutz@ics.u-strasbg.fr

Poly(ethylene oxide), (PEO) is an hydrophilic polymer which exists in various forms and structures1 and exhibits specific solution and solid state properties. Furthermore, the remarkable biocompatible properties of this polymer have already led to a wide number of biomedical applications. In particular, hydrogels based on PEO have been shown to be suitable materials for numerous applications. Among the different approaches to design such hydrogels, the homopolymerization of well-defined bifunctional PEO macromonomers2 represents a promising one. In such systems, crosslinking is achieved upon free radical polymerization of the methyl methacrylate units located at both chain ends.

The first part of the present work discusses the advantages of the free radical homopolymerization of α,ω−methacryloyloxy PEO macromonomers with respect to classical end-linking procedures. Gels were synthesized over a large range of molar masses of the macromonomer precursor. They were investigated swollen to equilibrium in THF or in water. The mechanical properties of the networks obtained in water are largely better than those of networks obtained in organic solvents under exactly the same conditions. This can be explained by the preferential formation in water of micellar structures containing high concentrations of polymerizable methyl methacrylate units. These results are self-consistent with the lower amount of extractable materials found for networks synthesized in water.2,3 The influence of the presence of an hydrophobic comonomer on the properties of the networks was also studied.4 The approach was extended to degradable PEO networks containing poly(1,3-dioxolane) segments.5

Poly(ethylene oxide) (PEO) hydrogels were also prepared by free radical polymerization in water, of multi methyl methacrylate substituted PEO star polymers.6 The PEO multiarm star polymers emanating from a hydrophilic hyperbranched poly(glycerol-b-propyleneoxide) core were prepared as previously reported. The weight swelling degrees in water and THF, uniaxial compression modulus and amount of extractable materials were measured. Strong aggregation of the hydrophobic methyl methacrylate moieties in water leads to an enhancement of the reaction rate and yield. Polymerization of multiarm star polymers partially substituted with polymerizable entities in the presence of linear macromonomers provides access to hydrogels still containing free hydroxyl groups. Incorporation of 10 wt.- % of PEO multiarm star polymer enhances weight swelling degree but does not affect the mechanical properties of the material with respect to pure linear macromonomer hydrogels. The dependence of the mechanical properties as well as weight swelling degrees upon the crosslinking concentrations were also examined.

Due to the uncontrolled polymerization process, the resulting hydrogels are rather heterogeneous which affects their physico-chemical properties. Recent developments in Atom Transfer Radical Polymerization (ATRP) made it possible to control the polymerization of methyl methacrylate to obtain polymers with controlled molar mass and narrow molar mass distributions. In addition, the presence of water has been shown to have no deleterious effects on the ATRP process: Matyjaswewski et al7 were able to control the polymerization of 2-hydroxyethyl acrylate in water at 90°C. Recently, Zhu et al8 reported on the ATRP of poly(ethylene glycol) dimethacrylates. Various bromide-based water dispersable macroinitiators were used in association with a transition metal complex CuBr / bipyridine catalyst to homopolymerize in water bifunctional poly(ethylene

oxide) macromonomers to hydrogels. Their properties were compared to those of networks resulting from classical free radical polymerization process9.

The third part of the present work is devoted to the application of these hydrogels as semi-permeable membranes or as a support for the growth of nervous cells. The immunoisolation of transplanted pancreatic islets of Langerhans, responsible for the insulin secretion in the body, represents a promising approach for treating the hormone deficiencies due to diabetes. Immunoisolation implies the presence of physical protection / isolation of the islets of Langerhans from immunological attack i.e. the presence of a membrane. This membrane has yet to be permeable to glucose and insulin. Therefore such a membrane has to fulfill several requirements: controlled pore size, biocompatibility, non-biodegradability and good mechanical stability.

Series of hydrogels were prepared as described previously.2 They were first tested in vitro for fibroblasts adsorption. They were also subcutaneously and intraperitonealy implanted in rats (during one year), and a section of the vena cava of a pig was replaced by an artificial vascular tubular implant based on PEO hydrogels. In vitro experiment results show that only few amounts of fibroblasts were adsorbed on the surface, and the spherical form of the adsorbed cells evidenced the weak interaction with the surface. Moreover these PEO hydrogels have not induced thrombotic or inflammation reactions even after long periods of implantation in direct contact with blood. The glucose diffusion properties of these membranes were studied by a lag time analysis method. As expected, glucose diffuses through the membranes. The diffusion coefficients were compared to free diffusion of the solute in water. Insulin diffusion was also studied. Higher values of diffusion coefficients correspond to longer PEO precursor chains.3 These properties were compared to those of PEO hydrogels grafted onto surfaces.

PEO hydrogels were also tested with respect to their ability to serve as a template for the survival and the growth of hepatoctytes.10 Two systems were considered : either the surface of pre-existing hydrogels, with selected structural parameters, were seeded with isolated rat hepatocytes or the hepatocytes were dispersed in physiological medium containing the macromonomer and the initiator, and heated to 37°C. In the first case, cells were examined at given times after spreading over two days. The results were compared to those observed for the dispersion of fibroblasts onto a surface of the same type of hydrogels. The effects of the structure of the surface of the hydrogels and its chemical nature on the extent of hepatocyte attachment and the morphology were investigated

These networks were also examined regarding their capacity to serve as a template for cell growth. Best results have been obtained with samples characterized by high equilibrium swelling degrees. Moreover, PEO hydrogels seem to give the opportunity to some neurons to extend neuritic growth processes without glial support.11

The author wishes to express his acknowledgments to all his colleagues and coworkers who have contributed to various topics dealing with macromonomer based hydrogels. References (1) Lutz, P.J. Macromol. Symp. 2001, 164, 277. (2) Schmitt, B.; Alexandre, E.; Boudjema, K.; Lutz, P.J. Macromol. Symp. 1995, 93, 117. (3) Schmitt, B.; Alexandre, E.; Boudjema, K.; Lutz, P.J. Macromol. Biosci. 2002, 2, 341. (4) Carrot, G.; Schmitt, B.; Lutz, P.J. Polym. Bull. 1998, 40, 181. (5) Naraghi, K.; Sahli, N.; Belbachir, M.; Franta, E.; Lutz, P.J. Polymer International 2002, 51, 912. (6) Knischka, R.; Lutz, P.J.; Sunder, A.; Frey, H. Polymeric Materials: Science & Engineering 2001, 84, 945. (7) Cosa, S.; Jasieczek, B.; Beers, K.L.; Matyjaswewski, K. J. Polym. Sci., Part A Polym. Chem. 1998, 36, 1417. (8) Yu, Q.; Zeng, F.;. Zhu, S. Macromolecules 2001, 34, 1612. (9) Buathong, S.; Peruch, F.; Isel, F.; Lutz P.J. in preparation. (10) Alexandre, E.; Cinqualbre, J.; Jaeck, D.; Richert, L.; Lutz, P.J. to be published in Macromol. Symp. (11) Naraghi, K.; Soussand, J.; Félix, J.M.; Schimchowitsch, S.; Lutz, P.J. Polym. Prep., Am. Chem. Soc. Div. Polym.

Chem. Boston, (USA). 1998, 39(2), 196.

Nanostructured Macromonomer Based Hydrogels

Designed for Biomedical Applications

International Workshop 5th Gel Symposium

Kashiwa 2003 Japan

Pierre J. Lutz

Institut Charles Sadron, UPR 22, CNRS, 6, Rue Boussingault, F-67083 Strasbourg

• Biocompatible Materials : not rejected once implanted,no degradation, no release of degradation products : PEO

• Materials of Controlled Pore Size Designed as Semi-Permeable Membrane, or as Template for the Growth of Cells

• Encapsulation of biological active Materials during crosslinking !

• Synthesis in Water, via simple polymerization process of Macromonomers based Hydrogels (water swellable materials)

• Study of Physico-chemical Properties : controlled structural parameters (Compatible with applications)

Aim

• General Remarks of PEO

• Poly(ethylene oxide) hydrogels : Macromonomer ApproachSynthesis and characterization

• Some Biomedical Applications of Macromonomer based PEO Hydrogels

Outline

Conclusion and some perspectives

General Remarks of PEO

POLY(ETHYLENE OXIDE)General Behavior

HO-CH2-CH2(O-CH2-CH2)n-O-CH2-CH2-OHPEG from 10 to 1000 ?

• A non-ionic water-soluble polymer (soluble without limits)• Exhibits a LCST in water• Solvates metal ions Li+, Na+, K+, Poor man ’s crown ether• Is soluble in various organic solvents (not in alcanes and ether)• Forms complexes with organic solvents at low temperature • (THF, acetone)• Exhibits a good chemical stability (not in strong acids)• Is crystalline when unsolvated Melting point 66°C• Forms spherolithes (high crystallization rates)

JC. Wittmann,ICS

POLY(ETHYLENE OXIDE)HO-CH2-CH2(O-CH2-CH2)n-O-CH2-CH2-OH

Biocompatibility : NON THROMBOGENIC MATERIALThe material of reference for biomedical applications

WHY : no capacity to bind biopolymers Therefore no cellular elements (red cells, platelets white cells)

PEO base line with reference to a adsorption

Some specific polymers bounded

to the hydroxyl end

Is soluble in blood, can not be used directly,

has to be CROSSLINKED OR GRAFTED ONTO SURFACES

POLY(ETHYLENE OXIDE) based Structures

SYNTHESIS OF THE PEO MACROMONOMERS

H3CO CH2CH2O H H CPh

PhK

CCH3

CO

ClH2CH3CO CH2CH2O CH2CH2O K

H3CO CH2CH2O CH2CH2O

H3CO CH2CH2O C CO

CH3

CH2

Kn

+n-1

nSB9 et SB8

n-1+

Mn= 2000

35°C

35°C4h

Monofunctional

Bifunctional

CHARACTERIZATION OF THE PEO MACROMONOMERS

-Molar Mass, SEC on line LS (determination of coupling ? )

-Functionality - Chemical Titration

-1H NMR

- Maldi-TOF

83,2%615010

89,7%106007

64,6%61506

95%61505

67%21004

103%206003

96,9%106002

89,7%61501

Funct.yield(UV)

Mn Macro.(g.mol-1)

Ref.

Bifunctional PEO Macromonomer

(CH2CH2OCH2O)n OHHO

K

(CH2CH2OCH2O)n O-K+K+O-

(CH2CH2O)m(CH2CH2OCH2O)n(CH2CH2O)m O-K+K+O-

OCl

(CH2CH2O)m(CH2CH2OCH2O)n(CH2CH2O)m

O

O

O

Cationic polymerizationof 1,3-Dioxolane

HOCH2CH2OHCF3SO3H

Anionic Polymerizationof oxirane

MacromonomerSynthesis

PEO or PEO-b-PDXL-b-PEO MACROMONOMER SYNTHESIS

CH2CCH3

COCH2CH2OCH3

OATRP CH2... C

C

CH3

OOCH2CH2OCH3

CH2 CCH3

CH2 CCH3

...C COCH2CH2OCH3

OCH2CH2OCH3

O O

n n n n

-Classical Free Radical PolymerizationWater has to preferred to organic solvents higher yieldsPolymerization in nano-structured medium (Ito et al)Control of molar mass and molar distribution ?

-Anionic Homopolymerization difficult (low temp, crystallization )

HOMOPOYMERIZATION OF MONOFUNCTIONAL MACROMONOMERS

Elution volume

RI macro

polymacro

SEC Diagram

ARTP- Possible in water (organic solvents ?)- Rapid- Sharp molar mass distribution- DP values around 20

+ A

+ B

Various Multifunctional IniatiatorsLiving poly(divinylbenzene) coresLiving poly(diisopropenylbenzene) cores

Hydrophobic Coremore or less Polydisperse

Other Initiators

Tris-alkoxidesModified Carbosilane dendrimers

Polyglycerol cores

Bifunctional coupling agent

Synthesis of core-first Star Shaped Polymers via Anionic Polymerization

Star Polymers via Anionic Polymerization, S. Plentz-Meneghetti, D. Rein, P.J. Lutz In: "Stars and hyperbranchedPolymers" Polymer Frontiers, M.K. Mishra and S. Kobayashi), pp. 27-57, Marcel Dekker Inc. New-York, Bâle (1999)

HO (CH2 CH2 O)n OH

OCNOCN

NCONCO

+H

H20 OH + H

1/2 H2

CH2 CH2 O

CH2 CH2 O

2 OH CH CH2 O

CH CH2 O

CH CH2 O

CH CH2 O

= ===

=

====

= === ==

POLY(ETHYLENE OXIDE)Crosslinking

Chem.

Irradiation

Rev. Cross (Temp. Conc…. )

a) Linear chains adsorbed of grafted b) Hydrogels

c) star-shaped hydrogels d) Block copolymers with hydrophobic sequences

POLY(ETHYLENE OXIDE) and SURFACES

Poly(ethylene oxide) hydrogels :

Synthesis and characterization

Crosslinking via Irradiation

Linear or Star-shaped PEO ’sdirectly in water

E. Merrill

- Only thin films- No control of the structure

+

Good Control of the structural parameters

- Not easy to perform- Not possible in water

End-linking crosslinking

Classical Ways to crosslink PEO chains

Linear or Star-shaped PEO ’s

Y. Gnanou

PEO HYDROGELS SYNTHESIS VIA HOMOPOLYMERIZATIONOF BIFUNCTIONAL PEO MACROMONOMERS

Solvent: Water Benzene Initiator: K2S2O

8 AIBNTemperature: 24°C 65°C

( CH2CH2O)n

O O

65°C 65°C

I1/I3

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.01.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

Hydrophobic end group concentration (expressed in mol.l-1) . 104

I1/I3 measurements for a 11500 g.mol-1molar mass macromonomer:

Influence of the macromonomerconcentration on the ratio I1/I3. Increasing the concentration leads to a decrease of the ratio I1/I3.

Fluorescence Emission Spectrum at various macromonomerconcentrations.

HYDROGELS SYNTHESIS VIA HOMOPOLYMERIZATIONOF BIFUNCTIONAL PEO MACROMONOMERS

STUDY OF THE MICELLAR BEHAVIOR

PREPARATION CONDITIONS AND PHYSICO-CHEMICAL CHARACTERISTICS OF HYDROGELS BASED ON MACROMONOMERS

Sample Mn

MacromonomerConcentration ε(%) Qw Eg (water) Eg (THF) Qp (THF)

CL1G2A 6000 10% 4% 15.08 16300 - -

CL1G2B 6000 20% 3.6% 8.77 91300 - -

CL1G2C 6000 30% 3.8% 6.98 129100 - -

CL2G2A 10000 10% 4% 20.09 12000 11400 14.05

CL2G2B 10000 20% 4% 11.14 58300 62800 7.42

CL2G2C 10000 30% 3.5% 8.79 116100 107200 6.04

CL3G1A 20000 10% 7.2% 36.51 3700 - -

CL3G1B 20000 20% 6.5% 16.54 22500 - -

CL3G1C 20000 30% 3.8% 13.80 56800 - -

Sample Mn

MacromonomerConcentration ε(%) Qw Eg (water) Eg (THF) Qp (THF)

CL1G2A 6000 10% 4% 15.08 16300 - -

CL1G2B 6000 20% 3.6% 8.77 91300 - -

CL1G2C 6000 30% 3.8% 6.98 129100 - -

CL2G2A 10000 10% 4% 20.09 12000 11400 14.05

Sample Mn

MacromonomerConcentration ε(%) Qw Eg (water) Eg (THF) Qp (THF)

CL1G2A 6000 10% 4% 15.08 16300 - -

CL1G2B 6000 20% 3.6% 8.77 91300 - -

CL1G2C 6000 30% 3.8% 6.98 129100 - -

CL2G2A 10000 10% 4% 20.09 12000 11400 14.05

CL2G2B 10000 20% 4% 11.14 58300 62800 7.42

CL2G2C 10000 30% 3.5% 8.79 116100 107200 6.04

CL3G1A 20000 10% 7.2% 36.51 3700 - -

CL3G1B 20000 20% 6.5% 16.54 22500 - -

CL3G1C 20000 30% 3.8% 13.80 56800 - -

0 5 10 15 20 250

20

40

60

80

100

120

140

160

E G (i

n Pa

). 10

-3

Reaction time in hours

water benzene

Uniaxial Compression

Hydrogel

Micrometric Screw

Evolution of the uniaxial compression modulus versus crosslinking time for gels synthesized in benzene or water

(Mn 3000 g.mol-1)

Volume degree of equilibrium swelling of networks, Qv,

Calculated from Weight degree of equilibrium swelling

G (G= (Weightdry gel + Weightsolvent) / Weight dry gel)) is :

Qv = 1 + (G - 1). vs / voUniaxial compression modulus (EG) is determined as :

where F/ Sg = σ corresponds to the uniaxial force exerted per unit area of hydrogel, Sg is the section of the gel in the swollen state, ν the number of elastic chains per volume unit of dry gel, EG the uniaxial compression modulus, (EG = σ / (Λx-Λx

-2), Q the equilibrium swelling

( )-2xvxvg Q/Q - Q RT F/S ΛΛ=ν

Characteristics of some PEO Hydrogels

PHYSICO-CHEMICAL PROPERTIES OF PEO HYDROGELS BASED ON MACROMONOMERS

Extractable Polymer•decreases with increasing Precursor concentration•lower in water

Swelling Behaviorfor a given Macromonomer Precursor Molar Mass•depends from the Macromonomer concentration•independent from the preparation solvent • Depends from the Macromonomer Molar Mass

Uniaxial Compression ModulusStrongly depends from the preparation solvent Higher in Water

Specific behavior for short precursor chains :increasing influence of the nodulus / McInfluence of the initiator

CONTROL IN ADVANCE OF THE STRUCTURAL PARAMETERS IN HYDROGELS OBTAINED BY POLYMERIZATION OF MACROMONOMERS IN WATER IS POSSIBLE

Homopolymerization or copolym

Degradable materials

Controlled free radical polym.

Incorporation of non linear PEOs

Sample MnMacromonomerConcentration

Nb ofstyrene

ε(%) QwEg

(water)Eg

(THF)Qp

(THF)

CL1G2B 6000 20% 0 3.6% 8.77 91300

CL1G2D 6000 20% 5 3.6% 8.06 87500 - -

CL1G2E 6000 20% 10 3.4% 7.46 114400 - -

CL2G2B 10000 20% 0 4% 11.14 58300 62800 7.42

CL2G2D 10000 20% 5 4.7% 10.45 34800 45100 7.58

CL2G2E 10000 20% 10 3.7% 9.81 40700 49000 7.73

CL3G1B 20000 20% 0 6.5% 16.54 22500

CL3G1D 20000 20% 5 10.25% 17.01 30800 - -

CL3G1E 20000 20% 10 14.63% 14.89 28700 - -

Hydrogels based on PEO Macromonomers : copolymerization with Styrene in water : Preparation conditions and Physico chemical properties (Potassium Persulfate 60°C)

Reference Μn,a)

Polymer

Conc.b) ε c) Preparation

solvent

QV THF d)

QVwater d)

EG

THF e)

EG

water e)

NET1 3400 0.132 1.8 THF 25.2 24.9 13500 65900

NET2 3400 0.160 2.14 THF 22.6 20.8 18400 115800

NET3 3400 0.186 2.54 THF 17.5 16.5 56800 146400

NET4 2500 0.140 0.45 water 20.9 19.2 27000 36300

NET5 3400 0.130 1.80 water 35.2 29.1 42000 71400

NET6 3400 0.162 1.30 water 29.8 24.3 61100 138600

NET7 3400 0.181 1.62 water 21.5 22.3 127500 183200

NET8 3400 0. 236 1.43 water 17.7 17.8 178000 271500

NET9 3400 0.280 1.15 water 10.5 16.7 190000 301100

NET10 6100 0. 300 3.10 water 22.4 28.1 66700 110000

NET11 9200 0. 400 4.22 water 24.1 37.4 45000 26900

a)Number average molar mass of the PDXL precusor chain b)Wt-% of macromonomer to be crosslinked c)Amount of extractable polymer in wt. % (polymerization time : 66 hours) d)Q

V THF and QVwater are the volume equilibrium swelling degrees in THF and in water, respectively e)EG THF and EG water are the uniaxial compression moduli in THF and in water, respectively, expressed in Pa The crosslinking reaction was conducted at 60°C in water or THF over 66 hours.

HYDROGELS SYNTHESIS PDXL-Macromonomers

Hydrogels based on PEO Macromonomers :

copolymerization with perdeuterated Styrene in water

Ref Mn [PEO] wt.-%

ε (%) water

Qw (water)

Qp (THF) Eg (water) Eg (THF)

1 10000 10% 12% 15.2 12 16 250 15370

2 10000 20% 9.4% 9.2 7 80 650 70290

3 10000 30% 9.5% 7.3 5.5 130 300 123010

4 6000 30% 3% 6.8 4.7 169 100 131440

5 20000 30% 10.4% 9.7 7 59700 53110

Asymptotic Behavior of the particle Scattering function of PEO Hydrogels

(T= 60°C) Potassium persulfateS.A.N.S Results : Networks swollen in THF

10 000 g.mol-1, 30 wt.-%

10 000 g.mol-1, 10 wt.-%

Coll. L. Lapp, LLB Saclay

q2.I(q)

q

HYDROGELS SYNTHESIS VIA PEO-b-PDXL-b-PEO Macromonomers : Degradation Studies of the PEO-b-PDXL-b-PEO precursor

22 24 26

copolymer 2h in CH2Cl2 , pH=6

copolymer2h water pH=3

Initial copolymer

Elution volume

Water pH=3

20 22 24 26

48h, Mn = 6000

24h, Mn = 6000

2h, Mn = 9500

Elution volume

SEC

Influence of the solvent Influence of the reaction time

Degradation followed by the evolution of the UniaxialCompression Modulus

0 200 400 600 8005,0x103

1,0x104

1,5x104

2,0x104

2,5x104

3,0x104Water pH 3

CH2Cl2 pH 6

Uni

axila

Com

pres

ion

MO

dulu

sde

com

pres

sion

(Pa)

Immersion time in (h)

Hydrogel Degradation followed by :

- NMR

- measurement of the amount of extratible polymer

0 20 40 60 80

0

20

40

60

80

100 free ChainConnected Chain

% c

haîn

Reaction time (h)

PEO-b-PDXL-b-PEO HYDROGEL DEGRADATION

K.Naraghi, N. Sahli, M. Belbachir, E. Franta, P. J.Lutz, Polymer International 51, 912-922 (2002)

Network Reaction time (mn)

[PEO] wt.-%

ε (%) water

Qw (water)

Eg (water)

A 40 30% 12% 14, 1 30 000

B 60 30% 9.4% 14,3 31 600

C 120 30% 9.5% - 31 600

D 240 30% 3% 14, 29 400

Evolution of Physico-Chemical Properties of PEO Hydrogels based on bifunctional PEO Macromonomers versus crosslinking time

C. Lux et al 2002

Why ? PEO hydrogels well suited for biomedical applications

Template for the Growth of Cells

- Incorporation of Cells difficult in existing hydrogels

- No cell survival when crosslinking at 60°C

- Potassium Persulfate at 37°C no crosslinking

Crosslinking of PEO Macromonomers

37°C, PEO/ Redox initiators

E. Alexandre, K.Boudjema, B. Schmitt, J. Cinqualbre, D. Jaeck, C.Lux, F. Isel,and P.J. Lutz

Polymeric Materials: Science & Engineering, 89, 240-241 (2003)

H3C OCH2CH2 OHEt3N

Br CO

CCH3

CH3

Br H3C OCH2CH2 O CO

CCH3

CH3

Brn

+THF, 4h n

SBA1 : Mn = 2110SBA2 : Mn = 508, n = 7SBA3 : Mn = 358, n = 4

+ HBr.NEt3

HYDROGELS SYNTHESIS VIA Atom Transfer Radical PolymerizationOF BIFUNCTIONAL PEO MACROMONOMERS :SYNTHESIS OF THE WATER SOLUBLE INITIATOR

Elution volume

SEC Diagram of PEO sample SBA1

RISBA1 : Mn 2110 SEC Sharp, easy to recorver

SBA2 : Mixture of oligomers !

SBA3 : only 4 units enough ?

once incorporated in the hydrogel influence of the properties ?

HYDROGELS SYNTHESIS VIA Atom Transfer Radical Polymerization OF BIFUNCTIONAL PEO MACROMONOMERS

Better control of the structure parameters with respect to classical free radical polymerization ?

O CH2CH2O C CO

CH3

CH2nCCO

H2CCH3

ATRP, 60°C, 24h, H2OHydrogel ?1) CuBr, 2,2’-bipyridine

2) Initiator

H3C CH

BrC OOCH2CH3

Initiators

H3C OCH2CH2 OHEt3N

Br CO

CCH3

CH3

Br H3C OCH2CH2 O CO

CCH3

CH3

Brn

+THF, 4h n

SBA1 : Mn = 2110SBA2 : Mn = 508, n = 7SBA3 : Mn = 358, n = 4

+ HBr.NEt3

Polymérisation

Solution Gel

HYDROGELS SYNTHESIS VIA Atom Transfer Radical Polymerization OF BIFUNCTIONAL PEO MACROMONOMERS

O CH2CH2O C CO

CH3

CH2nCCO

H2CCH3

ATRP, 60°C, 24h, H2O

1) CuBr, 2,2'-bipyridine2) Amorceur

Hydrogel ?

Polymerization

2)Initiator

-Rapid Crosslinking is possible !-Strong influence of various experimental parameters : -[Initiator] / [Double bond], -Toluene (no crosslinking)

56800430013,8035,53,817,530%20000SB3G1C22500170016,5448,56,525,420%20000SB3G1B

11610051008,7919,93,52530%10000SB2G258300350011,1428,8420,820%10000SB0G5

129100204006,9814,63,818,130%6000SB10G2C129100214006,9815,93,824,830%6000SB10G1C91300215008,7716,93,614,920%6000SB10G1B

16300100015,0851,4438,810%6000SB10G1A16300154015,0847,6438,710%6000SB5G9

EgK2S2O8 e)

EgATRP e)

QpK2S2O8 d)

QpATRP d)

ε(%)K2S2O8 c)

ε(%)ATRP c)

Wt. (%)b)

Mn a)Ref

PHYSICO-CHEMICAL PROPERTIES OF MACROMONOMER BASED PEO HYDROGELS

a)Number average molar mass of the PEO precusor chainb)Wt-% of macromonomer to be crosslinkedc)Amount of extractable polymer in wt. %d)Qp water is the weight equilibrium swelling degree in water, respectivelye) Eg water is the uniaxial compression modulus in water, expressed in Pa

-amount extractible polymer higher in ATRP-Higher equilibrium swelling degrees -Poor mechanical properties !

S. Buathong et al 2003

HYDROGEL SYNTHESIS

(Co-)polymerization in water of linearPEO macromonomers with star-shaped macromonomers (polyglycerol based)

• Advantage of the micellar polymerization in water: aggregation of the hydrophobicpart increases polymerization kinetics

• Control in Advance of the Structural Parameters possible ?

• Incorporation of the star PEO affects the the propertiesLow contents : no changes !

HYDROGELExperimental conditionssolvent: H2Oradical source: K2S2O8, 1 mol% per DBreaction temperature: 60°Creaction time: 24hPEO concentration: 30 w% in most cases

+ Macromonomer

Col . H. Frey et al Freiburg /Mainz (D) PSME 2002

Some Biomedical Applications of Macromonomer

based PEO Hydrogels

PEO HYDROGELS AND ENCAPSULATION OF ISLETS OF

LANGERHANS

Islets of Langerhans Semi-permeable MembraneVascular Device

PEO in the Form of Hydrogels or Grafted onto Surfaces

Micro (Macro) encapsulation !

Fibroblast culture on a PS Surface Fibroblast culture on a PEO Hydrogel

IN VITRO

STUDY OF THE BIOCOMPATIBILITY OF PEO HYDROGELS OBTAINED FROM PEO MACROMONOMERS

See photo article Macromol. Biosciences

STUDY OF THE BIOCOMPATIBILITY OF PEO HYDROGELS OBTAINED FROM PEO MACROMONOMERS

IN VIVO

SEM of the surface of a PTEFE sleeve after one month implantation in a pig

Membrane implanted in the intraperitoneal zone of rats and removed 20 days after implantation

SEM of the surface of an hydrogel after one month implantation in a pig

STUDY OF THE GLUCOSE DIFFUSION THROUGH PEO HYDROGELS

Experimental set-up

•Glucose diffusion coefficients were

determined by the lag time analysis

method described by Hannoun and

Stephanopoulos.

•From the total flux of the glucose versus time, the lag time t0 (i.e. the time required to be saturated with glucose) can be extrapolated. •The resolution of Fick’s law by the Fourrier analysis gives a simple relation between Da and t0. Da = e2 / 6t0 where Da is the absolute diffusion of glucose in the membrane (given in cm2s-1), and e is the thickness of the membrane (in cm).

Dr = DaDw

GLUCOSE DIFFUSION THROUGH PEO HYDROGELS

PEO Chain D a (cm 2 s -1 ) D r

3000 0.91.10 -6 0.13

6000 1.14.10 -6 0.17

10000 1.61.10 -6 0.24

Glucose diffusion curve as a function of molar mass and time

Insuline diffusion curve(Molar mass 11 000 g.mol-1)

SEM of FRESH ISOLATED RAT HEPATOCYTES

GROWTH OF ISOLATED RAT HEPATOCYTES ON THE SURFACE OF PEO HYDROGELS AFTER 4 HOURS IN CULTURE

• Hepatocytes attached and formed a monolayer on PS• Attachment rate was low on the hydrogels• The higher the PM of the hydrogels was, the lower the attachment of the hepatocytes was

INFLUENCE OF THE POLYMER CONCENTRATION IN THE HYDROGEL ON THE NEURONAL DEVELOPMENT

Influence of the polymer concentration in the hydrogelon the cellular development on the surface

Immunocitochemical labelling of neurons forMAP2c (green) and of glial cells forvimentin (bar 25mm)

Double labelling of MAP2c (green and viviment (red) showing neuriticextension growth in the absence of glialCells --- = 10 mm

Coculture of hypothalamic neurons and pituitary melanotrophs

S. Schimchowitsch et al

NO Growth in the Network ?

MICRO ENCAPSULATION OF CELLS IN HYDROGELS

Advantage : no diffusion problems, homogeneous ?

• Micro encapsulation of Islets of Langerhans (J. Hubbell et al)

PEO macromonomers / Hydrogels

• Crosslinking of PEO macromonomers in the presence of nervous cells at 37°C (S. Schimchowitsch et al )

Hydrogels ? Microgels ? but heterogeneous ?

• Polymerization of PEO Macromonomers in the presence of hepatic cells : with potassium persulfate no crosslinking at all at 37°C

Crosslinking of PEO Macromonomers with redox-initiators

(decomposition at 37°C, compatible with cell survival ?

E. Alexandre, J. Cinqualbre, D. Jaeck, L. Richert, P.J. Lutz sent to Macromol. Symp,

ENCAPSULATION OF ISOLATED RAT HEPATOCYTES DURING CROSSLINKING

After 5 hours in culture • Hepatocytes attached and formed a monolayer on PS• Hepatocytes are present at different levels in the hydrogels

PS Surface

PEO HydrogelM = 10000

PEO HydrogelM = 20000

Phase contrast micrographs of hepatocytes

encapsulated in PEO Hydrogels.

Macro 10000

3h 24 h

SEM of a PEO hydrogel containing hepatocytes

SEM of a PEO hydrogel containing hepatocytes

3h 24h

Precursor Macro 20 000

SEM of a PEO hydrogel containing hepatocytes

CONCLUSIONS AND PERSPECTIVES

The Possibility to prepare, by polymerization of PEO linear (or star-shaped) macromonomers, Hydrogels of controlled structural parameters opens new perspectives

• Hydrogel Synthesis possible directly in Water

• Many modifications of structural parameters (copolymerization,

degradable materials, functionalizable networks…. )

• Combination of Macromonomers

• Silsesquioxane based networks

• Incorporation of cells during during crosslinking : has to be improved

• Grafting of PEO Macromonomers on surfaces (Irradiation crosslinking

of star-shaped PEO’s could be achieved)

-Other approach for reversible PEO based Hydrogels, )

• Aknowledgments.

• S. Buathong, G. Carrot, C. Lux, K. Naraghi, B. Schmitt, Y. Ederlé, S. Grutke, S. Plentz-Meneghetti,

• F. Ise F, E. Franta,(ICS, Strasbourg (F)

• H. Frey,(Mainz) R. Knischka Freiburg (D)

• E. Alexandre, . Pr. D.Jaeck, Prof. L. Richert, Prof. J. Cinqualbre, Prof. K. Boudjema, Strasbourg, Rennes (F) Fondation Transplantation,Strasbourg, )

• N. Sahli, M. Belbachir (Oran, Algérie)

• CNRS, French Ministerium (ACI Technologie pour la Santé) • FICU (Canada)

SURFACES MODIFIED WITH STAR-SHAPED PEO HYDROGELSScanning Electron Microscopy

Unmodified with PEO Hydogel

NO MODIFICATION of Diffusion Properties with respect to Macromonomer based Hydrogels

Col E. W Merrill

Some References

"Free radical homopolymerization, in heterogeneous medium, of linear and star-shaped polymerizable amphiphilic poly(ethers): A new way to design hydrogels well suited for biomedical applications" P. J. Lutz, Macromol. Symp. 164, 277-292 (2001)

"Structured degradable poly(ether) hydrogels based on linear bifunctional macromonomers"K.Naraghi, N. Sahli, M. Belbachir, E. Franta, P. J.LutzPolymer International 51, 912-922 ((2002)

"Poly(ethylene oxide) Hydrogels as Semi-permeable Membranes for an Artificial Pancreas"B. Schmitt, E. Alexandre, K. Boudjema, P. J. Lutz Macromol. Biosci. 2, 341-351 (2002)

"Properties of Degradable Homo or co-polyether Hydrogels Based on Linear Bifunctional Macromonomers"

K. Naraghi, N. Sahli, M. Belbachir, E. Franta, P.J. Lutz

Polymer International

"Poly(ethylene oxide) Macromonomer based Hydrogels as a Template for the Culture of Hepatocytes"

E. Alexandre, J. Cinqualbre, D. Jaeck, L. Richert, P.J. Lutz

Macromol. Reactive Polymers in Inhomogeneous Systems,

in Melts and at Interfaces (Dresden, septembre 2003)

K. Naraghi, J. Soussand, J.M. Félix, S. Schimchowitsch, P.J. Lutz

Polym. Prep., Am. Chem. Soc. Div. Polym. Chem. Boston, (USA). 39(2), 196-197, (1998)

" Ralf Knischka, Pierre J. Lutz, Alexander Sunder, Holger Frey

Polymeric Materials: Science & Engineering 84, 945-946 (2001

E. Alexandre, K.Boudjema, B. Schmitt, J. Cinqualbre, D. Jaeck, C.Lux, F. Isel,and P.J. Lutz

Polymeric Materials: Science & Engineering, 89, 240-241 (2003)