Controlled release of proteins from selfassembling hydrogels based on oppositely charged dextran microspheres Sophie Van Tomme a Rene van Nostrum a, Stefaan De Smedt b, Wim Hennink a a Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences Utrecht University, The Netherlands b Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University, Belgium GPEN 2006, 25 th ctober 2006 University of Kansas, Lawrence Utrecht UIPS Institute for Pharmaceutical Sciences
utline Hydrogels general features crosslinking methods Aim of the project Approach Results: network characterization Conclusions protein release degradation
Introduction Hydrogel 3dimensional hydrophilic network Good biocompatibility minimal tissue irritation low tendency for proteins and cells to adhere Possible applications Drug delivery system Release can be tailored Scaffold for tissue engineering Degradation rate can be tailored Growth factors can be incorporated RGD peptides can be coupled to promote cell adhesion
Introduction 1. Chemical crosslinking physical crosslinking Radical polymerization Reaction of complementary groups (e.g. NH 2 /CH) Enzymes (e.g. proteases) Crystallization Hydrogen bonding Ionic interactions Covalent bonds Nonpermanent bonds high mechanical strength toxicity/denaturation selfassembly weaker network 2. Implants injectable matrix
Aim of the Project Synthesis, characterization and evaluation of an injectable, selfassembling hydrogel for pharmaceutical and tissue engineering applications
Design H H DexHEMA H methacrylic acid Size: 220 µm N dimethylaminoethyl methacrylate
Design DexHEMA H methacrylic acid H H Size: 220 µm dexhemamaa microspheres mixing dexhemadmaema microspheres network formation N dimethylaminoethyl methacrylate
Design DexHEMA H methacrylic acid H H dimethylaminoethyl methacrylate N Size: 220 µm dexhemamaa microspheres shear mixing dexhemadmaema microspheres network formation
Design DexHEMA H methacrylic acid H H dimethylaminoethyl methacrylate N Size: 220 µm dexhemamaa microspheres shear mixing in situ gelling at site of injection dexhemadmaema microspheres network formation
Microsphere preparation Radical polymerization of dexhema, emulsified in an aqueous PEG solution initiation by KPS (=potassium peroxodisulphate) ) and TEMED (=tetramethyl ethylene diamine) dexhema PEG dexhema PEG stirring radical polymerization waterin inwater emulsion
Network formation Rheology Study of flow and deformation Geometry: 20mm flat plate Gap: 500 µm G = Storage modulus (elasticity( elasticity) G = Loss modulus (viscosity( viscosity) tan δ= = G /G tan δ= = 0 fully elastic tan δ= Newtonian 0< tan δ< < 1 viscoelastic
Rheology: controlled strain 7000 6000 10 tan(δ) G' (Pa) G' (Pa) 5000 4000 3000 2000 1 0.1 tan(δ) 1000 0 0.01 5 10 15 20 25 30 % solid Strength (modulus) of the network can be tailored by the solid content c of the hydrogel
Rheology: creep % strain 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0 25 50 75 100 125 150 175 200 time (s) dexhema HEMAMAA/dexHEMADMAEMA mixture Elastic network
Rheology: creep % strain 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0 25 50 75 100 125 150 175 200 time (s) Newtonian liquid dexhema HEMAMAA MAA dispersion % strain dexhema HEMAMAA/dexHEMADMAEMA mixture Elastic network 2250 2000 1750 1500 1250 1000 750 500 250 0 0 25 50 75 100 125 150 175 200 time (s)
Injectability Reversibility of the network? G' and G" (Pa) 450 6.5 G' (Pa) 400 6.0 G" (Pa) 5.5 350 5.0 tan (δ) 300 4.5 4.0 250 3.5 200 3.0 2.5 150 2.0 100 1.5 50 1.0 0.5 0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 tan (δ) oscillatory stress (Pa) shear Network rebuilds
Degradation: influence of charge Swelling ratio S=L t /L 0 L H H swelling ratio 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 neutral 0 15 30 45 60 75 90 105 120 135 time (days)
Degradation: influence of charge Swelling ratio S=L t /L 0 L H H 3.5 3.0 swelling ratio 2.5 2.0 1.5 1.0 0/100 neutral 0.5 0.0 0 15 30 45 60 75 90 105 120 135 time (days)
Degradation: influence of charge Swelling ratio S=L t /L 0 L H H 3.5 3.0 swelling ratio 2.5 2.0 1.5 1.0 100/0 0/100 neutral 0.5 0.0 0 15 30 45 60 75 90 105 120 135 time (days)
Degradation: influence of charge Swelling ratio S=L t /L 0 L H H 3.5 3.0 swelling ratio 2.5 2.0 1.5 1.0 50/50 100/0 0/100 neutral 0.5 0.0 0 15 30 45 60 75 90 105 120 135 time (days) Ratio / degradation time
dexhema microspheres (R 1 ) ran H H H H normal situation dexhemamaa microspheres (R 2 ) ran H H repulsion dexhemadmaema microspheres (R 3 ) ran H N H H H attraction stabilization transition state
In vitro protein release protein solution dexhemamaa microspheres dexhemadmaema microspheres
In vitro protein release protein solution Possible release mechanisms dexhemamaa microspheres dexhemadmaema microspheres
In vitro protein release protein solution Possible release mechanisms dexhemamaa microspheres dexhemadmaema microspheres
In vitro protein release protein solution Possible release mechanisms dexhemamaa microspheres dexhemadmaema microspheres
In vitro protein release Hydrogels composed of 15% microspheres and 85% buffer lysozyme cumulative release (%) 100 90 80 70 60 50 40 30 lysozyme 20 BSA 10 0 IgG 0 10 20 30 40 50 60 70 time (days) cumulative release (%) BSA 60 IgG 50 40 30 20 10 0 0 1 2 3 4 5 6 time (days) Quantitative release of lysozyme and BSA Full preservation of enzymatic activity of released lysozyme
In vitro protein release diffusioncontrolled release
In vitro protein release diffusioncontrolled release degradationcontrolled release
In vitro rhodaminebdextran dextran release rhodaminebdextran release (%) 100 90 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35 40 45 50 time (days) 50 rho/50 50 rho/50
In vitro rhodaminebdextran dextran release rhodaminebdextran release (%) 100 90 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35 40 45 50 time (days) 50 rho/50 50 rho/50 rho 50 rho/50 Combination of release from positive and negative microspheres gives a zero order release
Conclusions Mainly elastic hydrogels are formed by mixing oppositely charged dextran microspheres Reversible gelation occurs Continuous diffusioncontrolled release of proteins Degradation behavior can be tailored Hydrogel properties can be tailored for various applications in drug delivery and tissue engineering
www.fip.org/pswc Abstract submission deadline 15 th December 2006 Travel grant requests: see website
Abstract submission deadline 15 th December 2006 www.fip.org/pswc