BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Lecture 10: Bioengineering applications of hydrogels: Molecular Imprinting and Drug Delivery Last Day polyelectrolyte gels Polyelectrolyte complexes and multilayers Applications in bioengineering Theory of ionic gel swelling Toda Molecular imprinting ug Supplementary Reading: S.R. Lustig and N.A. Peppas, Solute diffusion in swollen membranes. IX Scaling laws for solute diffusion in gels, J. App/. Polym. Sci. 36, 735-747(1988) T. Canal and N A. Peppas, "Correlation between mesh size and equilibrium degree of swelling of polymeric networks, J. Biomed Mater Res 23, 1183-1193 (1989) Molecular Imprinting oncepts of molecular imprinting Molecular imprinting is the design of polymer networks that can recognize a given target molecule and bind it preferentially in the presence of an excess of irrelevant molecules, some of which may have very similar molecular structures o Seeks to mimic specificity in biological recognition obtained through protein-protein interactions Steps to the preparation of molecularly-imprinted networks 1. mixing of binding monomers and target molecule o target can be mixed directly with liquid monomers in bulk or co-dissolved in a non-interfering solvent o monomers bind target non-covalent bonding metal coordination o mixture usually at high concentration(e.g. 50%W/vol solutions): enforces close interactions of target with binding monomers and leads to a tight network that holds the position of functional groups in position of template binding 2. polymerization of monomers in place sually photopolymerization(rapidly 'trap structure) 3. washing for removal of target molecule from network pockets Lecture 10-Bioengineering Applications of Hydrogels 1of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 10: Bioengineering applications of hydrogels: Molecular Imprinting and Drug Delivery Last Day: polyelectrolyte gels Polyelectrolyte complexes and multilayers Applications in bioengineering Theory of ionic gel swelling Today: Molecular imprinting Hydrogels in drug delivery Supplementary Reading: S.R. Lustig and N.A. Peppas, ‘Solute diffusion in swollen membranes. IX. Scaling laws for solute diffusion in gels,’ J. Appl. Polym. Sci. 36, 735-747 (1988) T. Canal and N.A. Peppas, ‘Correlation between mesh size and equilibrium degree of swelling of polymeric networks,’ J. Biomed. Mater. Res. 23, 1183-1193 (1989) Molecular Imprinting1,2 Concepts of molecular imprinting • Molecular imprinting is the design of polymer networks that can recognize a given target molecule and bind it preferentially in the presence of an excess of irrelevant molecules, some of which may have very similar molecular structures o Seeks to mimic specificity in biological recognition obtained through protein-protein interactions • Steps to the preparation of molecularly-imprinted networks: 1. mixing of binding monomers and target molecule o target can be mixed directly with liquid monomers in bulk or co-dissolved in a non-interfering solvent o monomers bind target covalent interactions non-covalent bonding metal coordination o mixture usually at high concentration (e.g. 50% w/vol solutions): enforces close interactions of target with binding monomers and leads to a tight network that holds the position of functional groups in position of template binding 2. polymerization of monomers in place o usually photopolymerization (rapidly ‘trap’ structure) 3. washing for removal of target molecule from network pockets Lecture 10 – Bioengineering Applications of Hydrogels 1 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 salyze Recognitive Prole Lectins fe Biemdlewle iie amino aed residues Biomolecule imvolved in specifie recognition) t Choos or Desien Reooemtive Polymer Syntess Resdue functional t D Wash 8武一 二冒生品 types of target molecules: 1 o small-molecule drugs o steroids o nucleic acids o amino acids o metal ions o proteins Structure of Molecularly-Imprinted Networks structure of molecularly-imprinted networks o imprinted networks can be confined to a thin surface layer or prepared in bulk o surface networks usually perform better for capture of large molecules like proteins simple synthetic components for recognition networks o monomers o itaconic acid o acrylamides 4-vinyl pyrrolidone o other designed mo cross-linker o ethylene glycol dimethacrylate o PEG dimethacrylate o 'chain effect o binding of monomers to macromolecular templates causes a reduction in chain termination and thus an overall increase in reaction rate Example of molecular recognition: molecular imprinting of D-glucose(Peppas) o Monomers chosen as analogs of the amino acid residues that bind to glucose in vivo O WHAT RECEPTORS BIND GLUCOSE? Aspartate Lecture 10-Bioengineering Applications of Hydrogels 20f12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 • types of target molecules: 1 o small-molecule drugs o steroids o nucleic acids o amino acids o metal ions o proteins Structure of Molecularly-Imprinted Networks • structure of molecularly-imprinted networks o imprinted networks can be confined to a thin surface layer or prepared in bulk o surface networks usually perform better for capture of large molecules like proteins • simple synthetic components for recognition networks o monomers: o methacrylic acid o itaconic acid o acrylamides o 4-vinyl pyrrolidone o β-cyclodextrin o other designed monomers o cross-linkers o ethylene glycol dimethacrylate o PEG dimethacrylate o ‘chain effect’3 o binding of monomers to macromolecular templates causes a reduction in chain termination and thus an overall increase in reaction rate • Example of molecular recognition: molecular imprinting of D-glucose (Peppas) o Monomers chosen as analogs of the amino acid residues that bind to glucose in vivo: o WHAT RECEPTORS BIND GLUCOSE? • Aspartate Lecture 10 – Bioengineering Applications of Hydrogels 2 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 o Draw structures on board o Simple synthetic monomers chosen to mimic the bonding interactions of these amino acids Hydroxyethyl methacrylate CH CH- OCH Acrylic acid TARGET: D-glucose Specificity of binding sues. 0.8 Tightly cross-linked networks hold functional group positions for better Bound 0 45 mu bound g cry polymer recognition but restrict entry of target into network 30 40 50 60 Limited complexity in recognition units Time(hrs) c时出 copolymers6 C贴 Mnomn.Fayme i wear:Ar把过用20 MA Copolymers wth67% Competive substrate pntd intensiy Nonimprinted intensity 3±11.81 5097±07 81±1383585 Flores Fluorescent analogue 5657±090 Improving recognition by surface templating(Ratner+5) o Protein adsorbed to mica surface, coated with disaccharide, then coated with C3 Fs film by radiofrequency glow-discharge plasma treatment o Sugar coating protects protein from denaturation on dehydration Lecture 10-Bioengineering Applications of Hydrogels 3of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 • Glutamate • Asparagines • Serine o Draw structures on board o Simple synthetic monomers chosen to mimic the bonding interactions of these amino acids: QuickTime™ and a Graphics decompressor are needed to see this picture. TARGET: D-glucose Hydroxyethyl methacrylate Acrylic acid acrylamide Specificity of binding: Issues: Tightly cross-linked networks hold functional group positions for better recognition but restrict entry of target into network Limited complexity in recognition units • Improving recognition by surface templating (Ratner 4,5) o Protein adsorbed to mica surface, coated with disaccharide, then coated with C3F6 film by radiofrequency glow-discharge plasma treatment o Sugar coating protects protein from denaturation on dehydration Lecture 10 – Bioengineering Applications of Hydrogels 3 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 HOCH 2H2 Trehalose(disaccharide) 是 pen es ad noted or p hash cbs ed ie nd ated prornt arace b. a tapia mom m mo of the t phomphstebufned asine CPHS. PH I4 A I-tom a drawing of tbnnogn. e Mechanims br the poc -6mn deming a 1-30nm fuoropoyner bocaue ots ma an ora song intact onc e回智doto The suing o Resulting recognition LSZ RNase LSZ IMP02±0.083 201-△ RNase IMp40±060 LSZ/RNese ratio LSZ in solution can exchange with LSZ= lysozyme imprinted LSZ, but Rnase cannot displace LSZ on surface o Utilizing in-situ formability of photopolymerized hydrogels for lab-on-a-chip applications o Photopolymerized Bulk templates(Peppas) Lecture 10-Bioengineering Applications of Hydrogels 4of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Trehalose (disaccharide) o Resulting recognition: LSZ RNase LSZ in solution can exchange with LSZ = lysozyme imprinted LSZ, but Rnase cannot displace LSZ on surface o Utilizing in-situ formability of photopolymerized hydrogels for lab-on-a-chip applications o Photopolymerized Bulk templates (Peppas): Lecture 10 – Bioengineering Applications of Hydrogels 4 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Silicon substrate Surface treated with organosilane Monomer applied agent to nduce bonding treated silicon substrate asked UV polymerization Mkregutterne pelynt 二, o Plasma-deposited surface templates patterned by microcontact printing(Ratner) PDMS stamp m o nm vidn (SA)n P8S ndw aber rinse Imprint surtace for -5s transterring a monolayer of streptawd in to mca ony tod mgons The m ca surface was then exposed to a bumin(BSA)n Fas. 10 ITImI baind i h w th a soon of botn BsA labeled with t0-nm lodai gold to Lecture 10-Bioengineering Applications of Hydrogels 5of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Plasma-deposited surface templates patterned by microcontact printing (Ratner): Lecture 10 – Bioengineering Applications of Hydrogels 5 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Hydrogels in drug delivery o What Control of drug release kinetics by hydrogel structure6, o Release from stable hydrogels is controlled by diffusion of solute through the network o Diffusion is described by Fick's second law o Recall the solution to Fick's second law for a semi-infinite slab contacting a perfect sink Egn 2 ID C(XI Increasing time enf(z) solution L o Diffusion of drugs through a network is controlled by the mesh size) Lecture 10-Bioengineering Applications of Hydrogels 6of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Hydrogels in drug delivery o What Control of drug release kinetics by hydrogel structure6,7 o Release from stable hydrogels is controlled by diffusion of solute through the network o Diffusion is described by Fick’s second law: ∂C ∂ 2 C Eqn 1 ∂t = Dgel ∂x 2 o Recall the solution to Fick’s second law for a semi-infinite slab contacting a perfect sink: Eqn 2 c0 − c(x) =1 − erf 2 tD x c0 c(x) c0 Increasing time erf(z) solution x o Diffusion of drugs through a network is controlled by the mesh size (ζ) Free s urface Lecture 10 – Bioengineering Applications of Hydrogels 6 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 o the mesh size is related to the network swelling Q and the end-to-end distance between cross-links Number of segments 2M Eqn 3 (2) o. assuming a polymer chain that has 2 carbon-carbon bonds per repeat unit o derived from random walk chain statistics Where /is the bond length in the polymer backbone Mc is the molecular weight between cross-links Mo is the molecular weight per repeat unit Where Cn is the characteristic ratio for the polymer chain 0o)=Cl0 Q is the degree of swelling= Vary polymer/s N is the degree of polymerization between cross-links The mesh size is related to the diffusion constant of a solute in the network Eyring theory of diffusion Ean 5 Lecture 10-Bioengineering Applications of Hydrogels 70f12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o The mesh size is related to the network swelling Q and the end-to-end distance between cross-links: ()1/2=Nc 1/2a statistical segment length Number of segments between cross-links Eqn 3 r0 2 1/ 2 2M c 1/ 2 C1/ 2 ( ) = l M 0 n o …assuming a polymer chain that has 2 carbon-carbon bonds per repeat unit o derived from random walk chain statistics Where l is the bond length in the polymer backbone Mc is the molecular weight between cross-links M0 is the molecular weight per repeat unit Where Cn is the characteristic ratio for the polymer chain ( )2 1/ 2 Eqn 4 ξ = r0 1/ 3 = Q1/ 3 ( ) r0 2 1/ 2 = Cn 1/ 2 Q1/ 3 N1/ 2 l φ2,s Q is the degree of swelling = Vdry polymer/Vswollen polymer N is the degree of polymerization between cross-links The mesh size is related to the diffusion constant of a solute in the network Eyring theory of diffusion: − ∆G* − ∆H * ∆S* Eqn 5 D = Tνe kT = Tνe kT e k Lecture 10 – Bioengineering Applications of Hydrogels 7 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Where△G′ is the activation energy,△H* is activation enthalpy,and△ S"is activation entropy o N= translational oscillating frequency of solute molecule jump rate! o T= temperature o k= Boltzman constant The ratio of diffusion constant in the gel to that in solution is D Eqn 6 o Where As gel is the activation entropy for diffusion in the gel and Aso is the activation entropy in for diffusion in the solvent o This assumes the activation enthalpy and oscillation frequencies for diffusion are approximately the same in the gel and pure solvent (reasonable for dilute and chemically inert systems The activation entropies are Ean 7 △Sae=knP*-knPo △So=knP*- k In Po n Pgel_pgel opering gel, volume. o Popening is the probability that the network has a solute-sized gap to jump through mp into o Where P"volume is the probability that a solute-sized volume of free space exists to jump into volume dr Egn 10 o Where r is the radius of the solute(drug) and E is the network mesh size The probability of a volume to jump into is an exponential of the ratio of the solute size to the available free volume per mole Eqn 11 Ean 12 o Where free is the specific free volume and v* is the volume of the solute(drug) o Refs for free volume theory applied here Lecture 10-Bioengineering Applications of Hydrogels 8of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Where ∆G* is the activation energy, ∆H* is activation enthalpy, and ∆S* is activation entropy o N = translational oscillating frequency of solute molecule (jump rate!) o T = temperature o k = Boltzman constant The ratio of diffusion constant in the gel to that in solution is: * ∆Sgel k ˆ Eqn 6 D = Dgel = e ∆S0 * D0 e k o Where ∆S*gel is the activation entropy for diffusion in the gel and ∆S*0 is the activation entropy in for diffusion in the solvent o This assumes the activation enthalpy and oscillation frequencies for diffusion are approximately the same in the gel and pure solvent (reasonable for dilute and chemically inert systems) The activation entropies are: Eqn 7 ∆S*gel = k ln P* - k ln P0 Eqn 8 ∆S*0 = k ln P*0 – k ln P0 * * * Eqn 9 D = Pgel = Pgel,openingPgel,volume ˆ * * P0 P0,volume o Where P*volume is the probability that a solute-sized volume of free space exists to jump into o P*opening is the probability that the network has a solute-sized gap to jump through P*gel,volume P*gel,opening drug drug * ξ − r =1 − r Eqn 10 Pgel,opening = ξ ξ o Where r is the radius of the solute (drug) and ξ is the network mesh size The probability of a volume to jump into is an exponential of the ratio of the solute size to the available free volume per mole: v* − * Eqn 11 Pgel,volume ~ e v free,gel v* − * Eqn 12 P0,volume ~ e v free,1 o Where vfree is the specific free volume and v* is the volume of the solute (drug) o Refs for free volume theory applied here: Lecture 10 – Bioengineering Applications of Hydrogels 8 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Yasuda et al. Makromol. Chem. 26, 177(1969) Now Eqn 13 gel, oume=e ('few gel "/eed The free volume in a swollen gel is approximately free, 1 since the free volume contribution from polymer is extremely low(2.5%even in solid polymers at 25C) Egn 14 Vree, gel=中ee+2mee I Therefore Ean 15 Vfreegel-91Vfree, 1=(1-02)Vree, 1=(1-1/Q)Vree. 1 o Where Q is the swelling degree Swollen ge vary gel=1/2 Therefore Eqn 16 _ge/ rohumeo-o red md) volume o V/free, 1-1 for most polymers, experimentally Eqn 17 And thus finally Ean o Insulin: MW-5900 g/mole; hydrodynamic radius =16 A Design of glucose-responsive drug delivery microgels for treatment of diabetes -10 Work by Podual and Peppas Immobilized glucose oxidase enzyme within pH-responsive polyelectrolyte gel network along with encapsulated o Network composed of DEAEM, PEGMA, and TEGDMA GOD covalently tethered to networ o Insulin entrapped in network polymerized gels as microspheres Lecture 10-Bioengineering Applications of Hydrogels 9of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Yasuda et al. Makromol. Chem. 26, 177 (1969) Peppas and Reinhart, J. Membrane Sci. 15, 275 (1983) Now: * v* v* Eqn 13 Pgel,volume = e − v free,gel − v free,1 * P0,volume The free volume in a swollen gel is approximately vfree,1 since the free volume contribution from polymer is extremely low (2.5% even in solid polymers at 25°C) Eqn 14 vfree,gel = φ1vfree,1 + φ2vfree,2 Therefore: Eqn 15 vfree,gel ~ φ1vfree,1 = (1-φ2)vfree,1 = (1-1/Q)vfree,1 o Where Q is the swelling degree = Vswollen gel/Vdry gel = 1/φ2 Therefore: − v* − v* * Q )v free,1 v free,1 − v* 1 1 Eqn 16 Pgel,volume = e (1− 1 = e v free,1 Q−1 ≈ e − Q−1 * P0,volume o v*/vfree,1 ~ 1 for most polymers, experimentally Therefore: −1 ˆ Eqn 17 D ≅ 1− r e(Q−1) ξ And thus finally: −1 Eqn 18 Dgel ≅ D0 1− r e(Q−1) ξ o Insulin: MW – 5900 g/mole; hydrodynamic radius = 16 Å Design of glucose-responsive drug delivery microgels for treatment of diabetes8-10 Work by Podual and Peppas Immobilized glucose oxidase enzyme within pH-responsive polyelectrolyte gel network along with encapsulated insulin o Network composed of DEAEM, PEGMA, and TEGDMA o GOD covalently tethered to network o Insulin entrapped in network o Polymerized gels as microspheres Lecture 10 – Bioengineering Applications of Hydrogels 9 of 12
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 nthesis of microgels? Glucose +C Glucose cidse Gluconic acid+ho Gluc Gluc Ins DEAEM PEGMA TEGDMA Glucose oxidase Hg 3 lec of crosslinking ratio on equlibrium weight swelling Etio of Gluc go polymer. The graphs shown are for x=Or o Fast variation in swelling due to microgel dimensions o Mesh size responds in a similar manner, using theory described above 3.2 1001 0153045607590105120 Fig 8. Pulsatile swelling response of P(DEAEM-g-EG)microspheres 200 to alternate pH changes between pH 7.4 and 3.2 shown at Fig 9. Change in the mesh size of P(DEAEM-g-EG)microspheres of the top of the plot The dynamie response was the fastest for the particle size= 160 um, E-3.3 x 10 PG=200 croparticles with x-0.02(.), followed by x-003() The par. to altemate pH changes between pH 7.4 and 3. 2. The mesh sizes ar density X=0.04(.) were the slowest in responding shown for hydrogels with x=0.02(Ox=0.03()and X=0. 04(9). to the changes in pH. The gels thus designed respond to concentrations of glucose in the surrounding medium, dynamically Lecture 10-Bioengineering Applications of Hydrogels 10of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Synthesis of microgels? o Fast variation in swelling due to microgel dimensions o Mesh size responds in a similar manner, using theory described above: • The gels thus designed respond to concentrations of glucose in the surrounding medium, dynamically: Gluc Gluc Gluc Gluc Gluc Glucose oxidase insulin Lecture 10 – Bioengineering Applications of Hydrogels 10 of 12
