BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Lecture 4: Degradable Materials with Biological Recognition(part Biological recognition in vivo Engineering biological recognition of biomaterials: adhesion/migration peptides Engineering biological recognition of biomaterials: enzymatic recognition and cytokine Reading S.E. Sakiyama-Elbert and J.A. Hubbell, Functional Biomaterials: Design of Novel biomaterials, Annu. Rev. Mater. Sci. 31, 183-201(2001) J.C. Schense et al., 'Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension, Nat. Biotech. 18, 415-419(2000) Supplementary Reading The Extracellular Matrix, pp. 1124-1150, Molecular Biology of the Cell, Lodish et al Recognition of Biomaterials by proteases: Engineering Enzyme-mediated degradation of polymers Basic concept: include peptide sequences in the polymer chain which are cleaved by enzymatic activity of serum proteins/cellular secreted products(active breakdown)[hydrolysis active but slow.] Amide bond-(NH)-(CO)-provides natural hydrolytic mechanism for degradation, but breaks down very slowly in physiological conditions Remodeling enzymes in vivo 1)binding of target by enzyme 2)specific site cleavage What is enzymatic cleavage used for in vivo?(Reading Ch 1 4 Voet and Voet"Enzymatic catalysis) o Remodling ECM, migration of cells through matrix o Removing functional groups from signaling molecules(phosphorylation/dephosphorylation) Utility in the design of biomaterials o Polymers can be designed to be surface-eroding easily (enzyme can't access interior) o Degradation could be localized to tissue where enzyme is produced Lecture 4- Biological Recognition pt. 2 1 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 4: Degradable Materials with Biological Recognition (part II) Last time: Biological recognition in vivo Engineering biological recognition of biomaterials: adhesion/migration peptides Today: Engineering biological recognition of biomaterials: enzymatic recognition and cytokine signaling Reading: S.E. Sakiyama-Elbert and J.A. Hubbell, ‘Functional Biomaterials: Design of Novel biomaterials,’ Annu. Rev. Mater. Sci. 31, 183-201 (2001) J.C. Schense et al., ‘Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension,’ Nat. Biotech. 18, 415-419 (2000) Supplementary Reading: ‘The Extracellular Matrix,’ pp. 1124-1150, Molecular Biology of the Cell, Lodish et al. Recognition of Biomaterials by Proteases: Engineering Enzyme-mediated degradation of polymers Basic concept: include peptide sequences in the polymer chain which are cleaved by enzymatic activity of serum proteins/cellular secreted products (active breakdown) [hydrolysis active but slow...] • Amide bond -(NH)-(CO)- provides natural hydrolytic mechanism for degradation, but breaks down very slowly in physiological conditions Remodeling enzymes in vivo: 1) binding of target by enzyme 2) specific site cleavage • What is enzymatic cleavage used for in vivo?1 (Reading Ch.1 4 Voet and Voet “Enzymatic catalysis”) o Remodling ECM, migration of cells through matrix o Removing functional groups from signaling molecules (phosphorylation/dephosphorylation) • Utility in the design of biomaterials: o Enzymatic cleavage can breakdown polymers more quickly than hydrolysis o Polymers can be designed to be surface-eroding easily (enzyme can’t access interior) o Degradation could be localized to tissue where enzyme is produced Lecture 4 – Biological Recognition pt. 2 1 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Reminder of breakdown mechanisms 1s BNOAESOR BARLE AND PIOFROHMLE MATERIALS Used for biodegradable hydrogels ?? Main mechanism exploited in solid polymers ( Ratner, Biomaterials Science) (SLIDE) Cleavage of synthetic Cell source Enzyme Native Degradation Result function Mechanism polyesters rotease polyesteramides Tritirachium album(mold)Proteinase K Protease oly (lactide) Monomers or dimers ammalian cells steranes ly(alkyl later -soluble cyanoacrylates) mammalian cells apain, pepsin proteases polyesteramides ntested Mammalian cells chymotrypsin erine protease Aromatic peptides in Untested polyesteramides Mammalian cells nastase protease polyesteramides ntested Pepsin: protease from papaya Papain: main protease in gastric juice of stomach Chymotrypsin: digestive enzyme Mold and bacterial proteases not relevant in vivo, but make these polymers also of significar environmentally-friendly packaging Of interest in the use of biodegradable materials in environmentally-friendly packaging but not a concern for in vivo applications Lecture 4- Biological Recognition pt. 2 2 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Reminder of breakdown mechanisms: Used for biodegradable hydrogels Main mechanism exploited in solid polymers (Ratner, Biomaterials Science) (SLIDE) Cell source Cleavage of synthetic polymers by enzymes Enzyme Native function Acts on Degradation Mechanism Result Various bacteria lipases protease Polyesters, polyesteramides III Monomers or dimers Tritirachium album (mold) Proteinase K Protease Poly(lactide) III Monomers or dimers Mammalian cells esterases protease Poly(alkyl cyanoacrylates) polyesteramides2 II Water-soluble polymers Mammalian cells Papain, pepsin proteases III Untested Mammalian cells α-chymotrypsin Serine protease Aromatic peptides in polyesteramides3 (e.g. Ala, Val, Leu) III Untested Mammalian cells elastase protease Polyesteramides III untested Pepsin: protease from papaya Papain: main protease in gastric juice of stomach Chymotrypsin: digestive enzyme Mold and bacterial proteases not relevant in vivo, but make these polymers also of significant interest for environmentally-friendly packaging • Of interest in the use of biodegradable materials in environmentally-friendly packaging, but not a concern for in vivo applications Lecture 4 – Biological Recognition pt. 2 2 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 o Data comparing in vitro and in vivo degradation rates indicates that enzymatic cleavage of most synthetic polymers is negligible o Polyesteramides have been synthesized with enzymatic recognition Enzymatic attack on polyesteramides Breakdown by mechanism Ill Enzymatic breakdown of polymers is fast relative to simple hydrolysis Polyesteramide breakdown by papain GHG2 hours of enzymatic hydrolysis Figure 10. Weight changes of three representative polymers during in uitro enzymatic degradation with papain in a 0.05M phosphate buffer. PGHG5 corre- sponds to the polymer of the studied poly(ester amide ries that has a faster degradation rate What does papain do in vivo? Esterase action on polyalkyl cyanoacrylates): +-(CH 2-C- Poly(akyl cyanoacrylates) formation of poly (2-cyanoacrylic acid Lecture 4- Biological Recognition pt. 2 3 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Data comparing in vitro and in vivo degradation rates indicates that enzymatic cleavage of most synthetic polymers is negligible o Polyesteramides have been synthesized with enzymatic recognition: Enzymatic attack on polyesteramides (SLIDE) • Breakdown by mechanism III • Enzymatic breakdown of polymers is fast relative to simple hydrolysis: Polyesteramide breakdown by papain: • What does papain do in vivo? Esterase action on poly(alkyl cyanoacrylates4 ): C≡N C≡N -(CH2-C-)n- H2O -(CH2-C-)n- C=O C=O O OPoly(akyl cyanoacrylates): R formation of poly(2-cyanoacrylic acid) _ _ _ _ _ _ _ Lecture 4 – Biological Recognition pt. 2 3 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Breakdown by mechanism Il (SLIDE) Active Site Enzyme concentration (mg/ml) Fig. 2. Butanol production with variation in cyme concentration Hg. IL. Enzyme catalysis of cst ng the eser hydrolysis of pely (buyl cyaneacybte) (pH7,37C. What does esterase do in vivo? Enzymatic activity in vivo on peptide sequences lavage Enzyme Functions in vivo Target amino acid sequences plasminogen activator Degradation of fibrin matrices. Jon fibrinogen: Arg1o4-Asp1o5, Arg10-Val111 urokinase or tissue-type angiogenesis, tumor progression Lys206-Met207, Arg42-Ala43, Lys130- plasminogen activator)/ urokinase can bind to cell surface Glu131, Lysg4-Sera5, Lysa7-Met plasminogen→> plasmin receptor atrix metalloproteinases Facilitate cell migration ype I collagen: Gly775-lle77e soluble and cell-surface): e.g In smaller peptides: Gly-Leu or Gly lle ibroblast Collagenase(MMP I) Elastase Elastin remodeling Poly(Ala)sequences REFS for MMP: (REF J Biol Chem. 256, 9511(1981);J Biol Chem. 264, 393 (1989); J Biol. I Chem. 266, 6747(1991)) Note that proteases often have complementary protease inhibitors e.g. plasminogen activators inhibited by plasminogen activator inhibitor type-1(a serine protease inhibitor) Lecture 4- Biological Recognition pt. 2 4 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 • Breakdown by mechanism II Mechanism: data on degradation of 250nm-diam. porous particles: (SLIDE) • What does esterase do in vivo? Enzymatic activity in vivo on peptide sequences: 5,6 Cleavage Enzyme Functions in vivo Target amino acid sequences Plasminogen activator (urokinase or tissue-type plasminogen activator) / plasminogen → plasmin Degradation of fibrin matrices, angiogenesis, tumor progression; urokinase can bind to cell surface receptor on fibrinogen: Arg104-Asp105, Arg110-Val111, Lys206-Met207, Arg42-Ala43, Lys130- Glu131, Lys84-Ser85, Lys87-Met88 Matrix metalloproteinases (soluble and cell-surface): e.g. Fibroblast Collagenase (MMP I) Facilitate cell migration Type I collagen: Gly775-Ile776 In smaller peptides: Gly-Leu or Gly Ile bonds Elastase Elastin remodeling Poly(Ala) sequences REFS for MMP: (REF J. Biol. Chem. 256, 9511 (1981); J. Biol. Chem. 264, 393 (1989); J. Biol.l Chem. 266, 6747 (1991)) Note that proteases often have complementary protease inhibitors: e.g. plasminogen activators inhibited by plasminogen activator inhibitor type-1 (a serine protease inhibitor) Lecture 4 – Biological Recognition pt. 2 4 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Examples of peptide sequences used to allow enzymatic cleavage in biomaterials (Work led by J. West and J.A. Hubbell) recognition by collagenase: -Ala-Pro-Gly-/-Leu recognition by plasmin -Val-Ala--Asn recognition by elastase: -Ala-Ala-Ala-Ala-Ala-(polyalanine sequence Example: poly(ethylene glycol) networks: acrylate-APGL-PEG-LGPA-acrylate (ON BOARD) APGL、PEG photopolymerization network structure formed by stitching together short strings of acrylate endgroups 二0 巴 50.5 100150 Time(hr Figure 1. Enzymatic degradation of a hydrogel material Time(hr) forned by photopolymerization of AcT- VRNI-PEC terial the presence of 2 U/mL plasmin(curve I)or 0.2 U/mL in(curve 2), but no Lecture 4- Biological Recognition pt. 2 5 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Examples of peptide sequences used to allow enzymatic cleavage in biomaterials (Work led by J. West and J.A. Hubbell7-9) recognition by collagenase: -Ala-Pro-Gly-/-Leurecognition by plasmin: -Val-Ala-/-Asnrecognition by elastase: -Ala-Ala-Ala-Ala-Ala- (polyalanine sequence) Example: poly(ethylene glycol) networks: acrylate-APGL-PEG-LGPA-acrylate (ON BOARD) Acrylate endgroups PEG collagenase sequence photopolymerization = = -APGL- -CH2CH2Ocollagenase -APGLcollagenase peptides network structure formed by stitching together short strings of acrylate endgroups Lecture 4 – Biological Recognition pt. 2 5 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Initially, swelling of network increases and wet weight goes up as first cross-links are broken, then as chains are freed and begin to diffuse out, weight goes down degradation rate of network depends on collagenase concentration(from Mann et al. ) 0.25 Fig 1. Degradation of GGLGPAGGK derivatized PEG hydrogels in of AAAAAAAAAK derivatized PEG hydrogels in lutions containing collagenase. (o) 2 mg/ml collagenase (O) solutio elastase(O)2 mg/ml elastase; (O)0.2 mg ml elas- 0. 2 mg/ ml collagenase;(A)no collagenase. note that these are hydrogels which has a major impact on the degradation rate degradation rate is controlled by enzyme concentration and is selective for the enzyme targeted Any examples tested in vivo? Recognition of Biomaterials by cytokine Resceptors: Engineering growth and differentiation of cells on biomaterials via cytokine peptides Growth factors small proteins(ca 50 aa) Can be immobilized to polymer chains present at surfaces of biomaterials much like adhesion peptides and presented to receptors of cells Many growth factors signal by DIMERIZATION and autophosphorylation Again, spatial distribution may be key in controlling signaling Cytokines immobilized in close proximity may favor signaling (Lodish Fig 20-32) Lecture 4- Biological Recognition pt. 2 6 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Initially, swelling of network increases and wet weight goes up as first cross-links are broken, then as chains are freed and begin to diffuse out, weight goes down degradation rate of network depends on collagenase concentration (from Mann et al.): • note that these are hydrogels which has a major impact on the degradation rate… • degradation rate is controlled by enzyme concentration and is selective for the enzyme targeted Any examples tested in vivo? Recognition of Biomaterials by Cytokine Resceptors: Engineering growth and differentiation of cells on biomaterials via cytokine peptides10-12 • Growth factors small proteins (ca. 50 aa) • Can be immobilized to polymer chains present at surfaces of biomaterials much like adhesion peptides and presented to receptors of cells • Many growth factors signal by DIMERIZATION and autophosphorylation Again, spatial distribution may be key in controlling signaling Cytokines immobilized in close proximity may favor signaling (Lodish Fig. 20-32) Lecture 4 – Biological Recognition pt. 2 6 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 INTERNALIZATION: used to down-regulate signals mobilized cytokines may show more potent signaling due to lack of this down-regulation pathway One of the first examples- Griffith lab work with epidermal growth factor immobilized on star polymers Glass slide Aminate Z.CHCH, OI g. 2 Tethered EGF produces same increase in DNA synthe sis as soluble EGF for primary rat hepatocytes. a, Soluble EGF case contained 10 ng/ml EGF(EGF)or 0 ng/ml EGF (EGF). Tethered EGF 180 and M, r1,000)as shown in 320 3. 2 ng/cm' covalently tethered EGF and 1.9 ng/cm'nonspecifi- ally adsorbed EGF (EGF)or o ng/cm covalently tethered EGF and 1.9 ng/cm nonspecifically ad sorbed EGF (EGF). Staining index represents the number of nuclei stained per area covered by cells, and represent the mean t s.d. for 200)as shown in Fig, 1b; surfaces present either 0. 4 ng/cm covalently tethered EGF and <0.006 ng/cm' nonspecifically adsorbed EGF (EGF)or O ng/cm covalently tethered EGF and <0.006 ng/cm nonspecifically adsorbed EGF(EGF). DNA synthesis was measured as a per. tage of the total nuclei that had synthesized DNA and each point represents the mean s d. for three separate surfaces with at least Lecture 4- Biological Recognition pt. 2 7 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 • INTERNALIZATION: used to down-regulate signals Immobilized cytokines may show more potent signaling due to lack of this down-regulation pathway • One of the first examples- Griffith lab work with epidermal growth factor immobilized on star polymers: Lecture 4 – Biological Recognition pt. 2 7 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 A second example, immobilized insulin( Ito) Ins-PSt 10 Amount of insulin (ug/well) (8N●(N (ns-PAA Fig. 2. Relative growth rate of mouse fibroblast STO cells in the presence of (A) native insulin,(4)Ins-POE (O)Ins-PAA, (O)Ins-PSt. Bars represent standard deviation. N=6. This data interestingly shows several biophysical effects o PEG-insulin not as good as free insulin Steric interference o PAA-insulin better than free insulin Multivalent o Surface-immobilized PAA-insulin better than all above Lack of internalization/signal downregulation? Issues faced in incorporation of cytokines in biomaterials: Protein stability (rugged, but not as good as peptides-may significant secondary structure to worry about Steric interference of tether/surface with receptor binding Growth factors that have been studied in biomaterials EGF Insulin(Y Ito) TGF阝(West) Lecture 4- Biological Recognition pt. 2 8 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 • A second example, immobilized insulin (Ito): This data interestingly shows several biophysical effects: o PEG-insulin not as good as free insulin Steric interference o PAA-insulin better than free insulin Multivalent o Surface-immobilized PAA-insulin better than all above Lack of internalization/signal downregulation? • Issues faced in incorporation of cytokines in biomaterials: Protein stability (rugged, but not as good as peptides- may significant secondary structure to worry about Steric interference of tether/surface with receptor binding • Growth factors that have been studied in biomaterials: EGF Insulin (Y. Ito) TGF-β (West) Lecture 4 – Biological Recognition pt. 2 8 of 9
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 References Voet & Voet in Biochemistry Paredes, N, Rodriguez, G. A& Puiggali, J. Synthesis and characterization of a family of biodegradable poly(ester amide)s derived from glycine Joumal of Polymer Science, Part A: Polymer Chemistry 36, 1271-1282 (1998) Fan,Y, Kobayashi, M.& Kise, H Synthesis and biodegradability of new polyesteramides containing peptide linkages. Polymer Journal 32, 817-822 (2000 4. O,SC& Birkinshaw, C Hydrolysis of poly(n-butylcyanoacrylate)nanoparticles using esterase Polymer Degradation and Stability 78, 7-15(2002) 5. Ekblom, P &Timpl, R Cell-to-cell contact and extracellular matrix. A multifaceted approach emerging. Curr Opin ce∥Bo/8,599-601(1996) 6. Chapman, H A Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration urr Opin Cell Biol 9, 714-24(1997) Mann, B K, Gobin, A S, Tsai, A. T, Schmedlen, R. H& West, J. L Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering. Biomaterials 22, 3045-51(2001) West, J. L& Hubbell, J. A. Polymeric biomaterials with degradation sites for proteases involved in cell migration Macromolecules 32, 241-244 (1999) 9. Gobin, A S& West, J. L Cell migration through defined, synthetic ECM analogs Faseb J16, 751-3(2002 10. Ito, Y. Tissue engineering by immobilized growth factors. Materials Science and Engineering C6, 267-274 (1998) 11. Ito, Y Regulation of cell functions by micropattern-immobilized biosignal molecules. Nanotechnology 9, 200-204 (1998) 12. KuhL, P. R& Griffith-Cima, L G. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat Med 2, 1022-7(1996) Lecture 4- Biological Recognition pt. 2 9 of 9
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 References 1. Voet & Voet. in Biochemistry. 2. Paredes, N., Rodriguez, G. A. & Puiggali, J. Synthesis and characterization of a family of biodegradable poly(ester amide)s derived from glycine. Journal of Polymer Science, Part A: Polymer Chemistry 36, 1271-1282 (1998). 3. Fan, Y., Kobayashi, M. & Kise, H. Synthesis and biodegradability of new polyesteramides containing peptide linkages. Polymer Journal 32, 817-822 (2000). 4. O, S. C. & Birkinshaw, C. Hydrolysis of poly (n-butylcyanoacrylate) nanoparticles using esterase. Polymer Degradation and Stability 78, 7-15 (2002). 5. Ekblom, P. & Timpl, R. Cell-to-cell contact and extracellular matrix. A multifaceted approach emerging. Curr Opin Cell Biol 8, 599-601 (1996). 6. Chapman, H. A. Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr Opin Cell Biol 9, 714-24 (1997). 7. Mann, B. K., Gobin, A. S., Tsai, A. T., Schmedlen, R. H. & West, J. L. Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering. Biomaterials 22, 3045-51 (2001). 8. West, J. L. & Hubbell, J. A. Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules 32, 241-244 (1999). 9. Gobin, A. S. & West, J. L. Cell migration through defined, synthetic ECM analogs. Faseb J 16, 751-3 (2002). 10. Ito, Y. Tissue engineering by immobilized growth factors. Materials Science and Engineering C 6, 267-274 (1998). 11. Ito, Y. Regulation of cell functions by micropattern-immobilized biosignal molecules. Nanotechnology 9, 200-204 (1998). 12. Kuhl, P. R. & Griffith-Cima, L. G. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat Med 2, 1022-7 (1996). Lecture 4 – Biological Recognition pt. 2 9 of 9