Layer-by-layer (LbL) polyelectrolyte coatings are intensively studied as reservoirs of bioactive proteins for modulating interactions between biomaterial surfaces and cells. Mild conditions for the incorporation of growth factors into delivery systems are required to maintain protein bioactivity. Here, we present LbL films composed of water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC), heparin (Hep), and tannic acid (TA) fabricated under physiological conditions with the ability to release heparin-binding proteins. Surface plasmon resonance analysis showed that the films formed on an anchoring HTCC/TA bilayer, with TA serving as a physical crosslinker, were more stable during their assembly, leading to increased film thickness and increased protein release. X-ray reflectivity measurements confirmed intermixing of the deposited layers. Protein release also increased when the proteins were present as an integral part of the Hep layers rather than as individual protein layers. The 4-week release pattern depended on the protein type; VEGF, CXCL12, and TGF-β1 exhibited a typical high initial release, whereas FGF-2 was sustainably released over 4 weeks. Notably, the films were nontoxic, and the released proteins retained their bioactivity, as demonstrated by the intensive chemotaxis of T-lymphocytes in response to the released CXCL12. Therefore, the proposed LbL films are promising biomaterial coating candidates for stimulating cellular responses.

Department of Physical Chemistry and Biophysics Pharmaceutical Faculty Wroclaw Medical University Borowska 211 50 556 Wrocław Poland

Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovsky Square 2 162 06 Prague Czech Republic

Jan Purkyňe University in Ústí nad Labem Faculty of Science Pasteurova 1 400 96 Ústí nad Labem Czech Republic

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Ferreira P.; Alves P.; Coimbra P.; Gil M. H. Improving polymeric surfaces for biomedical applications: a review. J. Coat. Technol. Res. 2015, 12, 463–475. 10.1007/s11998-015-9658-3. DOI

Costa R. R.; Mano J. F. Polyelectrolyte multilayered assemblies in biomedical technologies. Chem. Soc. Rev. 2014, 43, 3453–3479. 10.1039/c3cs60393h. PubMed DOI

Wang Z.; Wang Z.; Lu W. W.; Zhen W.; Yang D.; Peng S. Novel biomaterial strategies for controlled growth factor delivery for biomedical applications. NPG Asia Mater. 2017, 9, e43510.1038/am.2017.171. DOI

Bjelić D.; Finšgar M. The Role of Growth Factors in Bioactive Coatings. Pharmaceutics 2021, 13, 108310.3390/pharmaceutics13071083. PubMed DOI PMC

Zhang D.; Chen Q.; Shi C.; Chen M.; Ma K.; Wan J.; Liu R. Dealing with the Foreign-Body Response to Implanted Biomaterials: Strategies and Applications of New Materials. Adv. Funct. Mater. 2021, 31, 200722610.1002/adfm.202007226. DOI

Richardson J. J.; Cui J.; Björnmalm M.; Braunger J. A.; Ejima H.; Caruso F. Innovation in Layer-by-Layer Assembly. Chem. Rev. 2016, 116, 14828–14867. 10.1021/acs.chemrev.6b00627. PubMed DOI

Park S.; Han U.; Choi D.; Hong J. Layer-by-layer assembled polymeric thin films as prospective drug delivery carriers: design and applications. Biomater. Res. 2018, 22, 2910.1186/s40824-018-0139-5. PubMed DOI PMC

Hammond P. T. Building biomedical materials layer-by-layer. Mater. Today 2012, 15, 196–206. 10.1016/S1369-7021(12)70090-1. DOI

Han U.; Park H. H.; Kim Y. J.; Park T. H.; Park J. H.; Hong J. Efficient Encapsulation and Sustained Release of Basic Fibroblast Growth Factor in Nanofilm: Extension of the Feeding Cycle of Human Induced Pluripotent Stem Cell Culture. ACS Appl. Mater. Interfaces 2017, 9, 25087–25097. 10.1021/acsami.7b05519. PubMed DOI

Hsu B. B.; Hagerman S. R.; Jamieson K.; Veselinovic J.; O’Neill N.; Holler E.; Ljubimova J. Y.; Hammond P. T. Multilayer films assembled from naturally-derived materials for controlled protein release. Biomacromolecules 2014, 15, 2049–2057. 10.1021/bm5001839. PubMed DOI PMC

Naves A. F.; Motay M.; Merindol R.; Davi C. P.; Felix O.; Catalani L. H.; Decher G. Layer-by-Layer assembled growth factor reservoirs for steering the response of 3T3-cells. Colloids Surf., B 2016, 139, 79–86. 10.1016/j.colsurfb.2015.11.019. PubMed DOI

Zhang K.; Huang D.; Yan Z.; Wang C. Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering. J. Biomed. Mater. Res., Part A 2017, 105, 1900–1910. 10.1002/jbm.a.36053. PubMed DOI

Dalonneau F.; Liu X. Q.; Sadir R.; Almodovar J.; Mertani H. C.; Bruckert F.; Albiges-Rizo C.; Weidenhaupt M.; Lortat-Jacob H.; Picart C. The effect of delivering the chemokine SDF-1α in a matrix-bound manner on myogenesis. Biomaterials 2014, 35, 4525–4535. 10.1016/j.biomaterials.2014.02.008. PubMed DOI PMC

Antunes J. C.; Pereira C. L.; Molinos M.; Ferreira-da-Silva F.; Dessì M.; Gloria A.; Ambrosio L.; Gonçalves R. M.; Barbosa M. A. Layer-by-Layer Self-Assembly of Chitosan and Poly(γ-glutamic acid) into Polyelectrolyte Complexes. Biomacromolecules 2011, 12, 4183–4195. 10.1021/bm2008235. PubMed DOI

Köwitsch A.; Zhou G.; Groth T. Medical application of glycosaminoglycans: a review. J. Tissue Eng. Regener. Med. 2018, 12, e23–e41. 10.1002/term.2398. PubMed DOI

Hu B.; Guo Y.; Li H.; Liu X.; Fu Y.; Ding F. Recent advances in chitosan-based layer-by-layer biomaterials and their biomedical applications. Carbohydr. Polym. 2021, 271, 11842710.1016/j.carbpol.2021.118427. PubMed DOI

Ori A.; Wilkinson M. C.; Fernig D. G. A Systems Biology Approach for the Investigation of the Heparin/Heparan Sulfate Interactome. J. Biol. Chem. 2011, 286, 19892–19904. 10.1074/jbc.M111.228114. PubMed DOI PMC

Hachim D.; Whittaker T. E.; Kim H.; Stevens M. M. Glycosaminoglycan-based biomaterials for growth factor and cytokine delivery: Making the right choices. J. Controlled Release 2019, 313, 131–147. 10.1016/j.jconrel.2019.10.018. PubMed DOI PMC

Ye X. F.; Hu X.; Wang H. Z.; Liu J.; Zhao Q. Polyelectrolyte multilayer film on decellularized porcine aortic valve can reduce the adhesion of blood cells without affecting the growth of human circulating progenitor cells. Acta Biomater. 2012, 8, 1057–1067. 10.1016/j.actbio.2011.11.011. PubMed DOI

Zhang J.; Wang D. W.; Jiang X. F.; He L.; Fu L.; Zhao Y.; Wang Y. T.; Mo H.; Shen J. Multistructured vascular patches constructed via layer-by-layer self-assembly of heparin and chitosan for vascular tissue engineering applications. Chem. Eng. J. 2019, 370, 1057–1067. 10.1016/j.cej.2019.03.270. DOI

Meng S.; Liu Z.; Shen L.; Guo Z.; Chou L. L.; Zhong W.; Du Q.; Ge J. The effect of a layer-by-layer chitosan-heparin coating on the endothelialization and coagulation properties of a coronary stent system. Biomaterials 2009, 30, 2276–2283. 10.1016/j.biomaterials.2008.12.075. PubMed DOI

Li L.; Wang X.; Li D.; Qin J.; Zhang M.; Wang K.; Zhao J.; Zhang L. LBL deposition of chitosan/heparin bilayers for improving biological ability and reducing infection of nanofibers. Int. J. Biol. Macromol. 2020, 154, 999–1006. 10.1016/j.ijbiomac.2020.03.152. PubMed DOI

Bhalerao U. M.; Valiveti A. K.; Acharya J.; Halve A. K.; Kaushik M. P. Controlled release studies of antimalarial 1, 3, 5-trisubstituted-2-pyrazolines from biocompatible chitosan-heparin Layer-by-Layer (LbL) self assembled thin films. Colloid Surf., B 2015, 125, 151–159. 10.1016/j.colsurfb.2014.11.016. PubMed DOI

Delechiave G.; Naves A. F.; Kolanthai E.; da Silva R. A.; Vlasman R. C.; Petri D. F. S.; Torresi R. M.; Catalani L. H. Tuning protein delivery from different architectures of layer-by-layer assemblies on polymer films. Mater. Adv. 2020, 1, 2043–2056. 10.1039/d0ma00432d. DOI

Andreica B.-I.; Cheng X.; Marin L. Quaternary ammonium salts of chitosan. A critical overview on the synthesis and properties generated by quaternization. Eur. Polym. J. 2020, 139, 11001610.1016/j.eurpolymj.2020.110016. DOI

Freitas E. D.; Moura C. F. Jr.; Kerwald J.; Beppu M. M. An Overview of Current Knowledge on the Properties, Synthesis and Applications of Quaternary Chitosan Derivatives. Polymers 2020, 12, 287810.3390/polym12122878. PubMed DOI PMC

Li T.; Shi X. W.; Du Y. M.; Tang Y. F. Quaternized chitosan/alginate nanoparticles for protein delivery. J. Biomed. Mater. Res., Part A 2007, 83A, 383–390. 10.1002/jbm.a.31322. PubMed DOI

Zhao S.-H.; Wu X.-T.; Guo W.-C.; Du Y.-M.; Yu L.; Tang J. N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a novel delivery system for Parathyroid Hormone-Related Protein 1–34. Int. J. Pharm. 2010, 393, 269–273. 10.1016/j.ijpharm.2010.04.034. PubMed DOI

Yuan R. Q.; Yang N.; Fan S. W.; Huang Y. R.; You D.; Wang J. R.; Zhang Q. L.; Chu C. L.; Chen Z. Z.; Liu L.; Ge L. Q. Biomechanical Motion-Activated Endogenous Wound Healing through LBL Self-Powered Nanocomposite Repairer with pH-Responsive Anti-Inflammatory Effect. Small 2021, 17, 210399710.1002/smll.202103997. PubMed DOI

Huang W.; Li X.; Xue Y.; Huang R.; Deng H.; Ma Z. Antibacterial multilayer films fabricated by LBL immobilizing lysozyme and HTCC on nanofibrous mats. Int. J. Biol. Macromol. 2013, 53, 26–31. 10.1016/j.ijbiomac.2012.10.024. PubMed DOI

Kumorek M.; Kubies D.; Riedel T. Protein Interactions With Quaternized Chitosan/Heparin Multilayers. Physiol. Res. 2016, 65, S253–S261. 10.33549/physiolres.933427. PubMed DOI

Kumorek M.; Minisy I. M.; Krunclova T.; Vorsilakova M.; Venclikova K.; Chanova E. M.; Janouskova O.; Kubies D. pH-responsive and antibacterial properties of self-assembled multilayer films based on chitosan and tannic acid. Mater. Sci. Eng., C 2020, 109, 11049310.1016/j.msec.2019.110493. PubMed DOI

Crouzier T.; Ren K.; Nicolas C.; Roy C.; Picart C. Layer-by-layer films as a biomimetic reservoir for rhBMP-2 delivery: controlled differentiation of myoblasts to osteoblasts. Small 2009, 5, 598–608. 10.1002/smll.200800804. PubMed DOI

Shah N. J.; Macdonald M. L.; Beben Y. M.; Padera R. F.; Samuel R. E.; Hammond P. T. Tunable dual growth factor delivery from polyelectrolyte multilayer films. Biomaterials 2011, 32, 6183–6193. 10.1016/j.biomaterials.2011.04.036. PubMed DOI PMC

Gospodarowicz D.; Cheng J. Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol. 1986, 128, 475–484. 10.1002/jcp.1041280317. PubMed DOI

Adrian E.; Treľová D.; Filová E.; Kumorek M.; Lobaz V.; Poreba R.; Janoušková O.; Pop-Georgievski O.; Lacík I.; Kubies D. Complexation of CXCL12, FGF-2 and VEGF with Heparin Modulates the Protein Release from Alginate Microbeads. Int. J. Mol. Sci. 2021, 22, 1166610.3390/ijms222111666. PubMed DOI PMC

Chen C.; Yang H.; Yang X.; Ma Q. Tannic acid: a crosslinker leading to versatile functional polymeric networks: a review. RSC Adv. 2022, 12, 7689–7711. 10.1039/D1RA07657D. PubMed DOI PMC

Cho J.; Grant J.; Piquette-Miller M.; Allen C. Synthesis and Physicochemical and Dynamic Mechanical Properties of a Water-Soluble Chitosan Derivative as a Biomaterial. Biomacromolecules 2006, 7, 2845–2855. 10.1021/bm060436s. PubMed DOI

Cui D.; Szarpak A.; Pignot-Paintrand I.; Varrot A.; Boudou T.; Detrembleur C.; Jérôme C.; Picart C.; Auzély-Velty R. Contact-Killing Polyelectrolyte Microcapsules Based on Chitosan Derivatives. Adv. Funct. Mater. 2010, 20, 3303–3312. 10.1002/adfm.201000601. DOI

Hoque J.; Adhikary U.; Yadav V.; Samaddar S.; Konai M. M.; Prakash R. G.; Paramanandham K.; Shome B. R.; Sanyal K.; Haldar J. Chitosan Derivatives Active against Multidrug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials. Mol. Pharmaceutics 2016, 13, 3578–3589. 10.1021/acs.molpharmaceut.6b00764. PubMed DOI

Ruihua H.; Bingchao Y.; Zheng D.; Wang B. Preparation and characterization of a quaternized chitosan. J. Mater. Sci. 2012, 47, 845–851. 10.1007/s10853-011-5862-4. DOI

Richert L.; Lavalle P.; Payan E.; Shu X. Z.; D P. G.; Stoltz J. F.; Schaaf P.; Voegel J. C.; Picart C. Layer by Layer Buildup of Polysaccharide Films Physical Chemistry and Cellular Adhesion Aspects. Langmuir 2004, 20, 448–458. 10.1021/la035415n. PubMed DOI

Kujawa P.; Moraille P.; Sanchez J.; Badia A.; Winnik F. M. Effect of Molecular Weight on the Exponential Growth and Morphology of HyaluronanChitosan Multilayers A Surface Plasmon Resonance Spectroscopy and Atomic Force Microscopy Investigation. J. Am. Chem. Soc. 2005, 127, 9224–9234. 10.1021/ja044385n. PubMed DOI

Kovacevic D.; van der Burgh S.; de Keizer A.; Cohen Stuart M. A. Kinetics of Formation and Dissolution of Weak Polyelectrolyte Multilayers: Role of Salt and Free Polyions. Langmuir 2002, 18, 5607–5612. 10.1021/la025639q. DOI

Picart C.; Mutterer J.; Richert L.; Luo Y.; Prestwich G. D.; Schaaf P.; Voegel J. C.; Lavalle P. Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12531–12535. 10.1073/pnas.202486099. PubMed DOI PMC

Rivero S.; García M. A.; Pinotti A. Crosslinking capacity of tannic acid in plasticized chitosan films. Carbohydr. Polym. 2010, 82, 270–276. 10.1016/j.carbpol.2010.04.048. DOI

Sun Q.; Tong Z.; Wang C.; Ren B.; Liu X.; Zeng F. Charge density threshold for LbL self-assembly and small molecule diffusion in polyelectrolyte multilayer films. Polymer 2005, 46, 4958–4966. 10.1016/j.polymer.2005.03.091. DOI

Lavalle P.; Picart C.; Mutterer J.; Gergely C.; Reiss H.; Voegel J. C.; Senger B.; Schaaf P. Modeling the buildup of polyelectrolyte multilayer films having exponential growth. J. Phys.Chem. B 2004, 108, 635–648. 10.1021/jp035740j. DOI

Ghiselli G. Heparin Binding Proteins as Therapeutic Target: An Historical Account and Current Trends. Medicines 2019, 6, 8010.3390/medicines6030080. PubMed DOI PMC

Velk N.; Uhlig K.; Vikulina A.; Duschl C.; Volodkin D. Mobility of lysozyme in poly(l-lysine)/hyaluronic acid multilayer films. Colloids Surf., B 2016, 147, 343–350. 10.1016/j.colsurfb.2016.07.055. PubMed DOI

Vogt C.; Ball V.; Mutterer J.; Schaaf P.; Voegel J.-C.; Senger B.; Lavalle P. Mobility of Proteins in Highly Hydrated Polyelectrolyte Multilayer Films. J. Phys. Chem. B 2012, 116, 5269–5278. 10.1021/jp300028v. PubMed DOI

Kujawa P.; Sanchez J.; Badia A.; Winnik F. M. Probing the stability of biocompatible sodium hyaluronate/chitosan nanocoatings against changes in salinity and pH. J. Nanosci. Nanotechnol. 2006, 6, 1565–1574. 10.1166/jnn.2006.217. PubMed DOI

Almodóvar J.; Place L. W.; Gogolski J.; Erickson K.; Kipper M. J. Layer-by-layer assembly of polysaccharide-based polyelectrolyte multilayers: a spectroscopic study of hydrophilicity, composition, and ion pairing. Biomacromolecules 2011, 12, 2755–2765. 10.1021/bm200519y. PubMed DOI

Lundin M.; Blomberg E.; Tilton R. D. Polymer Dynamics in Layer-by-Layer Assemblies of Chitosan and Heparin. Langmuir 2010, 26, 3242–3251. 10.1021/la902968h. PubMed DOI

Lundin M.; Solaqa F.; Thormann E.; Macakova L.; Blomberg E. Layer-by-Layer Assemblies of Chitosan and Heparin: Effect of Solution Ionic Strength and pH. Langmuir 2011, 27, 7537–7548. 10.1021/la200441u. PubMed DOI

Wang Q. Z.; Chen X. G.; Liu N.; Wang S. X.; Liu C. S.; Meng X. H.; Liu C. G. Protonation constants of chitosan with different molecular weight and degree of deacetylation. Carbohydr. Polym. 2006, 65, 194–201. 10.1016/j.carbpol.2006.01.001. DOI

Izumrudov V.; Sukhishvili S. A. Ionization Controlled Stability of Polyelectrolyte Multilayers in Salt Solutions. Langmuir 2003, 19, 5188–5191. 10.1021/la034360m. DOI

Anouz R.; Repanas A.; Schwarz E.; Groth T. Novel Surface Coatings Using Oxidized Glycosaminoglycans as Delivery Systems of Bone Morphogenetic Protein 2 (BMP-2) for Bone Regeneration. Macromol. Biosci. 2018, 18, e180028310.1002/mabi.201800283. PubMed DOI

Juang R.-S.; Su X.; Lee I.-C. Feasibility Assessment of Parathyroid Hormone Adsorption by Using Polysaccharide-Based Multilayer Film Systems. Polymers 2021, 13, 207010.3390/polym13132070. PubMed DOI PMC

Lvov Y.; Ariga K.; Onda M.; Ichinose I.; Kunitake T. A careful examination of the adsorption step in the alternate layer-by-layer assembly of linear polyanion and polycation. Colloids Surf., A 1999, 146, 337–346. 10.1016/S0927-7757(98)00789-4. DOI

Chen J.; Luo G.; Cao W. The Study of Layer-by-Layer Ultrathin Films by the Dynamic Contact Angle Method. J. Colloid Interface Sci. 2001, 238, 62–69. 10.1006/jcis.2001.7460. PubMed DOI

Lourenço J. M.; Ribeiro P. A.; do Rego A. M. B.; Raposo M. Counterions in layer-by-layer films - Influence of the drying process. J. Colloid Interface Sci. 2007, 313, 26–33. 10.1016/j.jcis.2007.04.040. PubMed DOI

Silva H. S.; Uehara T. M.; Bergamaski K.; Miranda P. B. Molecular Ordering in Layer-by-Layer Polyelectrolyte Films Studied by Sum-Frequency Vibrational Spectroscopy: The Effects of Drying Procedures. J. Nanosci. Nanotechnol. 2008, 8, 3399–3405. 10.1166/jnn.2008.125. PubMed DOI

Björck M.; Andersson G. GenX: an extensible X-ray reflectivity refinement program utilizing differential evolution. J. Appl. Crystallogr. 2007, 40, 1174–1178. 10.1107/S0021889807045086. DOI

Decher G.; Lvov Y.; Schmitt J. Proof of multilayer structural organization in self-assembled polycation polyanion molecular films. Thin Solid Films 1994, 244, 772–777. 10.1016/0040-6090(94)90569-x. DOI

Picart C.; Lavalle P.; Hubert P.; Cuisinier F. J. G.; Decher G.; Schaaf P.; Voegel J. C. Buildup Mechanism for Poly(l-lysine)/Hyaluronic Acid Films onto a Solid Surface. Langmuir 2001, 17, 7414–7424. 10.1021/la010848g. DOI

Zhao N.; Yang C.; Wang Y.; Zhao B.; Bian F.; Li X.; Wang J. Probing the surface microstructure of layer-by-layer self-assembly chitosan/poly(l-glutamic acid) multilayers: A grazing-incidence small-angle X-ray scattering study. Mater. Sci. Eng., C 2016, 58, 352–358. 10.1016/j.msec.2015.08.048. PubMed DOI

Gonçalves R. M.; Antunes J. C.; Barbosa M. A. Mesenchymal stem cell recruitment by stromal derived factor-1-delivery systems based on chitosan/poly(γ-glutamic acid) polyelectrolyte complexes. Eur. Cells Mater. 2012, 23, 249–260. 10.22203/ecm.v023a19. PubMed DOI

Peysselon F.; Ricard-Blum S. Heparin–protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis. Matrix Biol. 2014, 35, 73–81. 10.1016/j.matbio.2013.11.001. PubMed DOI

Miller T.; Goude M. C.; McDevitt T. C.; Temenoff J. S. Molecular engineering of glycosaminoglycan chemistry for biomolecule delivery. Acta Biomater. 2014, 10, 1705–1719. 10.1016/j.actbio.2013.09.039. PubMed DOI PMC

Cardoso A. P.; Goncalves R. M.; Antunes J. C.; Pinto M. L.; Pinto A. T.; Castro F.; Monteiro C.; Barbosa M. A.; Oliveira M. J. An interferon-gamma-delivery system based on chitosan/poly(gamma-glutamic acid) polyelectrolyte complexes modulates macrophage-derived stimulation of cancer cell invasion in vitro. Acta Biomater. 2015, 23, 157–171. 10.1016/j.actbio.2015.05.022. PubMed DOI

Kulkarni A.; Diehl-Jones W.; Ghanbar S.; Liu S. Layer-by-layer assembly of epidermal growth factors on polyurethane films for wound closure. J. Biomater. Appl. 2014, 29, 278–290. 10.1177/0885328214523058. PubMed DOI

Macdonald M. L.; Rodriguez N. M.; Shah N. J.; Hammond P. T. Characterization of Tunable FGF-2 Releasing Polyelectrolyte Multilayers. Biomacromolecules 2010, 11, 2053–2059. 10.1021/bm100413w. PubMed DOI PMC

Song A.; Jiang A.; Xiong W.; Zhang C. The Role of CXCL12 in Kidney Diseases: A Friend or Foe?. Kidney Dis. 2021, 7, 176–185. 10.1159/000514913. PubMed DOI PMC

Ottoson N. C.; Pribila J. T.; Chan A. S.; Shimizu Y. Cutting edge: T cell migration regulated by CXCR4 chemokine receptor signaling to ZAP-70 tyrosine kinase. J. Immunol. 2001, 167, 1857–1861. 10.4049/jimmunol.167.4.1857. PubMed DOI

Hong J.; Kim B. S.; Char K.; Hammond P. T. Inherent Charge-Shifting Polyelectrolyte Multilayer Blends: A Facile Route for Tunable Protein Release from Surfaces. Biomacromolecules 2011, 12, 2975–2981. 10.1021/bm200566k. PubMed DOI