Biocompatible Materials Surface Modification (PDF)

376-1714-00L Biocompatible Materials Surface modification of (bio)materials 13.11.2024 Prof. Dr. Katharina Maniura, Empa, Biointerfaces/ D-HEST Dr. Markus Rottmar, Empa, Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, D-HEST, Tissue Engineering & Fabrication November 13, 2024...

376-1714-00L Biocompatible Materials Surface modification of (bio)materials 13.11.2024 Prof. Dr. Katharina Maniura, Empa, Biointerfaces/ D-HEST Dr. Markus Rottmar, Empa, Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, D-HEST, Tissue Engineering & Fabrication November 13, 2024 Katharina Maniura 1  Teaching objectives ▪ You know different techniques to control biomolecule / protein adsorption to biomaterial surfaces ▪ You can describe the advantages and disadvantages of physical adsorption compared to covalent protein coupling ▪ You can name different chemical pathways and relevant functional groups for conjugating biomolecules to surfaces ▪ You know the basic mechanisms guiding the degradation of biomaterial surface coatings BD Ratner et al., “Biomaterials Science”, 3rd edition, Elsevier 2013 P Ducheyne et al., “Comprehensive Biomaterials”, Elsevier 2011 November 13, 2024 Katharina Maniura 2 Surface modifications – key parameters and why shall we care? Specificity? Density? Stability? Activity? Chemical binding Physical adsorption ▪ defines specificity of interactions with biomolecules ▪ defines specific response (e.g. tissue growth) to implanted materials ▪ control over protein adsorption and cell interactions (e.g. adhesion) for in vitro studies November 13, 2024 Katharina Maniura 3  1. Bioactive surface functionalization ▪ Physical adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 4 Physical adsorption of biomolecules - I H Stutz, Electrophoresis 30: 2032-2061 (2009) November 13, 2024 Katharina Maniura 5 Physical adsorption of biomolecules - II All materials adsorb biomolecules in varying amounts due to: G = H − T S ▪ hydrophobic interactions (“lack of interaction with water”) ▪ electrostatic / coulomb interactions (proteins and surfaces are charged; 1/r – 1/r4) ▪ van der Waals interactions (dipole interactions; 1/r6) ▪ π-π bonding / stacking (between aromatic rings) ▪ ion bridging (via divalent metals, e.g. Ca2+, Zn2+) Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 November 13, 2024 Katharina Maniura 6 Control of physical adsorption of biomolecules How can protein adsorption be controlled and enhanced? ▪ electrostatic interactions ▪ introducing charges to biomaterial surface ▪ hydrophobic interaction / entropic interactions ▪ water release / changes in protein mobility ▪ making surface hydrophobic ▪ adding hydrophobic side chains to protein/linker ▪ affinity recognition ▪ adding relevant end group to surface (e.g. biotin->streptavidin) November 13, 2024 Katharina Maniura 7  Physical adsorption – electrostatic interactions I introducing charges to biomaterial surface 1. exposure to oxidizing agent, e.g. Piranha solution (H2O2 + H2SO4) - hydroxylates most surfaces (by adding –OH groups) 2. plasma treatment cleaning: - ionization of gas atoms that act as “molecular sandblast” and break down organic components on the surface activation / chemical reaction: - ionized gas reacts with the surface and creates radicals that can be used for further surface chemistry ▪ both methods increase the surface energy Diener electronic GmbH + Co. KG; plasma.com ▪ they further increase the number of –OH groups on the ▪ surface and therefore the negative charge ▪ this also renders the surface more hydrophilic November 13, 2024 Katharina Maniura 8  Physical adsorption – electrostatic interactions IIa What is a plasma? I+ e- P* e- R I+ I+ R P* e- I+ R R e- e- e- P* P* R a plasma is sometimes called the „fourth state of materials“ A plasma is a partially ionized gas formed at several thousand °C as a material is heated its molecules gain kinetic energy to melt and vaporize Ions and electrons of the plasma can transfer their energy to neutral as more energy is added inelastic collision events lead to molecules to trigger chemical reactions dissociation (homolytic) and later ionization November 13, 2024 Katharina Maniura 9  Physical adsorption – electrostatic interactions IIb functionali- polymerization zation crosslinking etching polymer ▪ plasmas trigger a variety of processes ▪ energy content, type of plasma gas and properties of polymers determine which process dominates ▪ however, plasma processes cannot be designed such that only the desired reaction takes place ▪ post plasma processes (aging) may cause further complications (peroxid formation, surface reorganization,...) November 13, 2024 Katharina Maniura 10 Physical adsorption – electrostatic interactions IIc Plasma surface treatment of biomaterials – examples for applications https://www.pvateplaamerica.c om/application/life-science/ (One RANDOMLY selected example company) November 13, 2024 Katharina Maniura 11 Physical adsorption – electrostatic interactions III introducing charges to biomaterial surface 3. coating with a charged polymer (e.g Poly-L-Lysine) protein - polymer comprised of repeating unit of basic amino acid lysine - positively charged amino groups in side chain bind to OH groups on surface and to negative residues on biopolymers and cell surfaces Matsunami Glass Co., Japan November 13, 2024 Katharina Maniura 12 Control of physical adsorption of biomolecules How can protein adsorption be controlled and enhanced? ▪ Electrostatic interactions ▪ introducing charges to surface (e.g. plasma treatment, charged molecules) ▪ Hydrophobic interaction (e.g. silanization => see next section) ▪ making surface hydrophobic ▪ adding hydrophobic side chains to protein/linker ▪ Affinity recognition (= bioactivation of surface) ▪ adding relevant end group to surface (e.g. biotin->streptavidin; antigen->antibody) to be recognized by specific receptors with high selectivity and affinity November 13, 2024 Katharina Maniura 13  Limitations of physical adsorption ▪ stability: Interaction of adsorbed biomolecules with surface is typically weak and reversible, cells “pull off” protein from surface ▪ specificity: Different molecules will adsorb when the surface is exposed to another medium (e.g. coating solution => serum) ▪ activity: Orientation of adsorbed biomolecules is typically random  need to increase affinity by binding biomolecules covalently, with specific conjugation and controlled orientation Comprehensive Biomaterials II, 2017, chapter 4.13 November 13, 2024 Katharina Maniura 14 1. Bioactive surface functionalization ▪ Physical protein adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 15  Covalent coupling of biomolecules to materials advantages: ▪ stable, specific immobilization ▪ applicable also for small oligopeptides that do not easily adsorb ▪ control of orientation possible disadvantages: ▪ requires appropriate functional groups on both, the material and the biomolecule surfaces ▪ more expensive and often low throughput November 13, 2024 Katharina Maniura 16 (Bio-)conjugation chemistry ▪ Relevant functional groups: ▪ Hydroxyl (-OH) ▪ Amino (-NH2) ▪ Carbonyl (-CO, -CHO) ▪ Carboxyl (-COOH) ▪ Thiol (-SH) ▪ Phosphate (-PO4) ▪ Vinyl (-CH=CH2) https://www.chemistryhelpcenter.org/ there are many possible combinations of surface groups, coupling agents, intermediates and reacting groups to covalently couple the same biomolecule => several books on Bioconjugation protocols available e.g. Bioconjugate Techniques, GT Hermanson, Elsevier November 13, 2024 Katharina Maniura 17 Methods to create reactive groups on surfaces ▪ ionizing radiation ▪ plasma gas discharge / plasma activation ▪ photochemistry ▪ ozone grafting ▪ chemical derivatization ▪ polymeric adlayers (e.g PLL) ▪ CVD, PVD deposition of other surface coatings (e.g metals) ▪ silanization November 13, 2024 Katharina Maniura 18 Silanization of biomaterials ▪ Coupling of Silane (Si compound) to hydroxylated (-OH) surface ▪ Glass, silicon, PDMS, aluminum, titanium, quartz are all rich in hydroxyl (-OH) groups ▪ Large variety of possible functionalities / silane linkers ▪ Simple, fast, low cost, and stable (can be hydrolyzed though) X leaves the compound after reaction R is the functionality (e.g. amino-group) November 13, 2024 Katharina Maniura 19 Silanization surface modification reaction November 13, 2024 Katharina Maniura 20 Surface coupling of biomolecules using a spacer ▪ bioreactive end group on one side ▪ covalent bonding to surface on other end advantages: ▪ steric freedom ▪ accessibility ▪ modularity ▪ tunable degradability typical spacer: variable-length PEG linker (more in 2nd part) November 13, 2024 Katharina Maniura 21 Comparison of surface immobilization strategies November 13, 2024 Katharina Maniura 22 Example: Albumin e.g. PLL T Hattori et al., Anal. Biochem. 295: 158-167 (2001) Physical Adsorption Covalent Tethering November 13, 2024 Katharina Maniura 23 Bioinspired strategies- using attraction to create 3D materials: nacre (mother of pearl) Tang Z, Kotov NA, Magonov S, Ozturk B. Nanostructured artificial nacre. Nat Mater. 2003;2(6):413–8. Layer-by-layer (LBL) deposition of clay (-) and PDDA (poly(diallydimethylammonium) chloride) polycation (+) to coat hydrophilic 3D polyacrylamide hydrogel structure with a stiff, transparent cell-adhesive surface. November 13, 2024 Katharina Maniura 24 Bioinspired strategies: mussle adhesion and DOPA 2013 Also for anti-fouling surfaces => second part November 13, 2024 Katharina Maniura 25 1. Bioactive surface functionalization ▪ Physical protein adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 26 Motivation – Surface fouling http://www.hypertextbookshop.com/biofilmbook/v004/r003/index.html November 13, 2024 Katharina Maniura 27 Motivation – what are anti-adhesive surfaces good for? November 13, 2024 Katharina Maniura 28 Surface passivation - facts ▪ ideal non-fouling, biopassive surfaces resist protein adsorption ▪ low protein adsorption means low cell adhesion ▪ hydrogels are typically non-fouling, whereas hydrophobic surfaces are typically fouling ▪ non-fouling surfaces often have strong interactions with water (strong hydration of surface or tightly bound water within polymer layer that shields the surface charges and extend hydrophilic polymer chains) ▪ most body surfaces (e.g. cell membranes, ECM, mucosae) adsorb very low amounts of proteins November 13, 2024 Katharina Maniura 29 How to avoid protein adsorption? ▪ reduce enthalpic interactions (mainly electrostatic, vdW, ionic) ▪ add entropic penalty to surface (mixing entropy; hydrophobic) How can Surface Chemistry How can Surfacehelp to Chemistry help to prevent protein adsorption? prevent protein adsorption? November 13, 2024 Katharina Maniura 30 1. Bioactive surface functionalization ▪ Physical protein adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 31 Self-assembled monolayers (SAM) spontaneous formation of alkanethiol layer as highly ordered structures (2D crystal) stabilized by van der Waals interactions between chains precise control over anchor and functional head group shields proteins from the surface combination with lipid layers possible compatible with patterning methods and textured material surfaces November 13, 2024 Katharina Maniura 32 1. Bioactive surface functionalization ▪ Physical protein adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 33 Supported lipid bilayers (SLB) Origin of passivating properties: zwitterionic nature of the lipid head groups, which make the SLB electrically neutral in a large pH range (3 < pH < 10) polar, hydrophilic lipid head group faces the liquid solution nonpolar, hydrophobic tail (e.g fatty acid for phospolipids) buried within bilayer Lipids in SLB are generally free to diffuse laterally as long as the temperature is above the phase transition temperature Low polarizability of hydrocarbon chains of the lipid molecules Castellana ET, Cremer PS. Solid supported lipid bilayers: From biophysical studies to sensor design. Surface Science Reports. 2006;61(10):429–44. November 13, 2024 Katharina Maniura 34 Fabrication of supported lipid bilayers Castellana ET, Cremer PS. Solid supported lipid bilayers: From biophysical studies to sensor design. Surface Science Reports. 2006;61(10 ):429–44. November 13, 2024 Katharina Maniura 35 1. Bioactive surface functionalization ▪ Physical protein adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 36 Polymer brushesbrushes Polymer The entropic penalty Further stretching: Loss of entropy of polymer chain conformations Compression and water exclusion: Loss of mixing entropy of polymer and water courtesy of Dr. R. Konradi November 13, 2024 Katharina Maniura 37  Polymer brushes PolymerDefinition brushes Terminally anchored polymer chains: three possibilities „mushroom" „pancake" „brush" D Anchor distance Polymer brush if D < 2Rg (anchor distance < polymer coil diameter) courtesy Oct. of Dr. R. Konradi 2nd 2012 November 13, 2024 Katharina Maniura 38 Polymer Polymer brushes brushes Synthesis: "grafting to" versus "grafting from" "grafting to" "grafting from“ = surface initiated polymerization (SIP) Kinetic adsorption barrier Only monomer diffuses to growing chain Rather low grafting densities end => no kinetic barrier Rather thin layers (< 5 nm) High grafting density Functional Polymers can be Thick layers (up to 2000 nm) synthesized before deposition Initiator synthesis necessary Separate polymerization for each surface courtesy of Dr. R. Konradi Oct. 2nd 2012 November 13, 2024 Katharina Maniura 39  PLL-g-PEG: Poly(L-Lysine)-graft-poly(ethylene glycol) Grafting to systems: self assembly graft copolymers h coatings opolymers ▪ detailed structure-property relationship protein-resistant known Antiadhesive polymer, e.g. polymer brush coatings Grafting to systems: Self-assembly graft copolymers poly(ethylene ▪ large variety of polymer variations glycol), PEG available protein-resistant ▪ backbone length O O polymer, e.g. poly(ethylene▪ backbone composition glycol), PEG ▪ PEG chain length _ _ ▪ side chain grafting density polycationic O O ▪ end-group functionalization backbone, e.g. poly(L-lysine), PLL for biofunctionalization vely charged surface + H3 N + H3 N + H3N _ _ _ _ _ ▪ ideally suited for surface patterning / H O H O + ily characterized and stored H3 N O N N H O N N H O OH passivation NH3+ NH3+ self-assembly on negatively charged surface ▪ easy ”dip-and-rinse” ed surfaces Pasche, de Paul, Vörös, Spencer, Textor. Langmuir, 2003, (19), 9216-9225 application on negatively charged bulk graft-copolymers can be readily characterized and stored surfaces possible November 13, 2024 simple dip-and-rinse procedure Katharina Maniura 40  PLL-g-PEG: Poly(L-Lysine)-graft-poly(ethylene glycol) Examples for applications: cell culture platforms https://www.youtube.com/watch?v=CLYKpSEEPd8 https://www.youtube.com/watch?v=0mWP6z8_TJA (RANDOMLY selected example companies) November 13, 2024 Katharina Maniura 41 PLL-g-PEG: Poly(L-Lysine)-graft-poly(ethylene glycol) November 13, 2024 Katharina Maniura 42 Other anti-adhesive polymer Other anti-adhesive architectures polymer architectures Brushes are not the only way Dalsin, Feller, Macromolecules, Macromolecules, 2005 2005 PEG SAMs Pluronics, PEG-PPS-PEG O O OH Wu, Coll.Surf.B, 2000 O O Zhang, Langmuir, 2003 O O Wu, Plasmas and Polymers, 2001 O O O O O Shen, J. Biomater. Sci. Polymer Edn, 2001 Siegers, Chem. Eur. J., 2004 PEG-like Plasma polymers Hyperbranched Polyglycerols Groll, Langmuir, 2005 O O O n O Oct. 2nd 2012 PEG Hydrogels Star PEG courtesy of Dr. R. Konradi November 13, 2024 Katharina Maniura 43 Overview of antifoulding strategies – different chemistries November 13, 2024 Katharina Maniura 44 Overview of antifouling strategies – different architecture November 13, 2024 Katharina Maniura 45 Summary on surface passivation ▪ important to consider both enthalpic as well as entropic contributions to bioadhesion as well as interaction with solvent ▪ PEG is “the” classical anti-fouling polymer ▪ self assembled monolayers (SAM) allow precise control over surface properties by functional head / end group ▪ supported Lipid Bilayers (SLB) mimic the cell membrane and are intrinsically anti- fouling ▪ polymer brushes can be coupled electrostatically or covalently, offer variable functionality, are relatively simple to make, and make use of both entropic and enthalpic forces to repel proteins November 13, 2024 Katharina Maniura 46 1. Bioactive surface functionalization ▪ Physical protein adsorption ▪ Chemical conjugation ▪ Bioinspired functionalization 2. Passivation: anti-fouling surfaces ▪ Self-assembled monolayers (SAM) ▪ Supported lipid bilayers (SLB) ▪ Polymer brushes 3. Stability and degradability November 13, 2024 Katharina Maniura 47 Stability of functionalized surfaces Degradation by passive desorption of the biomolecule: ▪ typically, by hydrolytic cleavage ▪ depends on accessibility of bonds by water Degradation by active processes (biodegradation) ▪ cells adhere and pull off adsorbed proteins ▪ enzymatic degradation of bonds ▪ can be controlled by introducing cleavable bonds Degradation of the bulk substrate can also lead to release of the biomolecules on the surface! November 13, 2024 Katharina Maniura 48  Thank you for your attention November 13, 2024 Katharina Maniura 49