Bio-functional Surfaces Biomaterial-Protein Interaction

1. Bio-functional Surfaces & Biomaterial-Protein Interaction Lecture 14 March 5, 2009

2. Lecture Topics Two parts to lecture I. Biologically functional materials (2.16) A distinct class of biomaterials created through physical and/or chemical synthesis of a synthetic material (usually a polymer) with biomolecules (enzymes, antibodies, drugs, cells, etc.) More details to material introduced in Lecture 9 II. Biomaterial-Protein Interactions (2.13, 3.1, 3.2) Proteins adsorb to materials almost instantly (<1sec), well before cells arrive When cells arrive, they see a protein layer, not the actual biomaterial surface Thus, protein films control the short term bioreactions to the implant

3. Biologically Functional Surfaces Generally polymer substrates are used Solid polymers and soluble polymers useful as immobilization supports for covalent binding of biomolecules Contain reactive groups or can be readily modified with reactive groups Polymers can be manufactured into many forms: Particulates Fibers Fabrics Membranes Hydrogels can be used to immobilize biomolecules within the aqueous pores of the polymer gel network

4. Immobilization Definition: Immobilization is a temporary or permanent localization of biomolecules on or within a support Physical or chemical immobilization can lead to permanent attachment Large biomolecules can be permanently physically immobilized If the support is biodegradable, the biomolecule can be released as the matrix erodes Drugs

5. Types of Biologically Active Molecules (I) Proteins/peptides: Substances composed of amino acids Enzymes, antibodies, antigens, Cell adhesion molecules, �Blocking� Proteins Saccharides: Carbohydrates, primarily sugars Sugars, Oligosaccharides, Polysaccharides Lipids: Any of a group of fats or fat-like substances, characterized by their insolubility in water and solubility in fat solvents such as alcohol, ether, and chloroform. Fatty acids, Phospholipids, Glycolipids Drugs: Antithrombogenic agents, anticancer agents, antibiotics, contraceptives, drug antagonists, peptide/protein drugs

6. Types of Biologically Active Molecules (II) Ligands: In immunology, small molecules that are bound to another chemical group or molecule Hormone receptors, cell surface receptors, avidin, biotin Nucleic acids, nucleotides: High MW substances with complex chemical structure formed of sugars, phosphoric acid, and nitrogen bases (purines and pyrimidines); found in all living things DNA, RNA Other: Conjugates or mixtures of any of the above In addition to molecules, living cells and microorganisms can be immobilized

7. Applications of Immobilized Biomolecules and Cells Enzymes: Bioreactors, Bioseparators, Biosensors, Diagnostic Assays, Biocompatible Surfaces Antibodies, peptides, and other affinity molecules: Biosensors, Diagnostic assays, Affinity separations, Targeted Drug Delivery, Cell Cultures Drugs: Thrombo-resistant surfaces, Drug delivery systems Lipids: Thrombo-resistant surfaces Nucleic acid derivatives and nucleotides: DNA probes, Gene Therapy Devices Cells: Bioreactors, Bioartificial organs, Biosensors

8. Methods of Immobilization Physical adsorption Van der Waals forces Electrostatic attraction Affinity Adsorbed and cross-linked Physical �Entrapment� Barrier systems Hydrogels Dispersed (matrix) systems Covalent attachment (next slides)

9. Covalent Attachment Surface must contain reactive groups (-COOH, -NH2, -OH, etc.) If the system does not have reactive surface groups, then it must first be modified A variety of coating methods can be used � Plasma gas discharge, chemical modification, etc. (Lect. 9) Types of substrate systems: Soluble polymers Solid surfaces (e.g. insoluble polymers) Hydrogels Multiple types of biomolecules can be attached to one supporting structure

10. Covalent Attachment Spacers Chemically immobilized biomolecules can also be attached via a spacer group (an �arm�, �leash�, or �tether�) These spacer �arms� possess reactive end groups amines, carboxylic acids, hydroxyl groups Spacer arms can enhance steric freedom of the attached biomolecule. Spacer �arms� can also be biodegradable, and release the biomolecule as they degrade.

11. Biomaterial-Protein Interaction Soluble proteins adsorb onto surfaces Often spherical or globular shapes (except for fibrinogen) Spatial arrangement results in hydrophobic regions �inside� the protein and hydrophilic regions �exposed� to the aqueous phase Soluble proteins present in biological fluids such as blood plasma and serum Fibrinogen Albumin Insoluble proteins such as collagen are normally not free to diffuse to implant surface

12. Nonfouling Surfaces (NFS) �Stealth� surfaces that resist protein adsorption or cell adhesion Generally, if proteins do not adsorb, cells will not adhere and vice versa Biofilms produced by bacteria can be problematic (e.g. biofilm protects from antibiotics) Blood contacting materials should not adsorb some proteins (clots) PEG or PEO (-CH2CH2O-) is commonly used on NFS (same polymer, different MW) Resistance to protein adsorption related to resistance of interface to release bound water Approaches involve making the surface more hydrophilic

13. Adsorption Adsorption - when a molecule prefers to localize at an interface (i.e. solid-liquid or air-liquid) Higher concentration at the interface than in either of the phases A consequence of surface energy Physisorption - physical adsorption, weak interactions Chemisorption - formation of a chemical (covalent) bond Example: water filters consist of carbon cartridges that adsorb contaminants Absorption is different Molecules move into pores of solid in absorption

14. Protein Adsorption Surface

15. Protein Monolayer Protein adsorption onto a surface is limited by the available space on the surface A monolayer (one protein thick) is formed Once surface is covered, other proteins do not generally attach to the monolayer More discussion in terms of kinetics and competition to come

16. Adhesion Proteins & Cellular Interactions Adsorption of a single protein is useful to study the interactions between cells and different proteins Preadsorption may promote or inhibit cell adhesion Fibronectin increases cell adhesion Preadsorption may increase cell spreading Cell spreading function of adsorbed protein concentration Depletion studies are more useful Bodily fluids contain many proteins Selective removal (depletion) of a single protein is more relevant to study protein-cell interactions Inhibition of protein receptors shows the interactions between cells and proteins (using antibodies)

17. Competitive Adsorption Competition for the available surface sites Monolayer of adsorbed protein limits amount that can be adsorbed Proteins differ in intrinsic �surface activity� � ability to adsorb to surfaces

18. Driving Force for Adsorption Two driving forces for adsorption: Relative bulk concentration of each protein Intrinsic surface activity Proteins have different affinities for each type of surface Surface composition is different than the bulk composition Depletion Studies Complex mixture e.g. plasma Remove a protein Immunoadsorption Chromatography Mutant individual Selective enzyme degradation Effect on cell adhesion

19. Protein-Surface Interaction Neutral, hydrophilic material surface shows little adsorption Hydrophobic surface has strong adsorption Strong hydrophobic-hydrophobic interactions in an aqueous biological environment Charged surfaces have variable adsorption depending on the electrostatic interaction of the protein and surface

20. Kinetics of Protein Adsorption 1. Very rapid initial phase that is diffusion limited Proteins arrive quickly to an empty surface 2. Slower phase approaching steady-state value More difficult for proteins to find an empty spot General kinetic models take into account rates of adsorption (ka), desorption (kd), conformational change and rearrangement (kG)

21. Langmuir Model Plateau in adsorption at 0.1 to 0.5 mg/cm2 of protein Concentration range for close-packed monolayer of protein

22. Selective Adsorption Binary mixtures (mixtures of two proteins) Behavior depends on proteins and surfaces Curves 1, 2 and 3 can represent either: 1) 3 different surfaces 2) 3 different pairs of proteins (A and B) �Vroman effect� � transient competition of protein adsorption to surfaces

23. Adsorption & Protein Denaturation Typically involves unfolding of the protein Disruption of secondary or tertiary structure May be reversible or irreversible Proteins with low thermodynamic stability more likely to adsorb to surface Soft (low thermodynamic stability) vs. hard proteins Demonstrated with mutant proteins (less stable) Mutant lysozyme adsorb to solid/liquid surface Mutant tryptophan synthase adsorb to air/water interface

24. Protein Denaturation

25. Denatured Proteins on Surfaces Changes to proteins affect Subsequent cellular interactions Material biocompatibility and immunogenicity

26. Effect of Denaturation on Surfaces Example: Fibrinogen Fibrinogen is a monoclonal antibody that binds only to fragment D of fibrinogen Antibody does not bind to fibrinogen when it is in solution Antibody does bind to fibrinogen when it is adsorbed to a surface Leads to platelet adhesion

27. Some Proteins Desorb Protein is removed from the surface and returns to solution Once the protein desorbs, its hydrophobic residues will be exposed to water and it will either aggregate or precipitate

28. Protein Spreading, Time, Albumin Fibrinogen as time passes the following occur: Reduced interactions with platelets and Abs Reduction in ability of SDS to displace it Albumin effect Adsorption of albumin inhibits the spreading of fibrinogen and further structural changes

29. Cell-Protein Interactions Sequence of events for cellular activity: Proteins adsorb on surface. Cells arrive at an implant surface Via diffusive, convective, or active (locomotion) mechanisms. At surface, cells adhere, release active compounds, recruit other cells, or grow. Structure of adsorbed protein layer determines in part the cellular response. (Responses can be desirable or undesirable) After cells arrive and attach at surfaces, they may multiply and organize into tissues A synthetic surface can interact with and disrupt living tissues The interfacial behavior of adsorbed proteins subsequent response of tissues to implanted synthetic materials Understanding adsorption behavior is important

30. Bioreactions: Short & Long Term Implant into soft tissue: 9 materials: Short-Term Reaction: Long-Term Reaction: Polyethylene 1. Different protein 1. Fibrous Hydroxyapatitie adsorption encapsulation Polyurethane 2. Varied activation of Silicone host response pHEMA PTFE Pyrolytic carbon Gold Titanium

31. Goal of Biomaterial Surfaces Does protein adsorption even matter if result is the same? Yes and no � depending on specific case Adsorption plays a role in Complement activation Coagulation activation Fouling of contact lenses Initial response to implants Transport applications (drug delivery) Goal is not just to understand adsorption of unmodified surfaces, but to design surface to mediate (or control) the adsorption process

32. Summary Many types of biological materials (or cells) can be immobilized for various application Materials: enzymes, proteins, polysaccharides, nucleic acids, etc Methods: physical adsorption, entrapment, covalent bonding Applications: sensors, drug delivery, gene therapy Monolayer adsorption and competition limit relative concentrations of adsorbed proteins Driving forces of adsorption intrinsic surface activity bulk phase concentration Exact surfaces and proteins involved both determine adsorption Biological activity of the adsorbed protein also varies on different surfaces