Surface Properties of Biomaterials

● Biomaterial surface: what biologic environment sees

● Protein adsorption
○ Adhesion of molecules to surface
○ Adsorbate is comprised of ions, water, and proteins
■ Cells interact with biomaterials via proteins
○ Controlling adsorption is a key aspect of biocompatibility
● Suface
○ Planar defect
○ Incomplete bonding
○ Often called surface tensions (ℽ)
■ Quantified using contact angle
● Why do proteins adhere?
○ Satisfies unfilled bonds
○ Minimizes surface tension
○ Decreases free energy of system
● What governs protein adsorption?
○ Thermodynamic principles
○ Compare system before and after adsorption
● Surface properties with the largest effect
○ Surface hydrophobicity
■ Increase hydrophobicity -> increase adsorption
○ Surface charge
■ Attract or repels charged areas of protiens
■ Depends on both charge of surface and protien
○ Other factors
■ Steric concerns
● I.e. spacial arrangements of ions
● Adding large/long polymer to surface would increase? Protein
adsorption?
■ Surface roughness
● Physics trapping
● Increase roughness ->> increase adsorption
● Surface Modification Techniques
○ How to modify surface to control adsorption (bulk characteristics are not altered)
○ Goals of surface modification
■ Thin
■ Resistant to delamination
■ Simple and robust
■ Discourage surface rearragnement
○ Classifications of surface treatments:
■ Physiochemical
● Physical principles or chemical reactions to alter surface
● Can be covalent or non-covalent
 ■ Biological
● Can be covalent or non-covalent
○ Physiochemical surface Coatings
■ Two examples:
● Plasma treatment
○ More common method
○ Plasma discharge - broader term that encompasses
several methods
○ Physiochemical treatment
● SAMs
■ Plasma Treatment:
● Inert gases react with “glow discharge”? To terminate free radicals
○ Crosslinking, etching, deposition, funtionalization
■ Self-assembled monolayers (SAMs)
● Entirely different basis for technique
○ No specialized equipment
○ Room temperature
○ Normal atmospheric pressure
● Thermodynamically favorable for coating molecules to align with
each other
○ Covalent bonds with surface
● SAMs are amphiphilic
○ Have bother hydrophilic (polar) and hydrophobic (non-
polar) areas
● Three key regions
○ Attachment group
○ Assembling structure (long hydrocarbon alkyl chain)
■ Intermolecular interactions create ordered
molecular orientation
○ Functional (polar) head group
● New “surface” is a functional group
○ Can alter hydrophobicity
○ Can be a point of further chemical reaction (i.e. to attach
biologically active molecules)
● How does this happen?
○ They self assemble
○ Driving force is a strong, exothermic reaction between
substrate and attachment group
○ VDW between SAMs molecules causes crystallization
○ Need high molecular mobility
Biological Surface Modificaitions
● Techniques
○ Attachemnt of biologically-active molecules
○ Interact with specific target areas on cells/tissues components
 ● Some possible molecules
○ Proteins
○ Sugars
○ Nucleic acids (RNA/DNA)
● Primary concerns
○ Will it remain attached while maintaining its biological activity?
○ Will it re-orient and rotate after coating?
● Covalent biological coatings
○ Need reactive substrate surface
■ I.e. hydroxyl (OH), carboxyl (COOH), or amine (NH2)
○ How to add these unnaturally?
● When do you want to attach it?
○ Post-fabrication reactions
○ Part of synthesis of polymers
● How to attach them to polymers:
○ Use binding agent to facilitate interaction post fabrication
■ Binding agent can remain as “glue” or catalyze reaction and be released
○ Combined cojugation/synthesis reactions
■ Option 1:
● Biomolecule bound to precursor (monomer)
● Polymerize in three-dimensions or as a surface coating
■ Option 2:
● Activated precursor containing groups with affinity for biomolecule
is polymerized
● Formed biomaterial exposed to molecule of interest
○ Can be bound directly to substrate or by using a spacer arm
■ Spacer arm -> inert molecule that provides physical space between
biomolecule and substrate
■ Why is this done?
● Allows for more rotational freedom
● Possible to design biodegradable spacer arms to release
biomolecule after implantation (drug delivery apps)

Techniques for Surface Characterization

● How do you know if your surface modification worked? How deep into the surface do
you want to characterize?
● ESCA/XPS
○ Spectroscopy
■ Interaction of energy w/ matter
■ Determines chemical structure of what is there
○ ESCA: Electron spectroscopy for chemical analysis
○ XPS: X-ray photoelectron spectroscopy (Same thing as ESCA)
○ Basic principles
■ X-ray is very high energy electromagnetic radiation
 ■ Irradiate sample surface
● Produces ejected free electrons with kinetic energy (Ek)
■ Record Ek
■ From Ek calculate binding energy (Eb) of electron
○ Binding energy
■ How tightly bound electron is to nucleus
● Closer to nucleus = larger binding energy
■ Changes with type of atom and interactions with nuclei of neighboring
atoms
■ More kinetic energy -> less binding
○ How it works
■ Sample bombarded with x-rays
■ Emitted electrons enter analyzer
■ Only electrons with a certain kinetic energy collected by detector (alter
voltage difference between walls of detector)
■ At end of voltage sweep, entire spectrum plotted for sample
■ Different orbitals give difference peaks in spectrum
○ Provided information
■ Chemistry of surface
■ Can detect all elements except hydrogen and helium
■ Compare against known values/standards
■ Also provides information about neighboring atoms to which an element is
bonded
○ Ex: Ethyl trifluoroacetate
■ Multiple peaks for carbon 1s
■ Occur at different kinetic energies
● AFM: atomic force microscopy
○ Forms 3d images of surface
○ Can’t distinguish chemical composition
○ Basic principles (contact mode)
■ Small tip is attached to cantilever and encounters material surface
■ Van der waals and electrostatic interactions
● Eventual attraction of tip to surface
● Cantilever bends
■ Feedback system keeps force constant by moving sample stage up and
down
■ Record change in stage position?
● Heigh data is in 3D
■ The smaller the surface features compared to the AFM tip dimensions,
the less accurate the sample image on the computer screen.
■ We want the tip to be smaller than the feature
● SIMS: Secondary Ion Mass Spectrometery
○ General theory
■ Ionize sample
 ■ Collect secondary ions using magnetic field
■ Analyze with mass spectrometer
■ Ionization: primary ions ejected from ion gun
● I.e. O2+, O2-, Ar+, Xe+
■ Strikes surface of sample like a pool ball
■ Causes ejection of secondary ions on surface layer (i.e. the balls the cue
ball hit)
■ Secondary ions drawn into analyzer for separation by mass
■ Speerate by mass - general theory
● Ions forced throuhg magnetic field and deflects them
● Lighter species deflect more than heavy
● Count them
● Separate based on mass
○ Information provided
■ SIMS spectra
● Y-axis: relative intensity or counts
● X-axis: mass/charge ratio (m/z)
■ Fibronectin coated polystyrene substrate example (see slides)
■ How to know what ion each peak corresponds to?
○ Static and dynamic methods
■ Static uses relatively low ion dose
■ Dynamic uses much larger ion dose
● Allows for indpeth profiling of specimens
○ Pros
■ Static SIMs causes minimal surface damage
● Outermost few A
■ High sensitivty with low concentration levels
○ Cons
■ Not all elements in all substrates can be analyzed
● Can’t tell difference between isotopes
■ Expensive $2-3M

Chapter 8: Protein Interactions with Biomaterials

● Proteins mediate interactions between biomaterial surface and cells
● Protein chemistry and structure
○ Proteins (or peptides)
○ Polymers of amino acids (basic subunit)
○ 20 standard amino acids
○ R-group = side chain
■ 4 diff R groups (polar, non-polar, positively charged, negatively charged)
○ Special amino acids
■ Proline: ring structure resticts conformation of peptide chain
■ Cysteine: two cysteines can form covalent disulfide bond which stablizes
proteins
○ 4 levels of protien structure
■ Primary: sequence of amino acids on chain, peptide bonds
● Directs all levels of strucutre
● Single substitution of amino acid can lead to entirely different
protein
■ Secondary
● Local interactions between amino acid residues
● Causes twists and turns in peptide chain
● Two standard shapes: alpha helix and beta pleated sheet
● Alpha helix
○ Spirals with side chains sticking out
○ Hydrogen bonding between AA’s stablize
● Beta sheet
○ Hydrogen bonding between muletiple peptide chains
■ Or parts of chains
○ Parallel sheets
○ Anti-parallel sheets
■ Tertiary structure
● 3d arrangement of entire polypeptide chain
● Interactions in AAs that are distant on same chain
○ Folding of secondary structure
● Side chain (r-group) interactions are important
● Called denaturing when protein loses this structure
● Effects on teritiary structure
○ Covalent bonds (disulfide bonds between systeines)
○ Ionic interactions between charged side groups
○ Hydrogen bodns between polar amino acids
○ Hydrophobic interactions
■ Keep hydrophobic parts away from aqueous
environment
■ Increase entropy by removing ordered water
■ Accomplished by putting non-polar residues near
each other
○ What is ordered water?
■ Interactions balance to create most
themodynamicall stable structure possible
■ Hydrophobic regions inside
■ Polar regions outside
■ Quaternary structure
● 3-d orientation of multiple polypeptide chains
● Interaction in R groups between different cgains
○ I.e. two hydrophobic patches can align
■ Protein structures are vert dynamic -> movement between several
thermodynamically stable conformations (e.g. can be pH dependent)
● Importance of protein-surface interactions
○ Modulate cell adhesion
○ Trigger biological cascade (foreign body response)
○ Used in diagnostic array/sensor device design
● Adsorption time scale
○ Observed <1 sec after implantation
○ Monolayer in seconds to minutes
○ Well in advance of cell arrival
● Big picture question:
○ Proteins are thermodtanmically happy
○ So why do they bond to surfaces?
● Thermodynamic System
○ Gibbs free energy of system must be negative for adsorption to occur
○ Enthapy, H
■ Energy in system available for mechanical work
○ Entropy, S
■ Disorder of system
○ Sefcond law of thermodynamics
■ Spontaneous proceosses occur to increase disorder of universe (increase
entropy)
■ ΔG = ΔH - TΔS
■ If G<0, energetically favorable
■ Compare before and after reaction within constant temp and pressure
■ Must combine ΔprotG, ΔGsolvent, and ΔG surface to get the overall ΔG
for adsorption reaction
● Qualitative analysis
○ What system properties govern protein adsorption?
■ Proteins are amphiphilic
■ Look at interactions between domains on biomaterial surface and protein
○ Look at 3 major system factors
■ Dehydration of surface and protein
■ Redistribution of charged groups of surface and protein
■ Structural rearrangement of protein
○ Dehydration
■ Water ordered at hydrophobic surface
● Either biomatieral surface or protein surface
■ If hydrophobic areas come together
● Reduce overall hydrophobic surface area
● Disorder water -> increase in entropy
● Must sses if protein can do this
■ In general, proteins more readily adsorb to hydrophobic surfaces
○ Hydrophilic surface
■ Proteins bind via hydrogen bonds or ionic interactions
■ Can lead to multi-layered adsorption
 ■ Relatively reversible -> less denaturing to do it
○ Hydrophobic surface
■ Proteins adhere via hydrophobic interactions
■ Usually mono-layer adsorption
■ Very irreversible
● Significant denaturation to do it
○ Charge
■ Protein region and surface
● Same charge -> repulsion, reduces adsorption
■ Surface and media
● Adsorb ions with opposite charge from the media
● Media is part of system
● But - need energy to adsorb ioins out of media
■ Which one happens?
● Protein adsorption favored when many areas of protein have
oppostei charge to surface
● Less transfer of solvent ions
○ Structural rearrangement
■ If protein is structurally unstable
● Preferentially adsorbs to surface
■ Rearragnemetn allows for optimal confirmation
● What does this mean?
● Proteins neither completely hydrophobic or hydrophilic no positive
or negative
■ Ex: biomaterial surface, contact angle 100 deg
● Hydrophobic or hydrophilic?
■ Ex: protein A has 10 cysteine residues, Protein B has 2
■ Which will absorb more? Why?
● Protein Transport