INTRODUCTION TO BIOADHESION CHRISTINE ORTIZ Associate Professor Department

INTRODUCTION TO BIOADHESION CHRISTINE ORTIZ, Associate Professor Department of Materials Science and Engineering, MIT WWW : http: //web. mit. edu/cortiz/www c D. Breger, used w/permission, http: //www. ldeo. columbia. edu/micro/images. section/pages/bloodclot. html

BIOADHESION : DEFINITION e term bioadhesion refers to the adhesion of synthetic and biological macrom very. Such drug delivery may be optimized at the site of action (e. g. , on the c mers to second-generation polymers and lectins. The nature of bioadhesive i bioadhesives, such as those used in wound management, surgery, and denti

Blood and Blood Vessels 40% cells in plasma or serum (p. H 7. 4, IS=0. 15 M) which contains 6 -8% proteins (over 3, 000 different types) in HOH, including : -58% albumins -38% globulins -4% fibrinogens

Synthetic Vascular Grafts or Prosthesis : prosthetic tube that acts either a permanent or resorbable artificial replacement for a segment of a damaged blood vessel (e. g. from athersclerosis, aneurysms, organ transplant, cancer, arteriovenous fistula, diabetes) : $200 million market worldwide http: //www. vascutek. com/ http: //www. artegraft. com/ http: //www. atriummedical. com/

Vascular Graft Materials • expanded polytetrafluoroethylene (Gore-Tex, e. PTFE) -fibrillated, open cell, microporous (pore size 0. 5 -30 mm), 70% air, nonbiodegradable, chemically stable, used for 26 yrs, hydrophobic/nonpolar, flexible • polyethylene terephthalate (Dacron, PET) -multifilamentous yarn fabricated by weaving/knitting, amphiphilic, smaller pores than e. PTFE Zhang, et al. J. Biomed. Mtls. Res. 60(3), 2002, 502. • polyurethane derivatives • bovine collagen -fibrous, hydrophilic www. vascutek. com

BLOOD FLOW blood plasma proteins PLATELETS! D. Gregory http: //medphoto. wellcome. ac. uk http: //www. rinshoken. or. jp/org/CR/photo-e. htm BLOOD PRESSURE+ ATTRACTIVE FORCES BLOOD CLOT! denatures -acute occlusive thrombosis - infection / inflammation - neointimal hyperplasia Solid-Liquid Interface adsorbs BIOMATERIAL SURFACE

WHAT CONTROLS PROTEIN ADSORPTION? Total Intersurface Force as a Function of Separation Distance : F(D) Many different components, both attractive (e. g. hydrogen, ionic, van der Waals, hydrophobic, electrostatic) and repulsive (e. g. configurational entropy, excluded volume, osmotic, enthalpic, electrostatic, hydration), can lead to complex interaction profiles. D ENDGRAFTED POLYMER “BRUSHES” ADSORBED POLYMER LAYERS BIOMATERIAL SURFACE

Direct Measurement of Protein Interactions with Poly(ethylene oxide) (PEO) Macromolecules lipid-bound HSA functionalized probe tip, RTIP~65 nm (SEM) F Rixman, et al. accepted, Langmuir 2003. Si 3 N 4 chemically end-grafted PEO 50 K “mushroom” Lcontour= 393 nm RF=8. 7 nm D sodium phosphate buffer ~35 -190 covalently solution proteins in maximum immobilized HSA IS=0. 01 M interaction area (D=0) ~10 nm p. H=7. 4 s = 62 ± 28 nm ~2. 5 PEO chains in maximum interaction area (D=0) Au-coated silicon chip

Chemical Attachment Scheme of Lipid-Bound HSA to Si 3 N 4 Probe Tip A. Vinkier; Heyvaert, I. ; D'Hoore, A. ; Mc. Kittrick, T. ; C. , V. H. ; Engelborghs, Y. ; Hellemans, I. Ultramicroscopy 1995, 57, 337. S. O. Vansteenkiste; Corneillie, S. I. ; Schacht, E. H. ; Chen, X. ; Davies, M. C. ; Moens, M. ; Van Vaeck, L. Langmuir 2000, 16, 3330. probe tip location Fluorescence micrograph of HSA-functionalized cantilever (courtesy of Irvine Lab-DMSE

Human Serum Albumin (HSA) M. O. Dayhoff Atlas of Protein Sequence and Structure; National Biomedical Foundation: Washington DC, 1972. S. Azegami; Tsuboi, A. ; Izumi, T. ; Hirata, M. ; Dubin, P. L. ; Wang, B. ; E. , K. Langmuir 1999, 15, 940947. III(C) I(N) II (*Steve Santoso (MIT-Biology) http: //pymol. sourceforge. net) II Ø The smallest and most abundant blood protein in the human body, HSA accounts for 55% of the total protein content in blood plasma Ø 3 -D structure consists of 3 homologous subdomains, each containing 5 principal domains and 6 helices. Ø Subdomains form hydrophobic channels placing basic and hydrophobic residues at the ends while the surface remains predominantly hydrophilic Ø Lcontour = 225 nm Ø Isoelectric point=4. 7 Ø 116 total acidic groups (98 carboxyl and 18 phenolic -OH) and 100 total basic groups (60 amino, 16 imidazolyl, 24 guanidyl).

“HEART SHAPED” STRUCTURE OF CRYSTALLIZED HSA (Curry, S. , H. Mandelkow, et al. Brookhaven Protein Databank. ) charge residue map - red, + blue hydrophilic-hydrophobic map C 8 nm PROPOSED ELLIPSOIDAL STRUCTURE OF HSA IN SOLUTION (Haynes, et al. (1994). Coll. Surf. B. : Biointerfaces 2: 517. ) 14 nm -9 e I (N) -8 e II +2 e III (C)

AFM Images of End-Grafted (Mono-Thiol) PEO 50 K Chains on Polygranular Gold Substrate (*contact mode, solvent=PBS buffer solution, IS=0. 15, p. H=5. 6) polygranular Au 100 nm 50 nm distance between polymer chains= <s>=62 26. 8 nm <G>=1/<s>2 =2. 6 • 10 -4 nm-2 Au-PEO 50 K 100 nm 50 nm

Poly(ethylene oxide) (PEO) In Aqueous Solution (Prog. Polym. Sci. 20, 1995, 1043) • hydrophilic & water soluble @RT low c<0. 5, high A 2=30 -60 cm 3 mol/g 2 (large excluded volume), q. W(A)=60 o intramolecular H- bond bridges between -Ogroups and HOH (tgt) t t • high flexibility, low s =1. 38 -1. 95 • high mobility, fast tc =15 -100 ps • locally (7/2) helical supramolecular structure (tgt axial repeat = 0. 278 nm) • low van der Waals attraction • neutral Nature 416, 409 - 413 (2002) t t • maintains some hydrophobic character t g t t 0. 278 nm t

DETERMINATION OF SURFACE INTERACTION AREA AND CONTACT AREA DMAX<100 nm, RTIP<100 nm ATIP(D=0) = 3000 -17, 000 nm 2 ~40 -180 proteins for a monolayer FMAX Rixman, et al. accepted, FMAX/protein<40 p. N Langmuir 2003. PROBE TIP RTIP surface interaction (tip and substrate not in contact) SUBSTRATE RTIP-DMAX r aqueous solution DMAX ACONTACT <3 nm 2 (tip and substrate in contact negligible substrate deformation)

“APPROACH” (COMPRESSION OR LOADING)

AVERAGE APPROACH CURVE : HSA PROBE TIP VERSUS PEO (SUBTRACTED AU INTERACTION) PBS, IS=0. 01 M, p. H=7. 4 F RF (PEO) Au • magnitude of force much larger than predicted by theory Rixman, et al. submitted, Langmuir 2003.

HSA versus PEO : Effect of Na. Cl IS Approach ● Na. Cl reduces the goodness of solvent for PEO (Armstrong, et al. 2001) : configurational entropy force expected↓ with ↑IS CONCLUSION: Electrostatic double layer and configurational entropy are outweighed by another interaction which increases with IS →possibly due to water interphase layer ● Salt screening : electrostatic double layer force expected↓ with ↑IS RF (PEO) Rixman, et al. 2003 unpublished data

HSA versus PEO : Effect of Solvent on Approach Isopropanol has been shown to block hydrophobic interaction forces (Jiang, et al 2002) RF (PEO) Rixman, et al. 2003 unpublished data

Poly(ethylene oxide) (PEO): REPULSIVE INTERACTIONS IN WATER • steric (large excluded volume) - - -- • electrostatic double layer forces • hydrophilic/ water soluble : hydration enthalpic penalties for disruption of supramolecular structure H-bonding with water • high flexibility & mobility : no local steric or charge • neutrality : won’t attract oppositely charged species

“RETRACT” (TENSION OR UNLOADING)

Quantities Used to Evaluate Nanoscale Adhesion • <FADHESION>, <FADHESION>/Radius, <DADHESION>= average maximum attractive force and corresponding separation distance within a dataset recorded for each point of pull-off and averaged over an entire data set • <Wexp>, <Uexp>/protein=effective adhesive interaction energy per unit area : BCP Theory (a=1. 4), JKR (a=1. 5), DMT Theory (a=2) : • <Ud>, <Ud>/ASUBSTRATE =energy dissipated during loadingunloading cycle Limitation : can’t use for curves exhibiting large adhesive forces followed by large cantilever instability regions (weak cantilever).

INDIVIDUAL APPROACH AND RETRACT CURVES, HSA PROBE TIP VERSUS PEO-AU SURFACE, PBS, IS=0. 01 M, p. H=7. 4 76% of total experiments F reversible decompression of the (net) repulsive surface interaction and no adhesion Au Au Rixman, et al. submitted, Langmuir 2003.

INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE, PBS, IS=0. 01 M, p. H=7. 4 17% of total experiments nonhysteretic repulsion F unknown desorption interaction profile long-range adhesion due to stretching of individual PEO chain nonspecific adsorption tether (net) repulsive surface interaction extension of individual PEO chain Au adhesive binding force FRUPTURE(Au-S) 2 -3 n. N Rixman, et al. submitted, Langmuir 2003.

INDIVIDUAL APPROACH AND RETRACT CURVES: HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0. 01 M, p. H=7. 4 7% of total experiments F Au extension of 2 PEO chains Rixman, et al. submitted, Langmuir 2003.

INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0. 01 M, p. H=7. 4 17% of total experiments nonhysteretic repulsion unknown desorption interaction profile long-range adhesion due to stretching of individual PEO chain <Fadhesion>=0. 16± 0. 18 n. N <Dadhesion>=265± 137 nm <Fadhesion>/Radius= 2. 46± 2. 76 m. N/m <Wexp> not calculated (DMT, JKR, BCP theories not applicable) <Ud>=1. 3 • 1 E 3 k. BT <Ud>/ASUBSTRATE=0. 5 m. J/m 2 adhesive binding force • one polymer chain Rixman, et al. submitted, Langmuir 2003.

INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0. 01 M, p. H=7. 4 CREATION OF MOLECULAR ELASTICITY MASTER CURVE (tgt) t t t g t t t 0. 278 nm strain-induced conformational transition (ttg ttt) • reduction in extensional force (*first reported by Oesterhelt, et al. 1999) reversible on experimental time scales

Fadhesion (n. N) <Dadhesion> (nm) <Fadhesion >(n. N) <Fadhesion>/Radius (m. N/m) Fadhesion/Radius (m. N/m) ADHESION FORCES AND DISTANCES FOR INDIVIDUAL RETRACT CURVES, HSA PROBE TIP VERSUS VARIOUS SURFACES : PBS, IS=0. 01 M, p. H=7. 4

SUMMARY OF RESULTS : PROTEIN-PEO INTERACTIONS • Large, long-range surface repulsion that can’t be explained by electrostatic and steric interactions alone (? WATER) • Elimination of surface adhesion (from ~1. 35 n. N) even at such low grafting densities • At high compressions, long range adhesion (<Fadhesion>=160 p. N) and stretching with an individual PEO 50 K chain allows the probing of short-range attractive contacts between surface functional groups and an individual PEO chain NH 2 OH • H-bonding

ADVANTAGEOUS MOLECULAR ATTRIBUTES FOR MAXIMUM BIOCOMPATIBILITY 1) maximum hydrophilicity and water solubility, i. e. molecules capable of strong hydrogen bonding such that there exists an enthalpic penalty to dehydration and disruption of supramolecular structure imposed by incoming protein molecules 2) a net neutral charge so that the surface will not attract proteins of net opposite charge or regions on a protein surface of opposite charge via electrostatic interaction. 3) for macromolecular surfaces, higher molecular weight, long chains with a large degree of backbone flexibility to produce maximum steric repulsion 4) Nontoxic HOW DO BLOOD VESSEL INTERIOR (LUMEN) SURFACES CONTROL NONSPECIFIC ADSORPTION?

Control of Nonspecific Adsorption In Blood Vessels Glycocalyx : External, Porous, Dynamic, Densely Carbohydrate Rich Region of Cell Membrane That Play a Role in Cell-Cell Recognition and Also Prevents Non-Specific Interactions , 500 nm thick (Vink, et al 1996 Circ. Res. 79, 581) Presumably, artificial biomaterial surfaces can be made more compatible if they are more similar in chemistry, morphology, and mechanical properties to the cell surface. http: //www. d. umn. edu/~sdowning/Membranes/

Glycocalyx-Mimetic Neutral Oligosaccharide Monolayers (Synthesized by Seeberger Lab, MIT-CHEM) chitobiose (CB) linear trimannoside (LT) oligomannose-9 (Man-9)

Glycocalyx-Mimetic Neutral Oligosaccharide Monolayers (Synthesized by Seeberger Lab, MIT-CHEM)

Plant Fibers cellulose