CHRISTINE ORTIZ, Associate Professor 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 Bioadhesion may be defined as the state in which two materials, at least one of which is biological in nature, are held together for extended periods of time by interfacial forces. In the context of their medical and pharmaceutical use, the term bioadhesion refers to the adhesion of synthetic and biological macromolecules to a biological tissue. The biological substrate may be cells, bone, dentine, or the mucus coating the surface of a tissue. If adhesive attachment is to a mucus coating, the phenomenon is sometimes referred to as mucoadhesion. Many examples of bioadhesion exist in nature, including such diverse events as cell-to-cell adhesion within a living tissue, barnacles binding to rocks, and bacteria binding to tooth enamel. In health care, bioadhesives were first used as wound dressings, skin adhesives, and denture fixatives. Over the last two decades, bioadhesives have been of interest within the pharmaceutical sciences for their potential to optimize drug delivery. Such drug delivery may be optimized at the site of action (e.g., on the cornea or within the oral cavity) or at the absorption site (e.g., in the small intestine or nasal cavity). Bioadhesives may also be used as therapeutic agents in their own right, to coat and protect damaged tissues (gastric ulcers or lesions of the oral mucosa) or to act as lubricating agents (in the oral cavity, eye, and vagina). Skin adhesives, tissue sealants, and dental and bone adhesives and cements are also defined as bioadhesives. This article first focuses on the types of muco/bioadhesives currently used in the pharmaceutical sciences, from first-generation hydrophilic polymers to second-generation polymers and lectins. The nature of bioadhesive interactions, types of bioadhesive formulations developed, and regions of the human body to which they may be administered are also considered. Other types of medical bioadhesives, such as those used in wound management, surgery, and dentistry, are also discussed.

Blood and Blood Vessels 40% cells in plasma or serum (pH7.4, 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.atriummedical.com/ http://www.artegraft.com/

Vascular Graft Materials Zhang, et al. J. Biomed. Mtls. Res.60(3), 2002, 502. • expanded polytetrafluoroethylene (Gore-Tex, ePTFE) -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 ePTFE • polyurethane derivatives • bovine collagen -fibrous, hydrophilic www.vascutek.com

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

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 END-GRAFTED POLYMER “BRUSHES” ADSORBED POLYMER LAYERS BIOMATERIAL SURFACE

Direct Measurement of Protein Interactions with Poly(ethylene oxide) (PEO) Macromolecules Rixman, et al. accepted, Langmuir 2003. lipid-bound HSA functionalized probe tip, RTIP~65 nm (SEM) F Si3N4 sodium phosphate buffer solution IS=0.01M pH=7.4 covalently immobilized HSA ~10 nm ~35-190 proteins in maximum interaction area (D=0) chemically end-grafted PEO50K “mushroom” Lcontour= 393 nm RF=8.7 nm D ~2.5 PEO chains in maximum interaction area (D=0) Au-coated silicon chip s = 62 ± 28 nm

Chemical Attachment Scheme of Lipid-Bound HSA to Si3N4 Probe Tip A. Vinkier; Heyvaert, I.; D'Hoore, A.; McKittrick, 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, 940-947. The smallest and most abundant blood protein in the human body, HSA accounts for 55% of the total protein content in blood plasma 3-D structure consists of 3 homologous subdomains, each containing 5 principal domains and 6 helices. Subdomains form hydrophobic channels placing basic and hydrophobic residues at the ends while the surface remains predominantly hydrophilic Lcontour = 225 nm Isoelectric point=4.7 116 total acidic groups (98 carboxyl and 18 phenolic -OH) and 100 total basic groups (60 amino, 16 imidazolyl, 24 guanidyl). III(C) I(N) II (*Steve Santoso (MIT-Biology) http://pymol.sourceforge.net) II

“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 4 nm -9e -8e +2e I (N) II III (C)

100 nm 100 nm AFM Images of End-Grafted (Mono-Thiol) PEO50K Chains on Polygranular Gold Substrate (*contact mode, solvent=PBS buffer solution, IS=0.15, pH=5.6) 100 nm 50 nm polygranular Au distance between polymer chains= <s>=6226.8 nm <G>=1/<s>2 =2.6•10-4 nm-2 100 nm 50 nm Au-PEO50K

Poly(ethylene oxide) (PEO) In Aqueous Solution (Prog. Polym. Sci. 20, 1995, 1043) • hydrophilic & water soluble @RT low c<0.5, high A2=30-60 cm3mol/g2 (large excluded volume), qW(A)=60o intramolecular H- bond bridges between -O- groups and HOH • maintains some hydrophobic character • 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 (tgt) t t t t t t g t t t g 0.278 nm Nature 416, 409 - 413 (2002)

DETERMINATION OF SURFACE INTERACTION AREA AND CONTACT AREA DMAX<100 nm, RTIP<100 nm ATIP(D=0) = 3000-17,000 nm2~40-180 proteins for a monolayer FMAX Rixman, et al. accepted, Langmuir 2003. FMAX/protein<40pN PROBE TIP aqueous solution RTIP RTIP-DMAX r surface interaction (tip and substrate not in contact) DMAX SUBSTRATE ACONTACT <3 nm2 (tip and substrate in contact negligible substrate deformation)

(COMPRESSION OR LOADING) “APPROACH” (COMPRESSION OR LOADING)

than predicted by theory AVERAGE APPROACH CURVE : HSA PROBE TIP VERSUS PEO (SUBTRACTED AU INTERACTION) PBS, IS=0.01M, pH=7.4 F RF (PEO) Au • magnitude of force much larger than predicted by theory Rixman, et al. submitted, Langmuir 2003.

Rixman, et al. 2003 unpublished data HSA versus PEO : Effect of NaCl IS Approach ● NaCl reduces the goodness of solvent for PEO (Armstrong, et al. 2001) : configurational entropy force expected↓ with ↑IS ● Salt screening : electrostatic double layer 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 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

(TENSION OR UNLOADING) “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 loading-unloading cycle Limitation : can’t use for curves exhibiting large adhesive forces followed by large cantilever instability regions (weak cantilever).

reversible decompression of the (net) repulsive INDIVIDUAL APPROACH AND RETRACT CURVES, HSA PROBE TIP VERSUS PEO-AU SURFACE, PBS, IS=0.01M, pH=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=0.01M, pH=7.4 17% of total experiments nonhysteretic repulsion F unknown desorption interaction profile nonspecific adsorption tether long-range adhesion due to stretching of individual PEO chain (net) repulsive surface interaction extension of individual PEO chain Au adhesive binding force FRUPTURE(Au-S)2-3 nN Rixman, et al. submitted, Langmuir 2003.

7% of total experiments F INDIVIDUAL APPROACH AND RETRACT CURVES: HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0.01M, pH=7.4 7% of total experiments F Au extension of 2 PEO chains Rixman, et al. submitted, Langmuir 2003.

• one polymer chain INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0.01M, pH=7.4 17% of total experiments nonhysteretic repulsion <Fadhesion>=0.16±0.18 nN <Dadhesion>=265±137nm <Fadhesion>/Radius= 2.46±2.76 mN/m <Wexp> not calculated (DMT, JKR, BCP theories not applicable) <Ud>=1.3•1E3 kBT <Ud>/ASUBSTRATE=0.5 mJ/m2 unknown desorption interaction profile long-range adhesion due to stretching of individual PEO chain adhesive binding force • one polymer chain Rixman, et al. submitted, Langmuir 2003.

(tgt) CREATION OF MOLECULAR ELASTICITY MASTER CURVE INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0.01M, pH=7.4 CREATION OF MOLECULAR ELASTICITY MASTER CURVE (tgt) t t t t t g t t t t g 0.278 nm strain-induced conformational transition (ttgttt) • reduction in extensional force reversible on experimental time scales (*first reported by Oesterhelt, et al. 1999)

<Fadhesion >(nN) <Dadhesion> (nm) ADHESION FORCES AND DISTANCES FOR INDIVIDUAL RETRACT CURVES, HSA PROBE TIP VERSUS VARIOUS SURFACES : PBS, IS=0.01M, pH=7.4 Fadhesion (nN) Fadhesion/Radius (mN/m) <Fadhesion >(nN) <Fadhesion>/Radius (mN/m) <Dadhesion> (nm)

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 nN) even at such low grafting densities • At high compressions, long range adhesion (<Fadhesion>=160 pN) and stretching with an individual PEO50K chain allows the probing of short-range attractive contacts between surface functional groups and an individual PEO chain NH2 • H-bonding OH

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) linear trimannoside (LT) chitobiose (CB) oligomannose-9 (Man-9)

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

Plant Fibers cellulose