Presentation on theme: "CHRISTINE ORTIZ, Associate Professor"— Presentation transcript:
1 CHRISTINE ORTIZ, Associate Professor INTRODUCTION TO BIOADHESIONCHRISTINE ORTIZ, Associate ProfessorDepartment of Materials Science and Engineering, MITWWW :cD. Breger, used w/permission,
2 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.
3 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
4 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
5 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 mm), 70% air,nonbiodegradable, chemicallystable, used for 26 yrs,hydrophobic/nonpolar, flexible• polyethylene terephthalate(Dacron, PET)-multifilamentous yarn fabricated byweaving/knitting, amphiphilic, smaller poresthan ePTFE• polyurethane derivatives• bovine collagen-fibrous, hydrophilic
7 WHAT CONTROLS PROTEIN ADSORPTION? Total Intersurface Force as a Function ofSeparation 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.DEND-GRAFTEDPOLYMER “BRUSHES”ADSORBED POLYMER LAYERSBIOMATERIAL SURFACE
8 Direct Measurement of Protein Interactions with Poly(ethylene oxide) (PEO) Macromolecules Rixman, et al. accepted, Langmuir 2003.lipid-bound HSA functionalizedprobe tip, RTIP~65 nm (SEM)FSi3N4sodium phosphate buffersolutionIS=0.01M pH=7.4covalentlyimmobilized HSA~10 nm~35-190proteins in maximuminteraction area (D=0)chemicallyend-graftedPEO50K “mushroom”Lcontour= 393 nmRF=8.7 nmD~2.5 PEO chains in maximum interaction area (D=0)Au-coatedsilicon chips = 62 ± 28 nm
9 Chemical Attachment Scheme of Lipid-Bound HSA to Si3N4 Probe TipA. 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,probetiplocationFluorescence micrographof HSA-functionalizedcantilever(courtesy of Irvine Lab-DMSE)
10 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,The smallest and most abundant blood protein in the human body, HSA accounts for 55% of the total protein content in blood plasma3-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 hydrophilicLcontour = 225 nmIsoelectric point=4.7116 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)II
11 “HEART SHAPED” STRUCTURE OF CRYSTALLIZED HSA (Curry, S., H. Mandelkow, et al. Brookhaven Protein Databank.)charge residue map - red, + bluehydrophilic-hydrophobic mapC8 nmPROPOSED ELLIPSOIDAL STRUCTURE OF HSA IN SOLUTION(Haynes, et al. (1994). Coll. Surf. B. : Biointerfaces 2: 517.)14 nm4 nm-9e-8e+2eI (N)IIIII (C)
13 Poly(ethylene oxide) (PEO) In Aqueous Solution (Prog. Polym. Sci. 20, 1995, 1043)• hydrophilic & waterlow 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 =• high mobility, fast tc = ps• locally (7/2) helical supramolecular structure (tgt axial repeat = nm)• low van der Waals attraction• neutral(tgt)ttttttgtttg0.278 nmNature 416, (2002)
14 DETERMINATION OF SURFACE INTERACTION AREA AND CONTACT AREA DMAX<100 nm, RTIP<100 nmATIP(D=0) = ,000 nm2~ proteins for a monolayerFMAXRixman, et al. accepted, Langmuir 2003.FMAX/protein<40pNPROBE TIPaqueoussolutionRTIPRTIP-DMAXrsurfaceinteraction(tip and substratenot in contact)DMAXSUBSTRATEACONTACT <3 nm2(tip and substrate in contact negligible substrate deformation)
15 (COMPRESSION OR LOADING) “APPROACH”(COMPRESSION OR LOADING)
16 than predicted by theory AVERAGE APPROACH CURVE : HSA PROBE TIP VERSUS PEO (SUBTRACTED AU INTERACTION) PBS, IS=0.01M, pH=7.4FRF (PEO)Au• magnitude of forcemuch largerthan predicted by theoryRixman, et al. submitted, Langmuir 2003.
17 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 ↑ISCONCLUSION: Electrostatic double layer and configurational entropy are outweighed by another interaction which increases with IS →possibly due to water interphase layerRF (PEO)Rixman, et al unpublished data
18 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 unpublished data
19 Poly(ethylene oxide) (PEO): REPULSIVE INTERACTIONS IN WATER ----• steric (large excluded volume)--• electrostaticdouble layer forces---------• hydrophilic/ water soluble :hydration enthalpic penalties fordisruption of supramolecularstructure H-bonding with water• high flexibility& mobility :no local steric or charge• neutrality : won’t attract oppositely charged species
20 (TENSION OR UNLOADING) “RETRACT”(TENSION OR UNLOADING)
21 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).
22 reversible decompression of the (net) repulsive INDIVIDUAL APPROACH AND RETRACT CURVES, HSA PROBE TIP VERSUS PEO-AU SURFACE, PBS, IS=0.01M, pH=7.476% of total experimentsFreversible decompression of the (net) repulsivesurface interactionand no adhesionAuAuRixman, et al. submitted, Langmuir 2003.
23 INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE, PBS, IS=0.01M, pH=7.417% of total experimentsnonhysteretic repulsionFunknowndesorptioninteraction profilenonspecificadsorptiontetherlong-range adhesiondue to stretching ofindividual PEO chain(net) repulsivesurfaceinteractionextension ofindividualPEO chainAuadhesive binding forceFRUPTURE(Au-S)2-3 nNRixman, et al. submitted, Langmuir 2003.
24 7% of total experiments F INDIVIDUAL APPROACH AND RETRACT CURVES: HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0.01M, pH=7.47% of total experimentsFAuextension of2 PEO chainsRixman, et al. submitted, Langmuir 2003.
25 • one polymer chain INDIVIDUAL APPROACH AND RETRACT CURVES : HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0.01M, pH=7.417% of total experimentsnonhysteretic 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/m2unknowndesorptioninteraction profilelong-range adhesiondue to stretching ofindividual PEO chainadhesive binding force• one polymer chainRixman, et al. submitted, Langmuir 2003.
26 (tgt) CREATION OF MOLECULAR ELASTICITY MASTER CURVE INDIVIDUAL APPROACH AND RETRACT CURVES :HSA PROBE TIP VERSUS PEO-AU SURFACE : PBS, IS=0.01M, pH=7.4CREATION OF MOLECULAR ELASTICITY MASTER CURVE(tgt)tttttgttttg0.278 nmstrain-induced conformational transition (ttgttt)• reduction in extensional forcereversible on experimental time scales(*first reported by Oesterhelt, et al. 1999)
27 <Fadhesion >(nN) <Dadhesion> (nm) ADHESION FORCES AND DISTANCES FOR INDIVIDUAL RETRACT CURVES, HSA PROBE TIP VERSUS VARIOUS SURFACES :PBS, IS=0.01M, pH=7.4Fadhesion (nN)Fadhesion/Radius (mN/m)<Fadhesion >(nN)<Fadhesion>/Radius (mN/m)<Dadhesion> (nm)
28 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 chainNH2• H-bondingOH
29 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 molecules2) 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 repulsion4) NontoxicHOW DO BLOOD VESSEL INTERIOR (LUMEN) SURFACES CONTROL NONSPECIFIC ADSORPTION?
30 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.