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
(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.artegraft.com/
http://www.atriummedical.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,
F F
nonbiodegradable, chemically
stable, used for 26 yrs,
hydrophobic/nonpolar, flexible
n
F F
• polyethylene terephthalate
(Dacron, PET)
-multifilamentous yarn fabricated by
weaving/knitting, amphiphilic, smaller pores
than ePTFE
O
O
O
O n
• 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.
Si3N4
chemically
end-grafted
PEO50K “mushroom”
Lcontour= 393 nm
RF=8.7 nm
D
sodium
phosphate buffer
~35-190
covalently
solution
proteins in maximum
immobilized HSA
IS=0.01M
interaction area (D=0)
~10 nm
pH=7.4
~2.5 PEO chains in
maximum interaction
area (D=0)
s = 62 ± 28 nm
Au-coated
silicon chip
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.
Si3N4 Probe Tip
Si
Si
Si
NH2
CH3
+ H3C O Si
Si OH
CH3
II
ABDMS
Si
O
O
Glutaraldehyde
N
Si
Si
N
III
Si
NH2
N
O
NH2
Si
N
CH3
Si
CH3
O
NH2
N
O
O
O
O
O
NH2
O
O
Si O
HSA
H2N
CH3
Si O Si
CH3
NH2 I
NH2
Si
NH2
NH2
CH3
Si
CH3
N
N
HSA
Fluorescence micrograph
of HSA-functionalized
cantilever
(courtesy of Irvine Lab-DMSE)
NH2
O
probe
tip
location
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.
III(C)
I(N)
II
(*Steve Santoso (MIT-Biology)
http://pymol.sourceforge.net)
II
 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).
“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
I (N)
-8e
II
+2e
III (C)
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)
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-PEO50K
100 nm
50 nm
Poly(ethylene oxide) (PEO) In Aqueous Solution
(Prog. Polym. Sci. 20, 1995, 1043)
• 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
O
H
H
O
O
t t
Nature 416, 409 - 413 (2002)
• 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 -Ogroups and HOH
g
t t
H
O
H
O
t g tO t
t
0.278 nm
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,
FMAX/protein<40pN
Langmuir 2003.
PROBE TIP
RTIP
surface
interaction
(tip and substrate
not in contact)
SUBSTRATE
RTIP-DMAX
r
aqueous
solution
DMAX
ACONTACT <3 nm2
(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=0.01M, pH=7.4
A
B
3 .2
C
HSA probe tip versus PEO
surface average
Electrostatic surface charge
model neutral surface
van der W aals
PEO/HOH/HSA
Dolan Edw ards steric
0 .2 5
RF (PEO50K)
2 .2
1.2
0 .15
A
B
5
Au
• magnitude of force
much larger
than predicted by theory
-0 .0 5
Rixman, et al. submitted,
-0 .8
0
RF (PEO)
0 .0 5
0 .2
C
Force (nN )
Force/ Radius (m N /m )
4 .2
10
15
20
D istanc e (nm )
25
30
Langmuir 2003.
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
3.5
0.3
0.25
RF (PEO)
2.5
0.2
1.0M
2
0.15
1.5 0.15M
0.1
1
0.01M
0.05
0.5
0
0
0
10
20
Distance (nm)
30
Rixman, et al. 2003 unpublished data
Force (nN)
3
Force/Radius (mN/m)
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
HSA versus PEO : Effect of Solvent on Approach
Isopropanol has been shown to block hydrophobic interaction forces
(Jiang, et al 2002)
5
0.19
3
0.09
1
-1 0
-0.02
10
20
-0.12
-3
-5
-7
30
0% Isopropanol
0.5% Isopropanol
5% Isopropanol
100% Isopropanol
0% F+sd
0% F-sd
Distance
(nm)
0.5%
F+sd
0.5% F-sd
Rixman, et al. 20035%
unpublished
F+sd data
-0.22
-0.32
Force (nN)
Force/Radius (mN/m)
RF (PEO)
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
O
O
H
H
H
O
O
H
O
O
• neutrality : won’t attract oppositely charged species
• high flexibility
& mobility :
no local steric or
charge
“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) :
 Fadhesion >
 Wexp >
a RTIP
• <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).
Ud  
0
D max(a )
Fapproach (D)dD  
D max(r)
0
Fretract (D)dD
INDIVIDUAL APPROACH AND RETRACT CURVES, HSA
PROBE TIP VERSUS PEO-AU SURFACE, PBS, IS=0.01M, pH=7.4
76% of total experiments
0.2
F
HSA probe tip versus PEO-Au
approach
HSA probe tip versus PEO-Au retract
0.1
0.9
0
-1.1
Force (nN)
Force/Radius (mN/m)
2.9
reversible decompression
of the (net) repulsive
surface interaction
and no adhesion
-0.1
-3.1
-0.2
0
100
200
300
Distance (nm)
400
500
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
0
unknown
desorption
interaction profile
-1.2
-3.2
-0.1
-0.2
long-range adhesion
due to stretching of
individual PEO chain
-5.2
-0.3
-0.4
cantilever instability region
approach
retract
-7.2
-0.5
adhesive binding force
-9.2
0
100
200
300
F
400
Distance (nm)
-0.6
500
600
Force (nN)
0.8
Force/Radius (mN/m)
0.1
nonhysteretic repulsion
nonspecific
adsorption
tether
(net) repulsive
surface
interaction
extension of
individual
PEO chain
Au
FRUPTURE(Au-S)2-3 nN
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
7% of total experiments
cantilever instability region
approach
retract
0.05
-0.05
-1.4
-0.15
-3.4
F
Force (nN)
Force/Radius (mN/m)
0.6
-0.25
Au
-5.4
-0.35
0
200
400
600
Distance (nm)
800
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=0.01M, pH=7.4
17% of total experiments
0
unknown
desorption
interaction profile
-1.2
-3.2
-0.1
-0.2
long-range adhesion
due to stretching of
individual PEO chain
-5.2
-0.3
-0.4
cantilever instability region
approach
retract
-7.2
0
100
200
300
400
Distance (nm)
<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
-0.5
adhesive binding force
-9.2
Force (nN)
0.8
Force/Radius (mN/m)
0.1
nonhysteretic repulsion
-0.6
500
600
• one polymer chain
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
CREATION OF MOLECULAR ELASTICITY MASTER CURVE
(tgt)
t
O
-1.2
H
H
O
O
-0.2
-3.2
-5.2
-0.4
Force (nN)
Force /Radius (mN/m)
0
t t
-0.6
0.4
0.6
0.8
0.278 nm
H
O
-9.2
1
Distance / Lcontour (nm)
• reduction in extensional force
(*first reported by Oesterhelt, et al. 1999)
t g tO t
t
H
Inextensible FJC
Markovian 2-state model for Si3N4 vs PEO
0.2
H
O
strain-induced conformational
transition (ttgttt)
-7.2
0
g
t t
O
H
(ttt)
O
H
H
O
O
O
O
H
0.358 nm
H
O
O
H
H
reversible on
experimental time scales
C12H23
Au
PEO
40
2
30
20
1
10
0
0
100
200
300
400
30
20
2
1.5
1
10
0
0.5
0
adhesion
2.5
<D<D
> (nm)
adhesion
adhesion > (nm)
Dadhesion (nm)
> (nN)
<F<F
adhesion >(nN)
(mN/m)
<F<F
>/Radius (mN/m)
adhesion>/Radius
adhesion
0
C12H23
Au
PEO
3
adhesion
50
F
(nN)
Fadhesion
(nN)
(mN/m)
Fadhesion
/Radius
Fadhesion
/ Radius (mN/m)
ADHESION FORCES AND DISTANCES FOR INDIVIDUAL RETRACT
CURVES, HSA PROBE TIP VERSUS VARIOUS SURFACES :
PBS, IS=0.01M, pH=7.4
500
400
300
200
100
0
C12H23
Au
PEO
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
O
NH2
OH
O
O
• 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)
OH
OH
OH
O
OH
O
O
HO
HO
chitobiose
(CB)
O
HO
OH
HN
HN
O
O
linear
trimannoside
(LT)
HO
HO
OH
O
O
HO
HO
OH
O
O
O
HO
HO
OH
OH
O
OH
OH
HO
HO
OH
O
O
OH
HO
HO
O
OH
O
O
O
O
oligomannose-9 (Man-9)
O
O
OH
O
HO
HO
HO
HO
HO
HO
O
HO
HO
OH
O
OH
OH
O
HO
OH
O
HO
O
OH
O
OH
O
HO
HO
O
O
O
O
HO
O
HO
HN
OH
O
O
HN
O
O
Glycocalyx-Mimetic Neutral Oligosaccharide
Monolayers (Synthesized by Seeberger Lab, MIT-CHEM)
COOH SAM
Mannose SAM
LTM SAM
HM SAM
NM SAM
COOH - sd
EG3 + sd
EG3 - sd
Mannose + sd
Mannose - sd
LTM + sd
LTM - sd
NM + sd
NM - sd
HM + sd
HM - sd
Force (nN)
0.3
0.2
0.1
0
0
20
40
60
Distance (nm)
80
Plant Fibers
lumen (0.11 mm)
tertiary wall or secondary wall S3
spiral
angle of
microfibrils
18o
inner secondary wall S2
outer secondary wall S1
(0.71 mm) helically arranged
crystalline cellulose microfibril
network
40o
primary wall :
(0.23 mm) disorderly arranged
crystalline cellulose microfibril
network
amorphous
region of lignin
and hemicellulose
HO
H
H
OH
H
H O
HO
HO
H
H
OH 1
H
H

H
OH
H

H
O
HO
O
O
HO
H
1
O
H
H
cellulose
OH
OH
O
HO
H OH
H
H

1
OH
H
H HO
n
H
O