Download Inhibition of cell adhesion by a synthetic polymer adsorbed to glass

Inhibition of cell adhesion by a synthetic polymer adsorbed to glass
shown under defined hydrodynamic stress
NORMAN F. OWENS, DAVID GINGELL
Department of Anatomy and Biology as Applied to Medicine, The Middlesex Hospital Medical School, Cleveland St, London W1P 6DB, L'K
and PAUL R. RUTTER
B.P. Research Laboratory, British Petroleum Pic, Snnbiin^ on Thames, Middlesex, UK
Summary
A co-polymer -with hydrophobic and hydrophilic
segments was allowed to adsorb from aqueous
solution onto glass previously made hydrophobic
by derivatization with octadecyl dimethylchlorosilane. The polymer is thought to adsorb via its
hydrophobic segments, leaving the hydrophilic
segments free to extend into the water. After
allowing cells to settle on the treated surface, the
shear stress at the chamber wall required to
remove red blood cells, Dictyosteliutn discoideum amoebae and Escherichia coli was determined in a calibrated laminar flow chamber. On
octadecyl glass a shear stress of 2-3 Nm~ 2 evicts
50% of adherent red cells and E. coli. No D.
discoideum amoebae could be removed at
5Nm~ 2 . In striking contrast, the lowest experimentally obtainable shear stress of 0*03 Nm~ z
removes 97-0-99-5% of cells of all three types
from the polymer-treated surface, even after a
cell residence time of l h without flow in the
absence of free polymer. The minimum shear
stress of 0-03 Nm~ 2 corresponds to only =20
times the force of gravity on a red cell. The
mechanism of action of the polymer and the
implications of the results are discussed.
Introduction
The relationship between surface physicochemical
properties and biocompatibility is not a trivial problem
and is still much disputed after more than two decades
(see the critical discussion by Ruckenstein & Gourisankar, 1984). In this period only limited progress has
been made towards the goal of a blood-compatible
interface for vascular prostheses or kidney dialysis
equipment (Brash, 1983).
Of the large number of polymers that have been
assessed for blood compatibility, polyethylene oxide
(PEO), either alone or as a component of a co-polymer,
has been found to have notable anti-adhesive properties. George (1972) described a dramatic reduction in
the adhesion of platelets to glass in the presence of
PEO. Earlier, Hiatt et al. (1971) had found that PEO
prevented the adsorption of rabies virus to glass.
Whicher & Brash (1978) and Brash & Uniyal (1979)
showed that little protein adsorption occurred on a
solid co-polymer composed of PEO and polyurethane.
Thrombin and platelet adsorption to similar copolymers was studied by Sa da Costa et al. (1980,
The prevention of cell adhesion to surfaces under
controlled conditions has long been the goal for the
development of surfaces that are non-adhesive to blood
components, marine microorganisms and bacteria.
The search has been for a material that would have
minimal attractive forces for cells and proteins, and
that would possess maximal repulsive forces. This
implies that the combined energies of van der Waals'
attraction, entropic attraction (i.e. 'hydrophobic bonding') and hydrogen bonding should be smaller than the
sum of electrostatic repulsion, hydration repulsion and
entropic repulsion due to thermal motion of flexible
molecular chains. It should, in addition, be mechanically and chemically stable. The search for such a
material has largely concentrated on synthetic
polymers (see Andrade, 1985a; Nordeei al. 1986) and
since their surface properties in a biological milieu will
be modified by protein adsorption, the latter process
has been extensively studied (see Andrade, 19856).
Key words: cell adhesion, blood compatible synthetic
polymer.
Journal of Cell Science 87, 667-675 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
667
1981), using thick layers of polymer on glass beads,
produced by evaporating the non-polar solvent. They
performed XPS analyses on the particle surface layers
and concluded, after comparing the results with XPS
of polyurethanes alone, that lack of cell and protein
adsorption was a function of the PEO moiety and
suggested that an amorphous network of strongly
hydrated PEO would be maximally anti-adhesive.
Merrill et al. (1982) further characterized the low
platelet adhesiveness of related co-polymers based on
polyethylene glycol and polyurethane. Similarly, Mori
et al. (1982) described the striking in vitro and in vivo
anti-thrombogenic properties of PEO-methacrylatePVC co-polymers, but detected substantial protein
adsorption by scanning electron microscopy (SEM)
after one day under in vivo conditions. They made
and tested polymers with PEO side-chains (arranged
herringbone fashion on a backbone of PVC) of various
lengths (5-100 monomers) and found the longest to be
the most blood-compatible.
Against the background of these results, we describe
a relatively simple and well-defined water-soluble
polymer consisting of two polyethylene oxide segments
spaced with a relatively hydrophobic polypropylene
sequence. The polymer adsorbs strongly to a hydrophobic glass surface, where it almost totally prevents
three very different cell types from adhering, as judged
by the criterion of liquid shear in a calibrated flow
system.
Materials and methods
Materials
The bifunctional polymer Pluronic (F108) was obtained from
ICI Pic. Penicillin-streptomycin (5000IU) from Gibco
Chemicals, Middlesex, UK was stored below 0°C. Concanavalin A (ConA) conjugated with tetramethylrhodamine B
isothiocyanate was purchased from Sigma Chemical Co.,
Poole, UK. A stock solution O-Smgml" 1 prepared in Tris
buffer at pH 7-4 was stored in 0-2 ml samples below 0°C until
required. Bacteriological peptone and yeast extract were
obtained from Oxoid Chemicals Ltd, Basingstoke, Hampshire, UK. Disodium hydrogen orthophosphate, sodium
dihydrogen orthophosphate, sodium chloride and D+-glucose
were all of AnalaR quality from BDH Chemical Co., Poole,
UK. The procedure for obtaining surface chemically pure
water by distillation from alkaline potassium permanganate
will be described elsewhere (Owens et al. 1987). Microscope
coverslips (40 mm X 22 mm X 0-17 mm) from Chance Propper Pic, Smethwick, UK were rendered hydrophobic by
overnight immersion in a 2 % solution of octadecyl dimethylchlorosilane (Sigma, Southampton, UK) in chloroform.
The treated coverslips were first rinsed with chloroform, then
in distilled water to remove HC1. If required for later use they
were stored in chloroform. Precision micrometer flow valves
(Nupro Co., types S and M) were purchased from North
London Valve & Fitting Co., London, UK.
668
vV. F. Owens et al.
Cultures
Vegetative cells of Dictvostelium discoideum (Ax2 strain)
were grown to the fully differentiated spore fruiting stage at
22°C on 2 % nutrient agar inoculated with Escherichia coli
B/r. The dormant spores were germinated in glucose-free
axenic medium containing 0-5 ml penicillin-streptomycin for
72 h in a rotary incubator at 22CC. This was followed by a
growth cycle in axenic medium supplemented with 86 mMglucose. Ax2 amoebae were isolated from shaken cultures
during log-phase growth ( l x 106cells ml" 1 ) by allowing the
cells to settle onto a clean glass surface rather than the usual
centrifugation routine. In so doing we avoided any tendency
for the cells to rupture during harvest. The adherent cells
were first washed with 20mM-NaCl and then resuspended in
the same electrolyte using gentle fluid flow from a Pasteur
pipette to dislodge cells from the glass surface. Streaming
flow experiments were done in unbuffered 20 mM-NaCl at the
pH of our distilled water (pH 5-8-6-2). The work was done
in a temperature-controlled laboratory at 20(±0-5)°C.
Human red blood cells were obtained by venipuncture into
citrate buffer, pH 6-8. The cells were washed three times by
centrifuging with Dulbecco A phosphate-buffered saline
(PBS), pH7-4 (Oxoid, UK) at 400 £. A 10ft! sample of the
packed cells was diluted with similar PBS to give a cell
density of 8 x l 0 6 m l ~ ' . This was kept at room temperature
until required. Streaming flow experiments were also done in
PBS at pH 7-4.
E. coli B/r were incubated in nutrient broth no.2 (Oxoid)
for 48 h at 37°C. A 30 ml suspension of bacteria was pelleted
by centrifugation at 1000 £f for lOmin. Bacteria were washed
by four successive 10-min centrifugations at 1000 g using
30 ml samples of PBS at pH7-4. The washed cells were
resuspended and stored in PBS until required. Streaming
flow experiments with E. coli were conducted in PBS,
pH7-4.
Methods
A laminar flow of electrolyte from an elevated reservoir was
passed through a rectangular conduit in which a derivatized
glass coverslip formed the upper wall of a closed parallel plate
channel 388 ftm wide. The wall shear TW exerted by the flow
against the solid surface was calculated for varying rates of
flow from the equation:
dP
b,
' d/
(1)
where P is the hydrostatic pressure drop across the conduit of
length / and wall separation 2b. The conduit design, flow
system and calibration procedure are described in detail
elsewhere (Owens et al. 1987) so only the salient features are
given here.
The components of the conduit are illustrated in Fig. 1. A
glass coverslip C was held in a recess on the upper face of a
polycarbonate block D by a stainless steel frame A. This was
secured by screws that passed through a silicone rubber
gasket B into a stainless steel baseplate E. The flow conduit
was formed between the coverslip and a flat-bottomed
channel across the polycarbonate block D. Fluid enters
through a cylindrical pipe integral with the end wall of block
D leading into a confusor of elliptical section and then into
the conduit. Fluid exit is via a symmetrical arrangement.
The baseplate E, which has a central hole for transmitted
illumination, locates the assembly on the microscope stage.
A Bowline of glass and silicone rubber tubing was connected to the chamber and the whole system filled with electrolyte, care being taken to exclude any air bubbles. Exchange of
electrolyte for 0 1 % polymer solution followed, to ensure
that the polymer-coated glass did not become exposed at any
stage to air. After 2 h the system was flushed with electrolyte
and the cells were injected into the inverted chamber on an
inverted microscope through a silicone rubber diaphragm in a
side arm, using a hypodermic syringe with an 8 cm needle.
Sedimentation of cells onto the derivatized glass wall was
essentially complete after 20min. Cells were observed with a
Zeiss 25 X oil-immersion objective lens under bright-field and
IRM illumination. The flow rate through the conduit was
varied by means of precision micrometer valves mounted in
parallel in the flowline. On polymer-treated surfaces, cells
were exposed to a minimal flow rate of 001 mis" 1 for 30 s in
contrast to those on control surfaces where the flow rate was
extended to 2-0 ml s~' over the same period. Cell-substratum
interactions under hydrodynamic shear were recorded on
closed-circuit TV using a Chubb surveillance camera connected to a video recorder (Sony U-Matic) and monitor. Later
experiments utilized a sensitive 'Falcon' SIT camera (Custom Camera Devices Ltd, Wells, UK).
Calibration of flow system
For a flow chamber of the type used, the Reynold's number is
given by R= \-32VnxM^b/rj, where Vmnx is the maximum
velocity of flow (along the central axis of the chamber), 2b is
the separation of the flat parallel walls and 7) is the absolute
viscosity. The critical Reynold's number Rc corresponds to a
critical value of Vnax below which flow is laminar and above
which it is turbulent (Eskinazi, 1975, p. 384). For a rectangular cuvette like ours where length > width *> depth,
Rc = 2300. In our system at maximum flow R = 200 < Rc so
that flow is laminar under all our experimental conditions.
Although flow in the cuvette will always be laminar, the
liquid must pass a certain distance along the channel before a
constant parabolic velocity profile is established. This establishment length Lc is a function of chamber shape and
Reynold's number, L e = 0013W? (Sparrow, 1955). For our
system Lc is less than 1 % of the conduit length so flow will be
fully developed over virtually the entire length of the conduit.
This conclusion is consistent with the findings of Van
Wagenen & Andrade (1980) for the measurement of streaming potentials generated by a laminar flow of electrolyte
between parallel glass plates. Fig. 2 shows a linear relationship between the wall shear, calculated from equation (1),
and the applied pressure difference across the chamber (P).
Results
Fig. 1. The flow chamber.
Numerical results for the behaviour of red blood cells,
D. discoideum and E. coli under hydrodynamic stress
on control octadecyl glass and F108-treated octadecyl
glass are shown in Table 1. On F108-treated glass
between 97-0 and 99-5 % of cells that were initially in
contact with the surface were removed at a wall shear
stress r w = 0-03 N m~ 2 . On the control surface 100-0 %
Inhibition of cell adhesion
669
300 400 500
Pressure p (Nm 2)
1(X)
600
700
800
Fig. 2. Calculated shear stress at the glass wall of the flow
chamber as a function of applied hydrostatic pressure
difference across the chamber. ( # ) Low flow rate valve;
(O) higher flow rate valve. Final point, both valves open.
Table 1. Behaviour of cells under hydrodynamic
stress
A. Polymer-treated glass
Number of
expts
Before
flow
RBC
5
E. coli
D. discoideum
4
1361
1070
137
2
After flow % Cells
(0-03 N m - 2 ) removed
98-8
99-5
97-0
17
5
4
RBC
E. coli
D. discoideum
2
1
3
186
360
29
186
360
29
0
126
29
°h Cells removed
(Nm-2)
0-03
5
0-0
0-0
0-0
100-0
65-0
0-0
For 50% removal in 30 s: red blood cells (RBC), 2-3 Nn-T 2 ;
E.coli, 3-0 Nm- 2 .
of all three cell types remained stuck at this shear stress.
Increasing the flow rate to give r w = 5 N m ~ caused
the removal of all red cells and 65-0% of E. coli but
failed to remove any D. discoideum amoebae. Nor were
any of the latter removed at 5-9Nm~ 2 , which is
currently our maximal obtainable shear stress. We
found that half the red cell population and also half
the bacteria were removed at 2-3 and 3'0Nm~ 2 ,
respectively.
Fig. 3A,B,C shows transmitted light images of red
cells, D. discoideum amoebae and E. coli on F108670
N. F. Owens et al.
We found that it was possible to define the boundary
zone between polymer-treated and untreated glass
using a fluorescent dye adsorption test. Rhodamineconjugated ConA was shown to adsorb strongly to
hydrophobic glass but failed to adsorb to glass treated
with F108, thus both providing a convenient practical
assay for polymer adsorption and showing that F108
vetoes protein adsorption as well as cell adhesion.
Discussion
B. Control octadecyl glass
After flow
Number
r2)
of
Before .
cxpts flow
0-03
5
treated glass before flow, taken from videotape. The
uneven background is due to light transmitted by the
non-optical plastic base of the chamber. Fig. 3D,E,F
shows typical corresponding fields at minimum flow,
giving O03Nm~ 2 wall shear stress. The 0-5 s photographic exposure time shows cells that have been
removed by flow as streaks or blurs, while the few static
cells are sharply focused. The behaviour of cells on the
control surface of octadecyl glass, before and after flow,
is shown in Fig. 4. We also made two tests on the
polymer-treated glass to assess its durability. In the
first, physiological saline was allowed to flow through
the chamber at full rate for 1 h. After this the surface
was as anti-adhesive to red blood cells as it was before
exposure to prolonged flow. In the second test the
polymer-coated glass was allowed to air dry for 30 min,
before being re-hydrated in physiological saline for
30 min. The prevention of cell adhesion by this surface
was indistinguishable from that of polymer-treated
glass kept rigorously out of contact with air.
The parallel plate hydrodynamic shearing technique
that we have used for assessing cell-to-substratum
adhesion is well known. It has been extensively
employed to investigate both the adsorption of cells
from flowing suspensions onto the chamber walls as
well as the removal of attached cells (Doroszewski et al.
1977, 1979; Forrester & Lackie, 1984; Hochmuthe/ al.
1973; Mohandasef al. 1973, 1974). The hydrodynamic
removal force on a cell attached to the chamber wall can
be determined from the wall shear stress r w and there is
reason to believe that this force is not significantly
dependent on the details of cell shape and the degree of
spreading (Hubbe, 1981) for any given cell type.
Our data for red cells on siliconized glass (Table 1)
can be directly compared with the work of Mohandas et
al. (1974) for the removal of red cells by hydrodynamic
shear in a saline solution. These authors report that
cells are removed during 300 s flow only when
2
T W > 0 - 3 5 Nm~ . Cell removal was found to depend on
both Tw and time. Their fig. 8 shows that half the cells
are removed in 300 min on exposure to 1 N m~ 2 and all
are gone in the same time at 1-5 N m~ 2 . We found that
in 30 s half the initially adherent cells are removed from
octadecyl glass at 3 N m~ and that all are removed at
*r
•
. •'••* ''•%. w
r
003
r i
'
I
•
s
••<•-!.."<
Nm2
•
./
003
Nm2
Fig. 3. Cells on F108-treated glass before flow (left column) and after flow at the minimum rate (right column). A,D. Red
cells; B,E, Dictyostelium; C,F, E. coli. Wall shear stress and scale inset.
5 N m 2 . Since our flow times differ substantially from
those of Mohandas et al. (1974) we can only state that
there is no evidence that the two sets of results are
inconsistent. In view of our results with F108, it is of
interest that Mohandas et al. (1974) found a large
decrease in percentage adhesion in the presence of
Inhibition of cell adhesion
671
fibrinogen. Unfortunately they did not measure protein adsorption to siliconized glass and give no shear
stress data. The analysis of red cell removal from
octadeeyl glass is considered in more detail elsewhere
(Owens et al. unpublished data).
Our data for bacterial adhesion can be compared
with those of Fowler & MacKay (1980), who used a
radial flow chamber to examine the growth of unidentified cocci in protein-free media. Although cell counts
are not given, cells are stated to be unable to attach and
multiply where the wall shear stress exceeds 2-6 N m~ 2
(units of Tw in their Tables 7.1 and 7.2 are not correctly
expressed). This is very similar to our value of
3-0 N m~ 2 for 50 % removal of E. coli, but owing to the
very different conditions employed, the similarity of
the two sets of results may be fortuitous. Taking the
dimension of E. coli as a cylinder of 0-5 jum radius and
3 jxra long, the bacterium is equivalent to a sphere of
0-8fim radius. Consequently, using
E=32r~2
(Hubbe, 1981) we obtain a characteristic removal force
of 1X10" 10 N per cell at 3 N m " 2 .
In the case of F108-treated glass, the minimal wall
shear stress used in our system, 0-03 Nm~ 2 , removes
between 97-0 and 99-5 % of attached cells, according to
cell type. In view of the widely different surface
compositions of these cells (prokaryote, eukaryote and
slime mould) this result argues persuasively that F108
is a very potent general anti-adhesive. Therefore its
mode of adsorption and mechanism of action are of
considerable interest.
Pluronic F108 consists of two hydrophilic polyoxyethylene chains separated by a central hydrophobic
polyoxypropylene chain and has the general formula:
H-(OCH 2 CH 2 ),,(OCH 2 CH(CH 3 )V
-(OCH 2 CH 2 ) n OH ,
where a is approximately 130 and b is approximately
45. The molecule adsorbs on hydrophobic surfaces in
such a way that the polyoxyethylene chains are oriented
003
. •
Fig. 4. Cells on control hydrophobed glass before flow (left) and after flow (right). A,C. Red cells; B,D, Dictvostelium.
672
N. F. Owens et al.
Water
CH,
Polyethylene oxide)
CH,
CH.
CH,
CH,
Poly(propylene glycol)
0-CH-CH,-0-CH-CH,-0
\ '
A '
A
CH
ACH
\
0-CH-CH,-0
A '
I
A ^
J
Fig. 5. Diagram of a molecule of the bifunctional polymer
F108 adsorbed at the interface between water and
hydrophobed glass.
away from the surface and into the aqueous phase (see
Fig. 5) to produce an adsorbed layer approximately
26 nm thick (Kayes & Rawlins, 1979). The high
solubility of polyoxyethylene is usually attributed to
the helical structure that it adopts in water, resulting in
an oxygen-oxygen distance of 0-288 nm, which is very
similar to that between these atoms in water itself. The
corresponding distance of 0-75 nm in polyoxypropylene is very different, rendering this material waterinsoluble (Tronel-Peyroz et al. 1983).
Since the molecular composition of F108 includes no
formal negative charges, Debye electrostatic repulsion
cannot be the mechanism of non-adhesiveness. It is
interesting that charge repulsion is in any case of
limited effectiveness
in preventing adhesion.
Trommler et al. (1985) found that although the proportion of red cells adhering to glass was reduced at low
ionic strength (when repulsion is substantially
increased) a significant proportion of cells nevertheless
made molecular contacts with the glass and became
strongly adherent.
Thus we seek another explanation for the action of
F108. The fact that red cells can remain indefinitely in
contact with a 0-01 % aqueous solution of the polymer,
or on a surface treated with polymer, without haemolysis shows that it does not disrupt the cell membrane.
There remain only two likely candidates for the repulsion observed, steric (or entropic) repulsion and water
structuring (hydration repulsion).
Steric repulsion is to be expected because of the
extension of the hydrophilic arms into the water. If so,
repulsion should begin when the glycocalyx of an
approaching cell begins to interdigitate with the hydrophilic segments of the adsorbed polymer, thus reducing
the available modes of gyration of the polymer chains
and decreasing entropy. A direct measurement of
intermolecular repulsion associated with PEO chains
has been obtained by Klein & Luckham (1982) using
the Israelachvili technique. They measured the force
between mica plates with adsorbed PEO in 0-1 MKNO3. Repulsion began at a mica-mica separation of
6R (R = radius of gyration of the polymer) and rose
monotonically as the separation was reduced. Little
evidence of desorption over 72h was found. Thus the
effective length of the individual polymer chains when
they began to repel mutually is 37?. This corresponds to
18-20 nm for one polymer (iV/r = 40000; degree of
polymerization, T Z ~ 9 0 0 ) or 33-38 for another
(Mr= 100000; « = 2300). Under conditions where
PEO can simultaneously adsorb to both mica plates,
cross-bridging forces could be measured (Klein, 1986).
These results suggest that repulsion in the case of the
PEO co-polymer that we used (;/ ~ 130) might begin at
a comparatively small effective extension distance, say
3nm.
Evidence for thermal flexibility of PEO chains in a
mixed polymer was obtained by Mori et al. (1982) who
related the width of the peak I3 C nuclear magnetic
resonance (n.m.r.) signal to the degree of polymerization (ti) of the PEO moiety. They found that increasing n to 100 decreases the width of the signal peak,
indicating high flexibility (no graph shown).
The other probable source of repulsion, water structuring, as an important component of interaction forces
is a relatively recent discovery (Le Neveu et al. 1977;
Parsegian et al. 1979; Parsegian & Rau, 1984; see also
Israelachvili, 1985). The electric field at a surface (due
to adsorbed ions, ionogenic groups or possibly dipoles)
alters the orientation and thus the structure of the
nearby water. The result is that water molecules within
about 2nm of a surface are to some extent associated
with the surface. Although the individual binding
energy per molecule is extremely small, the energy
needed to remove large numbers of them from any
macroscopic region between approaching surfaces is
very substantial. Between the lamellae of lecithin liquid
crystals the hydration pressure at 1 nm separation
exceeds 10 7 Nm~ 2 and rises to near 10 9 Nm~ 2 or 104
atmospheres at 0-2 nm (Parsegian et al. 1979). However, until the extent of water structuring associated
with F108 is measured it will not be possible to
appreciate fully how the PEO exerts its remarkable
anti-adhesive effects.
Inhibition of cell adhesion
673
The authors thank British Petroleum Pic for financial
support and for permission to publish this paper. D.G.
thanks the Wellcome Trust and the SERC for financial
support.
KLEIN, J. (1986). Surface interactions with adsorbed
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KLEIN, J. & LUCKHAM, P. (1982). Forces between two
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aqueous solvent. Nature, Land. 300, 429-431.
LE NEVEU, D. M., RAND, R. P., PARSEGIAN, V. A. &
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