Note: Descriptions are shown in the official language in which they were submitted.
CA 02226133 1998-01-02
Bioactive surface coatinq
The invention relates to a coating process of a
surface of a substrate, preferably of a plastic (or polymer)
substrate, by graft polymerization. An important property of
the coating applied by the process of the invention is high
compatibility with a body fluid and tissue. Depending on the
functionality of a coating monomer and/or on a molar ratio of
certain functional groups within the coating, the surface
additionally acquires either antibacterial and cell
proliferation inhibiting properties, or antibacterial and cell
proliferation promoting properties. The invention also
relates to an article with a surface coated by the process of
the invention and to the use of such article for medical or
biotechnical purposes.
The colonization and multiplication of bacteria on a
surface is a phenomenon which in general is unwanted and which
is frequently associated with adverse consequences. For
instance, in the drinking water and beverage industry,
bacterial populations can lead to a reduction in quality and
can pose a hazard to health. Bacteria on or in packaging
frequently brings about the decay of foods and can cause
infections in consumers. In biotechnical plants that are to
be operated under sterile conditions, bacteria alien to a
system constitute a considerable processing risk. Such
bacteria may be introduced with raw materials or may remain in
any parts of the plant if sterilization is inadequate. By
means of adhesion, portions of a bacterial population may
escape the normal liquid exchange entailed in rinsing and
cleaning and can then multiply within the system.
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Colonies of bacteria are also known in water
treatment plants (for example for membrane desalination) or
else in containers which are filled with dissolved or liquid
undiluted organic substances and which provide suitable
conditions for supporting bacterial populations. In general,
significant microbial colonization in a water treatment plant
can, to a considerable extent, lead to the blocking and/or
corrosive destruction of the plant.
Particular importance is attached to protecting
against bacteria adhesion and propagation in the areas of
nutrition, human care, especially care of the elderly, and
medicine. In the case of large-scale outlets serving food or
drinks, there are considerable risks especially when, as an
alternative to disposable tableware with its attendant problem
of wastage, reusable tableware is employed but is not
adequately cleaned. Also known is the harmful propagation of
bacteria in hoses and pipes which conduct foods, as is their
multiplication in storage containers, in textiles and in hot
and damp environments, for example in swimming baths.
Facilities such as these are preferred habitats for bacteria,
as are certain surfaces in areas with a high level of public
traffic, for example in public transport vehicles, hospitals,
telephone boxes and schools and especially in public toilets.
In the care of the sick and elderly, the often
reduced defenses of those affected necessitate careful
measures to counter infections, especially on intensive care
wards and at home.
Particular care is required in the use of medical
articles and instruments in the case of medical investi-
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CA 02226133 1998-01-02
gations, treatments and interventions, especially when such
instruments or articles come into contact with living tissue
or with body fluids. In the case of long-term or permanent
contact, especially in the case of implants, catheters,
stents, cardiac valves and pacemakers, bacterial contamination
can become a life-threatening risk to a patient.
Diverse attempts have already been made to suppress
the colonization and propagation of bacteria on surfaces. In
J. Microbiol. Chemoth. 31 (1993), 261-271, S.E. Tebbs and
T.S.J. Elliot describe paintlike coatings with quaternary
ammonium salts as antimicrobial components. It is known that
these salts are dissolved out of the coating material by
water, by aqueous or other polar media and by body fluids, and
that their action is therefore short-lived. This applies
equally to the incorporation of silver salts in coatings, as
described in WO 92/18098.
T. Ouchi and Y. Ohya, in Progr. Polym. Sci. 20
(1995), 211 ff., describe the immobilization of bactericidal
active substances on polymer surfaces by means of covalent
bonding or ionic interaction. In such cases, the microbicidal
actions are in many cases markedly reduced relative to the
pure active substance. Heteropolar bonds often prove to be of
insufficient stability. Furthermore, the killing of microbes
by this approach in general leads to unwanted deposits on the
surfaces, which mask further bactericidal action and form a
basis for subsequent bacterial colonization.
W. Kohnen et al., in ZBl. Bakt. Suppl. 26, Gustav
Fischer Verlag, Stuttgart-Jena-New York, 1994, pages 408 to
410, report that adhesion of Staphylococcus epidermidis to a
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polyurethane film is reduced if the film is pretreated by glow
discharge in the presence of oxygen and is then grafted with
acrylic acid.
As mentioned, when medical articles and instruments
are used in medical investigations, treatments and inter-
ventions, it is important to prevent bacterial contamination
of these articles and instruments. In the case of some of
these articles and instruments, which come into medium- or
long-term contact with living tissue or body fluids, adhesion
and propagation of endogenous cells is extremely undesirable.
Thus cell colonization in the case of catheters applied
intracorporally in the medium term is just as harmful as in
the case of cardiac valves or stents which are implanted in
the long term.
Furthermore, transparency of intraocular lenses
after implantation may undergo a continuous deterioration as a
result of cell colonization. There is a range of processes
aimed at avoiding cell colonization, for example incorporating
certain metals or metal salts into the mount of the
intraocular lens, although the incorporation lS usually
incomplete and not durable. For example, WO 94/16648
describes a process which is intended to prevent the
proliferation of cells on the surface of implanted ocular
lenses made from polymer material.
According to EP 0 431 213, polymers can be furnished
with cell-repelling properties by rendering their surface
hydrophilic with strong mineral acids. The subsequent
chemical modification of polymer surfaces, however, is in most
cases not uniform. In general, there remain sites which
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either remain untreated or are not sufficiently treated and
which constitute starting points for cell colonization. In
addition, the cell-repelling properties of surfaces treated in
this way in many cases do not last.
On the other hand, certain applications require
articles having surfaces which are repellent to bacteria but
which promote cell colonization. This applies, for example,
to a variety of instruments for medical investigations,
treatments and interventions and also to many prostheses which
are intended to grow into the tissue into which they have been
implanted. Such instruments and prostheses, for example
artificial hip joints or teeth, often consist of polymer-clad
materials such as titanium.
Finally, materials for instruments and devices which
come into contact with body fluids, such as blood or lymph, or
with tissue, must be compatible with their foreign environ-
ment. Blood compatibility in particular is an important
desired property. The materials must therefore as far as
possible have pronounced antithrombic properties.
There is therefore a variety of partially mutually
exclusive requirements with respect to the bioactive
properties of the surface of polymers which are intended for
medical uses. They are required always to be antibacterial
and compatible with body fluids and tissue but should have an
alternatively cell proliferation inhibiting or promoting
action.
A major object of the present invention is to
develop an improved process for coating a surface of a
substrate to make the surface antibacterial and compatible
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with a body fluid and tissue as well as cell proliferation-
inhibiting or promoting, without thereby altering the
mechanical properties of the treated substrate or giving rise
to any other major disadvantages.
It has surprisingly been found that a surface of a
substrate, especially a polymeric substrate can be made to
possess those properties mentioned above by coating according
to a process described hereinunder.
According to the process, at least one monomer of
the general formula (I):
R-(A)a (I)
(in which R is a mono- or diolefinically unsaturated
organic radical, for example a hydrocarbon
radical, having the valency a,
A is a carboxyl group -COOH, a sulfuric acid
group -OSO20H, a sulfonic acid group -SO3H, a
phosphoric acid group -OPO(OH)2, a phosphonic
acid group -PO(OH)2, phosphorous acid group
-OP(OH)2, a phenolic hydroxyl group, or a salt
or an ester of one of these groups, and
a is an integer from 1 to 6),
is subjected to graft-polymerization under radiative induction
on to an activated substrate surface, with the proviso that
when A is a carboxyl group -COOH or a salt or an ester of the
carboxyl group, the monomer contains at least one further
radical A which is a different one of those specified for A or
is used together with at least one further monomer of the
formula (I) in which A is a different one of those specified
for A.
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Among the salts of the groups specified for A,
preferred are physiologically acceptable salts including
alkali metal salts and, in particular, sodium salts.
The common feature of the monomers of formula (I) is
that they have one or two olefinic double bonds and also at
least one acidic group or a particular derivative, namely a
salt or an ester, of an acidic group.
Coatings produced on various substrates by a plasma-
induced graft polymerization of functional monomers are known,
for example, from B. Lassen et al., Clinical Materials 11
(1992), 99-103, and have been investigated for biocompati-
bility. No activating pretreatment is mentioned in this
literature.
Moreover, plasma is not an optimal polymerization
initiator. H. Yasuda refers accordingly in J. Polym. Sci.:
Macromolecular Review, Vol. 16 (1981), 199-293, to the
undefined and uncontrollable chemistry of plasma polymer-
ization. This may be acceptable for some purposes, but is
problematic for medical and biotechnical applications, since
reproducible coatings of consistently high quality are
required.
Surprisingly, the antibacterial properties of the
surface coated in accordance with the invention with a
carboxyl, carboxylate or carboxylic ester group-containing
monomer of formula (I) together with another monomer of
formula (I) are markedly more pronounced than is the case with
the modification with acrylic acid alone, according to
W. Kohnen et al., in ZBl. Bakt. Suppl. 26, Gustav Fischer
Verlag, Stuttgart-Jena-New York, 1994, pages 408 to 410, under
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comparable conditions.
A surface coated in accordance with the process of
the invention displays a remarkable combination of advan-
tageous properties and therefore outstanding physiological
compatibility. It is, in particular, highly compatible with
blood and reduces the adhesion and propagation of bacteria to
a high extent even over a prolonged period. Bacteria affected
by this action include Staphylococcus aureus, Staphylococcus
epidermidis, StrePtococcus pyoqenes, Klebsiella pneumoniae,
Pseudomonas aeruginosa and Escherichia coli. At the same time
there is also inhibition of the proliferation of cells in most
cases, for example of fibroblasts and endothelial cells, such
as human umbilical cord cells. The particular conditions
under which a coating has an antibacterial but cell
proliferation-promoting action are explained later. The
surface of a substrate coated in accordance with the process
of the invention is free from migratable and/or extractable
monomer and oligomer components. Unwanted side effects
resulting from released exogenous substrates or from dead
bacteria are avoided from the outset.
In the process according to the invention, the
substrate surface is first activated, as described in more
detail below, and then is coated under the action of
radiation, such as W light by non-aggressive graft
polymerization or graft copolymerization.
1. The mo~nm~r~
In the formula (I), R is a mono- or diolefinically
unsaturated organic radical. Preferably, the organic radical
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R is a hydrocarbon radical having 2 to 12 carbon atoms which
may have a substituent other than an acidic group and not
having negative effects upon living bodies, such as C1_4 alkyl
and -NH2. The hydrocarbon radical may preferably be one
represented by the formula CnH2n_q_x (in which n, q and x are
defined hereinunder), (C6H6-b-C-d~cnH2n-l-q-x) (in which b~
c, d, n, q and x are as defined hereinunder), R1 HC=CR1 -R2 _
(in which Rl and R2 are as defined hereinunder) or
R5HC=CRl -R2 (in which R5, Rl and R2 are as defined
hereinunder).
In the graft (co)polymerization, the monomer is
preferably a mixture of (1) a monomer of the formula (I)
having one or two carboxyl groups or a salt or ester thereof
and (2) a monomer of the formula (I) having one or two
sulfonic acid groups or a salt or ester thereof. More
preferably, such a mixture may be a mixture of monomers of the
general formulae (II) and (III):
(CnH2n_q_x)(COOR )x (II)
(CnH2n_q_x)(S03R )x (III).
In the formulae, which are within the scope of the
formula (I),
B independently at each occurrence is an integer from
2 to 6;
_ independently at each occurrence is 1 or 2;
independently at each occurrence is 0 or 2; and
Rl, independently at each occurrence, is hydrogen, an
equivalent of a metal ion, preferably an alkali metal ion, a
residue of an aliphatic, cycloaliphatic or araliphatic
alcohol, preferably of an alkanol having 1 to 8 carbon atoms,
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preferably having 1 to 4 carbon atoms, a residue of a
cycloalkanol having 5 to 12 carbon atoms, a residue of an
arylalkanol having 7 to 10 carbon atoms or a residue of an
alkanol having one or more of oxygen or nitrogen atoms in its
chain and up to 13 carbon atoms, such as -(CH2-CH2-O)d-H,
-(CH2-CH(CH3)-O)d-H, -(CH2-CH2-CH2-O)d-H or -(CH2)d-
NH2_e(R2)e, where R2 is -CH3 or -C2H5, d is 1, 2, 3 or 4 and e
is 0, 1 or 2.
In accordance with the definitions given, the
radical (CnH2n_q_x)~ independently at each occurrence is a
straight-chain or branched monovalent alkenyl radical (q=0,
x=1) or alkadienyl radical (q=2, x=1) or a divalent alkenylene
radical (q=0, x=2) or alkadienylene radical (q=2, x=2).
Instead of two monomers of formula (II) and (III),
it is also possible to employ only one monomer which includes
both -COOR1 and -SO3R1 groups in the same molecule.
In addition, another group of preferred monomers of
the formula (I) are benzene-derived monomers of the general
formula:
(C6H6-b-c-d)(B)v(R3)c(oH)d~ namely
~ ~ (~E~d
~R3
in which:
B independently at each occurrence is a straight-chain
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or branched radical of the formula (CnH2n_1_q_x)_
(COOR )x or (cnH2n-l-q-x)(so3Rl)xt where R
and _ are as defined above;
R3 independently at each occurrence is a monovalent
substituent such as C1_4 alkyl, -NH2, - COOH, -SO3H,
-OSO3H, -OPO(OH)2, -PO(OH)2, -OP(OH)2, -OPO(O-)OCH2-
CH2 N (CH3)3, -PO(O )O-CH2-CH2-N+(CH3)3, -OP(O-)-
OCH2-CH2-N+(CH3)3 or optionally a salt, especially
an alkali metal salt, or an ester of the acid group;
- 10 b is 1, 2 or 3;
c is 0, 1, 2 or 3; and
d is 0, 1, 2 or 3;
with the proviso that b + c + _ < 6, preferably c 4.
It is of course also possible to employ any suitable
mixture of monomers of the general formulae (II), (III) and
(IV) for the process according to the invention.
Other suitable monomers corresponding to formula (I)
can contain neutral or acidic sulfuric esters and salts of the
latter; sulfonic acids, their salts and esters; phosphonic
acids, their neutral or acidic salts, neutral or acidic esters
and salts of the acidic esters; phosphoric esters, their
neutral or acidic salts, neutral or acidic esters and salts of
the acidic esters; and phosphorous acids, their neutral or
acidic salts, neutral or acidic esters and salts of the acidic
esters. These monomers too can be used as a mixture with one
another and/or with the monomers of the general formulae (II),
(III) and (IV) for the process according to the invention.
Finally, mention may also be made of phenols of
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formula (I) having a valency (or basicity) of from 1 to 3, and
also their salts, as suitable monomers. These too are
optionally employed as a mixture with one another and/or with
the abovementioned monomers.
For the process according to the invention it has
proved worthwhile to use a combination of monomers of formulae
(I) to (IV) which leads to a coating which has both a carboxyl
and/or carboxylate group and a sulfonic acid and/or sulfonate
group. From the standpoint of compatibility with regard to
lo the groups, there are three possible two-way combinations,
namely carboxyl and sulfonic acid groups, carboxyl and
sulfonate groups, and carboxylate and sulfonate groups, and
also two three-way combinations, namely carboxyl, carboxylate
and sulfonate groups, and carboxyl, sulfoacid and sulfonate
groups. All of these combinations constitute useful monomers
for the process according to the invention. It is of course
also possible to employ monomers whose functional groups are
altered after the graft polymerization. Thus it is possible,
for example, to transform an acrylamide structural unit
subsequently, by hydroysis in an acidic medium, into the
acrylic acid structural unit. It is also possible to convert
carboxyl and sulfonic acid groups by neutralization (for
example in phosphate buffers), and carboxylic ester and
sulfonic ester groups by hydrolysis, into carboxylate and
sulfonate groups, respectively.
In the abovementioned combination, the molar ratio
of carboxyl and/or carboxylate groups to sulfonic acid and/or
sulfonate groups in the coating can fluctuate within wide
limits. Particularly pronounced cell proliferation inhibiting
12
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properties are obtained when the ratio is from 0.2 to 3,
preferably from 0.4 to 3 and, in particular, from 0.4 to 2.
The coated surface exhibits, in a remarkable manner,
antibacterial but cell proliferation promoting properties when
the molar ratio is from 2 to 10, preferably from 3 to 10 and,
in particular, from 3 to 5. A coating is cell proliferation
promoting for the purposes of the invention when the adhesion
and multiplication of mammalian cells is improved by the
coating, in comparison with the uncoated surface, or at least
is less strongly impaired than the adhesion and multiplication
of bacteria.
Of the suitable monomers of the general formulae (I)
to (IV) which contain one or more identical or different
radicals A in the molecule, examples include:
acrylic acid, sodium acrylate, isobutyl acrylate, 2-
ethylhexyl acrylate, 2-hydroxyethyl acrylate, 2-(2'-
hydroxyethoxy)ethyl acrylate, 2-hydroxy-1-methylethyl
acrylate, 2-N,N-dimethylaminoethyl acrylate, methacrylic
acid, sodium methacrylate, n-propyl methacrylate, 2-
hydroxyethyl methacrylate, 2-(2'-hydroxyethoxy)ethyl
methacrylate, 2-hydroxy-1-methylethyl methacrylate, 2-
N,N-dimethylaminoethyl methacrylate, diethylene glycol
dimethacrylate, triethylene glycol diacrylate, 4-
vinylsalicyclic acid, itaconic acid, vinylacetic acid,
cinnamic acid, 4-vinylbenzoic acid, 2-vinylbenzoic acid,
sorbic acid, caffeic acid, maleic acid, methylmaleic
acid, crotonic acid, isocrotonic acid, fumaric acid,
dimethylfumaric acid, methylfumaric acid, dihydroxymaleic
acid, allylacetic acid;
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sodium allyl sulfate, sodium allylsulfonate, sodium
methallyl sulfate, sodium methallylsulfonate, sodium
vinylsulfonate, 2-hydroxyethyl vinylsulfonate, 4-
vinylbenzenesulfonic acid, sodium styrenesulfonate,
sodium vinyltoluenesulfonate;
2-butene-1,4-diol diphosphate (i.e., 1,3-butadiene-1,4-
diol diphosphate), 2-butene-1,4-diol diphosphonate, the
disodium salts of these phosphates or phosphonates,
diallyl phosphite (meth)acryloyloxyethylphosphoryl-
chlorine;
2-vinylphenol, 2-allylhydroquinone, 4-vinylresorcinol,
m-hydroxystyrene, p-hydroxystyrene and carboxyl-vinyl-
benzene sulfonic acid.
Examples of the monomers of the formula (I) having
only one or more carboxyl groups and their derivatives, which
may not be used alone but may be used together with one or
more other monomers of the formula (I), include acrylic acid,
methacrylic acid, itaconic acid, vinylacetic acid, c; nn~m; C
acid, vinylbenzoic acid, sorbic acid, caffeic acid, maleic
acid, methylmaleic acid, crotonic acid, isocrotonic acid,
fumaric acid, dimethylfumaric acid, methylfumaric acid,
dihydroxymaleic acid, allylacetic acid and their salts and
esters.
Examples of the monomers of the formula (I) having a
carboxyl group and another acidic group and their derivatives,
which may be used alone or together with one or more other
monomers of the formula (I), include vinylsalicyclic acid
carboxy-vinylbenzenesulfonic acid, and their salts and esters.
Examples of the monomers of the formula (I) having a
14
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sulfonic or sulfuric acid group and their derivatives, which
may be used alone or together with one or more other monomers
of the formula (I), include allylsulfuric acid, allylsulfonic
acid, methallylsulfuric acid, methallylsulfonic acid,
vinylsulfonic acid, 2-hydroxyethylvinylsulfonic acid,
vinylbenzenesulfonic acid (i.e. styrenesulfonic acid),
vinyltoluenesulfonic acid and their salts and esters.
Examples of the monomers of the formula (I) having a
phenolic hydroxyl group and their derivatives, which may be
used alone or together with one or more monomers of the
formula (I), include vinylphenol (i.e. hydroxystyrene),
allylhydroquinone and vinylresorcinol.
In a further embodiment of the process according to
the invention, as the monomer (I) use may be made of a mixture
of monomers of the general formulae V and VI
cRl~ CHR1' CR1~ CHR5
~2' (V) and R2l (VI)
(HC R3' R4) COOR6
CH2 R3 R4
In the formulae (V) and (VI)
R1 is hydrogen or a methyl radical,
R2 is a divalent organic radical, preferably an
aliphatic, cycloaliphatic or aromatic hydrocarbon
radical, having up to 10 carbon atoms, or a single
bond,
R3 is -O- or -NH-,
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R4 is hydrogen or a -SO~3Na~ radical,
R5 is hydrogen, a methyl radical or a -R2 -COO~Na~
radical,
R6 is hydrogen or Na~ and
n' is 4 or 5;
with the proviso that at least one of substituents R4 is a
-SO33Na~ radical.
In preferred monomers (V) and (VI)
R1 is hydrogen,
R2 is a alkylene radical having 1 to 4 carbon atoms, a
phenylene radical or a single bond,
R3 is -O- or -NH-,
R4 is hydrogen or radical -SO~3Na~,
R5 is hydrogen or the radical -R2 -COO~ Na3,
R6 is hydrogen or Na~ and
n~ is 4.
The monomers (V) include modified sugar residues,
preferably from pentoses and, in particular, from arabinose.
The sugar residues comprise at least one of the radicals -O-
SO~3Na~ (O-sulfate) or -NH-SO~3Na~ (N-sulfate), preferably
adjacent to the radical R2 . They have preferably 1 to 4 of
these radicals. O-sulfate and N-sulfate radicals can be
present simultaneously in one sugar residue, in which case the
N-sulfate radical is preferably positioned adjacent to the
radical R2 . Alternatively, the sugar residue may comprise
exclusively one kind of these radicals, for example the O-
sulfate radical. Each of the species of formula (V) specified
(residues containing only O-sulfate and residues containing N-
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sulfate) is suitable, alone or together with other species of
formula (V). The mixing proportion is therefore from 0:100 to
100: 0 .
The quantitative proportion in which the monomers of
formulae (V) and (VI) are employed can fluctuate within wide
limits. Thus the molar ratio of the N-sulfate and/or O-
sulfate groups of the monomer of formula (V) to the carboxyl
and/or carboxylate groups of the monomer of formula (VI) can
be, for example, from 1:100 to 100:1. Preferred molar ratios
are between 1:20 and 20:1.
The preparation of the monomers of formula (V) is
described in detail for example, in German Patent Publication
197 20 369.8. It will be explained here on the basis of a
special case, which starts from D-glucono-1,5-lactone 1 and
leads to a monomer of formula (V) which is derived from a
pentose, namely d-arabinose. It is within the scope of the
skilled worker, however, to transfer the process readily to
other suitable precursors.
In a first stage the hydroxyl groups of the lactone
1 are protected by acetalization, for example with acetone in
methanol as solvent. In this procedure the lactone is cleaved
and an isomer mixture is obtained comprising methyl 3,4;5,6-
di-O-isopropylidene-D-gluconate 2 and methyl 2,3j5,6-di-O-
isopropylidene-D-gluconate 3. This mixture is reduced in a
second stage, for example with lithium aluminum hydride,
whereby the carboxylic ester function becomes the carbinol
function. An isomer mixture is obtained again, namely of 3,4;
5,6-di-O-isopropylidene-D-sorbitol 4 and 2,3j5,6-di-O-
isopropylidene-D-sorbitol 5. In a third ctage, this isomer
17
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mixture is oxidized with an oxidizing agent such as sodium
periodate and with cleavage of the carbon chain to give a
uniform product, the arabinose aldehyde 2,3;4,5-di-O-
isopropylidenealdehyde-D-arabinose 6. In the subsequent
fourth stage a vinyl function is introduced, for example by a
Grignard reaction with 4-vinylphenylmagnesium chloride. A
partially protected 4-vinylphenylpentanepentanol, 2,3;4,5-di-
O-isopropylidene-1-(4-vinylphenyl)-D-gluco(D-manno)pentitol 7,
is obtained, which is referred to below in shortened form as
arasty.
This sequence of stages 1 to 4 is illustrated by the
following reaction scheme:
CH20H COOCH3 - COOCH3
~H ~ o CH3OH4 H~ ~ OH
'- -' ~ - OH
OH _ o X - ~ X
1 2 3
- OH - - OH
_ o ~ _ OHN~04 o ~ ~ 4
- ~oX - oX- oX oX
4 5 6 7
This reaction sequence has been described in more
detail by H. Regeling et al., Recl. Trav. Chim. Pays-Bas 1987
(106), 461: D.Y. Jackson, Synth. Commun. 1988 (18), 337; and
G. Wulff et al., Macromol. Chem. Phys. 1996 (197), 1285.
18
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To prepare a compound which corresponds to the
arasty 7 and has an amino group in position 1, arasty can be
oxidized in a first stage to the corresponding ketone,
2,3;4,5-di-0-isopropylidene-D-arabino 4-vinylphenyl ketone 8.
This is transformed reductively in a second stage to the amine
l-amino-l-deoxy-2l3i4/5-di-o-isopropylidene-l-(4-vinylphenyl)
D-gluco(D-manno)pentitol 9. This reaction sequence is
illustrated by the following formula scheme:
~J
CHOH C-O CHNH2
o ~ DMSO/(C0~)2 ~ ~ NaGNBH3 0
_OX _OX _oX
7 8 9
In the first stage, arasty 7 can be oxidized, for
example, with a complex of oxalyl chloride and dimethyl
sulfoxide at a temperature of ~-50~C in an inert solvent. The
reductive amination in the second stage is advantageously
achieved using sodium cyanoborohydride as a reducing agent in
the presence of ammonium acetate in a solvent under anhydrous
conditions at room temperature.
Heparin contains unprotected hydroxyl groups and is
O-sulfated and N-sulfated. The compounds 7 and 9 are
therefore deprotected (deacetalized) in a first stage and 0-
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and N-sulfated in a second stage so that the polymer prepared
from them is substantially analogous to heparin. Deprotection
takes place in an acidic medium, in which ketals are not
stable. The protected compounds are heated, for example, with
dilute mineral acid or with an acidic ion exchanger to give,
from 7, 1-hydroxy-1-deoxy-1-(4-vinylphenyl)-D-gluco(D-
manno)pentitol 10 and, from 9, 1-amino-1-deoxy-1-(4-
vinylphenyl)-D-gluco(D-manno)pentitol 11. The deprotection
and subsequent sulfation are represented by the following
formula scheme:
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~CHOH ,~mh~tlit~?* IR 120 CHOH sulfa~ion CHOH
~~ H~ form HO--
/ \ --OR'
--o \ --OH
--OR'
--~X --OH --OR'
--~ --OH
7 10 12
R' - H OR SO 3 Na
J
CHNH2 CHNH2 CHNHR'
0.1 N HCI HO-- sulfati~n ~ R'O--
--OH --OR'
--OH --OR'
--O~
_0~\ --OH --OR'
9 11 13
R' = H OR SO~)3 Na(~3
The two compounds 10 and 11 are sulfated,
judiciously by means of a sulfur trioxide-pyridine complex.
Because of the deacetalization that has been performed, the
sulfation does not lead to a uniform product having one or
more sulfate groups in defined positions. However, the
primary hydroxyl groups and the amino groups should be
sulfated preferentially. By choosing an appropriate molar
*Trade-mark
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ratio of sulfur trioxide to hydroxyl and/or amino groups, the
degree of sulfation can be regulated. It is advantageous to
introduce on average more than one sulfate group per molecule,
since heparin contains about 2.7 sulfate groups per
disaccharide unit (corresponding to 1.35 sulfate groups per
molecule of monomer). Sulfation of the deprotected amine
compound 11 produces, simultaneously, O-sulfate and N-sulfate
groups in the molecule, which is desirable in view of the
sought-after analogy with heparin.
The sulfation is advantageously conducted at room
temperature in order to avoid premature polymerization. After
a prolonged period, for example up to 100 hours, the reaction
is nevertheless complete. As solvent it is possible, for
example, to use excess pyridine or an ether, such as
tetrahydrofuran. Since the sulfate groups of the reaction
products are unstable to acid, it is advisable to add a water-
binding agent, for example a molecular sieve, to the precursor
solution before the addition of the sulfur trioxide-pyridine
complex. For the same reason it is advisable, after the end
of the reaction, first of all to hydrolyze the reaction
mixture by adding water followed soon thereafter by a base
(which keeps the pH in the alkaline range). One example of a
suitable base is a saturated barium hydroxide solution, which
at the same time precipitates sulfate ions. Excess barium
ions can be precipitated by, for example, introducing carbon
dioxide, directly or after careful concentration with removal
of solvent. The barium carbonate is filtered off and the
filtrate is passed through an ion exchanger column in the Na+
form or is treated otherwise with the ion exchanger in order
22
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to exchange the barium ions for sodium ions. From the
subsequently concentrated solution it is possible by freeze-
drying to recover the products, O-sulfated l-hydroxy-l-deoxy-
1-(4-vinylphenyl)D-gluco(D-manno)pentitol 12 and N- and O-
sulfated l-amino-l-deoxy-1-(4-vinylphenyl)-D-gluco(D-
manno)pentitol 13, in each case in the form of the sodium
salt, as pulverulent solids. Both substances correspond to
the formula (V) and are suitable monomers for the present
invention.
The monomers of formula (VI) are known and readily
obtainable substances which contribute the carboxyl or
carboxylate groups that are required for the heparin-analogous
action. Suitable monomers of formula (VI) have one olefinic
double bond and one or two carboxyl and/or carboxylate
functions or functions which can be transformed into carboxyl
and carboxylate functions, such as carboxylic ester,
carboxamide or carboxylic anhydride groups. Sodium ions are
preferably used as counter ions to the carboxylate function.
Examples of suitable monomers of formula (VI) are
(meth)acrylic, crotonic, 4-vinylbenzoic, maleic, fumaric,
itaconic, vinylacetic, ci nn~mi C, isocrotonic, methylmaleic,
dimethylfumaric, methylfumaric, dihydroxymaleic and
allylacetic acids and their sodium salts.
2. Other monomers which can be used if desired
In addition to the monomers of formula (I) to (VI)
described, having the stated blood-compatibilizing and
antibacterial and/or cell proliferation inhibiting groups, it
is also possible at the same time to use other monomers whose
23
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activity in this respect is neutral or at most weak. Examples
include vinyl ethers, such as vinyl methyl ether and vinyl
butyl ether; vinyl ketones, such as vinyl ethyl ketone;
olefins and diolefins such as 1-butene, 1-hexene, 1,3-
butadiene, isoprene and chloroprene; acrylamide and
methacrylamide; vinylaromatic compounds, such as styrene,
vinyltoluene and divinylbenzene; and vinylsiloxanes. These
monomers can even be present in a predominant amount, for
example accounting for up to 90 mol~, preferably 30 mol~ or
less.
3. The aubstrate materials
Particularly suitable substrate materials are all
polymeric plastics, such as polyurethanes, polyamides,
polyesters and polyethers, polyether-block-amides, poly-
styrene, polyvinyl chloride, polycarbonates, polyorgano-
siloxanes, polyolefins, polysulfones, polyisoprene,
polychloroprene, polytetrafluoroethylene (PTFE), poly-
acrylates, polymethacryla'tes, corresponding copolymers and
blends and also natural and synthetic rubbers, with or without
radiation-sensitive groups. The process according to the
invention can also be applied to surfaces of painted or
otherwise polymer-coated metal, glass or wooden structures.
4. The activation of the substrate surfaces
The surface of the substrates can, in accordance
with the invention, be activated by a variety of methods.
They are judiciously freed beforehand in a known manner, by
means of a solvent, from oils, fats or other cont~m'n~nts.
24
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4.1 The activation of standard polymers without W -
sensitive groups can advantageously be effected by W
radiation, for example in the wavelength range from 100 to
400 nm, preferably from 125 to 310 nm. A suitable source of
radiation is, for example, a HERAEUS* Noblelight W excimer
device from Hanau, Germany. Mercury vapor lamps, however, are
also suitable for substrate activation provided they emit
considerable components of radiation within the stated ranges.
The exposure time is in general from 0.1 second to 20 minutes,
preferably from 1 second to 10 minutes. It has been found
that the presence of oxygen is advantageous. The preferred
oxygen pressures are between 2X10-5 and 2x10-2 bar. The
operation is conducted, for example, in a vacuum of from 10-4
to lo-l bar or using an inert gas, such as helium, nitrogen or
argon, with an oxygen content of from 0. 02 to 20 parts per
thousand.
4.2 Activation can also be achieved in accordance with
the invention by means of a high-frequency plasma or microwave
plasma (for example, Hexagon, Technics Plasma, 85551
Kirchheim, Germany) in air or a nitrogen or an argon
atmosphere. The exposure times are in general from 30 seconds
to 30 minutes, preferably from 2 to 10 minutes. The energy
employed in the case of laboratory devices is between 100 and
500 W, preferably between 200 and 300 W.
4.3 It is also possible to use corona discharge devices
(for example, SOFTAL, Hamburg, Germany) for activation. The
*Trade-mark
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exposure times in this case are in general
from 1 second to 10 minutes, preferably from 1 to 60 seconds.
4.4 Activation by electron beams or gamma rays (for
example from a cobalt 60 source) enables short exposure times
which are in general from 0.1 to 60 seconds.
4.5 Flame treatments of surfaces lead likewise to their
activation. Suitable devices, especially those having a
barrier flame front, can be constructed in a simple manner or
obtained, for example, from ARCOTEC, 71297 Monsheim, Germany.
They can be operated with hydrocarbons or hydrogen as
combustion gas. In every case, harmful heating of the
substrate must be avoided, which is easily achieved by means
of intimate contact with a cooled metal surface on the
substrate surface facing away from the side subject to flame
treatment. Activation by flame treatment is restricted,
accordingly, to relatively thin, flat substrates. The
exposure times amount in general to from 0.1 second to 1
minute, preferably from 0.5 to 2 seconds, the flames involved
being - without exception - nonluminous and the distances of
the substrate surfaces from the external flame front being
from 0.2 to 5 cm, preferably from 0.5 to 2 cm.
4.6 Furthermore, the substrate surfaces can also be
activated by treatment with strong acids or strong bases.
Suitable strong acids which may be mentioned are sulfuric
acid, nitric acid and hydrochloric acid. Polyamides, for
example, can be treated at room temperature with concentrated
26
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sulfuric acid for from 5 seconds to 1 minute. Particularly
suitable strong bases are alkali metal hydroxides in water or
an organic solvent. Thus, for example, dilute sodium
hydroxide solution can be allowed to act on the substrates at
from 20 to 80~C for from 1 to 60 minutes. Alternatively, for
example, polyamides can be activated by allowing 2~ strength
KOH in tetrahydrofuran to act on the surface for from 1 minute
to 30 minutes.
4.7 Finally, monomers having W-sensitive groups can be
incorporated by polymerization during the actual preparation
of the substrate polymers. Examples of suitable such monomers
are furyl or c;nn~moyl derivatives, which can be employed, for
example, in amounts of from 3 to 10 mol~. Highly suitable
monomers of this kind are c-nn~moylethyl acrylate and
methacrylate.
In some cases, for example with highly hydrophobic
polymers, it may be advisable to activate the substrate
surfaces by a combination of two or more of the methods
stated. Preferred activation methods are those specified
under 4.1 and 4.2.
5. Coatinq bY qraft (co)polymerization
If a substrate has been pretreated by one of the
methods described above, the activated surfaces are
judiciously exposed for from 1 to 20 minutes, preferably from
1 to 5 minutes, to the action of oxygen, for example in the
form of air.
Subsequently, the surfaces that have been activated
27
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(including those which have been activated in accordance with
4.7) are coated by known methods, such as dipping, spraying or
spreading, with solutions of the vinyl monomer(s) of the
formula (I) to be used in accordance with the invention.
Solvents which have been found preferably are water and water-
ethanol mixtures, although other solvents can also be used
provided they have sufficient solvency for the monomers and
provide good wetting of the substrate surfaces. Depending on
the solubility of the monomers and on the desired film
thickness of the finished coating, the concentrations of the
monomers in the solution may be from 0.1 to 50 percent by
weight. Solutions with monomer contents of from 3 to 10
percent by weight, for example with about 5 percent by weight,
have been found particular preferred in practice and give rise
in general and in one pass to coherent coatings which cover
the substrate surface and have film thicknesses which can be
more than O.l~m.
Following the evaporation of the solvent or even
during the evaporation, the polymerization or copolymerization
of the monomers applied to the activated surfaces is brought
about, by radiation preferably in the shortwave segment of the
visible light region or in the longwave segment of the W
region of the electromagnetic radiation. Highly preferable
radiation, for example, is that of a W excimer of the
wavelengths 250 to 500 nm, preferably from 290 to 320 nm.
Here again, mercury vapor lamps are preferred provided they
emit considerable fractions of radiation within the stated
ranges. The exposure times are in general from 10 seconds to
30 minutes, preferably from 2 to 15 minutes.
28
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In some cases it is desired to repeat the described
operations, including the activation, by means of such a
multicoat technique to ensure a hermetically sealed and/or
relatively thick coating. Alternatively, it is also possible
to immerse the activated substrate, if desired after the
oxygen treatment described, into the solution of the vinyl
monomer(s) of the formula (I) to be used in accordance with
the invention and to irradiate it in the immersed state. By
means of guideline experiments it is not difficult to
ascertain the irradiation times with a given radiation source
and the substrate/solution contact times, which may be
relatively long, required to achieve the desired film
thickness.
The process according to the invention for the
antibacterial and cell proliferation inhibiting coating of the
surface of substrates and, in particular, of polymeric
plastics permits the establishment of precise molar ratios of
different functional groups which are optimum for inhibiting
bacterial adhesion and/or propagation and cell proliferation.
Furthermore, the process offers the advantage that, if
appropriate activation methods are chosen, plastics which have
already become established can, while retaining their
mechanical properties and their form, be additionally modified
to make them antibacterial and cell proliferation inhibiting.
In general, no other treatments before or after are necessary.
Highly hydrophobic plastics may require a hydrophilicizing
pretreatment, for example by chemical etching with acids or
bases or by plasma treatment, in order to attain sufficient
wettability by the monomer solution. The highly hydrophobic
29
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plastics are then hydrophilicized at the same time and
activated in the sense of the present invention.
The examples which follow are given to illustrate
further the present invention. The monomers used therein are
representative of a large number of other compounds which come
under the general formulae (I) to (IV).
Examples
Monomers
5% strength by weight aqueous solutions of each of
the monomers listed in Table 1 were prepared under sterile
conditions.
Table 1 - Monomers employed - 5% by weight solution
Solution Mo~o~r Abbreviation
number
S 1 Sodium styrenesulfonate NaSS
S 2 Acrylic acid AAc
S 3 Methacrylic acid MAAc
S 4 Maleic acid MAc
S 5 2-[(N-dimethylamino)ethyl] acrylate DMAEA
S 6 Sodium vinylsulfonate VS
S 7 Methacryloyloxyethylphosphorylcholine, PCHEMA
CH2=C(CH3)-COO-CH2CH2OPO2 -CH2-CH2 N (CH3)3
S 8 Diethylene glycol methacrylate DEGMA
S 9 1,3-Butadiene-1,4-diol diphosphate BDDl,4DP
S 10 Caffeic acid KafAc
S 11~ 4-Vinylresorcinol 4VR
S 12 4-Vinylsalicylic acid 4VSAC
* 1% by weight solution
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Substrates
The investigations on the effects of the coatings according to
the invention on antibacterial behaviour were conducted on
sheets of the plastics listed in Table 2, each of which has a
thickness of 0.1 mm and a surface area, relevant for the
determination of 4 cm2. They were prepared both by dissolving
the powders in solvents, then pouring the solutions into Petri
dishes and drying them, and by calendering, extrusion,
compression molding or knife coating. In some cases, films
from the manufacturer were available.
Table 2 - Films employed
Film No. Plastic Name, source Preparation
F 1 Polyamide 12 VESTAMID~, HULS AG Extrusion
- F 2 Polystyrene VESTYRON~, HULS AG Compression
F 3 Polyurethane PELLETHANE~ 2363-A Extrusion
DOW CHEMICAL COMPANY
F 4 Polyether-block- VESTAMID~, HULS AG Extrusion
amide
F 5 Polyethylene VESTOLEN~ A, Extrusion
VESTOLEN GmbH
F 6 Polypropylene VESTOLEN~ P, Extrusion
VESTOLEN GmbH
F 7 Polyorgano- NG 37-52, Silicon GmbH, Knife coating
siloxane Nunchritz
F 8 Polyvinyl VESTOLIT~ + DEH Brabendering
chloride VESTOLIT Gm~H
F 9 PTFE HOSTAFLON~, Extrusion
HOECHST AG
Activation of the substrate surfaces
The films were first of all activated, alternatively
according to the conditions and techniques stated in Table 3.
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Table 3 - Activation conditions
Activation Activating technique Conditions
reference
A 1 W excimer rays 1 s - 20 min, 1 mbar
(= 172 nm) 4 cm distance
A 2 Microwave plasma (argon) 1 s - 30 min, 1 mbar
A 3 High-frequency plasma 1 s - 30 min, 6 mbar
(argon)
A 4 Corona 0.1s - 60s, 2 mm distance
A 5 Flame treatment CH4:air = 1:10:4 cm distance
A 6 ~-rays 1 Mrad
A 7 Electron beams 1 min
A 8 NaOH solution 1~, 5 min, 60~C
A 9 W excimer rays 10 s - 20 min
(= 308 nm)
Coatinq of the Substrate Surfaces by Graft (co)Polymerization
Following activation, the films, casting or other
substrates and also, in the case of industrial manufacture,
the extrudates or injection moldings, are coated with the
solutions S1 to S16, by the techniques indicated in Table 4.
Table 4 - Coating techniques
Coating reference Coating technique
T 1 Dipping
T 2 Spraying
T 3 Spreading
- During dipping or after dipping, spraying or
spreading, irradiation is carried out with rays in the range
250 - 500 nm, preferably 290 - 320 nm.
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Determination of Antibacterial Pro~erties
The test for adhesion of bacteria can be performed
with various strains. Particularly suitable for this purpose
are the bacteria listed in Table 5, since they occur
frequently in clinical isolates from infected catheters.
Table 5 - Strains of bacteria for measuring
the primary adhesion
Strain
B 1 Staphylococcus aureus
B 2 Staphylococcus epidermidis
B 3 Escherichia coli
B 4 Klebsiella pneumoniae
The method of determining the primary adhesion (i.e.
independently of subsequent multiplication) of these bacterial
strains is described below by way of example for Klebsiella
pneumoniae. The primary adhesion of the other strains (B1 to
B3) was determined in a similar way.
Determ;n~tion of PrimarY Bacteria Adhesion Under Static
Conditions
An overnight culture of the bacterial strain
Klebsiella pneumoniae in yeast extract-peptone-glucose
nutrient medium (1% + 1% + 1%) is centrifuged and the extract
is taken up again in phosphate-buffered saline (=PBS; 0.05 M
KH2PO4, pH 7.2 + 0.9% NaCl). The suspension is diluted with
PBS buffer to a cell concentration of 108 cells/ml. The
suspended bacteria are brought into contact for 3 h with the
section of film that is to be investigated. This is done by
33
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spiking circular film sections having a diameter of 1.6 cm
(=4.02 cm2), coated on both sides, onto a preparation needle
and shaking them with the cell suspension. Films coated on
one side, in the form of a circular, planar disk with a
diameter of 4.5 cm and with a supporting membrane of 2-3 cm
thick flexible PVC, are clamped into a membrane filter
apparatus. The cell suspension is applied to the upward
facing side, bearing the test coating, and is shaken for 3 h.
The membrane filter apparatus must be tightly sealed; in other
words, no cell suspension may flow out through leak sites.
After the contact time has expired the bacterial
suspension is drawn off under suction using a water jet pump
and the film sections are washed for 2 minutes by shaking them
in a 100 ml glass beaker with 20 ml of sterile PBS solution.
The film section is immersed again in sterile PBS solution and
then extracted in a boiling water bath for 2 minutes with 10
ml of heated TRIS/EDTA (0.lM trishydroxyethylaminomethane,
4 mM ethylenediaminetetra-acetic acid, adjusted to pH 7.8 with
HCl).
The extraction solution is used to fill small
Eppendorf cups and is immediately frozen at -20~C until the
extracted adenosine-triphosphate (ATP) is determined by
bioluminescence. The determination is carried out as follows:
100 ~l of reagent mix (bioluminescence test CLS II,
BOEHRINGER MANNHEIM GmbH) are placed in a transparent
polycarbonate tube, and the light pulses are integrated over a
period of 10 seconds in a light impulse meter LUMAT* LB 9501
(Laboratorien Prof. Berthold GmbH, 75323 Bad Wildbad,
*Trade-mark
34
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CA 02226133 1998-01-02
Germany). Then a 100 ~l sample is added and measurement is
repeated. The relative light units (RLU) are obtained by
subtracting the light pulses in the reagent mix from the
number of light pulses measured in the complete batch. This
value is related to the number of bacteria which have adhered
to the film. The conversion factor between the RLU value and
the bacterial count is determined by extracting an aliquot of
0.1 ml of the bacterial suspension containing 108 cells/ml in
10 ml of hot TRIS/EDTA and then determining the ATP content.
For Klebsiella ~neumoniae the value found is 1.74
104 RLU = 1 107 cells in the ATP extract. With a measured
value of 4.7 104 RLU from 4 cm2 of film, this gave a value
for the primary adhesion per cm2 of film surface of
4.7 104
= 1.175 104 RLU/cm2 - 6.8 106 cells/cm2
Resul tg
Compiled in Tables 6a and 6b below are the various
conditions and the results of a total of 27 experiments and
comparison experiments without prior activation (14, 16, 18,
20, 22, 24, 26).
O.Z. 5192
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CA 02226133 1998-01-02
m r_
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36
O,Z. 5192
23443-625
CA 02226133 1998-01-02
o\~ a~ ~ , ~ , ~ , In , C~ , ~
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--) o _N ~ r~ ~ '~
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rnrn u~ n rn rn rn
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37
O.Z. 5192
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Tables 6a and 6b show that the coatings obtained by
the process according to the invention lead to a considerable
reduction in bacterial adhesion. The reductions are markedly
over 50~ in comparison with the uncoated substrates.
Furthermore, the comparison examples (experiments 3, 4 and 15)
reveal that, by activating the substrate surfaces with W
excimer rays of wavelength 172 nm (Al) or plasma treatment
(A2, A3), higher inhibitions of bacterial adhesion (290~) are
- surprisingly - achieved than with other activating means,
such as electron beams (A7, experiment 13) or NaOH solutions
(A8, experiment 17).
The results reproduced in the tables also show that
it is possible to operate successfully with a single monomer
(S 7, experiment 25) but that copolymers of two monomers,
namely of sodium styrenesulfonate and acrylic acid (S 1 + S 2:
experiment 15), sodium styrenesulfonate and maleic acid (S 1 +
S 4: experiment 17), sodium vinylsulfonate and caffeic acid (S
6 + S 10: experiment 21) and acrylic acid and sodium
vinylsulfonate (S 2 + S 6: experiment 27) also give coatings
having good antibacterial properties.
O.Z. 5192
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