DE19720370A1 - Bioactive coating of surfaces - Google Patents

Description

The invention relates to a method for bioactive coating the surface of substrates, preferably plastic (or Polymer) substrates by graft polymerization. An important property The coating applied according to the invention is theirs good tolerance in contact with body fluids and tissue. Depending on the functionality of the coating monomers or the Molar ratio of certain functional groups in the coating the surfaces are also bacteria-repellent and cell anti-proliferative or bacterial and cell proliferative promotional. The invention further relates to objects with surfaces coated in this way and the use of these counterparts stands for medical or biotechnical purposes.

The settlement and multiplication of bacteria on surfaces is a usually undesirable phenomenon, which often has night effects consequences. So in the drinking water and Beverage technology bacterial populations become a health hazard lead to a reduction in quality. Bacteria on or in packaging genes often cause or spoil food even infections in the consumer. To be operated in sterile condition Biotechnical plants raise foreign bacteria ches process engineering risk. Such bacteria can with raw substances are entered or in the event of poor sterilization in remain in all parts of the system. Parts of the bacterial population can adhere to normal fluid exchange during adhesion Remove rinsing and cleaning and multiply in the system.

There are also bacterial settlements in water treatment plants (e.g. for desalination through membranes) or in containers knows that with dissolved or liquid undiluted organic Substances are filled and beneficial for bacterial populations  Have conditions. Such microbial coverings can be found in considerable extent for blocking and / or corrosive destruction the system.

Protection against bacterial adhesion and -Distribution in nutrition, care, especially in the Geriatric care, and in medicine too. For mass catering or - There are considerable risks when it comes to serving Avoiding waste of disposable tableware and one the reusable dishes are not cleaned properly. The harmful spread of bacteria in food-carrying Hoses and pipes are just as well known as the propagation in Storage containers and in textiles in damp and warm surroundings, e.g. B. in bathrooms. Such facilities are preferred habitats for bacteria, as well as certain surfaces in areas with high public traffic, e.g. B. in public transport, Hospitals, telephone booths, schools and especially in public toilets.

In elderly and nursing care, the often reduced require Defenses of those affected take careful measures against infection nen, especially in intensive care units and in home care ge.

The use of medical objects requires special care and devices for medical examinations, treatments and a grabbed, especially if such devices or objects with living tissue or body fluids. in the In the case of long-term or permanent contacts, for example with Implan acts, catheters, stents, heart valves and pacemakers, can bacterial contamination pose a life-threatening risk for the patient.

Settlement has already been attempted in many different ways and prevent the spread of bacteria on surfaces. In J. Microbiol. Chemoth. 31 (1993), 261-271 describe S.E. Tebbs and T.S.J. Elliott lacquer-like coatings with quaternary ammonium salt as antimicrobial components. It is known,  that these salts of water, aqueous or other polar media as well as body fluids from the coating material can be solved and their effect 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 describe in Progr. Polym. Sci. 20: 211, 1995 ff., the immobilization of bactericidal agents on polymer surfaces through covalent bonding or ionic interactions. In such cases, the germicidal effects are often opposite the pure active ingredient is significantly reduced. Heteropolar bonds often prove to be insufficiently stable. In addition leads the germ killing usually leads to undesirable deposits on the Surfaces that mask the further bactericidal effect and that Form the basis for subsequent bacterial colonization.

W. Kohnen et al. report in ZBl. Bakt. Suppl. 26, Gustav Fischer Verlag, Stuttgart-Jena-New York, 1994, pages 408 to 410 that the Adhesion of Staphylococcus epidermidis on a polyurethane film is diminished if the film is counter-glow were pre-treated with oxygen and then grafted with acrylic acid becomes.

As mentioned, it is important that when using medical Ge objects and devices for medical examinations, treatments and interventions bacterial contamination of these objects and Devices is prevented. With some of these items and devices, the medium or long term with living tissue or body fluids coming into contact is also a liability and spread extremely undesirable from the body's own cells. So are Zellbe Settlements of catheters applied intracorporeally in the medium term just as harmful as with long-term implanted stents or Heart valves.

Furthermore, the transparency of intraocular lenses due to Cellular population continuously decrease after implantation. A A series of procedures aims at cell colonization for example by incorporating certain metals or metal  to avoid salts in the holder of the intraocular lens, taking the However, the effect is usually incomplete and unsustainable. Also WO 94/16648 describes a method by which the Proli feration of cells on the surface of implanted eyepiece should be prevented from polymer material.

According to EP 0 431 213, polymers with cell-repellent properties can be endowed with strong mineral acids is hydrophilized. The subsequent chemical modification of polymer surfaces, however, is usually not uniform. It remains usually not or insufficiently treated areas back, which are the starting points for cell colonization. Next the cell-repellent properties of the upper treated in this way areas are often not permanent.

On the other hand, for certain uses, items with upper areas that are bacteria-repellent, but the colonies are desirable Promote cells. That applies e.g. B. for a number of devices for medical examinations. Treatments and interventions and also for some prostheses that should grow into the tissue, into which they were implanted. Such devices and prostheses, e.g. B. Artificial hip joints or teeth often consist of polymer encased materials such as titanium.

Finally, materials for equipment and devices that with body fluids, such as blood or lymph, or with tissue in Come in contact, be compatible with their foreign environment. In particular In particular, blood tolerance is an important characteristic shaft. The materials must therefore be pronounced anti have thrombic properties.

So there are different, partially mutually exclusive requirements changes to the bioactive properties of the surface of Polyme that are intended for medical use. You should always bacteria-repellent and compatible with body fluids and tissue, but either cell proliferation-inhibiting or - have a positive effect.

The object of the present invention was to improve it to develop a method for coating surfaces with the surfaces are bioactive, namely bacteria-repellent and ver inert for body fluids and tissues as well as optionally cell can be coated to inhibit or promote proliferation, oh ne that the mechanical properties of the treated materials be changed or other significant disadvantages occur ten.

It has surprisingly been found that the surface of substrates, in particular polymeric substrates, can advantageously be bioactive coated if at least one monomer of the general formula is used

Formula I: R- (A) _a

in the
R denotes a mono- or di-olefinically unsaturated organic radical, for example a hydrocarbon radical, with the valence a,
A is a carboxyl group -COOH, sulfuric acid group -OSO ₂ OH, sulfonic acid group -SO ₃ H, phosphoric acid group -OPO (OH) ₂ phosphonic acid group -PO (OH) ₂ phosphoric acid group -OP (OH) ₂ , phenolic hydroxyl group or a salt or an ester of one of the designated groups, and
a represents an integer from 1 to 6,
radiation-induced graft-polymerized on an activated substrate surface, with the proviso that a monomer I in which A is the carboxyl group -COOH or a salt or an ester of the carboxyl group either has at least one further radical A with another of the meanings mentioned for A. contains or is used together with at least one further monomer I in which A has another of the meanings mentioned for A.

Among the salts of the groups mentioned for A are the alkali ze and particularly preferred the sodium salts.  

The common characteristic of the monomers of formula I is that they 1 or 2 olefinic double bonds and at least one acidic one Group or a specific derivative, namely a salt or a Have esters, an acidic group.

By plasma-induced graft polymerization of functional mono are produced coatings on various substrates e.g. B. B. Lassen et al., 1992, Clinical Materials 11, 99-103, be known and examined for biocompatibility. Activate one de No pretreatment is mentioned. In addition, plasma is not optimal Polymerization initiator. H. Yasuda speaks accordingly in J. Polym. Sci .: Macromolecular Review, Vol. 16 (1981), 199-293, by the undefined and uncontrollable chemistry of the plasma Po lymerisation. This may be acceptable for some purposes, but it is but problematic for medical and biotechnical applications, because right here on reproducible coatings of the same consistently high quality is particularly important.

Surprisingly, the bacteria-repellent properties of the according to the invention with a carboxyl, carboxylate or carbonic ester group-containing monomers I together with another monomer I coated surfaces much more pronounced than this the modification with acrylic acid according to W. Kohnen et al., loc. cit., is the case under comparable conditions.

The surfaces coated according to the invention show a dimer noteworthy combination of advantageous properties and therefore a excellent physiological tolerance. You are special well tolerated by the blood and reduce the adhesion and increase of Bacteria to a high degree even over a long period. Of this Bacteria affected may include a. Staphylococcus aureus, Sta phylococcus epidermidis, Streptococcus pyogenes, Klebsiella pneumo niae, Pseudomonas aeroginosa and Escherichia coli. At the same time the proliferation of cells is usually inhibited, at for example of fibroblasts and endothelial cells, such as human Umbilical cord cells. The special conditions under which one Coating that repels bacteria, but promotes cell proliferation  are explained later. The surfaces of the invention coated substrates are free of migration and / or extractable monomer and oligomer fractions. Unwanted side effects through released foreign substances or through killed killed bacteria are avoided from the outset.

In the method according to the invention, the substrate surfaces first, as described in more detail below, activated and on closing under the influence of UV light through gentle grafting lymerization or copolymerization coated.

1. The monomers

In the graft (co) polymerization, z. B. as monomer I before a mixture of monomers of general formulas II or III

Formula II: (C _n H _2n-qx ) (COOR ¹ ) _x

Formula III: (C _n H _2n-qx ) (SO ₃ R ¹ ) _x

a. The formulas that are included in the general formula I state

n each independently for an integer from 2 to 6 inclusive;
x each independently for 1 or 2;
q each independently for 0 or 2; and

the radical R ¹ each independently denotes -H, one equivalent of a metal ion, in particular an alkali metal ion, a radical of an aliphatic, cycloaliphatic or araliphatic alcohol, preferably an alkanol with 1 to 6, in particular re with 1 to 4 carbon atoms, a cycloalkanol with 5 up to 12 carbon atoms, an arylalkanol with 7 to 10 carbon atoms or an alkanol with oxygen and / or nitrogen atoms in the chain and up to 12 carbon atoms,
such as - (CH ₂ -CH ₂ -O) _d -H, - (CH ₂ -CH (CH ₃ ) -O) _d -H, - (CH ₂ -CH ₂ -CH ₂ -O) _d -H
or - (CH ₂ ) _d -NH _2-e (R ² ) _e , where R ^{2 is} -CH ₃ or -C ₂ H ₅ , d is 1, 2, 3 or 4 and e is 0, 1 or 2 .

According to the given definitions, the radical (C _n H _2n-qx ) each independently means 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 alkadiene radical (q = 2, x = 2).

Instead of two monomers II and III, it is also possible to use only one monomer (II + III) which contains the groups -COOR ¹ and -SO ₃ R ¹ in the same molecule.

Also monomers of the general formula derived from benzene

Formula IV: (C ₆ H _6-bcd ) B _b R ³ _c (OH) _d

can be used in which
B each independently _denotes a mono- or divalent straight-chain or branched radical of the formulas (C _n H _2n-1-qx ) (COOR ¹ ) _x or (C _n H _2n-1-qx ) (SO ₃ R ¹ ) _x , wherein R ¹ , n , q and x are as previously defined;
R ³ each independently C _1-4 alkyl, -NH ₂ , -COOH, -SO ₃ H, -OSO ₃ H, -OPO (OH) ₂ , -PO (OH) ₂ , -OP (OH) ₂ , - OPO (O⁻) OCH ₂ -CH ₂ -N⁺ (CH ₃ ) ₃ , -PO (O⁻) O-CH ₂ -CH ₂ -N⁺ (CH ₃ ) ₃ , -OP (O⁻) OCH ₂ - CH ₂ -N⁺ (CH ₃ ) ₃ or optionally a salt, in particular an alkali salt, or an ester of the groups mentioned means:

b represents 1, 2 or 3;
c represents 0, 1, 2, or 3; and
d represents 0, 1, 2, or 3;

with the proviso that b + c + d ≦ 6, advantageously ≦ 4.

Of course, any mixtures of monomers of the general my formulas II, III and IV for the process according to the invention be used.

Other suitable monomers are those corresponding to formula I which are neutral or acidic sulfuric acid 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 latter; Phos phosphoric acid esters, their neutral or acidic salts, neutral or acidic re esters and salts of the latter; and phosphorous acids, their neutral or acidic salts, neutral or acidic esters and salts the latter. These monomers can also be mixed with one another and / or with the monomers of the general formulas II, III and IV can be used for the method according to the invention.

Finally, 1 to 3-valent (or basic) phenols as their salts, which correspond to the formula I, as suitable mono mere mentioned. If necessary, they are also mixed together of and / or used with the aforementioned monomers.

A combination of has been found for the method according to the invention Proven monomers I to IV, which leads to coatings which egg on the one hand carboxyl and / or carboxylate groups and on the other hand sul have fonic acid and / or sulfonate groups. There is under the Aspect of compatibility with regard to the three groups mentioned possible combinations of two, namely carboxylic and sulfonic acid groups, carboxyl and sulfonate groups as well as carboxylate and sulfo natgruppen, further two combinations of three, namely carboxyl, Carboxylate and sulfonate groups as well as carboxyl, sulfonic acid and Sulfonate groups. Characterize all these combinations bare embodiments of the method according to the invention. Naturally it is also possible to use monomers whose functional group pen can be changed after the graft polymerization. So you can e.g. B. the acrylamide building block by hydrolysis in acid Convert the medium into the acrylic acid building block. Furthermore you can Carboxyl and sulfonic acid groups by neutralizing (e.g. in Phosphate buffers) and carbonic ester and sulfonic acid ester groups converted into carboxylate or sulfonate groups by hydrolysis.  

In the combination mentioned, the molar ratio of carb oxyl and / or carboxylate groups to sulfonic acid and / or sulfonate groups in the coating fluctuate within wide limits. Especially pronounced cell proliferation-inhibiting properties he will aims if the said ratio is 0.2 to 3, advantageously 0.4 to 3 and in particular 0.4 to 2. The coated surfaces remarkably show bacteria-repellent but cell-pro liferative properties when said molar ver Ratio 2 to 10, advantageously 3 to 10 and in particular 3 to 5 be wearing. Cell proliferation in the sense of the invention is one Coating when the adhesion and multiplication of mammals cells through the coating compared to the uncoated Surface improved or at least less affected is called the adhesion and multiplication of bacteria.

Examples of suitable monomers of the general formulas I to IV which contain one or more identical or different radicals A in the molecule are:
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 2-hydroxyethyl methacrylate, 2- (2'-hydroxyethoxy) ethyl methacrylate, 2-hydroxy-1-methyl ethyl methacrylate, 2-N, N-dimethylaminoethyl methacrylate,
Diethylenglykolmethacrylat, triethylene glycol, 4-Vinylsali cylsäure acid itaconic acid, vinyl acetic acid, cinnamic acid, 4-Vinylbenzoe, 2-vinylbenzoic acid, thylfumarsäure sorbic acid, caffeic acid, maleic acid, methylmaleic acid, crotonic acid, isocrotonic acid, fumaric acid, dime, methylfumaric acid, dihydroxymaleic acid Allylessig;
Sodium allyl sulfate, sodium allyl sulfonate, sodium methallyl sulfate, sodium methallyl sulfonate, sodium vinyl sulfonate, vinyl sulfonic acid re-2-hydroxyethyl ester, 4-vinylbenzenesulfonic acid, sodium styrene sulfonate, sodium vinyl toluene sulfonate;
Butene (2) diol (1,4) diphosphate, butene (2) diol (1,4) diphosphonate, the disodium salts of the two phosphates or phosphonates, diallyl phosphite;
2-vinylphenol, 2-allylhydroquinone, 4-vinylresorcinol, m-hydroxystyrene, o-hydroxystyrene, p-hydroxystyrene, carboxyl-vinylbenzenesulfonic acid.

In a further embodiment of the process according to the invention, the monomer I used is a mixture of monomers of the general formulas V and VI

In formulas I and II mean
R ^{1 is} hydrogen or the methyl radical,
R ^{2 is} a divalent organic radical, preferably an aliphatic, cycloaliphatic or aromatic hydrocarbon radical with up to 10 carbon atoms, or a CC single bond,
R ³ -O- or -NH-,
R ^{4 is} hydrogen or the radical -SO _3⁻ Na⁺,
R ^{5 is} hydrogen, the methyl radical or the radical -R ² -COO⁻Na⁺,
R ^{6 is} hydrogen or Na and
n 4 or 5;
with the proviso that at least one of the substituents R ^{4 is} a radical -SO _3⁻ Na⁺.

In preferred monomers V and VI means
R ^{1 is} hydrogen,
R ^{2 is} an alkylene radical with 1 to 4 carbon atoms, a phenylene radical or a CC single bond,
R ³ -O- or -NH-,
R ^{4 is} hydrogen or the radical -SO _3⁻ Na⁺,
R ^{5 is} hydrogen or the radical -R ² -COO⁻Na⁺
R ^{6 is} hydrogen or Na and is
n for 4.

The monomers V contain modified sugar residues, preferably from pentoses and in particular from arabinose. The sugar residues contain at least one of the residues -O-SO _3⁻ Na⁺ (O-sulfate) or -NH-SO _3⁻ Na⁺ (N-sulfate), preferably adjacent to the residue R ² . They preferably have 1 to 4 of these residues. O-sulfate and N-sulfate residues can be present simultaneously in a sugar residue, in which case the N-sulfate residue is preferably positioned adjacent to the residue R ² . Alternatively, the sugar residue can also contain only one type of these residues, e.g. B. only O-sulfate residues. Each of the named species (only O-sulfate-containing and N-sulfate-containing radicals) is suitable alone or together with the other species as monomer V. The mixing ratio is therefore 0: 100 to 100: 0.

The ratio in which the monomers V and VI are used that can fluctuate within wide limits. So can the molar ratio the N-sulfate and / or O-sulfate groups of the monomer V to the carb Oxyl and / or carboxylate groups of the monomer VI, for example 1: 100 to 100: 1. Preferred molar ratios are between 1: 20 and 20: 1.

The preparation of the monomers V is in the German patent application dung. . . (O.Z. 5195) described in detail. she be explained here using a special case, that of D-Glu cono-1,5-lactone 1 starts and leads to a monomer V, which from a Pentose, namely the D-arabinose, is derived. The specialist will however, the process is readily applicable to other suitable starting materials can transmit.

In a first stage, the hydroxyl groups of lactone 1 protected by acetalization, e.g. B. with acetone in methanol as Solvents. The lactone is split and you get one Isomer mixture of methyl 3,4; 5,6-di-O-isopropylidene-D-gluconate 2 and methyl 2,3; 5,6-di-O-isopropylidene-D-gluconate 3. This mixture is reduced in a second stage, e.g. B. with lithium aluminum hy drid, whereby the carboxylic ester function becomes the carbinol function. An isomer mixture is again obtained, namely 3,4; 5,6-di-O-iso propylidene-D-sorbitol 4 and 2,3; 5,6-di-O-isopropylidene-D-sorbitol 5. In  A third stage is this mixture of isomers with an oxide tion agents, such as sodium periodate, with cleavage of the carbon chain to a single product, the arabinosealdehyde 2,3; 4,5-di-O-iso propylidene aldehyde-D-arabinose 6 oxidized. In the subsequent the fourth stage, a vinyl function is introduced, e.g. B. by a Grignard reaction with 4-vinylphenylmagnesium chloride. Man he holds a partially protected 4-vinylphenylpentanpentaol, 2,3; 4,5-di-O-iso propylidene-1- (4-vinylphenyl) -D-gluco (D-manno) pentitol 7, which in subsequently referred to as Arasty.

This sequence of steps 1 to 4 is illustrated by the following reaction scheme:

This sequence of reactions is described 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 closer have been described.

To prepare a compound corresponding to Arasty 7 with an amino group in the 1-position, Arasty can be converted in a first step to the corresponding ketone, (2,3; 4,5-di-O-isopropylidene-D-arabi no) - (4- vinylphenyl) ketone 8, oxidize. In a second stage, this reductively becomes the amine 1-amino-1-deoxy-2,3; 4,5-di-O-isopropylidene-1- (4-vinylphenyl) -D-gluco (D-manno) pentitol 9 converted. This sequence of reactions is illustrated by the following formula:

In the first stage, Arasty Z z. B. with the complex (?) Of oxa lyl chloride and dimethyl sulfoxide at a temperature of <-50 ° C in be oxidized with an inert solvent. The reductive amination the second stage is advantageously achieved with sodium cyanobor hydride as a reducing agent in the presence of ammonium acetate in egg solvent with exclusion of water 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 / or N-sulfated in a second stage, so that the polymer produced from them is as heparin-analogous as possible. Deprotection is achieved in an acidic medium in which ketals are not stable. The protected compounds are heated, for. B. with dilute mineral acid or egg nem acidic ion exchanger and obtained from 7 1-hydroxy-1-deoxy-1- (4-vi nylphenyl) -D-gluco (D-manno) pentitol 10 and from 9 the 1-amino no -1-deoxy-1- (4-vinylphenyl) -D-gluco (D-manno) -pentitol 11. The deprotection and the subsequent sulfation are represented by the following formula:

The two compounds 10 and 11 are sulfated, appropriately using a sulfur trioxide-pyridine complex. Because of the advance Deacetalization does not occur during sulfation a single product with one or more sulfate groups in defined positions. However, it should be the primary hydroxyl groups and the amino groups are preferably sulfated. By Selection of a suitable molar ratio of sulfur trioxide to The degree of sulfation can be regulated by hydroxyl or amino groups will. On average, more than one sulfate group is advantageous pe introduced per molecule, because heparin about 2.7 sulfate groups per Disaccharide unit (corresponding to 1.35 sulfate groups per molecule Monomer I) contains. By sulfating the deprotected amine compound manure 11, O-sulfate and N-sulfate groups are obtained simultaneously in the Molecule what in terms of the targeted heparin analogy is desired.  

The sulfation is advantageously carried out at room temperature, to avoid premature polymerization. After a long time, e.g. B. up to 100 hours, the reaction is still complete. As Solvents can be used e.g. B. excess pyridine or an ether, such as tetrahydrofuran. Because the sulfate groups of the reaction products are acid-labile, it is advisable to precede the educt solution the addition of the sulfur trioxide-pyridine complex to bind water add the agent, e.g. B. a molecular sieve. For the same reason it is recommended to stop the reaction after the reaction mix first by adding water and soon afterwards a ba hydrolysis (which keeps the pH in the alkaline range). A suitable base is e.g. B. a saturated barium hydroxide solution, the sulfate ions also fail. Excess barium ions can e.g. B. be precipitated by introducing carbon dioxide if after careful concentration with removal of solvent. The barium carbonate is filtered off and the filtrate through a Given ion exchange column in the Na⁺ form or otherwise with treated the ion exchanger to the barium ions against sodium ion exchange. From the further narrowed solution you can go through Freeze-dry the products, O-sulfated 1-hydroxy-1-deoxy-1- (4-vi nylphenyl) -D-gluco (D-manno) -pentitol 12 or N- and O-sulfa tated 1-amino-1-deoxy-1- (4vinylphenyl) -D-gluco (D-manno) pentitol 13 each in the form of the sodium salt as powdery solids win. Both substances correspond to the formula V and are for the present invention suitable monomers.

The monomers VI are known and easily accessible substances that are necessary for the heparin-analogous effect Contribute carboxyl or carboxylate groups. Suitable monomers VI have an olefinic double bond and one or two carboxyl or Carboxylate functions or functions that are in carboxyl or Carboxylate functions can be converted, such as carboxylic ester, Carbonamide or carboxylic anhydride groups. As counterions to Sodium ions serve carboxylate function. Examples of suitable ones Monomers II are the acids (meth) acrylic acid, crotonic acid, 4-vinyl benzoic acid, maleic acid, fumaric acid, itaconic acid, vinyl acetic acid, Cinnamic acid, crotonic acid, isocrotonic acid, methyl maleic acid, dime thyl fumaric acid, methyl fumaric acid, dihydroxymaleic acid, allyl vinegar  acid and its sodium salts.

2. Other monomers which may also be used

In addition to the described monomers I to V with the listed blood-tolerant and bacteria-repellent or cell proliferation ration-inhibiting groups can also other monomers also use that are neutral or at most weakly effective are. These include e.g. B. vinyl ethers such as vinyl methyl ether and vinyl butyl ether; Vinyl ketones such as vinyl ethyl ketone; Olefins and diolefi ne, such as 1-butene, 1-hexene, 1,3-butadiene, isoprene and chloroprene; Acrylic and methacrylamide; Vinyl aromatics such as styrene, vinyl toluene and divinylbenzene; and vinyl siloxanes. These monomers can even be present in predominant quantities, e.g. B. up to 90 mol% chen.

3. The substrate materials

All polymeric art are particularly suitable as substrate materials substances such as polyurethanes, polyamides, polyesters and ethers, poly ether block amides, polystyrene, polyvinyl chloride, polycarbonates, Polyorganosiloxanes, polyolefins, polysulfones, polyisoprene, poly-chloro roprene, polytetrafluoroethylene (PTFE), polyacrylates, polymeth acrylates, corresponding copolymers and blends as well as natural and synthetic rubbers, with or without radiation-sensitive groups. The method according to the invention can also be used on surfaces of lacquered or otherwise coated with plastic, Use glass or wooden bodies.

3. The activation of the substrate surfaces

The surfaces of the substrates can be according to the invention Set of methods to be activated. They are expedient in known manner by means of a solvent of oils, fats or their impurities freed.

3.1 The activation of standard polymers without UV-radiation-sensitive groups can advantageously by UV radiation, for. B. in the wavelength range from 100 to 400 nm, preferably from 125 to 310 nm. A suitable radiation source is e.g. B. a UV excimer Ge advises HERAEUS Noblelight, Hanau, Germany. But mercury vapor lamps are also suitable for substrate activation, provided that they emit significant amounts of radiation in the areas mentioned. The exposure time is generally 0.1 seconds to 20 minutes, preferably 1 second to 10 minutes. It has been shown that the presence of oxygen is advantageous. The preferred oxygen pressures are between 2 × 10⁻ ⁵ and 2 × 10⁻ ² bar. For example, one works in a vacuum of ⁴ to 10⁻ 10⁻ ¹ 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.

3.2 According to the invention, the activation can also be carried out by a high frequency quenz or microwave plasma (Hexagon, Technics Plasma, 85 551 Kirchheim, Germany) in air, nitrogen or argon atmosphere can be achieved. The exposure times are generally 30 Seconds to 30 minutes, preferably 2 to 10 minutes. The Ener The entry for laboratory devices is between 100 and 500 W, preferably between 200 and 300 W.

3.3 Furthermore, corona devices (SOFTAL Ham castle, Germany) for activation. The exposure times in this case are usually 1 second to 10 minutes, preferably 1 to 60 seconds.

3.4 Activation by electron or gamma rays (e.g. from a cobalt 60 source) enables short exposure times generally 0.1 to 60 seconds.

3.5 Flaming surfaces also leads to their action crossing. Suitable devices, especially those with a barrier flame menfront, can be easily built or for example sourced from ARCOTEC, 71297 Mönsheim, Germany. You can use hydrocarbons or hydrogen as the fuel gas operate. In any case, harmful overheating of the Substrate can be avoided, which is due to intimate contact with a ge cooled metal surface on the side facing away from the flame side  Substrate surface is easily reached. Activation by Flame is accordingly relatively thin, flat Limited substrates. The exposure times are generally mean 0.1 seconds to 1 minute, preferably 0.5 to 2 seconds the, which is, without exception, non-glowing flames and the distances of the substrate surfaces from the outer flame front 0.2 to 5 cm, preferably 0.5 to 2 cm.

3.6 Furthermore, the substrate surfaces can also be activate action with strong acids or strong bases. Of the suitable strong acids are sulfuric acid, nitric acid and Called hydrochloric acid. You can e.g. B. Polyamides 5 seconds to 1 minute treat with concentrated sulfuric acid at room temperature. As strong bases are particularly suitable for alkali metal hydroxides in water or an organic solvent. So you can z. B. diluted Sodium hydroxide solution on the substrates at 20 to 80 ° C for 1 to 60 minutes let it work. Alternatively, for example, polyamides can be activated by mixing 2% KOH in tetrahydrofuran for 1 minute to 30 Minutes on the surface.

3.7 Finally, already in the manufacture of the substrate po polymeric monomers with UV-sensitive groups be settled. As such, z. B. furyl or cinnamoylde derivatives, e.g. B. can be applied in amounts of 3 to 10 mol% nen. Suitable monomers of this type are cinnamoyl ethyl acrylate and methacrylate.

In some cases, e.g. B. with highly hydrophobic polymers, it can be advisable to combine the substrate surfaces by a combination to activate from two or more of the methods mentioned. Preferred Activation methods are those mentioned under 3.1 and 3.2.

4. The coating by graft (co) polymerization

If the substrates according to one of the described in 3.1 to 3.6 Methods that have been pretreated are the activated surfaces expediently 1 to 20 minutes, preferably 1 to 5 minutes of on effect of oxygen, e.g. B. exposed in the form of air.  

Then the activated ones (if necessary also according to 3.7) Surfaces according to known methods, such as dipping, spraying or Brush to use with solutions according to the invention coated the vinyl monomers. Water has become a solvent and water-ethanol mixtures are tried and tested, but other solvents are also can be used provided that they have sufficient solvent power for the Have monomers and wet the substrate surfaces well. Each according to the solubility of the monomers and the desired layer thickness finished coating can the concentrations of the monomers in the solution be 0.1 to 50 percent by weight. Solutions with mono content of 3 to 10% by weight, for example about 5% by weight, have proven themselves in practice and generally result connected in one pass, the substrate surface be opaque coatings with layer thicknesses of more than 0.1 µm can.

After evaporation of the solvent or even during the Evaporation is the polymerization or copolymerization of the the activated surfaces applied monomers expedient by rays in the short-wave segment of the visible range or in the long-wave segment of the UV range of the electromagnetic radiation. Z. B. the radiation of a UV excimers of wavelengths from 250 to 500 nm, preferably from 290 up to 320 nm. Mercury vapor lamps are also suitable here, provided that they emit considerable amounts of radiation in the areas mentioned animals. The exposure times are generally 10 seconds to 30 minutes, preferably 2 to 15 minutes.

Sometimes it is useful to follow the steps described and finally to repeat the activation in order to use a sol Chen multilayer technology a hermetically sealed and / or ensure thicker coating. Alternatively, it is also possible the activated substrate, optionally after the sow described erstoffverarbeitung, in the solution of the or according to the invention immersed using vinyl monomers and in the immersed state to irradiate. Orientation tests make it easy determine at what radiation times with a given Radiation source and at which, possibly longer contact time  the desired layer thickness is reached becomes.

The method according to the invention for bacteria-repellent and cell proliferation-inhibiting coating of the surface of Sub strate and in particular of polymer plastics allows precise adjustment of molar ratios of different functional Groups used to inhibit bacterial adhesion and / or out spread and cell proliferation are optimal. Furthermore the method offers the advantage that when choosing appropriate Akti methods already proven plastics while maintaining their mechanical properties and their shape rejectable and modifiable to inhibit cell proliferation. In general there are no further pre- or post-treatments required. Highly hydrophobic plastics may be required a hydrophilizing pretreatment, e.g. B. by chemical etching with acids or bases or by plasma treatment to get one To achieve wettability through the monomer solution. The highly hydrophobic plastics are then simultaneously hydrophilized and activated in the sense of the present invention.

To further explain the present invention, the fol given examples. The monomers used therein are representative of a variety of others among the general Formulas I to IV falling compounds.

Examples The monomers used

Of the monomers listed in Table 1 were in each case 5 wt .-% aqueous solutions prepared under sterile conditions.  

Monomers used - 5% by weight solution

Monomers used - 5% by weight solution

The substrates to be coated

The investigations into the effects of the coatings according to the invention on bacteria-repellent behavior were carried out on films made of the plastics listed in Table 2, each having a thickness of 0.1 mm and a surface area of 4 cm ² relevant for the determination. They were produced by dissolving the powders in solvents, then pouring them into petri dishes and drying, and also by calendering, extruding, pressing or knife coating. In some cases, films were available from the manufacturer.

Foils used

Foils used

Foils used (continued)

Foils used (continued)

The activation of the substrate surfaces

The foils were first activated, optionally according to Table 3 specified procedures and conditions.  

Activation conditions

Activation conditions

Coating of the substrate surfaces by graft (co) polymerization

After activation, the foils, castings or others Substrate body and in the case of technical production, the extrudates or injection molded body coated with solutions S1 to S16, and according to the procedures given in Table 4.

Coating process

Coating process

During diving or after diving, spraying or Is coated with rays in the range of 250-500 nm,  preferably irradiated 290-320 nm.

Determination of the bacteria-repellent properties

Testing for bacterial adhesion can be done with different strains men can be made. The ones in the table are particularly suitable for this 5 bacteria listed, since they are in clinical isolates of infi decorated catheters are common.

Strains of bacteria to measure primary adhesion

Strains of bacteria to measure primary adhesion

The method for determining the primary adhesion (i.e. independent of later multiplication) of these bacterial strains is exemplary for Klebsiella pneumoniae described below. The primary ad The adhesion of the other strains (B1 to B3) was determined analogously.

Determination of the primary bacterial adhesion under static conditions

An overnight culture of the bacterial strain Klebsiella pneumoniae in yeast extract-peptone-glucose culture medium (1% + 1% + 1%) is centrifuged off and in phosphate-buffered saline (= PBS; 0.05 m KH ₂ PO ₄ , pH 7.2 + 0.9% NaCl) resumed. It is diluted with PBS buffer to a cell concentration of 108 cells / ml. The suspended bacteria are brought into contact with the piece of film to be examined for 3 hours. For this purpose, double-sided coated circular pieces of film with a diameter of 1.6 cm (= 4.02 cm ² ) are placed on a dissecting needle and shaken with the cell suspension. Films coated on one side are clamped in a membrane filter apparatus in the form of a round, flat disc of 4.5 cm in diameter and with a support membrane made of 2-3 cm thick soft PVC. The cell suspension is applied to the upward-facing side with the coating to be tested and shaken for 3 hours. The membrane filter apparatus must be leak-tight, ie no cell suspension may leak through leaky cells.

After the contact time has elapsed, the bacterial suspension is treated with a Aspirated water jet pump and the pieces of film become Wash with 20 ml sterile PBS solution in a 100 ml beaker Shaken for 2 minutes. The piece of film is again in sterile PBS solution solution and then immersed in 10 ml of heated TRIS / EDTA (0.1M Tris hydroxyethylaminomethane, 4 mM ethylenediaminetetraacetic acid, with HCl adjusted to pH 7.8) for 2 min in a boiling water bath here.

Small Eppendorf cups are filled with the extraction solution and frozen immediately at -20 ° C until the bioluminescence determination of the extracted adenosine triphosphate (ATP). The determination is carried out as follows: 100 .mu.l of reagent mix (bioluminescence test CLS II, from BOEHRINGER MANNHEIM GmbH) is placed in a transparent tube made of polycarbonate, and over a period of 10 sec. In a light pulse measuring device LUMAT LB9501 (laboratories Prof. Berthold GmbH, 75323 Bad Wildbad, Germany) integrated the light impulses. Then a 100 µl sample is added and measured again. The relative light units (RLU) are obtained by subtracting the light pulses in the reagent mix from the number of measured light pulses in the complete batch. This value is related to the number of bacteria adhering to the film. The conversion factor between the RLU value and the number of bacteria is determined by extracting an aliquot of 0.1 ml of the bacterial suspension with 10 ⁸ cells / ml in 10 ml of hot TRIS / EDTA and then determining the ATP content.

For Klebsiella pneumoniae there is a value of 1.74.10 ⁴ RLU = 1.10 ⁷ cells in the ATP extraction batch. With a measured value of 4.7.10 ⁴ RLU of 4 cm ³ film were per cm ² film surface

adheres primarily.

Results

In the following tables 6a and 6b are the different conditions conditions and the results of a total of 27 experiments and ver try to equalize without prior activation (14, 16, 18, 20, 22, 24, 26).  

Tables 6a and 6b illustrate that according to the invention ß process coatings are obtained that lead to a considerable lead to a reduction in bacterial adhesion. The reductions lay well over 50% compared to the uncoated substrates Furthermore, the comparative examples (Experiments 3, 4 and 15) recognize that by activating the substrate surfaces with UV excimer rays with a wavelength of 172 nm (A1) or plasma treatment (A2, A3) surprisingly, higher inhibitions of the bacterial ad adhesion (≧ 90%) can be achieved than with other activating agents, such as electron beams (A7, experiment 13) or NaOH solutions (A8, Experiment 17).

The results shown in the tables further show that one can work successfully with a single monomer (S7, Ver such 25), but also copolymers of two monomers, namely from sodium styrene sulfonate and acrylic acid (S1 + S2; experiment 15), Sodium styrene sulfonate and maleic acid (S1 + S4; experiment 17), Sodium vinyl sulfonate and caffeic acid (S6 + S10; experiment 21) see above such as acrylic acid and sodium vinyl sulfonate (S2 + S6; experiment 27) Coatings with good bacteria-repellent properties result.

Claims (22)

1. A method for bioactive coating of the surface of sub strates, characterized in that at least one monomer of the general formula
Formula I: R- (A) _a
in which R denotes a mono- or di-olefinically unsaturated organic radical with the valence a,
A is a carboxyl group -COOH, sulfuric acid group -OSO ₂ OH, sulfonic acid group -SO ₃ H, phosphoric acid group -OPO (OH) ₂ phosphonic acid group -PO (OH) ₂ phosphoric acid group -OP (OH) ₂ , phenolic hydroxyl group or a salt or an ester of one of the designated groups and
a represents an integer from 1 to 6,
radiation-induced graft-polymerized on an activated substrate surface, with the proviso that a monomer I in which A is a carboxyl group -COOH or a salt or an ester of the carboxyl group either has at least one further radical A with another of the meanings mentioned for A. contains or is used together with at least one further monomer I in which A has another of the meanings mentioned for A. 2. The method according to claim 1, characterized in that one as a monomer I is a mixture of monomers of the general formulas II or III
Formula II: (C _n H _2n-qx ) (COOR ¹ ) _x
Formula III: (C _n H _2n-qx ) (SO ₃ R ¹ ) _x
used, where
n each independently for an integer from 2 to 6 inclusive;
x each independently for 1 or 2;
q each independently represents 0 or 2 and
the radical R ¹ each independently represents -H, an equivalent of a metal ion or a radical of an aliphatic, cycloaliphatic or araliphatic alcohol. 3. The method according to claim 1, characterized in that one as monomer I is a benzene-derived monomer of the general formula
Formula IV: (C ₆ H _6-bcd ) B _b R ³ _c (OH) _d
used in the
B each independently _denotes a mono- or divalent straight-chain or branched radical of the formulas (C _n H _2n-1-qx ) (COOR ¹ ) _x or (C _n H _2n-1-qx ) (SO ₃ R ¹ ) _x , wherein R ¹ , n , q and x are as defined in claim 2;
R ³ each independently is C _1-4 alkyl, -NH ₂ , -COOH, -SO ₃ H, -OSO ₃ H, -OPO (OH) ₂ -PO (OH) ₂ , -OP (OH) ₂ , -OPO (O⁻) OCH ₂ -CH ₂ -N⁺ (CH ₃ ) ₃ , -PO (O⁻) O-CH ₂ -CH ₂ -N⁺ (CH ₃ ) ₃ , -OP (O⁻) OCH ₂ -CH ₂ -N⁺ (CH ₃ ) ₃ or optionally a salt or an ester of the groups mentioned means:
b represents 1, 2 or 3;
c represents 0, 1, 2, or 3; and
d represents 0, 1, 2, or 3;
with the proviso that b + c + d ≦ 6. 4. The method according to claim 1, characterized in that the monomer I is a mixture of monomers of the general formulas V and VI
used in which
R ^{1 is} hydrogen or the methyl radical,
R ^{2 is} a divalent organic radical or a CC single bond
R ³ -O- or -NH-,
R ^{4 is} hydrogen or the radical -SO _3⁻ Na⁺
R ^{5 is} hydrogen, the methyl radical or the radical -R ² -COOR ⁶ ,
R ^{6 is} hydrogen or Na and
n is 4 or 5;
with the proviso that at least one of the substituents R ^{4 is} a radical -SO _3⁻ Na⁺. 5. The method according to claim 2, characterized in that the Monomers II and III are chosen so that the grafted polymer layer (iii) carboxyl and sulfonic acid groups, (iv) carboxyl and Sulfonate groups, (v) carboxylate and sulfonate groups, (vi) carb oxyl, carboxylate and sulfonate groups or carboxyl, sulfonic acid and Contains sulfonate groups. 6. The method according to claim 5, characterized in that the mo lare ratio of carboxyl and / or carboxylate groups to sulfone acid and / or sulfonate groups is 0.2 to 3. 7. The method according to claim 5, characterized in that the mo lare ratio of carboxyl and / or carboxylate groups to sulfone acid and / or sulfonate groups is 0.4 to 3. 8. The method according to claim 5, characterized in that the mo lare ratio of carboxyl and / or carboxylate groups to sulfone acid and / or sulfonate groups is 0.4 to 2. 9. The method according to claim 5, characterized in that the mo lare ratio of carboxyl and / or carboxylate groups to sulfone acid and / or sulfonate groups is 2 to 10. 10. The method according to claim 5, characterized in that the mo lare ratio of carboxyl and / or carboxylate groups to sulfone acid and / or sulfonate groups is 3 to 10.   11. The method according to claim 5, characterized in that the mo lare ratio of carboxyl and / or carboxylate groups to sulfone acid and / or sulfonate groups is 3 to 5. 12. The method according to claim 1, characterized in that the Monomer I or the monomers I are chosen so that the grafted te polymer layer phosphoric acid groups or phosphonic acid groups or their salts or esters. 13. The method according to any one of claims 1 to 12, characterized characterizes that the activation of the substrate surfaces by monomers copolymerized into the substrate polymer with a UV radiation sensitive group, by UV radiation, plasma treatment treatment, corona treatment, electron beam treatment, flame treatment and or or treatment with a strong acid or strong base follows. 14. The method according to any one of claims 1 to 12, characterized records that the activation of the substrate surface by UV radiation in the wavelength range from 100 to 400 nm at an Expo sition time from 0.1 seconds to 20 minutes. 15. The method according to any one of claims 1 to 12, characterized records that the activation of the substrate surface by UV radiation in the wavelength range from 125 to 310 nm at an Expo sition time from 1 second to 10 minutes. 16. The method according to any one of claims 1 to 12, characterized records that the activation of the substrate surface by Hochfre quenz or microwave plasma with an exposure time of 30 Se customers up to 30 minutes. 17. The method according to any one of claims 1 to 14, characterized records that the activated surfaces before coating Exposed to oxygen for 1 to 20 minutes. 18. The method according to claim 17, characterized in that one let the oxygen act for 1 to 5 minutes.   19. The method according to any one of claims 1 to 18, characterized records that the polymerization of the monomers by radiation effected in the range of 250 to 500 nm. 20. The method according to any one of claims 1 to 18, characterized in records that the polymerization of the monomers by UV radiation len in the range from 290 to 320 nm. 21. Use of substrates with be according to claims 1 to 20 layered surfaces according to claims 1 to 18 for the manufacture treatment of medical or biotechnical articles, storage containers or packaging. 22. Use according to claim 21, characterized in that the Articles are catheters, tubing or tubing.