DE10216639A1 - Stellate pre-polymers containing reactive functional groups on the free ends useful for the production of ultra-thin coatings forming hydrogels and showing non-specific protein absorption and for antifouling coatings - Google Patents

Abstract

The use of stellate pre-polymers having on average at least 4 polymer branches (A), soluble in water, with reactive functional groups (R) on the free ends, which can react with complementary functional groups (R') or with each other to form bonds for production of ultra-thin hydrogel forming coatings is new. An Independent claim is also included for preparation of these coatings by: (i) application of the pre-polymer with free functional end groups (R) to the surface to be coated; and (ii) subjection of the reactive groups to a linkage reaction.

Description

The present invention relates to the use of star-shaped Polymers with hydrophilic polymer arms at their free ends carry a reactive functional group R for the preparation ultra-thin, hydrogel-forming coatings. The received Hydrogel-forming coatings effectively suppress non-specific ones Protein absorption on surfaces equipped with it.

The specific and nonspecific interaction of proteins and Cells with artificial surfaces form the basis of many medical, biochemical and biotechnological applications. to Prevention of unwanted deposits, also as biofouling and plaque referred to, as well as for the stimulation of a desired Cell colonization requires an unspecific protein and cell adsorption to prevent. Because biological systems are their environment (surfaces) To be able to actively modify yourself, you have to reduce it Nonspecific protein and cell adsorption prevent the cells from growing Environment through membrane proteins or extracellular matrix proteins in a way to condition that is an unspecific cell population prepared.

The use of specific adsorption processes for analysis naturally requires, type, quantity and conformation unspecific to control adsorbed molecules precisely, or even better completely prevent unspecific adsorption. For the detection of Biomolecules, which are only present in extremely small quantities, have to be be sure that the substances to be detected are on the way to Detection is not lost through unspecific adsorption.

Suppression of unspecific protein adsorption and Cell colonization in the field of medical technology, e.g. B. catheters, Contact lenses or prostheses are also important. For prostheses and Implants make the targeted suppression of non-specific Adsorption processes are a prerequisite for the integration of ligands, Signal substances and growth factors have a specific coupling of To enable molecules and cells that are healing and that Ingrowth of biological tissue enables.

Furthermore, the prevention of biological deposits plays out Proteins or bacteria play an essential role in hygiene and when keeping surfaces clean, which are not permanent can be cleaned without residue. Likewise, unwanted, biological deposits on extremely large wetted surfaces such as Ship hulls, water basins and the like as well as deposits inaccessible places, such as large pipe systems, a large one economic problem. The currently used Antiplaque coatings are mostly toxic or organometallic compounds lose their bactericidal effect very quickly.

Plaque formation and biofouling are therefore considerable economic and ecological burden that has not yet existed could be solved satisfactorily.

There have been various polymeric, especially hydrogel-forming Coatings to avoid unspecific protein adsorption Surfaces suggested. Hydrogel-forming coatings are Coatings that are swollen by water. So decrease Adsorbate layers made of polyethylene glycol (PEG) a subsequent adsorption of proteins from biological media (see, for example: Merrill E. W., in Poly (ethylene glycol) Chemistry, editor J. M. Harris, p. 199-220, Plenum Press, New York: 1992; C.-G. Gölander, Jamea N. Herron, Kap Lim, P. Claesson, P. Stenius, J.D. Andrade, in Poly (ethylene glycol) Chemistry, edited by J. M. Harris, Plenum Press, New York: 1992). Polymer surfaces coated with poly (ethylene oxide) to reduce protein adsorption on implant materials modified, have been intensively investigated in recent years (Paine et al. Macromolecules 1990, 23, p. 3104).

US 5,993,890 describes triblock copolymers which have one Polysaccharide block, e.g. B. a heparin or dextran block and hydrophobic Have hydrocarbon residues. The polymers are especially for Prevention of protein adsorption on hydrophobic surfaces suitable.

EP 272842 A2 describes coatings with low affinity Proteins made up by applying compositions Polymers containing hydroxyl groups, e.g. B. cellulose derivatives and Crosslinkers, e.g. B. copolymers of acrylic acid and N-methylolacrylamide, on microporous substrates and subsequent crosslinking of the Coating can be produced.

EP 335308 A2 describes the use of prepolymers Polyethylene oxide diols and triols, the terminal OH groups with Polyisocyanates have been implemented for the production of coatings with low non-specific protein adsorption.

No. 6,087,415 discloses antimicrobial coatings for biomedical articles such as contact lenses, which are produced by coupling polymers having carboxyl groups to the OH or NH ₂ groups on the surface of the biomedical article.

Furthermore, US 6,150,459 and 6,207,749 are used for this purpose synthetic comb polymers are proposed which are hydrophobic Polymer backbone, e.g. B. a polylactide or one of methyl methacrylate derived polymer, and grafted hydrophilic polymer chains thereon preferably derived from polyethylene glycols or polyacrylic acid are.

J. Ruhe et al. (Prucker O. and Rühe J. Langmuir 1998, 14 (24), 6893-6898; Macromolecules, 1998, 31 (3), 592-601; Macromolecules, 1998, 31 (3), 602-613.) Describe a coating process in which first modified a surface with a monolayer of an initiator becomes. This then triggers a radical one Polymerization used. There are very dense layers, the Layer thickness can grow up to several hundred nanometers thick.

Cruise et al. (Biomaterials 1998, 19, 1287-1294) and and Han et al. (Macromolecules 1997, 30, 6077-6083.0) describe the use of Acrylate-terminated prepolymers, either from PEG diols or PEG triplets were made for the production of Hydrogel. The acrylate is terminated to connect to the surface Exposed prepolymer on a correspondingly pretreated substrate, whereby the prepolymer terminated alone or with acrylate Glycerol triol with the addition of benzene dimethyl ketal to a hydrogel network. In this way, hydrogel layers with layer thicknesses obtained between 135 microns and 180 microns. As areas of application for this Hydrogel layers are used in vivo, e.g. B. for suppression postoperative adhesion processes, the use as Diffusion barrier, for bonding or sealing tissues in vivo Medication or use as a direct implant, e.g. B. in Form of a hydrogel cell suspension, peptide hydrogel or one Growth factor hydrogel, proposed.

Although the hydrogel-forming agents known from the prior art Coatings a reduction in non-specific cell and The effect of protein adsorption is often the long-term stability of Coatings unsatisfactory. In other cases it is the barrier effect for proteins is insufficient. In many cases prevent complicated manufacturing processes for these coatings wide applicability, especially to irregularly shaped ones Surfaces. There is therefore a need for suitable methods for Manufacture of extremely thin, hydrogel-forming coatings that form reproducible dense and controllable layers, one sufficiently high long-term resistance to protein and cell adsorption show and can be used universally, i.e. for a wide range of uses Allow material coatings. Furthermore, it should be possible specific, functional molecules defined in the layers to integrate or anchor on the surface. Desirably should the coatings in the application fields mentioned can be applied as ultra-thin, molecularly smooth films. It is also desirable to have defined in these coatings To bring in functionalities. In addition, that should Coating processes advantageously also take place from aqueous preparations can. As far as possible, existing procedures for the Manufacture of substrates and components using Hydrogel coatings are not affected and the applicability in different fields of application.

It has been found that this task can be solved by Coatings based on interlinked star-shaped Prepolymers which have on average at least four polymer arms A, which in themselves are soluble in water and those on their free ends carry a reactive functional group R, which with a complementary reactive functional groups R 'or with reacting to themselves by forming bonds.

Accordingly, the present invention relates to the use of star-shaped prepolymers which have on average at least four polymer arms A have, which are seen as soluble in water and on their free ends carry a reactive functional group R, which with complementary groups R 'or with themselves under React bond formation to produce ultra-thin, hydrogel-forming Coatings.

• a) eine Lösung eines sternförmigen Präpolymers, das im Mittel wenigstens vier Polymerarme A aufweist, die für sich gesehen in Wasser löslich sind und an ihren freien Enden eine reaktive funktionelle Gruppe R tragen, auf die zu beschichtende Oberfläche aufbringt und
• b) anschließend eine Verknüpfungsreaktion der reaktiven Gruppen untereinander durchführt.

The present invention also relates to a method for producing ultra-thin, hydrogel-forming coatings, which is characterized in that

• a) a solution of a star-shaped prepolymer, which has on average at least four polymer arms A, which are in themselves soluble in water and carry a reactive functional group R at their free ends, applied to the surface to be coated and
• b) subsequently carrying out a linking reaction between the reactive groups.

A person skilled in the art understands a hydrogel-forming coating such coatings that are swollen by water as a result the intercalation of water molecules in the coating.

Star-shaped polymers are understood to mean those polymers which have a plurality of polymer chains bonded to a low-molecular central unit, the low-molecular central unit generally having 4 to 100 skeletal atoms, such as C atoms, N atoms or O atoms. Accordingly, the star-shaped polymers used according to the invention can be described by the following general formula I:

Z (ABR) _n (I)

wherein
n is an integer with a value of at least 4, e.g. B. 4 to 12, in particular 5 to 12 and especially 6 to 8;
Z stands for a low molecular weight n-valent organic radical as central unit, which generally has 4 to 100, preferably 5 to 50 framework atoms, in particular 6 to 30 framework atoms. The central unit can have both aliphatic and aromatic groups. It stands for example for one of at least 4-valent alcohol, e.g. B. a 4- to 12-valent, preferably an at least 5-valent, especially a 6- to 8-valent alcohol, e.g. B. pentaerythritol, dipentaerythritol, a sugar alcohol such as erythritol, xylitol, mannitol, sorbitol, maltitol, isomaltulose, isomaltitol, trehalulose, or the like derived residue;
A is a hydrophilic polymer chain which as such is water soluble;
B is a chemical bond or a divalent, low molecular weight organic radical with preferably 1 to 20, in particular 2 to 10 C atoms, for example a C ₂ -C ₁₀ alkylene group, a phenylene or a naphthylene group or a C ₅ -C ₁₀ cycloalkylene group , wherein the phenylene, naphthylene and the cycloalkylene group additionally one or more, for. B. 1, 2, 3, 4, 5, or 6 substituents, e.g. B. may have alkyl groups with 1 to 4 carbon atoms, alkoxy groups with 1 to 4 carbon atoms or halogen; and
R is a reactive group that can react with a complementary reactive functional group R 'or with itself to form bonds.

Reactive groups R in this sense are those with nucleophiles react in an addition or substitution reaction, e.g. B. Isocyanate groups, (meth) acrylic groups, oxirane groups, oxazoline groups, Carboxylic acid groups, carboxylic acid ester and carboxylic anhydride groups, Carboxylic acid and sulfonic acid halide groups, but also those complementary groups reacting as nucleophiles such as alcoholic OH groups, primary and secondary amino groups, thiol groups and like. In particular, examples of carboxylic acid ester groups are so-called active ester groups of the formula -C (O) O-X are preferred, in which X for pentafluorophenyl, pyrrolidin-2,5-dion-1-yl, benzo-1,2,3-triazole 1-yl or a carboxamidine residue.

Free radicals are also suitable as reactive groups R. polymerizable C = C double bonds, e.g. B. in addition to the above (Meth) acrylic groups as well as vinyl ether and vinyl ester groups activated C = C double bonds, activated C ~ C triple bond and N = N double bonds with allyl groups in the sense of an en reaction or with conjugated diolefin groups in the sense of a Diels-Alder React reaction. Examples of groups with allyl groups in the In the sense of an en reaction or with serving in the sense of a Diels-Alder Reaction are maleic acid and fumaric acid groups, Maleic acid ester and fumaric acid ester groups, cinnamic acid ester groups, Propiolic acid (ester) groups, maleic acid amide and fumaric acid amide Groups, maleimide groups, azodicarboxylic acid ester groups and 1,3,4- Triazolin-2,5-dione groups.

In a preferred embodiment, the star-shaped Prepolymer on such functional groups that an addition or Substitution reaction accessible by nucleophiles. Which includes also groups that react in the sense of a Michael reaction. Examples are in particular isocyanate groups, (meth) acrylic groups (react in the sense of a Michael reaction), oxirane groups or Carbonsäureestergruppen. A particularly preferred embodiment relates star-shaped prepolymers which are reactive groups R isocyanate Have groups.

In another embodiment, the prepolymer is ethylenic Unsaturated, radical polymerizable double bonds as reactive Groups R on.

To achieve layers that are as compact as possible, it is necessary that the star-shaped prepolymer averaged at least 4, e.g. B. 4 to 12, preferably at least 5 and in particular 6 to 8 polymer arms having. The number average molecular weight of the polymer arms is preferably in the range from 300 to 3000 g / mol, and in particular in Range from 500 to 2000 g / mol. Accordingly, that star-shaped prepolymer has a number average molecular weight in the range of 2000 to 20000 g / mol, in particular 2500 to 15000 g / mol.

The molecular weight can be determined in a manner known per se by means of gel permeation chromatography, whereby on commercially available columns, detectors and evaluation software can. If the number of end groups per polymer molecule is known, one can determine the molecular weight also by titration of the end groups, in the case of isocyanate groups z. B. by implementation with a defined amount of a secondary amine such as dibutylamine and subsequent titration of the excess amine with acid.

The sufficient swellability of the coating by water is guaranteed by the water solubility of the polymer arms A. A sufficient swellability of the coating by water is in the Usually guaranteed if the molecular structure, i.e. H. at least the type of repetition units, preferably also that Molecular weight of the polymer arm corresponds to a polymer whose Solubility in water at least 1% by weight and preferably at least 5 wt .-% (at 25 ° C and 1 bar).

Examples of such polymers with sufficient water solubility are poly-C ₂ -C ₄ -alkylene oxides, polyoxazolines, polyvinyl alcohols, homo- and copolymers which contain at least 50% by weight of copolymerized N-vinylpyrrolidone, homo- and copolymers which have at least 30% by weight. % Copolymerized hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, acrylamide, methacrylamide, acrylic acid and / or methacrylic acid, hydroxylated polydienes and the like.

In a preferred embodiment, the polymer arms A are derived from poly-C ₂ -C ₄ -alkylene oxides and are selected in particular from polyethylene oxide, polypropylene oxide and polyethylene oxide / polypropylene oxide copolymers, which can have a block or a statistical arrangement of the repeating units. Star-shaped prepolymers whose polymer arms A are derived from polyethylene oxides or from polyethylene oxide / polypropylene oxide copolymers with a propylene oxide content of not more than 50% are particularly preferred.

The prepolymers used according to the invention are e.g. T. known, e.g. B. from WO 98/20060, US 6,162,862 (Polyether star polymers), Chujo Y. et al., Polym. J. 1992, 24 (11), 1301-1306 (star-shaped polyoxazolines), WO 01/55360 (star-shaped polyvinyl alcohols, copolymers containing star-shaped vinyl pyrrolidone) or can according to the methods described there.

The star-shaped prepolymers used according to the invention are generally produced by functionalizing suitable star-shaped prepolymer precursors which already have the prepolymer structure described above, ie at least 4 water-soluble polymer arms, and which each have a functional group R "at the ends of the polymer arms, which can be converted into one of the aforementioned reactive groups R. The groups R "include halogen atoms bonded to aliphatic or aromatic carbon atoms, preferably to a primary aliphatic carbon atom, in particular chlorine, bromine or iodine, to an aliphatic or aromatic OH groups, thiol groups and NHR ² groups with R ² = hydrogen or C ₁ -C ₄ alkyl and in particular bound to a primary, aliphatic C atom.

Such prepolymer precursors are known from the prior art, for. B. from US 3,865,806, US 5,872,086, US 6,162,862, Polym. J. 1992, 24 (11), 1301-1306, WO 01/55360 and commercially available, e.g. B. in the case of star-shaped poly-C ₂ -C ₄ -alkylene oxides under the trade names VORANOL®, TERRALOX®, SYNALOX® and DOWFAX® from Dow-Chemical Corporation, SORBETH® from Glyco-Chemicals Inc. and GLUCAM® from Amerchol Corp. or can be prepared by known methods in polymer chemistry by polymerizing suitable monomers in the presence of polyfunctional initiators, e.g. B. by "living" polymerization (see: Hsieh, HL; Quirk, RP Anionic polymerization: Principles and Practical Applications, New York, Marcel Decker, 1996; Matyjaszewski, K. Controlled / Living radical polymerization: Progress in ATRP, NMP, and RAFT, Washington, DC: American Chemical Society, 2000) or in particular in the case of ethylenically unsaturated monomers by atom transfer radical polymerization (ATRP) according to the processes described in WO 98/40415.

The star-shaped prepolymer precursors can be functionalized basically in analogy to known functionalization processes state of the art.

For the preparation of prepolymers which carry amino groups at the ends of the polymer arms A, those prepolymer precursors which have A OH groups at the ends of the polymer arms A are particularly suitable as starting materials. The conversion of OH groups into amino groups can e.g. B. in analogy to that of Skarzewski, J. et al. Monatsh. Chem. 1983, 114, 1071-1077 method described. To this end, the OH groups in a conventional manner (be z. B. by Organikum, 15th ed., VEB, Berlin 1981 et seq .; S. 241 J. March, Advanced Organic Synthesis ^3rd ed. S. 382 ff . See also Example 12) with a halogenating agent such as thionyl chloride, sulfuryl chloride, thionyl bromide, phosphorus tribromide, phosphorus oxychloride, oxalyl chloride and the like, optionally in the presence of an auxiliary base such as pyridine or triethylamine, in the corresponding halide, or with methanesulfonyl chloride in the corresponding mesylate. The halogen compound or the mesylate thus obtained is then converted into the corresponding azide using an alkali metal azide, preferably in an aprotic polar solvent such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide or N-methylpyrrolidone. The azide is then converted to the amino compound with hydrogen in the presence of a transition metal catalyst or with a complex hydride such as lithium aluminum hydride.

The preparation of prepolymers which are at the ends of the polymer arms A carry oxirane groups, for example, by reacting Prepolymer precursors that are attached to the ends of the polymer arms A OH groups exhibit with glycidyl chloride.

Prepolymers which carry A (meth) acrylic groups at the ends of the polymer arms can be prepared, for example, by esterifying prepolymer precursors which carry A OH groups at the ends of the polymer arms with acrylic acid or methacrylic acid or by reacting the OH- Groups with acrylic chloride or with methacryl chloride in analogy to known processes. Alternatively, in prepolymer precursors which carry A NH ₂ groups at the ends of the polymer arms, the NH ₂ groups can be reacted with acrylic acid or methacrylic acid or with their acid chlorides. The preparation of (meth) acrylate-terminated prepolymers can e.g. B. in analogy to that of Cruise et al. Biomaterials 1998, 19, 1287-1294 and Han et al. Macromolecules 1997, 30, 6077-6083 for the modification of polyether diols and triols described methods take place.

The preparation of prepolymers which are at the ends of the polymer arms A carry thiol groups, for example, by reacting Prepolymer precursors attached to the ends of the polymer arms A halogen atoms wear, with thioacetic acid and subsequent hydrolysis in analogy the one in Houben-Weyl, Methods of Organic Chemistry, Ed. E. Müller, 4th ed., Vol. 9, p. 749, G. Thieme, Stuttgart 1955 Method.

The preparation of prepolymers which are at the ends of the polymer arms A carry isocyanate, preferably by adding one low molecular weight diisocyanate on prepolymer precursors at the ends of the Polymer-poor A have OH, SH, or NHR "groups (R '= H, or an aliphatic residue). Star-shaped polyols are preferably used OH end groups used.

Both aromatic diisocyanates and toluene 2,4-diisocyanate, toluene-2,6-diisocyanate, commercially available Mixture of toluene-2,4- and -2,6-diisocyanate (TDI), m-phenylene diisocyanate, 3,3'-diphenyl-4,4'-biphenylene diisocyanate, 4,4'-biphenylene diisocyanate, 4,4'-diphenylmethane diisocyanate, 3,3'-dichloro-4,4'- biphenylene diisocyanate, cumen-2,4-diisocyanate, 1,5-naphthalene diisocyanate, p-xylylene diisocyanate, p-phenylene diisocyanate, 4-methoxy 1,3-phenylene diisocyanate, 4-chloro-1,3-phenylene diisocyanate, 4-bromo 1,3-phenylene diisocyanate, 4-ethoxy-1,3-phenylene diisocyanate, 2,4- Dimethyl-1,3-phenylene, 5,6-dimethyl-1,3-phenylene diisocyanate, 2,4-diisocyanatodiphenyl ether, benzidine diisocyanate, 4,6- Dimethyl-1,3-phenylene diisocyanate, 9,10-anthracene diisocyanate, 4,4'- Diisocyanatodibenzyl, 3,3'-dimethyl-4,4'-diisocyanatodiphenylmethane, 2,6-dimethyl-4,4'-diisocyantodiphenyl, 2,4-diisocyanatostilbene, 3,3'- Dimethoxy-4,4'-diisocyantodiphenyl, 1,4-anthracene diisocyanate, 2,5- Fluoro diisocyanate, 1,8-naphthalene diisocyanate, 2,6-diisocyanatobenzofuran, as well as aliphatic and cycloaliphatic diisocyanates such as isophorone diisocyanate (IPDI), ethylene diisocyanate, Ethylidene diisocyanate, propylene-1,2-diisocyanate, cyclohexylene-1,2-diisocyanate, Cyclohexylene-1,4-diisocyanate, 1,6-hexamethylene diisocyanate, 1,4- Tetramethylene diisocyanate, 1,10-decamethylene diisocyanate and Dicyclohexylmethane.

Preference is given to diisocyanates whose isocyanate groups are in their Differentiate reactivity, such as toluene-2,4-diisocyanate, toluene-2,6- diisocyanate, and a mixture of toluene-2,4- and -2,6-diisocyanate and cis and trans isophorone diisocyanate.

Star-shaped prepolymers with aliphatic are particularly preferred Diisocyanate end groups, especially those as added by IPDI on the chain ends of OH group terminated star-shaped prepolymer precursors can be obtained.

The star polymers are naturally reacted with the diisocyanate in such a way that at each chain end of the star molecules a diisocyanate unit is added, the second isocyanate group of the diisocyanate being free remains. In this way, each end group of the star molecules is over provide a urethane linkage with a free isocyanate group. Procedures for this are e.g. B. from US 5,808,131, WO 98/20060 and US 6,162,862 and Bartelink C.F. et al. J. Polymer Science 2000, 38, 2555-2565.

As a rule, the prepolymer precursor is used for this purpose Avoiding the formation of multimeric adducts, d. H. Adducts in which two or more prepolymers with one another via diisocyanate units are linked, give an excess of the diisocyanate. In the As a rule, the excess is at least 10 mol%, based on the Stoichiometry of the reaction, i. H. you put at least 1.1 moles, preferably at least 2 moles and in particular at least 5 moles Diisocyanate and especially at least 10 moles of diisocyanate per mole functional group in the prepolymer precursor. This is preferably done Reaction under controlled reaction conditions, i.e. H. the addition the prepolymer precursor is so slow under reaction conditions that that heating of the reactor contents by more than 20 K is avoided becomes. The prepolymer precursor is preferably reacted with the diisocyanate in the absence of a solution or Diluent.

The reaction can be carried out in the absence or in the presence of small amounts conventional catalysts that promote the formation of urethanes, respectively. Suitable catalysts are, for example, tertiary amines such as diazabicyclooctane (DABCO) and organotin compounds, e.g. B. Dialkyltin (IV) salts of aliphatic carboxylic acids such as Dibutyltin dilaurate and dibutyltin dioctoate. The amount of catalyst is in usually not more than 0.5 wt .-%, based on the Prepolymer precursor, e.g. B. 0.01 to 0.5 wt .-%, in particular 0.02 to 0.3 wt .-%. In a preferred procedure, no catalyst is used.

The required reaction temperatures naturally depend on the Reactivity of the prepolymer precursor used, the diisocyanate, and if used by the type and amount of used Catalyst. It is usually in the range of 20 to 100 ° C and especially in the range of 35 to 80 ° C. It goes without saying that the implementation of the prepolymer precursor with the diisocyanate in Absence of moisture (<2000 ppm, preferably <500 ppm) he follows.

The reaction mixture thus obtained is worked up in the Rule by distilling off the excess diisocyanate, preferably at reduced pressure. The reaction products obtained predominantly contain the star-shaped prepolymer, the has isocyanate groups at the ends of the polymer arms. The amount of the star-shaped prepolymer is usually at least 70% by weight, preferably at least 80% by weight of the reaction product. The remaining components of the reaction product are essentially Dimers and trimers in small amounts in these amounts also suitable for producing the coatings according to the invention are.

The with the star-shaped prepolymers according to the invention coating substrates are generally not subject to any restrictions. The substrates can be regular or irregularly shaped, smooth or have porous surfaces. Examples of suitable ones Surface materials are oxidic surfaces, e.g. B. silicates such as glass, Quartz, silicon dioxide as in silica gels, or ceramics continue Semimetals such as silicon, semiconductor materials, metals and Metal alloys such as steel, polymers such as polyvinyl chloride, polyethylene, Polymethylpentene, polypropylene, polyester, fluoropolymer (e.g. Teflon®), polyamides, polyurethanes, poly (meth) acrylates, blends and Composites of the above materials surfaces, including cellulose and natural fibers such as cotton fibers and wool. The polymers can it is woven or non-woven material.

The production of ultra-thin, hydrogel-forming coatings takes place according to the invention by separating the star-shaped Prepolymers on the surface to be coated from a solution of Prepolymers according to known methods and subsequent crosslinking the reactive groups of the prepolymers. The steps of deposition and crosslinking can be repeated if desired become. This leads to thicker layers.

Examples of deposition processes are the immersion of the coating surface in the solution of the prepolymer as well Spin coating - the solution of the prepolymer is applied to the high speed rotating surface to be coated applied. It goes without saying that one can manufacture ultrathin coatings usually take the coating measures dust-free conditions.

In the immersion process, the substrates are immersed in a solution of the star polymer in a suitable solvent and leaves then drain the solution so that a thin film of liquid remains on the substrate with as uniform a thickness as possible. This is then dried. The resulting film thickness depends on the concentration of the star polymer solution. Then will the networking triggered.

As a rule, spin coating does not initially rotate Complete the substrate with the solution of the star-shaped prepolymer wetted. Then the substrate to be coated with high RPMs, preferably above 1000 rpm, z. B. 1000 to 10000 rpm set in rotation, the solution largely is thrown off and a thin coating film on the surface of the substrate remains. Then there will also be networking triggered.

The concentration of the prepolymer in the solution is usually a value of 100 mg / ml, preferably 50 mg / ml and in particular Do not exceed 20 mg / ml. The concentration is usually at least 0.001 mg / ml, preferably at least 0.005 and in particular at least 0.01 mg / ml. Concentration can be used naturally control the thickness of the coating, being very small Concentration usually to monolayers of the star polymers on the coated surfaces.

As a rule, the coating measures are chosen so that the coating thickness (measured by means of ellipsometry according to the method described in Guide to using WVASE 32 ^TH , JA Woollam Co. Ind., Lincoln NE USA 1998) has a value of 500 nm, preferably 200 nm and in particular does not exceed 100 nm. The process also enables the production of monolayers with thicknesses below 2 nm, e.g. B. 0.5 to 2 nm. For many applications, layer thicknesses in the range from 1 to 100 nm, in particular in the range from 2 to 50 nm, are preferred.

In principle, all solvents are suitable as solvents, which have little or no reactivity to the have functional groups R of the prepolymer. Among them are such preferred, which have a high vapor pressure and are therefore easy have it removed. Preferred solvents are therefore those at Normal pressure a boiling temperature below 150 ° C and preferably have below 120 ° C. Examples of suitable solvents are aprotic solvents, e.g. B. ethers such as tetrahydrofuran (THF), Dioxane, diethyl ether, tert-butyl methyl ether, aromatic Hydrocarbons such as xylenes and toluene, also acetonitrile, propionitrile and mixtures of these solvents. In the case of prepolymers with OH, SH, carboxyl, (meth) acrylic and oxirane groups are also protic solvents such as water or alcohols, e.g. B. methanol, ethanol, n-propanol, isopropanol, n-butanol and tert-butanol, and their Mixtures with aprotic solvents are suitable. In case of In addition to the abovementioned aprotic ones, prepolymers with isocyanate groups are Solvents surprisingly also water and mixtures of Suitable water with aprotic solvents, since probably the Degradation of the isocyanate groups in the prepolymers is comparatively slow he follows.

The type of networking can be done in different ways.

In one embodiment of the invention, you will usually with the uncrosslinked prepolymer coated article with a Treat crosslinking agents.

In principle, all polyfunctional compounds whose functional groups react with the functional groups of the prepolymer to form bonds are suitable as crosslinking agents. These functional groups are also referred to below as complementary functional groups R '. Table 1 provides an overview of complementary functional groups R ', the reactive groups R of the prepolymer being indicated in the first row and the groups R' complementary thereto being given in the first column: Table 1 Complementary functional groups

Accordingly, one embodiment of the invention relates to a method in which the linkage of the reactive groups R by addition triggers a connection V1 which has at least two reactive groups R ' per molecule, which with the reactive groups R des star-shaped prepolymer reacted with bond formation.

The polyfunctional connections V1 can be act low molecular weight compounds, e.g. B. aliphatic or cycloaliphatic diols, triols and tetraols, e.g. B. ethylene glycol, butanediol, Diethylene glycol, triethylene glycol, trimethylolpropane, pentaerythritol and the like, aliphatic or cycloaliphatic diamines, Triamine or Tetramine, e.g. B. ethylenediamine, diethylenetriamine, Triethylene tetramine, tetraethylene pentamine, 1,8-diamino-3,6-dioxaoctane, Diaminocyclohexane, isophoronediamine and the like, amino alcohols such as Ethanolamine, diethanolamine, aliphatic or cycloaliphatic dithiols, to dicarboxylic acids or tricarboxylic acids such as sebacic acid, glutaric acid, Adipic acid, phthalic acid, isophthalic acid, or to the aforementioned Diisocyanates, depending on the type of reactive groups that Has prepolymer. The low molecular weight polyfunctional compounds in contrast to the prepolymers, as a rule Molecular weight <500 g / mol.

The polyfunctional connection V1 can already be found in the solution of the Contain prepolymers that are used for coating. In the initially created coating of largely uncrosslinked Prepolymers then react, e.g. B. when drying or heating of the coating, the reactive groups R 'of the crosslinking agent the reactive groups R of the prepolymer and form in this way a layer of cross-linked prepolymers.

If the reactive groups R of the prepolymer conjugated diene, the connection V1 becomes accordingly have at least two dienophilic groups and vice versa. If the Prepolymer reactive groups have an en reaction come in, the compound V1 becomes at least two allylic Have double bonds. As a rule, one is at such Systems for making the coatings use the solutions contain both the prepolymer and the compounds V1. The Crosslinking then takes place when the primarily obtained is dried Coating, if necessary after heating.

Basically, polyfunctional compounds are also V1 Suitable prepolymers which have at least four polymer arms A, which seen as soluble in water and at their free ends have a reactive functional group R 'with the reactive Groups R of the prepolymer react to form bonds. With in other words, solutions can also be used for the method according to the invention of at least two different prepolymers are used, wherein one prepolymer reactive groups R and the other has complementary reactive groups R '. That way too receive a layer of cross-linked prepolymers.

In another embodiment of the invention the link of the reactive groups R triggered by a sufficient amount a compound V2 admits that with some of the reactive groups R reacts to form reactive groups R ', which react with the remaining reactive groups R reacted to form bonds. in the For example, in the case of prepolymers with isocyanate groups Trigger networking by using the coated object Water treated e.g. B. by storage in a humid atmosphere or under water. Here, some of the isocyanate groups react Formation of amino groups, which in turn with the remaining Isocyanate groups react to form bonds, forming a layer arises from interlinked prepolymers. The Crosslinking agent V2 is water here.

In a further embodiment of the invention the group R selected from ethylenically unsaturated, radical polymerizable double bonds. In this case, networking takes place on thermal or photochemical way, d. H. by irradiation with UV Radiation or with electron radiation. In the case of photochemical Crosslinking by UV radiation will result in the solution of the prepolymer usually add suitable photoinitiators. The type and amount of Photoinitiator used to trigger photochemical crosslinking is required is the specialist from the technology radiation-curing lacquers common.

In a further embodiment of the invention, a star-shaped prepolymer, preferably as a monolayer, is first applied to the surface to be coated in the manner described above, if appropriate a partial crosslinking of the reactive groups R is carried out and then at least one further star-shaped prepolymer 2 is applied to the one treated in this way Applies surface that has at least four polymer arms A, which are in themselves soluble in water and have at their free ends a reactive functional group R ', which have a complementary to the reactive groups R of the prepolymer 1 reactivity. If necessary, the remaining reactive groups R 'are then crosslinked again. This process can be repeated one or more times. In this way it is possible to produce multilayer coatings in a targeted manner. This procedure is also referred to below as the layer-by-layer method. The layer-by-layer process can be implemented in a particularly elegant manner with those prepolymers which have reactive groups R which react with compounds V2 to form reactive groups R 'which have a reactivity which is complementary to the groups R. Examples of groups R are isocyanate groups. In this case, the connection V2 is water. NH ₂ groups then form as complementarily reactive groups R '. If the crosslinking of the first layer with compound V2 is triggered, the coating obtained has free groups R '(eg amino groups) on its surface. These then react with the groups R (for example isocyanate groups) of the prepolymers applied in a second coating process to form bonds. The second coating process is preferably also controlled such that a monolayer of prepolymers is deposited on the first coating. Cross-linking of the second layer by means of compound V2 (e.g. water) and repetition of this procedure thus allows the production of highly cross-linked, highly ordered layers, in particular if the amount of coating in the individual coating stages has been chosen so that monolayers are obtained.

The coatings can also be made with mixtures of star-shaped Prepolymers and water-soluble polysaccharides, such as hyaluronates, Heparins, alginates or e.g. B. dextran. at Use of prepolymers with reactive towards OH functions functional groups, e.g. B. isocyanate groups then acts the polysaccharide as a crosslinker.

The method according to the invention also allows the targeted Incorporation of foreign materials, i.e. materials that do not Form hydrogel-forming coatings in the coating. Which includes bioactive materials such as drugs, oligonucleotides, peptides, Proteins, signaling substances, growth factors, cells, carbohydrates and Lipids, inorganic components such as apatites and hydroxyapatides, quaternary ammonium salt compounds, compounds from bisguanidines, quaternary pyridinium salt compounds, compounds from phosphonium salts, Thiazoylbenzimidazoles, sulfonyl compounds, salicyl compounds or organometallic compounds. Installation is preferably done by coadsorption from solutions containing the prepolymer and the Foreign component included. In addition, the prepolymers with the mentioned bioactive materials are implemented before adsorption or as Mixture with unmodified prepolymers on the surface Response. Of course it is also possible targeted by physisorption or chemisorption on the finished Apply hydrogel coating.

When coating according to the layer-by-layer method biological components also in the form of a not completely covering Intermediate layer can be introduced.

This is done by using the top layer of the star-shaped Prepolymers usually still carry reactive groups, which also include mild conditions (in aqueous solution, at room temperature) specifically with common chemical groups of biomolecules react. Examples are the reaction of on the surface of the Polymer layer NCO groups with alcohol, thiol or Amino groups present in proteins and peptides or after Prior art methods easily into many biomolecules can be introduced. Another example is Michael addition of thiol or amino groups on acrylates and acrylamides in the top layer of coating. Another example is the implementation of activated esters in the top layer of the coating Alcohol or amino groups of the biomolecules.

Examples of suitable biological components that are included in the after Hydrogel coatings produced according to the method of the invention medications, e.g. B. heparins, Antibiotics such as streptomycin, gentomycin, penicillin, neomycin, Acriflavin, Ampillicin, Chitin, Chitosan and their derivatives, as well as others bactericidal substances, growth factors such as BMPs (bone morphogenic proteins), HGHs (human growth hormons), GMCSF (macrophage colony stimulating factors), heparin-binding factors such as FGFs, VGF, TGFs, communication and architecture mediating signal substances like BHL, HHL, OHL, DHLs, OHHL, OOHL, ODHL, OdDHL, HBHL, HtDHL and other integrin-mediating signaling molecules, proteins such as fibronectin, Laminin, Vitronectin, Collagen, Thrombospondin and others adhesion-promoting proteins, mechanically or otherwise physically modulated Proteins such as stretched fibronectin, adhesion-promoting Peptide sequences such as RGD, RGDS, RGDV, RDT, LRGDN, LDV, REDV, IKVAV, YIGSR, PDSGR, DGEA, peptide sequences that are changed only by one differ in conformation, e.g. B. Cyclic peptide sequences Amino acid sequences and oligonucleotides that are molecular Like sequences from RNA or DNA, carbohydrates can be recognized and lipids such as sugar and long chain hydrocarbon compounds which allow interaction with the cell membrane, cells or Cellular assemblies of fibroblasts, osteoblasts, chondrocytes and others Cell types, but also pluripotent cell material.

It is often advantageous to have the surface to be coated in one Way to pretreat that they have an increased number (areal density) has functional groups R 'with the functional groups R of the prepolymer can react to form bonds.

To do this, the surfaces of inert materials are often exposed to the Activate the coating chemically. This can be done, for example, by Treatment of the surface to be coated with acid or alkalis, by oxidation (flaming), by electron radiation or by a plasma treatment with an oxygen-containing plasma as described by P. Chevallier et al. J. Phys. Chem. B 2001, 105 (50), 12,490 to 12,497; in JP 09302118 A2; in DE 100 11 275; or by D. Klee. et al. Adv. Polym. Sci. 1999, 149, 1-57.

The surface to be coated can also be coated treat that is known to have good adhesion to the surface have and also have functional groups R ', with the functional groups R of the star-shaped prepolymer complementary are. Suitable groups R 'are dependent on the reactive group R of the prepolymer: isocyanate, amino, hydroxyl and epoxy groups, groups that react in the sense of a Michael addition, dienophile groups that enter into Diels-Alder addition reactions, electron-poor double bonds in a Diels-Alder addition or react the ene reaction with allylic double bonds; activated ester groups; oxazoline; as well as vinyl groups and thiols specifically enter into a free radical addition.

The type of group that has a liability on the to be coated Surface effects naturally depends on the chemical nature of the coating surface. In the case of oxide, such as ceramic and glass-like surfaces, as well as in the case of metallic surfaces Connections have proven themselves as adhesion-promoting groups Have silane groups, especially trialkoxysilane groups. Examples for such compounds are trialkoxyaminoalkylsilanes such as Triethoxyaminopropylsilane and N [(3-triethoxysilyl) propyl] ethylenediamine, trialkoxyalkyl-3-glycidyl ether silanes such as triethoxypropyl-3- glycidyl ether silane, trialkoxyalkyl mercaptans such as Triethoxypropyl mercaptan, trialkoxyallylsilanes such as allyltrimethoxysilane and Trialkoxysilylacryloxyalkanes and acrylamidoalkanes such as 1-triethoxysilyl-3- acryloxypropan. For oxidic and plastic materials are also polyfunctional as adhesion-promoting groups Suitable polyammonium groups. Examples of such compounds are Polyammonium compound with free primary amine groups such as z. B. from J. Scheerder, J.F. J Engbersen, and D. N. Reinhoudt, Recl. Trav. Chim. Pays-Bas 1996, 115 (6), 307-320, and von Decher, Science 1997, 277, 1232-1237 are described for this purpose.

The aforementioned compounds are preferred as a monolayer on the applied surface to be coated. Leave such monolayers themselves in a manner known per se by treating those to be coated Reach surfaces with dilute solutions of the compounds, e.g. B. according to the immersion process described above or by means of Spin coating. Solvents and concentrations correspond to those for the Application of the prepolymers given information. It is often recommended treat the surfaces with the aforementioned compounds, which are known to have good adhesion to the surface, in Following activation by flame, by Electron irradiation or by plasma treatment.

The surfaces coated according to this invention swell in direct contact with water, aqueous solutions and moist gases forming very stable hydrogels. The coatings are in Difference to many hydrogel-forming coatings of the stand technology is stable even after prolonged contact with aqueous solutions can be used several times because of the deposits simply rinse with water. The invention The coatings obtained effectively prevent unspecific adsorption of proteins and cells over a long period of time and are in this point the hydrogel coatings of the prior art think. The coatings are due to their chemical Composition biocompatible and non-toxic.

The use of star-shaped prepolymers with reactive end groups advantageously allows easier use Coating processes such as immersion, spin coating (rotary spin processes) and in particular a layer-by-layer structure.

The peculiarity of the star-shaped prepolymers with reactive End groups also allow, in a very simple and controlled way, thin to ultra-thin hydrogel coatings with a layer thickness below 100 nm, preferably below 50 nm and if desired below 10 nm manufacture. The coatings prevent a non-specific Protein adsorption as well as the adhesion of cells and so can Prevent bacterial colonization. The thin thickness of the coating is it is particularly advantageous since the macroscopic properties and the appearance of the underlying material is practically unchanged stay.

The use of star-shaped prepolymers with reactive end groups allows you to easily order ordered mono and Generate multi-layers, their structure and properties like Water absorption, penetrability and flexibility for each Application can be set very precisely.

The method according to the invention also allows a unique Way, different functions z. B. biological agents, Functional centers, signal substances, growth factors, etc. in the store individual hydrogel layers and thus a biofunctional one Transfer coating to substrates. The suppression of one nonspecific bacterial colonization can be achieved by incorporating Bactericides, biological signaling substances and colloidal particles a diameter preferably less than 100 nm. The On the other hand, the method according to the invention also opens up the possibility of through the integration of biological signaling molecules and ligands targeted colonization with specific bacteria and cells promote.

Due to the properties of those obtained according to the invention Coatings is an application of the method according to the invention for Manufacture of microsensors and microanalysis systems for coating of microcannulas for the introduction of genetic material into Cells and of capillary systems in which the adsorption of biological connections on the capillary surfaces a big problem represents and significantly impair the analytical sensitivity can, possible. In other words, the use of star-shaped Prepolymers opened for the production of hydrogel coatings Areas of application in which conventional polymers and hydrogels none Can find application or because of their insufficient Protein resistance has not been applied.

The method according to the invention is in particular also for coating of objects that are in direct contact with living Matter are like implants. The coatings can on a wide range of laboratory equipment, medical products and Instruments, but also applied to surfaces in the semi-technical area that are extremely clean, so as free of protein and cells as possible must be kept or difficult for cleaning steps are accessible.

The coatings obtainable according to the invention are also Particularly advantageous where only extremely thin coatings are possible are. For example, the method according to the invention for Manufacture of ultra-thin coatings on the inner walls of hose and Tube systems are used, which in turn are only special small diameter, e.g. B. in the µm range, have (implantable Pump systems, thin catheters, microbiological and genetic engineering Laboratory equipment). The method according to the invention is also for Coating of extremely large areas (ship hulls, technical Pipe systems, swimming pools, operating theaters, etc.) are suitable.

Fig. 1, the ellipsometric certain layer thickness shows a coating according to the invention in dependence on the number of coating operations.

Fig. 2 shows the increase in the thickness of a dried coating of the invention, is exposed to humidity (determined by ellipsometry).

FIG. 3 shows a light micrograph of a glass plate coated according to the invention (thickness of the coating about 50 nm), which had been incubated with a fibroblast suspension.

FIGS. 4a and 4b show light micrographs of glass plates which have on one half a coating according to the invention and on the other half a polystyrene coating and were incubated with a fibroblast suspension.

FIG. 5 shows a light micrograph of a glass plate which was half coated with a mixture of star prepolymers and dextran (1: 1) and the other half was uncoated and which was incubated with a fibroblast suspension.

The following examples are intended to explain the invention further.

I. Manufacturing examples 1. Six-armed isocyanate-terminated polyethers

In all cases, a commercially available isocyanate was used Isophorone diisocyanate (IPDI: 72% cis and 28% trans isomer)

The prepolymer precursors used are commercially available 6-armed polyalkylene ethers (hereinafter polyols), by anionic ring-opening polymerization from ethylene oxide and / or propylene oxide using sorbitol as Initiator were made. The polyol used was before use dried to a residual water content of less than 350 ppm. Remains of the used for the production of the polyols Alkali hydroxide was obtained by neutralizing with phosphoric acid bound.

In all manufacturing examples, the polyol was pumped slowly added (approx. 80 ml / h) so that the reaction temperature not deviate more than 10 K from the specified temperature.

The determination of the molecular weight (indicated is that Number average) was carried out by gel permeation chromatography Room temperature on three columns connected in series (column 1: Waters µ-Styragel 1000 angstroms, l = 30 cm; Pillar 2: Waters µ- Styragel 100 angstroms, l = 30 cm; Pillar 3: PSS SDV 50 angstroms, l = 60 cm) using tetrahydrofuran as eluent, a refractometry detector (Waters RI 2410) and below Use of the evaluation software PSS Win-GPC V 4.02. The Elution diagrams were adjusted for evaluation on two Gaussian curves, the proportion of bi- / trimer over the area ratios was determined.

The characterization of the end groups more functionalized Prepolymers as well as the functionalization yield took place where specified, also elemental analysis (sulfur, nitrogen), by means of IR spectroscopy (V SH, C = O, NH) or by titration (not converted OH groups).

The unreacted OH groups were determined by Implementation of prepolymers with acetic anhydride in pyridine and Titration of excess acid (hydrolysis of the unreacted Acetic anhydrides) with NaOH.

Production Example 1

The polyol used is a 6 arm statistical Poly (ethylene / propylene oxide) with an EO / PO ratio of 80/20 with a molecular weight of 3100 g / mol. Before implementation 0.05% by weight of phosphoric acid was added to the polyol and heated under Stir for 1 h at 80 ° C in a vacuum.

262 g of IPDI (1.18 mol) were placed in a reactor and heated to 50 ° C under a protective gas atmosphere. Then you gave in the dried and degassed polyol (50 g, 0.016 mol) slowly with the help of a peristaltic pump (approx. 80 ml / h). After the addition was complete, the reaction mixture stirred at 50 ° C for a further 60 hours. Excess IPDI was at 130 ° C and a pressure of 0.025 mbar using a Thin-film distillation apparatus completely distilled off.

Production Example 2

The polyol used corresponds to the polyol Production Example 1.

210 g of IPDI (0.94 mol) and 0.06 g were placed in a reactor (0.1 wt%) diazabicyclooctane (DABCO) and heated under Shielding gas atmosphere to 50 ° C. Then you gave under violent Stir in the dried and degassed polyol (58 g, 0.019 mol) Slowly with the help of a peristaltic pump (approx. 80 ml / h). To The addition was completed at 50 ° C stirred for a further 60 hours. Excess IPDI was at 130 ° C and a pressure of 0.025 mbar using a Distilled off thin film distillation apparatus.

Production Example 3

The polyol used is a 6 arm statistical Poly (ethylene / propylene oxide) with an EO / PO ratio of 80/20 with a molecular weight of 10000 g / mol. Before implementation 0.05% by weight of phosphoric acid was added to the polyol and heated with stirring for 1 h at 80 ° C in a vacuum.

IPDI (100 g, 0.45 mol) was placed in a reactor and heated to 50 ° C. in a protective gas atmosphere. Added to that the dried and degassed polyol is stirred vigorously (50 g, 0.005 mol) slowly using a peristaltic pump to (approx. 80 ml / h). After the addition was complete, the Reaction mixture stirred at 50 ° C for a further 60 hours. After Thin-film distillation (100 ° C, 0.025 mbar) was obtained Isocyanate-terminated star prepolymers.

Production Example 4

The polyol used is a 6-armed polypropylene oxide a molecular weight of 3000 g / mol. Before the implementation one gave 0.05 wt% phosphoric acid to the polyol and heated for 1 h 80 ° C in a vacuum.

480 g (0.154 mol) of the polyol were under an inert gas atmosphere with the help of a peristaltic pump slowly (80 ml / h) to a at 50 ° C, vigorously stirred mixture of 840 g IPDI (3.78 mol) and 0.99 g of dibutyltin dilaurate (DBTL). To upon completion of the addition, the reaction mixture was at 50 ° C stirred for a further 48 hours. After the thin film Distillation (160 ° C, 0.01 mbar) gave the isocyanate terminated star prepolymers.

The prepolymers of Production examples Nos. 5, 6, 7 and 9 are produced. Analogous Preparation Example 2 were the prepolymers of Production examples 8 and 10 are produced. The molar ratio of diisocyanate Table 2 shows the OH groups in the polyol (NCO / 2 / OH). Table 2 also shows the proportions of monomolecular star-shaped polymers (mono-star) and on bi- and trimolecular ones Reaction products (bi- and tri-star) indicated how were determined by means of gel permeation chromatography.

Production Example 11

Analogously to Preparation Example 1, 30 g of a 6-arm statistical poly (ethylene oxide / propylene oxide) with an EO / PO ratio of 80/20 with a number average molecular weight of 12,000 were reacted with 50 g (0.23 mol) of IPDI. After thin-layer distillation (100 ° C / 0.001 mbar), the isocyanate-terminated star prepolymer was obtained. Table 2 Production examples

2. Production of star polymers with thiol, acrylate, acrylamide or NH ₂ groups Production Example 12 Thiol-terminated star polyether

A commercially available 6-arm polyethylene oxide, which had OH groups at the ends of the polyether chains, was brominated with PBr ₃ using a conventional method (Mills et al., J. Chem. Soc. Perkin Trans. 2, (4), 697-706 , 1995, see also Tetrahedron 44 (5), 1988, pp. 1553-1558 and Inorg. Chim. Acta 97 (2) 1985, pp. 143-150 and in general J. March, Advances in Organic Synthesis 3rd ed. J. Wiley & Sons, New York 1985, p. 383), whereby a 6-arm polyethylene oxide was obtained which had bromine atoms at the ends of the polyether chains. This was predried in the manner described for Example 1. A solution of 15 eq was then slowly dripped in at 100 ° C. Thioacetic acid in 200 ml ethanol with 15 eq. Ethoxide. After a further 24 h the thioacetate was hydrolyzed with 1 N aqueous HCl. After hydrolysis of the thioacetate obtained as an intermediate, the solution was stirred under nitrogen at 78 ° C. for a further two hours. The desired thiol group-containing product was separated off in a thin-film distillation apparatus with exclusion of air.

An IR spectrum of the product obtained showed a weak band at 2558 cm ^-1 , which is attributable to the SH vibration. The sulfur content determined by elemental analysis corresponds to a degree of functionalization of 5.1.

Production Example 13 Acrylate-terminated star polyether

A solution of 40 g (12.9 mmol) of the in Preparation Example 1 used, pre-dried polyether in 400 ml dichloromethane was cooled to 0 ° C and with a solution for several hours of 465 mmol pyridine and 465 mmol acryloyl chloride in 30 ml Dichloromethane added. The reaction solution became another six Hours at 0 ° C and then another 30 h at Room temperature stirred. The precipitated salt was filtered off and that The filtrate was first diluted with hydrochloric acid and then with aqueous sodium bicarbonate solution washed. The organic phase was dried over magnesium sulfate. The product was precipitated by adding diethyl ether, filtered off and in Vacuum dried.

An IR spectrum of the prepolymer showed an intense characteristic band at 1730 cm ^-1 , which can be assigned to the C = O vibration. Titrimetric determination of the OH group content showed a degree of functionalization of> 5.

Production Example 14 Amine-terminated star polyether

A saturated solution of 100 g (32.3 mmol) of the predried polyether used in Preparation Example 1 in 400 ml of dichloromethane was cooled to 0 ° C. under a nitrogen atmosphere. For this purpose, 580 mmol pyridine were first added very slowly and then 580 mmol methanesulfonyl chloride. After 24 h, the precipitate was filtered off and the mesylate formed was precipitated by adding diethyl ether. The mesylate obtained was dissolved in dimethylformamide and stirred with 1.16 mol sodium azide at 60 ° C. for 24 hours. After cooling, the mixture was diluted with water and extracted with dichloromethane. The organic phase was then dried over magnesium sulfate, the product was precipitated by adding diethyl ether, filtered and the product was dried in vacuo. The polyether thus functionalized with azide groups was dissolved in dry tetrahydrofuran and the solution was added dropwise to a suspension of 290 mmol LiAlH ₄ in tetrahydrofuran. The mixture was stirred at 60 ° C. for 16 h and, after cooling, 15% sodium hydroxide solution was slowly added until a white granular precipitate formed. It was then filtered through silica. The amine-terminated polyether was precipitated from the solution thus obtained by adding diethyl ether and dried in vacuo.

A sharp band at 3310 cm ^-1 can be seen in the IR spectrum of the prepolymer, which is attributable to the NH vibration. The nitrogen content determined by elemental analysis corresponds to a degree of functionalization> 5.

Production Example 15 Acrylamide-terminated star polyether

A solution of 30 g (approx. 9.6 mmol) of the predried amine terminated polyether from preparation example 14 in 300 ml Toluene was cooled to 0 ° C. under a nitrogen atmosphere and diluted with dichloromethane until a clear solution was obtained. For this purpose, 320 mmol of pyridine were added and then over several Hours 320 mmol acryloyl chloride. After the addition is complete the mixture was stabilized with BHT, at least for more Stirred for 6 hours at 0 ° C with exclusion of light and to 200 ml concentrated. The precipitated salt was filtered off and that Filtrate added to 500 ml of cold diethyl ether. It occurred to me Solid which was dissolved in 200 ml of distilled water. 10 g of NaCl were added to the solution. By adding 1 N Na OH the pH was adjusted to 7 and the neutralized Solution extracted several times with dichloromethane. The organic Phases are combined with diethyl ether, with a Solid failed. This was a second time in dichloromethane recorded, mixed with a stabilizer and in again 500 ml of cold diethyl ether precipitated, filtered off and in vacuo dried. In this way, one obtained the one with acrylamide groups terminated star polyether.

A strong band at 1670 cm ^-1 can be seen in the IR spectrum, which can be assigned to the amide EO group. The nitrogen value determined by elemental analysis corresponds to a degree of functionalization> 5.

II. Production of the hydrogel coatings

Glass platelets (floated glass, quartz glass, Standard glass) and hydrophilic silicon wafers (Si [100]). The substrates were in the Ultrasonic bath first in acetone, then in Millipore water and then cleaned in isopropanol. All substrates were basically to avoid contamination by dust and Fat droplets from the air in suitable protective containers under stored in a layer of liquid.

The water used was basically desalinated (18 MΩ-cm or better). All solutions were made with a 0.05 µ filter from Dust and particulate contaminants cleaned. filtered Deionized water is also referred to below as Millipore Called water.

Coatings with the exclusion of water were carried out in a glove box (brown) under an atmosphere with a water content of less than 1 ppm H ₂ O / O ₂ .

1. Amino functionalization of the substrates

• 1.1.1. The cleaned substrates were treated in a plasma system of the TePla 100-E type from Plasma Systeme for 10 minutes in oxygen plasma (pressure: 0.15 mbar). The substrates treated in this way were then stored in deionized water.
  For amino functionalization, the substrate surface was first coated with an aminosilian monolayer as a promoter. For this purpose, the sample taken from the water and blown dry with nitrogen was transferred to a glove box. There the substrates were stored in a 0.4% (v / v) solution of N- (3- (trimethoxysilyl) propyl) ethylenediamine in dry toluene for 16 h, then washed thoroughly with toluene and, before use, in a glove box in a nitrogen atmosphere a filtered nitrogen stream dried.
• 2. 1.2 Alternatively, the substrates were treated with oxygen under a 40 W UV lamp for 10 min (distance between substrate surface and light source 2 mm) and then placed in Millipore water (MP-H ₂ O). The sample taken from the MP-H ₂ O and blown dry with nitrogen was then treated as in 1.1 with (trimethoxysilyl) propyl) ethylenediamine.

2. Coating the substrates

All measures were carried out under dust-free conditions (Clean room conditions).

2.1 Coating through immersion General regulation a)

The substrates functionalized according to the instructions under 1.1 are immersed in a solution of the respective prepolymer in dry THF (0.05 mg / ml to 5 mg / ml). The solution is allowed to drain carefully so that a thin liquid film of uniform thickness remains on the substrate. This is dried. The resulting film thickness depends on the concentration of the star polymer solution. The samples are then removed from the glovebox, protected from dust, and reacted in a moist atmosphere. The samples are placed in MP-H ₂ O until use / examination.

General regulation b)

The substrates functionalized according to the instructions under 1.1 are immersed in a freshly prepared solution of the prepolymer (0.005 mg / ml to 20 mg / ml in 1: 1 THF: MP-H ₂ O). The solution is allowed to drain carefully so that a thin liquid film of uniform thickness remains on the substrate and is dried. After the film has reacted, the samples are placed in MP-H ₂ O until use.

Table 3 lists tests and resulting layer thicknesses which were obtained by an immersion coating. The layer thicknesses were determined by ellipsometry ("Guide to using WVASE32 ™", JA Woollam Co., Ind., Lincoln, NE, USA, 1998). Table 3

2.2 Coating by means of spin coating (rotary spin process) a thin polymer layer (general rule)

The coating of 1 amine-functionalized substrates produced was carried out using a spin coater (model: WS-400A-6TFM / lite from the company: SPS). For this purpose, the non-rotating, dried substrate is first completely wetted with the prepolymer solution (0.05 mg / ml to 5 mg / ml star polymer in THF) before the solution at acceleration level "5" and a final speed of 5000 rpm for 40 Is flung for seconds. The sample is then dry. The substrates coated in this way were stored in the absence of dust overnight at an air humidity of 50 to 80%. The samples treated in this way can then be used immediately for the desired application or placed in MP-H ₂ O until they are used.

In a variant of the method described above, this is followed 1 prepared amine-functionalized substrate on the Spincoater rinsed with salt-free water while rotating (3000 rpm) before coating with a freshly prepared one water-containing star polymer solution in THF. Achieved in this way better wetting of the substrate and a very thin one Polymer film.

The results are summarized in Table 4. The layer thickness was determined ellipsometrically. Table 4

3. Coating layer by layer 3.1 General regulation for the production of monolayers by means of spin coating

The substrates functionalized and washed according to point 1 are carefully blown dry on the spin coater in a filtered nitrogen stream. The substrate is slightly warmed with the nitrogen stream preheated to 50 ° C. before a solution of the prepolymer from preparation example 1 (in THF; 0.5 to 5 mg / ml, see III-1) is spun on with the exclusion of moisture (spin coater) Model: WS-400A- 6TFM / lite from the company: PLC, acceleration level "2" and a speed of 1500 rpm). Even before the film has dried completely, the surface of the still rotating substrate is washed several times by applying a drop of anhydrous dichloromethane in order to remove excess prepolymer. The procedure is repeated to achieve a dense monofilm. The sample coated in this way is then crosslinked by placing it in MP-H ₂ O. The procedure is repeated according to the number of monolayers to be applied.

3.2 General regulation for the production of monolayers by means of immersion

The substrates pretreated according to item 1 are placed for one hour with exclusion of moisture in a solution of the prepolymer in anhydrous THF. The samples are then washed several times with anhydrous THF to rinse off non-chemically bound prepolymer molecules. The samples are then placed in MP-H ₂ O for one hour. The sample is dried and then placed in a THF bath to remove water from the coating. The samples are then dried in a dust-free atmosphere or in a filtered nitrogen stream. To apply another monofilm, this procedure is repeated, starting with immersing the sample in the anhydrous solution of the prepolymer in THF. The samples coated in this way can be used immediately for the desired application or placed in MP-H ₂ O until they are used.

III. Examination of the polymer films 1. Investigation of the concentration dependence of the thickness of a monolayer

Silicon wafers manufactured according to the regulation under II-1. were pretreated, were placed in a solution of the prepolymer from preparation example 1 in anhydrous THF. After immersion, the samples were washed several times with anhydrous THF. After the subsequent one-hour soak in MP-H ₂ O, they were removed and dried as described under II-3.2. The layer thickness of the hydrogel films obtained was determined by means of ellipsometry. The values are given in Table 5: Table 5

The values in Table 5 show that the layer thickness with increasing concentration of the prepolymer solution first increases sharply and a limit at high concentrations (e.g. 5 mg / ml) approaches. It is believed that at low concentrations due to the lower degree of surface coverage, more NCO Groups with reactive groups on the surface covalent Make bonds. In contrast, a higher one Concentration probably to a tightly packed adsorbate layer of Star molecules with less binding via the NCO groups the surface.

2. Examination layer thickness as a function of the number of applied layers

For this purpose, a monolayer was first prepared as described under 1., using a solution of the prepolymer from Preparation Example 1 in THF with a concentration of 1.0 mg / ml. The substrate coated in this way was then placed in a solution of the same prepolymer in anhydrous THF (concentration 1.0 mg / ml). The sample was then washed several times with anhydrous THF. After then placing them in MP-H ₂ O for one hour, they were removed and dried as described above. This procedure was repeated a total of five times, the layer thickness being determined by means of ellipsometry after each application of a further layer. The results are shown in Fig. 1.

As can be seen from FIG. 1, the layer thickness increases linearly with increasing number of layers, the slope of the regression line is approximately 4.7 Å / layer, which corresponds to the thickness of a monolayer.

3. Determination of the water swelling of a thin hydrogel layer

The swelling behavior was determined by means of ellipsometry on a thin Coating examined. The change in layer thickness that the Directly reflects the swelling behavior of the hydrogel layer measured in situ.

The samples were pretreated as described under II.-1. After drying the sample in a filtered nitrogen stream, a solution of the polymer from preparation example 1 (5 mg / ml in dry THF) was spin-coated at a speed of 6000 rpm. (40 sec.) As described under II-2.2. The sample thus prepared was then stored in water for one hour and, after drying in a filtered nitrogen stream, was dried for 30 minutes at 90 ° C./0.1. The dried sample was then exposed to the laboratory atmosphere (20 ° C., 60% relative atmospheric humidity) and the change in layer thickness caused by water absorption was examined in situ using ellipsometry. The results of the investigation are shown in Fig. 2.

As can be seen from FIG. 2, the layer thickness of the dried sample initially increases continuously through contact with the moist air. After about 10 h no further increase in layer thickness can be seen. The total relative increase in layer thickness is approx. 2%, which corresponds to an absolute increase of a few angstroms.

4. Investigation of the adsorption of biopolymers by observation the diffusion of single molecules

Glass slides with a recess were used as substrates cover slips (d = 170 µm) are also used and initially as below II -1. described pretreated. Then the used substrates with the prepolymer from preparation example 3 in coated in the manner described for II-2.2 (spin coating a solution of 5 mg / ml prepolymer in dry THF with a Speed of 5000 rpm).

The investigation was carried out by means of confocal laser microscopy and by means of confocal fluorescence correlation spectroscopy (FCS) using the fluorescent dye MR 121. The dye was in PBS buffer (140 mM NaCl, 10 mM KCl, 6.4 mM Na ₂ HPO ₉ × 2H ₂ O, 2 mM KH ₂ PO ₄ ) diluted to a concentration of 10 ^-10 M. 120 µl of the solution were pipetted into the approx. 100 µl well of the above slide and covered with the 170 µm coated cover slip. An uncoated glass slide and an uncoated cover slip were used as references.

The slide was used for confocal laser microscopy the sample with the cover slip facing down over the Microscope objective positioned. By moving the lens the Laser focus approx. 10 µm above the cover slip in the Trial solution focused.

In this way, the interaction of the dye (MR121), but also the oligonucleotides labeled with the dye (MR 121-IPs) in solutions of 10 ^-9 -10 ^-10 M in a volume of approx. 1 µm ³ directly on the glass surface to be watched. In order to observe the conditions directly on the surface, the excitation light of the laser was focused on the glass surface in such a way that the reflection caused by the glass-water transition was maximum. It was found that the labeled oligonucleotides from the PBS (additionally with 5 mM MgCl ₂ ) solution adsorbed at the reference and were then no longer freely movable. The individual molecules could then be located on the surface in the microscopic images. In contrast, in the sample space coated according to the invention, the oligonucleotides are free to move - similarly as in solution and cannot be localized as in solution, so that an increased background fluorescence was measured for the entire image.

Confocal fluorescence correlation spectroscopy (FCS) enables the interaction of the dye oligonucleotide (MR 121-IPs) of 10 ^-9 -10 ^-10 M with the surface to be measured quantitatively (see also M. Sauer et al., Anal Chem. 2000, 72, 3717-3724). FCS curves of homogeneous solutions, i.e. all fluorophores behave in the same way and can be described by neglecting tripletters by a sigmoidal fit. Its inflection point corresponds to an average diffusion time for the movement of a fluorophore through the detection volume. The higher the mean diffusion time, the less the interaction of the fluorophore with its surroundings or, the higher the diffusion times of the fluorophore, the stronger the adhesion of the labeled molecule on the surfaces examined. This can be used as a quality feature of a non-adhesive coating.

If you describe the FCS curve for the sample of MR 121-IP (T30) in the area of the uncoated glass surface (glass, focused on 0 µm) with a sigmoid curve, you get a turning point of approx. 1 second. This indicates a very strong interaction of the dye with the glass surface. In contrast, the FCS curve for a sample of MR 121-IP (T30) on the surface coated according to the invention (hydrogel, focused on 0 μm) has a turning point at 238 μs, which almost corresponds to that of a freely diffusing MR 121-IP. If the detection volume is shifted 4 μm into the solution, no more surface influence can be determined in the case of the glass coated according to the invention. In the case of glass surfaces, it can be seen that the FCS curve (glass, focused on 4 µm) shows a turning point at 23.5 ms, which in turn indicates very clear interactions of the fluorescent-labeled oligonucleotide with the surface. Only when the detection volume has been shifted by 40 µm into the solution, both curves approach the freely moving MR 121-IP. The diffusion times obtained for MR 121-IP (T30) are summarized in Table 6. Table 6 Average diffusion times of MR 121 on and at different heights above a glass or hydrogel surface

5. Cell adhesion experiments

The cell adhesion experiments were carried out with GFP-Actin 3T3 fibroblasts (chicken) and MC3T3-E1 osteoblasts (chicken). The cells were stored in 50 ml PMMA cell culture boxes at 37 ° C. and 5% CO ₂ in a water vapor-saturated atmosphere in an incubator and replicated. The cell media used are standard cell media. For fibroblasts, the cell medium consisted of an aqueous solution of 88 vol% DMEM (Dulbecco's modified eagle's medium, Biochrom KG) with 10 vol% fetal calf serum (FCS, Invitrogen), 1 vol% penecillin solution (penstrep, Sigma), 1 vol% glutamine ( Invitrogen) and 0.5 mg / ml antibiotic, Geneticin (Sigma), while for the osteoblasts the cell medium from an aqueous solution of 94 vol% α-MEM (α-modified eagle's medium) with 5 vol% calf serum (FCS, Invitrogen) and 1 vol% glutamine (Invitrogen) was produced. The cells were split regularly to guarantee maximum growth and to maintain GFP expression.

Adhesion experiments were carried out in sterile PS petri dishes (50 mm Diameter). Before adding the cells to the Petri dishes were made with 2 ml trypsin per petri dish removed the cell culture boxes and then in a Centrifuge (10 min. At 1000 rpm) separated as sediment. Again diluted with medium, the cell suspension was then applied to the substrates using pipettes and then incubated. If the cells adhere to the substrate, they form an even layer that can be easily seen in the light microscope is. If the cells do not stick to the substrate can, i.e. the adhesion of the cell to the substrate is suppressed, the cells die and form a small, round cluster of cells to recognize the surface.

In experiment 1, a glass plate coated according to the invention was used as the substrate, which was spin-coated according to the instructions given in II-2.2 with a solution of the prepolymer from preparation example 3 (5.0 mg / ml), one according to II-1. pretreated glass plate was produced. The thickness of the coating was approximately 50 nm. The sample was then treated with the cell suspension in the manner described above and examined in a light microscope. The results of the study shows FIG. 3.

Fig. 3 shows that the coating is resistant cell, because the cells have died and recognizable as small round cell clusters on the surface. The surface shows no cell growth even after 120 h, which demonstrates the long-term effect and stability of the coating. Similar results can also be found with thinner coatings which have a thickness of approximately 5 nm.

In experiments 2 and 2a, glass plates were used as substrates which had been coated by spin coating according to the instructions given in II-2.2 with a solution of the prepolymer from preparation example 3 (5.0 mg / ml or 1 mg / ml) (d = 15 nm or d = 5 nm). One half was then dip-coated with polystyrene (1.0 mg / ml or 2.5 mg / ml in THF). The sample was then treated with the cell suspension in the manner described above and examined in a light microscope. The results of the investigation are shown in FIGS. 4a and 4b.

From FIGS. 4a and 4b shows that the cells colonize on the treated polystyrene surface and die on the inventively coated surface.

In experiment 3, glass plates were used as the substrate, which was dip-coated on one half according to the instructions given in II-2.1b with a mixture of prepolymers from preparation example 3 (5.0 mg / ml) and dextran dissolved in water (20 mg / ml, d = 30 nm) had been coated. The sample was then treated with the cell suspension in the manner described above and examined in a light microscope. The results of Fig. 5 shows.

From Fig. 5 shows that the cells colonize on the untreated surface and die on the inventively coated surface.

Claims (22)

1. Use of star-shaped prepolymers, on average have at least four polymer arms A, which by themselves are soluble in water and have one at their free ends reactive functional group R wear with this complementary reactive functional groups R 'or with itself can react themselves to form bonds ultra-thin, hydrogel-forming coatings. 2. Use according to claim 1, characterized in that the reactive group R is selected from isocyanate groups, (Meth) acrylic groups, oxirane groups, carboxylic ester groups and the reactive group R 'is selected from primary and secondary amino groups, thiol groups, carboxyl groups and Hydroxyl groups. 3. Use according to claim 1, characterized in that the reactive group R is selected from ethylenic unsaturated, free-radically polymerizable double bonds. 4. Use according to any one of the preceding claims, wherein the star-shaped prepolymer with an average of 6 to 8 polymer arms having. 5. Use according to any one of the preceding claims, wherein the star-shaped prepolymer has a number average molecular weight in the range from 2000 to 20,000 g / mol. 6. Use according to any one of the preceding claims, wherein the Polymer arms have a number average molecular weight in the range have from 300 to 3000 g / mol. 7. Use according to one of the preceding claims, wherein the polymer arms A are selected from poly-C ₂ -C ₄ -alkylene oxides, polyoxazolidones, polyvinyl alcohols, homo- and copolymers which contain at least 50% by weight of copolymerized N-vinylpyrrolidone, homo- and copolymers which contain at least 30% by weight of copolymerized acrylamide and / or methacrylamide, homopolymers and copolymers which contain at least 30% by weight of copolymerized acrylic acid and / or methacrylic acid. 8. Use according to claim 7, wherein the polymer arms A are selected from polyethylene oxide, polypropylene oxide and Polyethylene oxide / polypropylene oxide block copolymers. 9. Use according to any one of the preceding claims, wherein the star-shaped prepolymer in the form of an aqueous preparation is present. 10. A process for producing ultra-thin, hydrogel-forming coatings, characterized in that a) a solution of a star-shaped prepolymer, which has on average at least four polymer arms A, which are in themselves soluble in water and carry a reactive functional group R at their free ends, applied to the surface to be coated and b) subsequently carrying out a linking reaction between the reactive groups. 11. The method according to claim 10, characterized in that the amount of prepolymer applied to the surface is chosen that a coating in a thickness of less than 50 nm results. 12. The method according to claim 10 or 11, characterized in that the concentration of prepolymer in the solution is 0.001 mg / ml is up to 100 mg / ml. 13. The method according to any one of claims 10 to 12, characterized characterized that the surface to be coated in a way was pretreated that an increased number of functional R 'groups. 14. The method according to claim 13, characterized in that the Surface treated in an oxygen-containing plasma has been. 15. The method according to claim 13 or 14, characterized in that the surface with a silane compound, the one has functional group R ', was pretreated. 16. The method according to any one of claims 10 to 15, characterized characterized in that the linking of the reactive groups R by adding a compound V1 that triggers at least two has reactive groups R 'per molecule which with the reactive groups R of the star-shaped polymer under Bond formation reacts. 17. The method according to any one of claims 10 to 15, characterized characterized that you can link the reactive Triggers groups R by using a sufficient amount of one Connection V2 admits that with part of the reactive Groups R reacted to form reactive groups R ', which with the remaining reactive groups R below React bond formation. 18. The method according to claim 17, characterized in that the reactive groups R are isocyanate groups and compound V2 Water. 19. The method according to claim 10, characterized in that the Group R is selected from ethylenically unsaturated, radical polymerizable double bonds and the Crosslinking is triggered thermally or photochemically. 20. The method according to any one of claims 10 to 16, characterized in that a) first a star-shaped prepolymer 1, which has at least four polymer arms A, which are in themselves soluble in water and have a reactive functional group R at their free ends, applied to the surface to be coated, optionally a partial crosslinking of the reactive groups R. carries out, whereby a coating is obtained which still has reactive groups R on its surface, b) then at least one further star-shaped prepolymer 2, which has at least four polymer arms A, which are in themselves soluble in water and have a reactive functional group R 'at their free ends, which react with the reactive groups R of the star-shaped polymer to form bonds , applied to the surface treated in this way, possibly triggering crosslinking of the groups R ' and optionally repeating steps a) and b) one or more times. 21. The method according to claim 17 or 18, characterized in that one a) a star-shaped prepolymer 1 which has at least four polymer arms A, which are in themselves soluble in water and have a reactive functional group R at their free ends, applied to the surface to be coated, b) a sufficient amount of a compound V2 is added, which reacts with a portion of the reactive groups R to form reactive groups R ', which react with the remaining reactive groups R to form bonds, whereby a coating is obtained which has functional groups R ' having, c) subsequently applying at least one further star-shaped prepolymer 1 to the surface treated in this way and again triggering crosslinking of the groups R by adding a compound V2 and optionally repeating steps b) and c) one or more times. 22. The method according to any one of claims 10 to 21, characterized characterized that you have a biologically active in the coating Material and / or cell material incorporated.