CA2226131C - Preparation of polymeric materials having cell poliferation-promoting properties - Google Patents
Preparation of polymeric materials having cell poliferation-promoting properties Download PDFInfo
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- CA2226131C CA2226131C CA002226131A CA2226131A CA2226131C CA 2226131 C CA2226131 C CA 2226131C CA 002226131 A CA002226131 A CA 002226131A CA 2226131 A CA2226131 A CA 2226131A CA 2226131 C CA2226131 C CA 2226131C
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F246/00—Copolymers in which the nature of only the monomers in minority is defined
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/06—Use of macromolecular materials
- A61L33/064—Use of macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/04—Acids; Metal salts or ammonium salts thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F228/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a bond to sulfur or by a heterocyclic ring containing sulfur
Abstract
Disclosed is a water-insoluble polymer which promotes cell proliferation, is produced by a radical copolymerization and is composed of: (I) repeating units of at least one aliphatically unsaturated monomer containing a carboxylate group; (II) repeating units of at least one aliphatically unsaturated monomer containing a sulfonate group; and (III) repeating units of at least one aliphatically unsaturated monomer other than the components (I) or (II).
The polymer is useful for producing an article, especially a medical article such as an artificial blood vessel.
The polymer is useful for producing an article, especially a medical article such as an artificial blood vessel.
Description
' CA 02226131 1998-O1-02 PREPARATION OF POLYMERIC MATERIALS HAVING CELL PROLIFERATION-PROMOTING PROPERTIES
Field of the Invention The invention relates to a water-insoluble polymer which promotes cell proliferation and to a process for the preparation thereof.
The invention furthermore relates to the use of the water-insoluble polymer for the production of a product having a surface which promotes cell proliferation and for the production of a product having a coating of the polymer, which promotes cell proliferation.
Background of the Invention For numerous medical applications of materials such as polymers, ceramics and metals, for example as suture materials, stems, implants or prostheses, good compatibilities with the immune and complement system and the blood must be ensured.
This property, which is often called biocompatibility, includes avoidance of degradation phenomena of the materials by physiological components, such as enzymes and macrophages.
Improved biocompatibility of substitute materials employed medically can be achieved in principle by colonization with human cells. The process described in EP 0 290 642 initially requires covalent bonding of an intermediate layer of so-called biopolymers onto polymer surfaces functionalized by carboxyl, amino and hydroxyl groups. The biocompatibility sought in the material is then O.Z. 5141 achieved by careful, extracorporeal colonization of the intermediate layer with endothelial cells.
WO 90/02145 describes, with the same aim, a process in which acrylic acid is grafted onto a fluorine-containing polymer substrate by irradiation with a 60Co source or a laser. After a series of chemical processes on the surface, controlled absorption of proteins takes place, which is followed by colonization with endothelial cells to establish the biocompatibility.
These processes are extremely time-consuming and cost-intensive and require the greatest care for a medical application, so that the endothelial cell layers applied extracorporeally are not damaged. It is furthermore not possible to allow the cell colonization and cell growth (cell proliferation) to be carried out by the body itself in vivo, since an undesirable thrombic reaction starts before the cell colonization.
A subsequent chemical modification of a surface of a polymeric material with an RGD (arginine-glycine-aspartic acid) sequence is usually not uniform and/or standard.
Untreated areas often remain, which are no longer available as starting points for cell colonization of the surface (G.
Muller, Angewandte Chemie, 104 (1992) 341 et seq.).
From another technical field, according to U.S.
Patent No. 5,278,200, polymers which contain carboxylate and sulfonate groups in a ratio comparable to that of naturally occurring heparin are known. These polymers have anticoagulating properties with respect to platelets in the blood.
O.Z. 5I41 An ob~ect of the present invention is therefore to improve cell proliferation on a surface of an article.
It has now been found, surprisingly, that a water-insoluble polymer which contains both carboxylate and sulfonate groups and is obtainable by free radical copolymerization of:
(I) at least one aliphatically unsaturated monomer containing a carboxylate group, or a correspondingly functionalized derivative thereof, (II) at least one aliphatically unsaturated monomer containing a sulfonate group, or a correspondingly functionalized derivative thereof, (III) at Ieast one aliphatically unsaturated monomer other than the monomers (I) and (II), and where required converting the correspondingly functionalized derivative into carboxylate or sulfonate group after the copolymerization, is capable of promoting cell proliferation.
The adhesion and the growth of cells is thus improved in a physiologically tolerated manner on the polymer according to the invention.
The polymer according to the invention is thus particularly suitable for the production of implants in which growth of endogenous or non-differentiated cells is desired.
The present invention therefore, in a first aspect, provides a water-insoluble polymer which promotes cell proliferation, is produced by a radical copolymerization and is composed of:
(I) repeating units of at least one aliphatically unsaturated monomer containing a carboxylate group, O.Z. 5141 (II) repeating units of at least one aliphatically unsaturated monomer containing a sulfonate group, and (III} repeating units of at least one aliphatically unsaturated monomer other than the repeating units (I) or (II) defined above.
The present invention, in a second aspect, provides a process for the preparation of the above-described water-insoluble polymer which comprises a free radial copolymer-izat ion of (I) at least one aliphatically unsaturated monomer containing a carboxylate group, or a correspondingly functionalized derivative thereof, (II} at least one aliphatically unsaturated monomer containing a sulfonate group, or a correspondingly functionalized derivative thereof, and (III) at least one aliphatically unsaturated monomer other than the monomers (I} or (II}, and where required, converting the correspondingly functionalized derivative or derivatives into a carboxylate group, or a sulfonate group or both after the copolymerization.
The present invention, in a third aspect, provides a product having a surface which promotes cell proliferation and is made of the above-described water-insoluble polymer which promotes cell proliferation and a product having a coating, of the polymer, which promotes cell proliferation. Preferably, the article is a medical article, in particular an artificial blood vessel, having a surface which promotes cell proliferation. Preferably, the medical article is made of plastics, ceramics or a metal and has a coating, of the O.Z. 5141 polymer, which promotes cell proliferation.
Description of Preferred Embodiments The polymer according to the invention is prepared by copolymerization of generally three components.
For the polymer according to the invention and for the process according to the invention, at least one aliphatically unsaturated monomer containing both carboxylate and sulfonate groups or a correspondingly functionalized derivative of the monomer can also be employed as the component (I).
For the polymer according to the invention and for the process according to the invention, at least one aliphatically unsaturated monomer containing both carboxylate and sulfonate groups or a correspondingly functionalized derivative of the monomer can furthermore also be employed as the component (II).
In such a particular embodiment, the component (I) may be identical to the component (II) for the polymer according to the invention and for the process according to the invention.
The aliphatically unsaturated monamers to be employed for the polymers according to the invention may contain both a double bond and a triple bond. The monomers preferably have one or two double bonds.
For the introduction of a carboxylate group into the polymer according to the invention any polymerizable compound of the following formula 1), or a mixture thereof, may preferably be used as the component (I):
O.Z. 5141 1): (CnH2n-q-x)(COORk)x (preferably (CnH2n-x~COOH}x, more preferably (CnH2n-1-~-(-COOH) ) where Rk = -(CH2-CH2-O)d-H, -(CH2-CH(CH3}-0}d-H, -(CH2-CH2-CH2-0)d-H or -(CH2)d-NH3_e(Rm)e, Rm = -CH3 or -C2H5, d - 0, l, 2, 3 or 4, a - 0, 1, 2 or 3, n - 2, 3, 4, 5 or 6, q - 0 or 2, and x = 1 or 2.
Examples of those compounds of the formula 1}
include (meth)acrylic acid, vinylacetic acid, cinnamic acid, itaconic acid, malefic acid, fumaric acid and their esters (i.e. those in which d is other than 0).
The ester groups if present are hydrolyzed after the polymerization and are thus converted to an ionic form. The aliphatically unsaturated monomer may be either straight-chain or branched.
Another group of monomers that may be used for introducing a carboxylate group are benzene derivatives represented by the empirical formula:
(C6H6-a-b-c)AaBb(OH}c i.e.
(B~
(O~c A = (CnH2n-q-x-1)(COORk)x O.Z. 5141 where Rk = -(CH2-CH2-O)d-H, -(CH2-CH(CH3)-O)d-H, -(CH2-CH2-CH2-O)d-H or -(CH2}d-NH3-e(Rm)e, in which Rm = -CH3 or -C2H5 a - 0, 1, 2 or 3, b - 0, 1, 2 or 3, c - 0, 1, 2 or 3, d - 0, 1, 2, 3 or 4, a - 0, 1, 2 or 3, n - 2, 3, 4, 5 or 6, q - 0 or 2, x - 0, 1 or 2, provided that a + b + c s 6, and B - -COOH, S03H, -NH2, -N+(CH3)3, -O-P03H ; -OS03H
or -O-PO-2-O-CH2-CH2-N+(CH3)3. When x is 0, then b is other than 0 and B includes -COOH.
In this specification, the "carboxylate group"
preferably is a free carboxyl group (-COOH) or a physiologically acceptable salt thereof, (preferably an alkali metal salt).
A sulfonate group can be introduced into the polymer according to the invention by using a compound of the formula 2) or a mixture thereof as the component (II):
2): (CmH2m_s_y)(S03R1)y (preferably (CmH2m-1)(S03H)) where Rl = -(CH2-CH2-0)d-H, -(CH2-CH(CH3)-O)d-H, -(CH2-CH2-CH2-O)d-H or -(CH2)d-NH3-e(Rm)e, Rm = -CH3 or -C2H5 d - 0, l, 2, 3 or 4, a - 0, 1, 2 or 3, O.Z. 5141 _ g _ m - 0, 2, 3, 4, 5 or 6, preferably other than 0, s - 0 or 2, and y - 1 or 2.
Examples of these compounds of the formula 2) include vinyl sulfonic acid and allylsulfonic acid.
The ester groups if present are hydrolyzed after the polymerization and are thus converted to an ionic form (i.e., a salt). The aliphatically unsaturated monomer may be either straight-chain or branched.
Another group of monomers that may be used for introducing a sulfonate group are benzene derivatives represented by the empirical formula:
(CSH6_k_i_p)RkLi(OH)p i.e., (~2m-1 SOgH
(OH)p (CmH2m-s-y-1)(S03R1)y~
i = 0, 1, 2 or 3, k = 0, 1, 2 or 3, m = 0, 2, 3, 4, 5 or 6, preferably other than 0, p = 0, 1, 2 or 3, preferably 0 or 1, s = 0 or 2, y = 0, 1 or 2, provided that i + k + p _< 6, and L = -COOH, -S03H, -NH2, -N+(CH3)3, -O-P03H-, -OS03H
or -O-PO-2-O-CH2-CH2-N+(CH3)3. When y is 0, then i is other than 0 and L includes -S03H. Examples of the monomers include styrenesulfonic acid, vinyl toluenesulfonic acid and hydroxystyrenesulfonic acid.
O.Z. 5141 _ g -In this specification, the "sulfonate group" is preferably a free sulfonic acid group (-S03H) or a physiologially acceptable salt such as an alkali metal salt.
More preferably, it is in the physiologically acceptable salt form. The sum of the proportions of the component (I) and the component (II) for the polymer according to the invention and for the process according to the invention is preferably 5 to 30 mol%, particularly preferably 15 to 20 mol%, based on the total of the components (I), (II) and (III).
According to the invention, the molar ratio of the carboxylate group to the sulfonate group contained in the polymer is 3 to 10, particularly preferably 3 to 5.
The copolymerization of the abovementioned monomers (I) and (II) is carried out according to the invention with one or more other aliphatically unsaturated monomers (III).
Usually, the component (III) does not contain an acidic group or a salt thereof.
A nonionic monomer is preferably used as the component (III). These monomers include, for example, vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes, unsaturated halogenated hydrocarbons and correspondingly functionalized derivatives thereof. Preferred as the component (III) are esters (especially Cl-C10 alkyl or C3-C8 cycloalkyl esters) of (meth)acrylic acid, styrene and (meth)acrylamide.
The polymer according to the invention may be prepared, for example, by an emulsion polymerization which is generally well known in the art (see, for example Hans-Georg Elias, Makromolekule [Macromolecules], Huthig & Wepf Verlag, O.Z. 5141 Heidelberg, 1981, p.603 et seq.).
For the preparation of the polymer according to the invention, the components (I), (II) and (III) may also be copolymerized by a solution or bulk polymerization which is also generally well known in the art (see, for example Hans-Georg Elias, Makromolekule [Macromolecules], Huthig &
Wepf Verlag, Heidelberg, 1981, p.602 et seq.).
It is desirable to use a solvent. The following solvents, for example, can be employed for copolymerization of the components (I), (II) and (III) in solution:
water, acetone, methyl ethyl ketone, butanone, cyclohexanone, diethyl ether, tetrahydrofuran, dioxane, methanol, ethanol, propanol, butanol, cyclohexanol, dimethylacetamide, dimethyl sulfoxide, dimethyl formamide, heptane, cyclohexane, benzene, toluene, dichloromethane, trichloromethane, ethyl acetate, propyl acetate, amyl acetate and acetonitrile.
Azonitriles, alkyl peroxides, acyl peroxides, hydroperoxides, peroxoketones, peresters and peroxocarbonates, peroxodisulfate, persulfate and all customary photoinitiators can be used, inter alia, as polymerization initiators.
Alternatively, the polymerization can be initiated by heat or by electromagnetic radiation, such as, for example, UV light or ~-radiation.
If no monomer containing a carboxylate or sulfonate group but instead functionalized derivatives thereof, such as, for example, a carboxylic acid ester instead of a carboxylic acid, are used for the preparation of the polymers according to the invention, the functionalized derivatives must be O.Z. 5141 - l0a -converted into carboxylate or sulfonate groups after the polymerization. In the case of the carboxylic acid ester, this can be carried out preferably by means of a base-catalyzed hydrolysis. The derivatization of polymeric material can be carried out by generally known processes (Hans Beyer, Lehrbuch der organischen Chemie [Textbook of organic chemistry], S. Hirzel Verlag, Stuttgart, 1988, p.260 et set.}.
In a preferred embodiment of the process, the component (I} is a free carboxylic acid and the component (II}
is an alkali metal salt of a sulfonic acid. In this case, no step f_or converting an ester as the component (I} to the free carboxylic acid or salt thereof is required.
A product (i.e., article} having a surface which promotes cell proliferation may be produced directly from the water-insoluble polymer according to the invention. However, the polymer according to the invention may also be applied, if appropriate as a solution in a suitable solvent, as a thin layer to a substrate of a proper material, e.g., standard polymer by any appropriate application techniques such as spraying, painting, dipping, knife-coating or coating or by multilayer injection molding, coextrusion or calendering and lamination.
It is furthermore possible to fix the water-soluble polymer according to the invention on a standard polymer, which may have been activated if appropriate, by a primer layer or an intermediate layer of a bifunctional compound.
Such standard polymers include, for example, polyvinyl chloride (PVC}, polystyrene, polyurethane, polyacrylate, polymethacrylate, polyester, polyether, O.Z. 5141 - lOb -polyether-block amide, polyamide, polycarbonate, polyolefin, silicone and polytetrafluoroethylene.
The measurement method for determination of the cell proliferation is described below.
Preparation of a cell suspension Human fibroblasts of the cell line ATCC CRL 1696 (American Type Culture Collection, Rockville, Maryland, USA) are cultured in DMEM (Dulbecco's Modified Eagles Medium) with the addit ion of ant ibiot ics, L-glut amine and 10 % of a fetal calf serum in culture bottles at 37°C under an atmosphere of 5% C02 and 95% air. After incubation, the nutrient medium is removed and the cell line is treated with 0.05% trypsin/0.02%
EDTA for 5 minutes. The cells are then washed with DMEM and suspended in the same nutrient medium.
Measurement of the cell proliferation In a 250 ml conical flask, a polymer sample 2 x 2 cm in size is pricked onto a dissecting needle and sterilized with ethylene oxide, and 20 ml of the abovementioned nutrient medium are added. The polymer sample is then inoculated with 105 cells from the freshly prepared cell suspension and O.Z. 5141 incubated for 8 days. The polymer sample is removed and rinsed with sterile PBS buffer solution. Adenosine triphosphate is then extracted from the cells with the aid of hot Tris/EDTA solution and determined quantitatively with the bioluminescence reagent CLSII (Boehringer Mannheim GmbH, Mann-heim).
Samples which were obtained by polymerization of the component (III) of the particular polymer according to the invention and were prepared in the same manner were used as reference sample. In a control experiment, a polymer sample was rinsed immediately after inoculation with the cell suspension and the cells rinsed off were determined quantitatively by the method described above. The pro~ticn of cell proliferation is expressed as the oercentaae quotient of the ATP concentration of the cells which have grown on the polymers according to the invention divided by the correspond-ing value of the reference sample.
The measurement results given in the following examples show that the cell proliferation increases between 60% and 110% on polymers according to the invention.
The following examples are intended to illustrate the invention in more detail.
Preparation of samples of the polymers according to the invention 2 o Example 1:
223.2 g of methyl methacrylate, 12.1 g of methacrylic acid and 4.9 g of sodium styrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring.
2.3 g of azobisisobutyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then 2 5 added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 16 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
O.Z. 5141 Subsequent analysis of the composition by'H-NMR gives:
Methacrylic acid: 14 mol%
Sodium styrenesulfonate: 4 mol%
Methyl methacrylate: 82 mol%
A ratio of carboxylate groups to sulfonate groups of 3.4 results from these values.
Example 2:
201.6 g of methyl methacrylate, 25.9 g of acrylic acid and 4.9 g of sodium styrenesulfonate are dissolved in 500 mi of dimethyl sulfoxide in a nitrogen 1o atmosphere. The solution is heated to 70°C, while stirring. 2 g of azobisiso-butyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 16 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
Subsequent analysis of the composition by'H-NMR gives:
Acrylic acid: 18 mol%
Sodium styrenesulfonate: 5 mol%
Methyl methacrylate: 77 mol%
A ratio of carboxylate groups to sulfonate groups of 3.6 results from these values.
Example 3:
244.0 g of styrene, 2.6 g of methacrylic acid and 4.9 g of sodium styrenesul-fonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphe-re. The solution is heated to 70°C, while stirring. 2.3 g of azobisisobutyroni-trite, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 20 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
O.Z. 5141 Subsequent analysis of the composition by ' H-NMR gives:
Methacrylic acid: 10 mol%
Sodium styrenesulfonate: 3 mot%
Styrene: 87 mol%
A ratio of carboxylate groups to sulfonate groups of 3.3 results from these values.
Example 4:
225 g of styrene, 14.2 g of acrylic acid and 9.9 g of sodium styrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring. 2.3 g of azobisisobutyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 20 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
Subsequent analysis of the composition by'H-NMR gives:
Acrylic acid: 21 mol%
Sodium styrenesulfonate: 5 mol%
Styrene: 74 mol%
2o A ratio of carboxylate groups to sulfonate groups of 4.2 results from these values.
Example 5:
316.3 g of n-butyl methacrylate, 12.5 g of methacrylic acid and 4.9 g of sodiumstyrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring.
2.3 g of azobisisobutyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 20 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
O.Z. 5141 Subsequent analysis of the composition by'H-NMR gives:
Methacrylic acid: 16 mol%
Sodium styrenesulfonate: 4 mvl%
n-Butylmethacrylate: 81 mol%
A ratio of carboxylate groups to sulfonate groups of 4.0 results from these values.
Example 6:
317 g of n-butyl methacrylate, 11.2 g of acrylic acid and 2.5 g of sodium styrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring. 2.3 g of azobisiso-butyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 16 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
Subsequent analysis of the composition by'H-NMR gives:
Acrylic acid: 9 mol%
Sodium styrenesulfonate: 2 mol%
n-Butyl methacrylate: 89 mol%
2 0 A ratio of carboxylate groups to sulfonate groups of 4.5 results from these values.
Production of membranes from polymers according to the invention Example 7:
A 5% strength dimethyl sulfoxide solution of the polymers according to the 2 5 invention according to Examples 1, 2 and 5 is prepared. The solution is poured into a Petri dish and the solvent is removed from the sample at 80°C
under reduced pressure. The membrane thus produced is then broken up into pieces of 2 cm x 2 cm each and extracted with water for 24 hours. Before the subsequent biological analyses, the membrane pieces are washed in a 3~ Michaelis buffer solution (pH = 7.33) three times for three hours each time O.Z. 5141 and stored at -4°C until analyzed further.
Production of coatings of polymers according to the invention Example 8:
A 5% strength methyl ethyl ketone solution of the polymer according to the invention according to Example 3 is prepared. A polyamide film 10 cm x 8 cm x 0.04 cm in size is dipped into this solution for 10 seconds. The film is removed and dried at 50°C under reduced pressure for 10 hours. The film coated with the polymer according to the invention is then broken up into pieces of 2 cm x 2 cm each and extracted with water for 24 hours. Before the l0 subsequent biological analyses, the samples are washed in a Michaelis buffer solution (pH = 7.33) three times for three hours each time and kept at -4°C until analyzed further.
Example 9 A 5% strength acetone solution of the polymer according to the invention according to Example 4 is prepared. A polyethylene film 10 cm x 8 cm x 0.03 cm in size, the surface of which has been activated beforehand by irradiation with the 172 nm radiation of an excimer emitter for 3 minutes, is immersed in this solution for 15 seconds. The film is removed and dried at 50°C
under reduced pressure for 10 hours. The coated film is then broken up into pieces 2 0 of 2 cm x 2 cm each and extracted with water for 24 hours. Before the subsequent biological analyses, the samples are washed in a Michaelis buffer solution (pH = 7.33) three times for three hours each time and kept at -4°C until analyzed further.
Example 10 2 5 A 5% strength acetone solution of the polymer according to the invention according to Example 6 is prepared. A polyether-block-amide film 10 cm x 8 cm x 0.04 cm in size is immersed in this solution for 10 seconds. The film is removed and dried at 50°C under reduced pressure for 10 hours. The coated film is then broken up into pieces of 2 cm x 2 cm each and extracted with 30 water for 24 hours. Before the subsequent biological analyses, the samples O.Z. 5141 are washed in a Michaelis buffer solution (pH = 7.33) three times for three hours each time and kept at -4°C until analyzed further.
Conditioning of the samples of polymers according to the invention Example 11:
The membranes according to Example 7 and the films according to Examples 8 to 10 coated with the polymers according to the invention are sterilized by irradiation with ultraviolet light for 15 minutes. The samples pretreated in this way are then kept in a 0.15 molar sodium chloride solution three times for three hours each time and then washed With distilled water for 3 hours. In the l0 subsequent purification step, they are placed in a phosphate buffer solution of the following composition three times for three hours each time:
CaCl2*H20 0.132 g/l KCI 0.2 g/l KHZP04 0.2 g/l MgClz*6H20 0.1 gll NaCI 8 gll Na2HP04 1.15 gll Thereafter, the samples are irradiated with ultraviolet light again for 15 minutes. The samples thus present are kept in a DMEM solution (Dulbecco's Modified Eagles Medium) at 37°C for about 16 hours. Finally, the samples are kept in a DMEM solution, to which antibiotics, L-glutamine and 10% by volume of a fetal calf serum have been added, at 37°C under an atmosphere of 5% COZ and 95% air for a further 16 hours.
The polymers according to the invention produced according to Examples 1, 2 and 5 were processed to membranes (Example 7). Polymers according to the invention according to Examples 3, 4 and 6 were applied to standard polymers (Examples 8 to 10). These samples were then conditioned according to Example 11 and the cell proliferation was determined by the process described.
O.Z. 5141 The following table shows the relative colonization of the polymers according to the invention by human fibroblasts.
Polymer according Reference Relative to the invention polymer colonization according to in % (reference Exam le of mer = 100 1 Polymethyl 165 methac Iry ate 2 Polymethyl 181 methac late 3 Pol s rene 161 4 Pol s rene 217 5 Poly-n-butyl 173 methac late 6 Poly-n-butyl 162 methac late Control sample, Polystyrene 1.8 t=Oh O.Z. 5141
Field of the Invention The invention relates to a water-insoluble polymer which promotes cell proliferation and to a process for the preparation thereof.
The invention furthermore relates to the use of the water-insoluble polymer for the production of a product having a surface which promotes cell proliferation and for the production of a product having a coating of the polymer, which promotes cell proliferation.
Background of the Invention For numerous medical applications of materials such as polymers, ceramics and metals, for example as suture materials, stems, implants or prostheses, good compatibilities with the immune and complement system and the blood must be ensured.
This property, which is often called biocompatibility, includes avoidance of degradation phenomena of the materials by physiological components, such as enzymes and macrophages.
Improved biocompatibility of substitute materials employed medically can be achieved in principle by colonization with human cells. The process described in EP 0 290 642 initially requires covalent bonding of an intermediate layer of so-called biopolymers onto polymer surfaces functionalized by carboxyl, amino and hydroxyl groups. The biocompatibility sought in the material is then O.Z. 5141 achieved by careful, extracorporeal colonization of the intermediate layer with endothelial cells.
WO 90/02145 describes, with the same aim, a process in which acrylic acid is grafted onto a fluorine-containing polymer substrate by irradiation with a 60Co source or a laser. After a series of chemical processes on the surface, controlled absorption of proteins takes place, which is followed by colonization with endothelial cells to establish the biocompatibility.
These processes are extremely time-consuming and cost-intensive and require the greatest care for a medical application, so that the endothelial cell layers applied extracorporeally are not damaged. It is furthermore not possible to allow the cell colonization and cell growth (cell proliferation) to be carried out by the body itself in vivo, since an undesirable thrombic reaction starts before the cell colonization.
A subsequent chemical modification of a surface of a polymeric material with an RGD (arginine-glycine-aspartic acid) sequence is usually not uniform and/or standard.
Untreated areas often remain, which are no longer available as starting points for cell colonization of the surface (G.
Muller, Angewandte Chemie, 104 (1992) 341 et seq.).
From another technical field, according to U.S.
Patent No. 5,278,200, polymers which contain carboxylate and sulfonate groups in a ratio comparable to that of naturally occurring heparin are known. These polymers have anticoagulating properties with respect to platelets in the blood.
O.Z. 5I41 An ob~ect of the present invention is therefore to improve cell proliferation on a surface of an article.
It has now been found, surprisingly, that a water-insoluble polymer which contains both carboxylate and sulfonate groups and is obtainable by free radical copolymerization of:
(I) at least one aliphatically unsaturated monomer containing a carboxylate group, or a correspondingly functionalized derivative thereof, (II) at least one aliphatically unsaturated monomer containing a sulfonate group, or a correspondingly functionalized derivative thereof, (III) at Ieast one aliphatically unsaturated monomer other than the monomers (I) and (II), and where required converting the correspondingly functionalized derivative into carboxylate or sulfonate group after the copolymerization, is capable of promoting cell proliferation.
The adhesion and the growth of cells is thus improved in a physiologically tolerated manner on the polymer according to the invention.
The polymer according to the invention is thus particularly suitable for the production of implants in which growth of endogenous or non-differentiated cells is desired.
The present invention therefore, in a first aspect, provides a water-insoluble polymer which promotes cell proliferation, is produced by a radical copolymerization and is composed of:
(I) repeating units of at least one aliphatically unsaturated monomer containing a carboxylate group, O.Z. 5141 (II) repeating units of at least one aliphatically unsaturated monomer containing a sulfonate group, and (III} repeating units of at least one aliphatically unsaturated monomer other than the repeating units (I) or (II) defined above.
The present invention, in a second aspect, provides a process for the preparation of the above-described water-insoluble polymer which comprises a free radial copolymer-izat ion of (I) at least one aliphatically unsaturated monomer containing a carboxylate group, or a correspondingly functionalized derivative thereof, (II} at least one aliphatically unsaturated monomer containing a sulfonate group, or a correspondingly functionalized derivative thereof, and (III) at least one aliphatically unsaturated monomer other than the monomers (I} or (II}, and where required, converting the correspondingly functionalized derivative or derivatives into a carboxylate group, or a sulfonate group or both after the copolymerization.
The present invention, in a third aspect, provides a product having a surface which promotes cell proliferation and is made of the above-described water-insoluble polymer which promotes cell proliferation and a product having a coating, of the polymer, which promotes cell proliferation. Preferably, the article is a medical article, in particular an artificial blood vessel, having a surface which promotes cell proliferation. Preferably, the medical article is made of plastics, ceramics or a metal and has a coating, of the O.Z. 5141 polymer, which promotes cell proliferation.
Description of Preferred Embodiments The polymer according to the invention is prepared by copolymerization of generally three components.
For the polymer according to the invention and for the process according to the invention, at least one aliphatically unsaturated monomer containing both carboxylate and sulfonate groups or a correspondingly functionalized derivative of the monomer can also be employed as the component (I).
For the polymer according to the invention and for the process according to the invention, at least one aliphatically unsaturated monomer containing both carboxylate and sulfonate groups or a correspondingly functionalized derivative of the monomer can furthermore also be employed as the component (II).
In such a particular embodiment, the component (I) may be identical to the component (II) for the polymer according to the invention and for the process according to the invention.
The aliphatically unsaturated monamers to be employed for the polymers according to the invention may contain both a double bond and a triple bond. The monomers preferably have one or two double bonds.
For the introduction of a carboxylate group into the polymer according to the invention any polymerizable compound of the following formula 1), or a mixture thereof, may preferably be used as the component (I):
O.Z. 5141 1): (CnH2n-q-x)(COORk)x (preferably (CnH2n-x~COOH}x, more preferably (CnH2n-1-~-(-COOH) ) where Rk = -(CH2-CH2-O)d-H, -(CH2-CH(CH3}-0}d-H, -(CH2-CH2-CH2-0)d-H or -(CH2)d-NH3_e(Rm)e, Rm = -CH3 or -C2H5, d - 0, l, 2, 3 or 4, a - 0, 1, 2 or 3, n - 2, 3, 4, 5 or 6, q - 0 or 2, and x = 1 or 2.
Examples of those compounds of the formula 1}
include (meth)acrylic acid, vinylacetic acid, cinnamic acid, itaconic acid, malefic acid, fumaric acid and their esters (i.e. those in which d is other than 0).
The ester groups if present are hydrolyzed after the polymerization and are thus converted to an ionic form. The aliphatically unsaturated monomer may be either straight-chain or branched.
Another group of monomers that may be used for introducing a carboxylate group are benzene derivatives represented by the empirical formula:
(C6H6-a-b-c)AaBb(OH}c i.e.
(B~
(O~c A = (CnH2n-q-x-1)(COORk)x O.Z. 5141 where Rk = -(CH2-CH2-O)d-H, -(CH2-CH(CH3)-O)d-H, -(CH2-CH2-CH2-O)d-H or -(CH2}d-NH3-e(Rm)e, in which Rm = -CH3 or -C2H5 a - 0, 1, 2 or 3, b - 0, 1, 2 or 3, c - 0, 1, 2 or 3, d - 0, 1, 2, 3 or 4, a - 0, 1, 2 or 3, n - 2, 3, 4, 5 or 6, q - 0 or 2, x - 0, 1 or 2, provided that a + b + c s 6, and B - -COOH, S03H, -NH2, -N+(CH3)3, -O-P03H ; -OS03H
or -O-PO-2-O-CH2-CH2-N+(CH3)3. When x is 0, then b is other than 0 and B includes -COOH.
In this specification, the "carboxylate group"
preferably is a free carboxyl group (-COOH) or a physiologically acceptable salt thereof, (preferably an alkali metal salt).
A sulfonate group can be introduced into the polymer according to the invention by using a compound of the formula 2) or a mixture thereof as the component (II):
2): (CmH2m_s_y)(S03R1)y (preferably (CmH2m-1)(S03H)) where Rl = -(CH2-CH2-0)d-H, -(CH2-CH(CH3)-O)d-H, -(CH2-CH2-CH2-O)d-H or -(CH2)d-NH3-e(Rm)e, Rm = -CH3 or -C2H5 d - 0, l, 2, 3 or 4, a - 0, 1, 2 or 3, O.Z. 5141 _ g _ m - 0, 2, 3, 4, 5 or 6, preferably other than 0, s - 0 or 2, and y - 1 or 2.
Examples of these compounds of the formula 2) include vinyl sulfonic acid and allylsulfonic acid.
The ester groups if present are hydrolyzed after the polymerization and are thus converted to an ionic form (i.e., a salt). The aliphatically unsaturated monomer may be either straight-chain or branched.
Another group of monomers that may be used for introducing a sulfonate group are benzene derivatives represented by the empirical formula:
(CSH6_k_i_p)RkLi(OH)p i.e., (~2m-1 SOgH
(OH)p (CmH2m-s-y-1)(S03R1)y~
i = 0, 1, 2 or 3, k = 0, 1, 2 or 3, m = 0, 2, 3, 4, 5 or 6, preferably other than 0, p = 0, 1, 2 or 3, preferably 0 or 1, s = 0 or 2, y = 0, 1 or 2, provided that i + k + p _< 6, and L = -COOH, -S03H, -NH2, -N+(CH3)3, -O-P03H-, -OS03H
or -O-PO-2-O-CH2-CH2-N+(CH3)3. When y is 0, then i is other than 0 and L includes -S03H. Examples of the monomers include styrenesulfonic acid, vinyl toluenesulfonic acid and hydroxystyrenesulfonic acid.
O.Z. 5141 _ g -In this specification, the "sulfonate group" is preferably a free sulfonic acid group (-S03H) or a physiologially acceptable salt such as an alkali metal salt.
More preferably, it is in the physiologically acceptable salt form. The sum of the proportions of the component (I) and the component (II) for the polymer according to the invention and for the process according to the invention is preferably 5 to 30 mol%, particularly preferably 15 to 20 mol%, based on the total of the components (I), (II) and (III).
According to the invention, the molar ratio of the carboxylate group to the sulfonate group contained in the polymer is 3 to 10, particularly preferably 3 to 5.
The copolymerization of the abovementioned monomers (I) and (II) is carried out according to the invention with one or more other aliphatically unsaturated monomers (III).
Usually, the component (III) does not contain an acidic group or a salt thereof.
A nonionic monomer is preferably used as the component (III). These monomers include, for example, vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes, unsaturated halogenated hydrocarbons and correspondingly functionalized derivatives thereof. Preferred as the component (III) are esters (especially Cl-C10 alkyl or C3-C8 cycloalkyl esters) of (meth)acrylic acid, styrene and (meth)acrylamide.
The polymer according to the invention may be prepared, for example, by an emulsion polymerization which is generally well known in the art (see, for example Hans-Georg Elias, Makromolekule [Macromolecules], Huthig & Wepf Verlag, O.Z. 5141 Heidelberg, 1981, p.603 et seq.).
For the preparation of the polymer according to the invention, the components (I), (II) and (III) may also be copolymerized by a solution or bulk polymerization which is also generally well known in the art (see, for example Hans-Georg Elias, Makromolekule [Macromolecules], Huthig &
Wepf Verlag, Heidelberg, 1981, p.602 et seq.).
It is desirable to use a solvent. The following solvents, for example, can be employed for copolymerization of the components (I), (II) and (III) in solution:
water, acetone, methyl ethyl ketone, butanone, cyclohexanone, diethyl ether, tetrahydrofuran, dioxane, methanol, ethanol, propanol, butanol, cyclohexanol, dimethylacetamide, dimethyl sulfoxide, dimethyl formamide, heptane, cyclohexane, benzene, toluene, dichloromethane, trichloromethane, ethyl acetate, propyl acetate, amyl acetate and acetonitrile.
Azonitriles, alkyl peroxides, acyl peroxides, hydroperoxides, peroxoketones, peresters and peroxocarbonates, peroxodisulfate, persulfate and all customary photoinitiators can be used, inter alia, as polymerization initiators.
Alternatively, the polymerization can be initiated by heat or by electromagnetic radiation, such as, for example, UV light or ~-radiation.
If no monomer containing a carboxylate or sulfonate group but instead functionalized derivatives thereof, such as, for example, a carboxylic acid ester instead of a carboxylic acid, are used for the preparation of the polymers according to the invention, the functionalized derivatives must be O.Z. 5141 - l0a -converted into carboxylate or sulfonate groups after the polymerization. In the case of the carboxylic acid ester, this can be carried out preferably by means of a base-catalyzed hydrolysis. The derivatization of polymeric material can be carried out by generally known processes (Hans Beyer, Lehrbuch der organischen Chemie [Textbook of organic chemistry], S. Hirzel Verlag, Stuttgart, 1988, p.260 et set.}.
In a preferred embodiment of the process, the component (I} is a free carboxylic acid and the component (II}
is an alkali metal salt of a sulfonic acid. In this case, no step f_or converting an ester as the component (I} to the free carboxylic acid or salt thereof is required.
A product (i.e., article} having a surface which promotes cell proliferation may be produced directly from the water-insoluble polymer according to the invention. However, the polymer according to the invention may also be applied, if appropriate as a solution in a suitable solvent, as a thin layer to a substrate of a proper material, e.g., standard polymer by any appropriate application techniques such as spraying, painting, dipping, knife-coating or coating or by multilayer injection molding, coextrusion or calendering and lamination.
It is furthermore possible to fix the water-soluble polymer according to the invention on a standard polymer, which may have been activated if appropriate, by a primer layer or an intermediate layer of a bifunctional compound.
Such standard polymers include, for example, polyvinyl chloride (PVC}, polystyrene, polyurethane, polyacrylate, polymethacrylate, polyester, polyether, O.Z. 5141 - lOb -polyether-block amide, polyamide, polycarbonate, polyolefin, silicone and polytetrafluoroethylene.
The measurement method for determination of the cell proliferation is described below.
Preparation of a cell suspension Human fibroblasts of the cell line ATCC CRL 1696 (American Type Culture Collection, Rockville, Maryland, USA) are cultured in DMEM (Dulbecco's Modified Eagles Medium) with the addit ion of ant ibiot ics, L-glut amine and 10 % of a fetal calf serum in culture bottles at 37°C under an atmosphere of 5% C02 and 95% air. After incubation, the nutrient medium is removed and the cell line is treated with 0.05% trypsin/0.02%
EDTA for 5 minutes. The cells are then washed with DMEM and suspended in the same nutrient medium.
Measurement of the cell proliferation In a 250 ml conical flask, a polymer sample 2 x 2 cm in size is pricked onto a dissecting needle and sterilized with ethylene oxide, and 20 ml of the abovementioned nutrient medium are added. The polymer sample is then inoculated with 105 cells from the freshly prepared cell suspension and O.Z. 5141 incubated for 8 days. The polymer sample is removed and rinsed with sterile PBS buffer solution. Adenosine triphosphate is then extracted from the cells with the aid of hot Tris/EDTA solution and determined quantitatively with the bioluminescence reagent CLSII (Boehringer Mannheim GmbH, Mann-heim).
Samples which were obtained by polymerization of the component (III) of the particular polymer according to the invention and were prepared in the same manner were used as reference sample. In a control experiment, a polymer sample was rinsed immediately after inoculation with the cell suspension and the cells rinsed off were determined quantitatively by the method described above. The pro~ticn of cell proliferation is expressed as the oercentaae quotient of the ATP concentration of the cells which have grown on the polymers according to the invention divided by the correspond-ing value of the reference sample.
The measurement results given in the following examples show that the cell proliferation increases between 60% and 110% on polymers according to the invention.
The following examples are intended to illustrate the invention in more detail.
Preparation of samples of the polymers according to the invention 2 o Example 1:
223.2 g of methyl methacrylate, 12.1 g of methacrylic acid and 4.9 g of sodium styrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring.
2.3 g of azobisisobutyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then 2 5 added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 16 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
O.Z. 5141 Subsequent analysis of the composition by'H-NMR gives:
Methacrylic acid: 14 mol%
Sodium styrenesulfonate: 4 mol%
Methyl methacrylate: 82 mol%
A ratio of carboxylate groups to sulfonate groups of 3.4 results from these values.
Example 2:
201.6 g of methyl methacrylate, 25.9 g of acrylic acid and 4.9 g of sodium styrenesulfonate are dissolved in 500 mi of dimethyl sulfoxide in a nitrogen 1o atmosphere. The solution is heated to 70°C, while stirring. 2 g of azobisiso-butyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 16 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
Subsequent analysis of the composition by'H-NMR gives:
Acrylic acid: 18 mol%
Sodium styrenesulfonate: 5 mol%
Methyl methacrylate: 77 mol%
A ratio of carboxylate groups to sulfonate groups of 3.6 results from these values.
Example 3:
244.0 g of styrene, 2.6 g of methacrylic acid and 4.9 g of sodium styrenesul-fonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphe-re. The solution is heated to 70°C, while stirring. 2.3 g of azobisisobutyroni-trite, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 20 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
O.Z. 5141 Subsequent analysis of the composition by ' H-NMR gives:
Methacrylic acid: 10 mol%
Sodium styrenesulfonate: 3 mot%
Styrene: 87 mol%
A ratio of carboxylate groups to sulfonate groups of 3.3 results from these values.
Example 4:
225 g of styrene, 14.2 g of acrylic acid and 9.9 g of sodium styrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring. 2.3 g of azobisisobutyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 20 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
Subsequent analysis of the composition by'H-NMR gives:
Acrylic acid: 21 mol%
Sodium styrenesulfonate: 5 mol%
Styrene: 74 mol%
2o A ratio of carboxylate groups to sulfonate groups of 4.2 results from these values.
Example 5:
316.3 g of n-butyl methacrylate, 12.5 g of methacrylic acid and 4.9 g of sodiumstyrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring.
2.3 g of azobisisobutyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 20 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
O.Z. 5141 Subsequent analysis of the composition by'H-NMR gives:
Methacrylic acid: 16 mol%
Sodium styrenesulfonate: 4 mvl%
n-Butylmethacrylate: 81 mol%
A ratio of carboxylate groups to sulfonate groups of 4.0 results from these values.
Example 6:
317 g of n-butyl methacrylate, 11.2 g of acrylic acid and 2.5 g of sodium styrenesulfonate are dissolved in 500 ml of dimethyl sulfoxide in a nitrogen atmosphere. The solution is heated to 70°C, while stirring. 2.3 g of azobisiso-butyronitrile, dissolved in 30 ml of dimethyl sulfoxide, are then added dropwise in the course of 2 minutes. The polymerization is carried out at 70°C over a period of 16 hours. Thereafter, the product which has formed is precipitated in a fourfold excess of ice-water, subsequently extracted in a Soxhlet with water for 24 hours and dried at 50°C in vacuo.
Subsequent analysis of the composition by'H-NMR gives:
Acrylic acid: 9 mol%
Sodium styrenesulfonate: 2 mol%
n-Butyl methacrylate: 89 mol%
2 0 A ratio of carboxylate groups to sulfonate groups of 4.5 results from these values.
Production of membranes from polymers according to the invention Example 7:
A 5% strength dimethyl sulfoxide solution of the polymers according to the 2 5 invention according to Examples 1, 2 and 5 is prepared. The solution is poured into a Petri dish and the solvent is removed from the sample at 80°C
under reduced pressure. The membrane thus produced is then broken up into pieces of 2 cm x 2 cm each and extracted with water for 24 hours. Before the subsequent biological analyses, the membrane pieces are washed in a 3~ Michaelis buffer solution (pH = 7.33) three times for three hours each time O.Z. 5141 and stored at -4°C until analyzed further.
Production of coatings of polymers according to the invention Example 8:
A 5% strength methyl ethyl ketone solution of the polymer according to the invention according to Example 3 is prepared. A polyamide film 10 cm x 8 cm x 0.04 cm in size is dipped into this solution for 10 seconds. The film is removed and dried at 50°C under reduced pressure for 10 hours. The film coated with the polymer according to the invention is then broken up into pieces of 2 cm x 2 cm each and extracted with water for 24 hours. Before the l0 subsequent biological analyses, the samples are washed in a Michaelis buffer solution (pH = 7.33) three times for three hours each time and kept at -4°C until analyzed further.
Example 9 A 5% strength acetone solution of the polymer according to the invention according to Example 4 is prepared. A polyethylene film 10 cm x 8 cm x 0.03 cm in size, the surface of which has been activated beforehand by irradiation with the 172 nm radiation of an excimer emitter for 3 minutes, is immersed in this solution for 15 seconds. The film is removed and dried at 50°C
under reduced pressure for 10 hours. The coated film is then broken up into pieces 2 0 of 2 cm x 2 cm each and extracted with water for 24 hours. Before the subsequent biological analyses, the samples are washed in a Michaelis buffer solution (pH = 7.33) three times for three hours each time and kept at -4°C until analyzed further.
Example 10 2 5 A 5% strength acetone solution of the polymer according to the invention according to Example 6 is prepared. A polyether-block-amide film 10 cm x 8 cm x 0.04 cm in size is immersed in this solution for 10 seconds. The film is removed and dried at 50°C under reduced pressure for 10 hours. The coated film is then broken up into pieces of 2 cm x 2 cm each and extracted with 30 water for 24 hours. Before the subsequent biological analyses, the samples O.Z. 5141 are washed in a Michaelis buffer solution (pH = 7.33) three times for three hours each time and kept at -4°C until analyzed further.
Conditioning of the samples of polymers according to the invention Example 11:
The membranes according to Example 7 and the films according to Examples 8 to 10 coated with the polymers according to the invention are sterilized by irradiation with ultraviolet light for 15 minutes. The samples pretreated in this way are then kept in a 0.15 molar sodium chloride solution three times for three hours each time and then washed With distilled water for 3 hours. In the l0 subsequent purification step, they are placed in a phosphate buffer solution of the following composition three times for three hours each time:
CaCl2*H20 0.132 g/l KCI 0.2 g/l KHZP04 0.2 g/l MgClz*6H20 0.1 gll NaCI 8 gll Na2HP04 1.15 gll Thereafter, the samples are irradiated with ultraviolet light again for 15 minutes. The samples thus present are kept in a DMEM solution (Dulbecco's Modified Eagles Medium) at 37°C for about 16 hours. Finally, the samples are kept in a DMEM solution, to which antibiotics, L-glutamine and 10% by volume of a fetal calf serum have been added, at 37°C under an atmosphere of 5% COZ and 95% air for a further 16 hours.
The polymers according to the invention produced according to Examples 1, 2 and 5 were processed to membranes (Example 7). Polymers according to the invention according to Examples 3, 4 and 6 were applied to standard polymers (Examples 8 to 10). These samples were then conditioned according to Example 11 and the cell proliferation was determined by the process described.
O.Z. 5141 The following table shows the relative colonization of the polymers according to the invention by human fibroblasts.
Polymer according Reference Relative to the invention polymer colonization according to in % (reference Exam le of mer = 100 1 Polymethyl 165 methac Iry ate 2 Polymethyl 181 methac late 3 Pol s rene 161 4 Pol s rene 217 5 Poly-n-butyl 173 methac late 6 Poly-n-butyl 162 methac late Control sample, Polystyrene 1.8 t=Oh O.Z. 5141
Claims (29)
1. A water-insoluble polymer which promotes cell proliferation, is produced by a radical copolymerization and is composed of:
(I) repeating units of at least one aliphatically unsaturated monomer containing a carboxylate group;
(II repeating units of at least one aliphatically unsaturated monomer containing a sulfonate group, with a molar ratio of the carboxylate group to the sulfonate group in the range of 3 to 10, and (III) repeating units of at least one aliphatically unsaturated monomer other than the components (I) or (II).
(I) repeating units of at least one aliphatically unsaturated monomer containing a carboxylate group;
(II repeating units of at least one aliphatically unsaturated monomer containing a sulfonate group, with a molar ratio of the carboxylate group to the sulfonate group in the range of 3 to 10, and (III) repeating units of at least one aliphatically unsaturated monomer other than the components (I) or (II).
2. The polymer as claimed in claim 1, wherein the carboxylate group of the repeating units (I) is a free carboxyl group (-COOH) or a physiologically acceptable salt thereof; and the sulfonate group of the repeating units (II) is a physiologically acceptable salt of a sulfonic acid group (-SO3H).
3. The polymer as claimed in claim 1 or 2, which has a molar ratio of the carboxylate group to the sulfonate group of 3 to 5.
4. The polymer as claimed in claim 3, wherein the molar ratio is 3 to 4.5.
5. The polymer as claimed in any one of claims 1 to 4, which contains the repeating units (I) and (II) in a total amount of 5 to 30 mol% based on the total amount of the repeating units (I), (II) and (III).
6. The polymer as claimed in claim 5, wherein the total amount of the repeating units (I) and (II) is 15 to 20 mol%.
7. The polymer as claimed in any one of claims 1 to 6, wherein the carboxylate group of the repeating units (I) is a free carboxyl group or an alkali metal salt thereof; the sulfonate group of the repeating units (II) is an alkali metal salt of a sulfonic acid group; and the repeating units (III) are nonionic.
8. The polymer as claimed in claim 7, wherein:
the repeating units (I) are of (meth)acrylic acid or an alkali metal salt thereof;
the repeating units (II) are of an alkali metal salt of styrenesulfonic acid; and the repeating units (III) are of at least one nonionic monomer selected from the group consisting of vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes and unsaturated halogenated hydrocarbons.
the repeating units (I) are of (meth)acrylic acid or an alkali metal salt thereof;
the repeating units (II) are of an alkali metal salt of styrenesulfonic acid; and the repeating units (III) are of at least one nonionic monomer selected from the group consisting of vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes and unsaturated halogenated hydrocarbons.
9. The polymer as claimed in claim 8, wherein the repeating units (III) are of a C1-C10 alkyl ester of (meth)acrylic acid or styrene.
10. The polymer as claimed in claim 7, wherein:
the repeating units (I) are of a free carboxylic acid of the formula (C n H2n-1) COOH (in which n is 2, 3, 4, 5 or 6) or an alkali metal salt thereof;
the repeating units (II) are of an alkali metal salt of a sulfonic acid of the formula:
(in which m is 2, 3, 4, 5 or 6 and p is 0, 1, 2 or 3); and the repeating units are of at least one nonionic monomer selected from the group consisting of vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes and unsaturated halogenated hydrocarbons.
the repeating units (I) are of a free carboxylic acid of the formula (C n H2n-1) COOH (in which n is 2, 3, 4, 5 or 6) or an alkali metal salt thereof;
the repeating units (II) are of an alkali metal salt of a sulfonic acid of the formula:
(in which m is 2, 3, 4, 5 or 6 and p is 0, 1, 2 or 3); and the repeating units are of at least one nonionic monomer selected from the group consisting of vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes and unsaturated halogenated hydrocarbons.
11. A process for producing the water-insoluble polymer as defined in any one of claims 1 to 10, which comprises:
a free radical copolymerization of:
(I) at least one aliphatically unsaturated monomer containing a carboxylate group or a corresponding functionalized derivative thereof, (II) at least one aliphatically unsaturated monomer containing a sulfonate group or a corresponding functionalized derivative thereof, and (III) at least one aliphatically unsaturated monomer other than the monomers (I) or (II), to form a polymer, and when the polymer has the functionalized derivative or derivatives and a carboxylate group, a sulfonate group or -20a-both are required, converting the functionalized derivative or derivatives into the carboxylate group, the sulfonate group or both after the copolymerization.
a free radical copolymerization of:
(I) at least one aliphatically unsaturated monomer containing a carboxylate group or a corresponding functionalized derivative thereof, (II) at least one aliphatically unsaturated monomer containing a sulfonate group or a corresponding functionalized derivative thereof, and (III) at least one aliphatically unsaturated monomer other than the monomers (I) or (II), to form a polymer, and when the polymer has the functionalized derivative or derivatives and a carboxylate group, a sulfonate group or -20a-both are required, converting the functionalized derivative or derivatives into the carboxylate group, the sulfonate group or both after the copolymerization.
12. The process as claimed in claim 11, wherein the monomer (I) is a free carboxylic acid; the monomer (II) is an alkali metal salt of a sulfonic acid, whereby the water-insoluble polymer is produced by the radical polymerization alone in which the repeating units (I) are of the free carboxylic acid and the repeating units (II) are of the alkali metal salt of the sulfonic acid.
13. The process as claimed in claim 11 or 12, which is conducted in a solvent selected from the group consisting of acetone, methyl ethyl ketone, butanone, cyclohexanone, diethyl ether, tetrahydrofuran, dioxane, methanol, ethanol, propanol, butanol, dimethylacetamide, dimethylsulfoxide, dimethyl formamide, dichloromethane, trichloromethane, ethyl acetate and acetonitrile.
14. The process as claimed in claim 12, which is conducted in dimethyl sulfoxide.
15. A use of the water-insoluble polymer as defined in any one of claims 1 to 10, for the production of a product having a surface which promotes cell proliferation.
16. The use as claimed in claim 15, wherein the product is a medical article.
17. The use as claimed in claim 15, wherein the medical article is an artificial blood vessel.
18. A use of the water-insoluble polymer as defined in any one of claims 1 to 10, for the production of a product having a coating which is made of the water-insoluble polymer and which promotes cell proliferation.
19. The use as claimed in claim 18, wherein the product is a medical article made of a plastic, ceramic or metal material having the coating.
20. The use as claimed in claim 19, wherein the medical article is an artificial blood vessel made of a plastic material having the coating.
21. An article having a substrate and a coating layer which promotes cell proliferation and is made of the water-insoluble polymer as claimed in any one of claims 1 to 10.
22. The article as claimed in claim 21, which is a medical article.
23. The article as claimed in claim 22, wherein the substrate is made of plastic, ceramic or metal.
24. The article as claimed in claim 22 or 23, which is an artificial blood vessel.
25. An article having a surface which promotes cell proliferation, the article being made of the water-insoluble polymer as claimed in any one of claims 1 to 10.
26. The article as claimed in claim 25, which is a medical article.
27. The article as claimed in claim 26, which is an artificial blood vessel.
28. An article for medical application having a surface which promotes proliferation of human cells when placed in a human body, the article itself being formed of a water-insoluble polymer or the article having a coating of a water-insoluble polymer on a substrate, wherein the water-insoluble polymer is a radical copolymerization product and is composed of:
(I) repeating units of acrylic acid, methacrylic acid or a physiologically acceptable salt thereof;
(II) repeating units of a physiologically acceptable salt of styrenesulfonic acid; and (III) repeating units of a nonionic monomer selected from the group consisting of vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes and unsaturated halogenated hydrocarbons, in which the total content of the repeating units (I) and (II) is 5 to 30 mol% based on the total amount of the repeating units (I), (II) and (III) and a molar ratio of the repeating units (I) to the repeating units (II) is 3 to 10.
(I) repeating units of acrylic acid, methacrylic acid or a physiologically acceptable salt thereof;
(II) repeating units of a physiologically acceptable salt of styrenesulfonic acid; and (III) repeating units of a nonionic monomer selected from the group consisting of vinyl compounds, allyl compounds, acrylic compounds, olefins, dimes and unsaturated halogenated hydrocarbons, in which the total content of the repeating units (I) and (II) is 5 to 30 mol% based on the total amount of the repeating units (I), (II) and (III) and a molar ratio of the repeating units (I) to the repeating units (II) is 3 to 10.
29. The article as claimed in claim 28, wherein the nonionic monomer of the repeating units (III) is at least one member selected from the group consisting of C1-C10 alkyl esters of acrylic acid or methacrylic acid and styrene.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19700078A DE19700078A1 (en) | 1997-01-03 | 1997-01-03 | Production of polymeric materials with cell proliferation-promoting properties |
| DE19700078.9 | 1997-01-03 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2226131A1 CA2226131A1 (en) | 1998-07-03 |
| CA2226131C true CA2226131C (en) | 2006-09-19 |
Family
ID=7816789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002226131A Expired - Fee Related CA2226131C (en) | 1997-01-03 | 1998-01-02 | Preparation of polymeric materials having cell poliferation-promoting properties |
Country Status (8)
| Country | Link |
|---|---|
| US (2) | US6365692B1 (en) |
| EP (1) | EP0852237B1 (en) |
| JP (1) | JPH10195143A (en) |
| AT (1) | ATE219502T1 (en) |
| CA (1) | CA2226131C (en) |
| DE (2) | DE19700078A1 (en) |
| DK (1) | DK0852237T3 (en) |
| NO (1) | NO315706B1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19908318A1 (en) * | 1999-02-26 | 2000-08-31 | Michael Hoffmann | Hemocompatible surfaces and methods of making them |
| US7056868B2 (en) * | 2001-07-30 | 2006-06-06 | Cabot Corporation | Hydrophobe associative polymers and compositions and methods employing them |
| FR2850107B1 (en) * | 2003-01-17 | 2007-03-30 | L A R S Laboratoire D Applic E | PROCESS FOR TREATING POLYMER SURFACES |
| US20040208983A1 (en) * | 2003-04-16 | 2004-10-21 | Hill Gregory A. | Antimicrobial coatings for ophthalmic devices |
| US7968650B2 (en) | 2006-10-31 | 2011-06-28 | Johnson & Johnson Vision Care, Inc. | Polymeric compositions comprising at least one volume excluding polymer |
| WO2012092566A1 (en) * | 2010-12-30 | 2012-07-05 | Surmodics, Inc. | Cell attachment coatings and methods |
| EP2866849B1 (en) | 2012-06-29 | 2020-02-19 | SurModics, Inc. | Cell attachment coatings and methods using phosphorous-containing photoreagent |
| FR3013584B1 (en) | 2013-11-22 | 2015-11-20 | L A R S Laboratoire D Applic Et De Rech Scient | BIOMIMETIC PROOFHETIC RESISTANCE LIGAMENT |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4056517A (en) * | 1971-12-28 | 1977-11-01 | Hoechst Aktiengesellschaft | Modacryl filaments and fibers and process for their manufacture |
| JPS5846242B2 (en) * | 1980-07-30 | 1983-10-15 | 東ソー株式会社 | Method for producing polymer emulsion |
| HU187669B (en) * | 1982-03-31 | 1986-02-28 | Richter Gedeon Vegyeszet | Process for producing new copolymeres and salts |
| JPS6136309A (en) * | 1984-07-30 | 1986-02-21 | Nippon Shokubai Kagaku Kogyo Co Ltd | Water-absorbent resistant to salt and light |
| DE3788062D1 (en) * | 1987-05-12 | 1993-12-09 | Christian Dr Med Mittermayer | Process for colonizing a polymer surface with human vascular skin cells. |
| AU605217B2 (en) | 1987-05-12 | 1991-01-10 | Ecolab Inc. | Disinfectant polymeric coatings for hard surfaces |
| EP0408673A4 (en) * | 1988-08-22 | 1992-03-11 | The Commonwealth Scientific And Industrial Research Organisation | Acid treated polyacrylic acid grafted fluorocarbon polymer surface for cell attachment |
| US5278200A (en) * | 1992-10-30 | 1994-01-11 | Medtronic, Inc. | Thromboresistant material and articles |
| TW228529B (en) * | 1992-12-23 | 1994-08-21 | Ciba Geigy | |
| US5783502A (en) * | 1995-06-07 | 1998-07-21 | Bsi Corporation | Virus inactivating coatings |
| US5756625A (en) * | 1996-10-11 | 1998-05-26 | Minnesota Mining And Manufacturing Company | Stabilized adhesive microspheres |
-
1997
- 1997-01-03 DE DE19700078A patent/DE19700078A1/en not_active Withdrawn
- 1997-11-19 AT AT97120234T patent/ATE219502T1/en not_active IP Right Cessation
- 1997-11-19 DK DK97120234T patent/DK0852237T3/en active
- 1997-11-19 DE DE59707555T patent/DE59707555D1/en not_active Expired - Fee Related
- 1997-11-19 EP EP97120234A patent/EP0852237B1/en not_active Expired - Lifetime
- 1997-12-26 JP JP9359951A patent/JPH10195143A/en not_active Ceased
- 1997-12-30 US US09/001,143 patent/US6365692B1/en not_active Expired - Fee Related
- 1997-12-30 NO NO19976153A patent/NO315706B1/en not_active IP Right Cessation
-
1998
- 1998-01-02 CA CA002226131A patent/CA2226131C/en not_active Expired - Fee Related
-
2001
- 2001-11-15 US US09/999,295 patent/US6417000B1/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US6365692B1 (en) | 2002-04-02 |
| DK0852237T3 (en) | 2002-10-14 |
| US20020068804A1 (en) | 2002-06-06 |
| ATE219502T1 (en) | 2002-07-15 |
| NO315706B1 (en) | 2003-10-13 |
| JPH10195143A (en) | 1998-07-28 |
| DE19700078A1 (en) | 1998-07-09 |
| NO976153L (en) | 1998-07-06 |
| EP0852237A2 (en) | 1998-07-08 |
| US6417000B1 (en) | 2002-07-09 |
| EP0852237A3 (en) | 1998-09-16 |
| DE59707555D1 (en) | 2002-07-25 |
| EP0852237B1 (en) | 2002-06-19 |
| CA2226131A1 (en) | 1998-07-03 |
| NO976153D0 (en) | 1997-12-30 |
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