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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/20—Small organic molecules
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers

Definitions

• the invention deals with polymer carriers with covalently bonded saccharides for temporary immobilization of cells with receptors for mannose or galactose.
• the invention is also aimed at the preparation of these polymer carriers. Their main application is the use of keratinocyte receptors for cultivation of these cells and transfer of the cells onto burns.
• the invention is a continuation of the patents of J. Labsky et al. "Polymer carrier for keratinocyte cultivation" (Czech Patent Applications PV 1998-1803, PV 1999-1946, PV 1999-1947, WO 99645631 (PCT/CZ/99/00017)).
• Biological systems such as some cell types, lectins and cell toxins, possess receptors, which enable temporary immobilization on polymer matrix surface with appropriate covalently bonded ligands.
• the temporarily formed receptor-ligand bond enables manipulation with the thus formed complex, such as separation, purification, cultivation or transport.
• Appropriate ligands for biological receptors are chemically bounded saccharides, such as mannose, galactose or disaccharides (lactose).
• lactose lactose
• the almost identical hydroxy groups in molecules of mono- or disaccharides require special techniques for the preparation of polymerizable derivatives or activation of saccharide molecules for modification of suitable polymer carriers.
• polymerizable derivatives of mono- or disaccharides where the hydroxy groups are protected, e.g., by acetylation, be ⁇ zoylation or benzylation.
• Acetal derivatives of saccharides or analogous isopropylidene derivatives prepared by condensation with acetone can be converted by a suitable chemical modification to polymerizable derivatives.
• Monomers are described where the polymerizable group is bonded to the saccharide molecule in positions 2 or 3 (S. Kimura et al: Makr ⁇ mol. Chem. 58, 232, 1962; S. Kimura et al: Makromol. Chem. 50, 155, 1961) or 6 (M. Imoto et al: Makromol. Chem. 53, 2120, 1962; N.P. Ivanova et al: Zh. Prikl. Khim. 71, 114-118, 1998; W.A.P. Black et al.: Makromol. Chem. 117, 210-14, 1968; W.A.P. Black: Carbohydr. Res. 5, 362-5, 1967; J. Klein: Makromol. Chem. Rapid Commun. 6, 675, 1985; S. Kimura, M. Imoto: Makromol. Chem 50, 155 (1961), 53, 210, 1962).
• Amino derivatives of saccharides, where the amino group is bonded in various positions are also often used for the preparation of polymerizable derivatives (E. Fanton, C. Fayet: Carbohydr. Res., 240, 143-52, 1993, J. Klein: Makromol. Chem., Rapid Commun. 6, 675, 1985, B. Badley et al: Macromol. Chem. Phys. 198, 945-57, 1997, Suk Hyeong Cho et al: Korea Polym. J. 6, 188-192, 1998, R.L. Whistler et al: J. Org. Chem. 26, 1583, 1961).
• Oxazolines which can be prepared from 2-acetamido-2-deoxysaccharides, are easily converted to 2-acetamido-2-deoxyglucosides, where a polymerizable group can be built-in in the aglycon.
• a variety of saccharide derivatives are described in literature, where the oxazoline skeleton of a saccharide can be transformed into a suitable precursor for the preparation of polymer glycosides (M. Okada, K. Aoi: Jpn. Kokai Tokkyo Koho JP 08259587, 1996), Jun-Ichi Kadokawa et al: Macromolecules 30, 8212-17, 1997).
• a saccharose derivative is given as an example of disaccharides with one or more polymerizable groups (T. Furuike et al: Macromolecules 1995, 28, 7241-7, J. Chen et al: WO 98/51694), N. D. Sachinvala et al: Carbohydr. Res. 218, 1991, 237-45).
• An aminosaccharide can be attached to a soluble polymer by the reaction with an active ester, e.g. according to J. F. Bridges, R. Duncan, J. Kopecek: Int. J. Pharm. 44, 213-223, 1988.
• a distinct disadvantage of polymer carriers utilized as cultivation supports for immobilization or separation of biological substances and formed as copolymers of saccharide monomers with protected hydroxy groups and hydrophilic or hydrophobic comonomers is the fact that the protecting groups must be removed.
• the polymers or copolymers prepared using the saccharides " where the protecting groups were removed prior to the polymerization show also problems - poor solubility of saccharide monomers in organic solvents and comonomers, the necessity to polymerize mostly in water, poor mechanical properties of the formed copolymers.
• a variable content of saccharide units in the polymer significantly affects mechanical properties of the polymers to such extent that they cannot be used as cultivation supports.
• Another disadvantage is that the removal of protecting groups is often not quantitative and the residual groups may affect the polymer structure.
• the present invention suggests the use of hydrophilic materials as matrices, both synthetic and natural, where specific ligands are bonded on modification of the carrier surface or activation of suitable saccharide derivatives and subsequent reaction with the matrix. Under these conditions, the reaction proceeds in surface layers of the matrix and mechanical properties of -the polymer formed are almost identical with those of the starting material and the amount of surface-bonded saccharides can be controlled.
• the subject of the present invention is the polymer carriers with bonded saccharides mannose, galactose or disaccharides, where at the nonreducing end of the disaccharide is mannose or galactose, which are covalently bonded to polymer matrix using various types of spacers, whereby polymer matrices are made suitable for cultivation of keratinocytes or for temporary immobilization of biological systems with receptors for mannose and galactose of the general formula
• PM is a hydrophilic crosslinked polymer prepared by radical polymerization of a polymerization mixture containing: (a) 1-99 wt.% of a monomer or monomer mixture, (b) 0.1-10 wt.% of a crosslinker or crosslinker mixture, (c) 0.01-10 wt.% of a radical initiator and/or (d) 0.1 -40 wt.% of a solvent or solvent mixture
• R is independently selected from the group: covalent bond, -(CH 2 ) a -, -OCH 2 CH 2 -, -
• R l is independently selected from the group: covalent bond, -(CH 2 ) a -, -OCH 2 CH 2 -, -C 6 H 4 -O-, -(OCH 2 CH 2 ) b -O-, -C 6 H 4 -CO-, -NH-C 6 H 4 -CO-,
• Y is independently selected from the group: covalent bond, -N(COCH 3 )-, -NH-CS-NH-C 6 H 4 -O-, -NH-CS-NH-(CH 2 )a-C6H 4 -O-,-NH-CS-NH-(CH 2 ) a -O-C 6 H 4 -O-, -NH-CS- ⁇ -(CH 2 ) a -C ⁇ H 4 -O-, -NH-CeHU-O-, -NH ⁇ C 6 H 4 -CO-
• subscript a ranges from 1 to 12
• b from 1 to 200
• p is 0-20
• subscript n is selected to ensure the saccharide concentration in the range from lxl0 "4 to 0.3 g per gram of polymer matrix
• Z is independently selected from the group of mannose, galactose and lactose derivatives, whose structure formulae are given; the wavy line shows the attachment of the saccharide molecule.
• the mannose derivatives are as follows
• the galactose derivatives are as follows.
• lactose derivatives are as follows.
• the basic materials for polymer carriers according to the invention are hydrophilic polymer matrices, which are surface-activated and, subsequently, saccharides are activated.
• activation of surface layers of polymer carriers is associated with application of spacers because in interactions of polymer systems with biological materials the antenna effect mostly operates, which enables the receptor contact with a polymer ligand.
• Polymer carriers suitable for preparation of cultivation supports by their surface modification may be of natural (cellulose, alginates) or synthetic origin (poly(vinyl alcohol), poly(2- hydroxyethyl methacrylate) etc.).
• the suitability of a polymer is determined by several parameters such as swelling, mechanical properties and, last but not least, by the presence of reactive groups such as hydroxy, amino or carboxy groups.
• Biocompatibility, chemical inertness, stability in biological media, insolubility, sterilizability and zero extractability of polymer matrices are other necessary requirements for their applications.
• Suitable monomers for preparation of polymer carriers according to the invention are 2- hydroxyethyl acrylate, 2-hydroxyethyl methacrylate (HEMA), 2-(2-hydroxyethoxy)ethyl acrylate, 2-(2-hydroxyethoxy)ethyl methacrylate (DEGMA), tri-, tetra- and poly(ethylene glycol) monoacrylates and methacrylates ; glycerol acrylate and methacrylate, 2- hydroxypropyl acrylate and methacrylate, ⁇ -hydroxyalkyl acrylates and methacrylates, N-( ⁇ - hydroxyalkyl)acrylamides and -methacrylamides, N-( ⁇ -aminoalkyl)acrylamides and -methacrylamides, glycidyl acrylate and methacrylate, N-[2-hydroxy-l , l - bis(hydroxymethyl)ethyl]acrylamide and -methacrylamide, acrylic and methacrylic acid, ⁇ -
• crosslinkers can be used for the preparation of polymer matrices according to the invention: ethylene diacrylate and dimethacrylate, diethylene glycol and oligo(ethylene glycol) diacrylates and dimethacrylates, N,N-ethylenediacrylamide and N,N- ethylenedimethacrylamide, 1,3-divinylurea, 1,1 '-divinyl-3,3 '-(ethane-1, l-diyl)di(pyrrolidin-2- one), 2,3-dihydroxybutan-l,4-diyl diacrylate or dimethacrylate and also other crosslinkers usually used in the field.
• thermal radical initiators are suitable such as azo compounds, diacyl peroxides and other types of peroxo compounds, photoinitiators generating radical by the action of UN radiation or redox initiators, which generate radicals by an oxidation-reduction reaction.
• a solvent in the preparation of polymer matrix such as water, alcohols (methanol, ethanol, glycol, glycerol), dimethylformamide, dimethyl sulfoxide, poly(ethylene glycol)s, esters of aliphatic acids, ethylene glycol monomethyl or dimethyl ether and also other common solvents used in the field.
• polymer matrices may be different.
• Common cultivation supports are prepared in moulds for film casting, where polymerisations can be performed also in the presence of solvents. For some purposes it is more convenient to prepare spongy polymers, foams, microparticles or beads. The techniques are well known and commonly used in the field and, therefore, are not further discussed here.
• polymer matrix is formed with the idealized structure PM-(R a ) p , where PM represents the structure of the crosslinked carbon chain formed by radical polymerization and the R a group is any functional group, which is a constituent of the used monomers or comonomers.
• the structure of this part of the matrix corresponds to the polymerization mixture composition and the functional groups correspond to the structure of the starting monomer.
• R" is most frequently -OH, -COOH, -CH 2 CH 2 OH, -CH 2 CH(OH)CH 3 , -NH 2 , -CH 2 H 2> -C 6 H 4 COOH, -C 6 H 4 OH, -C 6 H 4 OCH 2 CH 2 OH, -CONH(CH 2 ) n COOH, -C 6 H 4 NH 2 , -COO(CH 2 ) personallyCOOH, -CONH(CH 2 ) crampNH 2 .
• This list of reactive groups is only illustrative; a variety of other possible groups are available, which must be included in the list.
• R a All the functional groups denoted R a are not utilized for modification reactions leading to binding of spacers and subsequent reaction with activated saccharides.
• the reactions occur in matrix surface layers and do not significantly affect mechanical properties of the matrix.
• a surface phenomenon appears called the orange effect. The effect consists in that on attachment of substances of the nature pronouncedly different from the matrix structure (saccharide), the matrix surface bursts during swelling due to different swelling characteristics of the ligand and matrix.
• the modified matrix has the idealized structure PM-(R -X ) x , where the general formula
• -R b -X b represents the matrix surface structure after modification reaction and, clearly, its concentration is lower than the original introduced groups generally denoted -R a .
• X b represents reactive groups or a spacer as described below. Possible repetition of the modification reaction using the same or different compounds (e.g., diazotation and azo coupling, formation of an active ester) is included in the general representation.
• Activation of amino and hydroxy groups can be accomplished by the reaction with bifunctional reactive systems, e.g. dichlorides or chloride-esters of dicarboxylic acids, such as dicarboxylic acids of the general formula HOOC-Q-COOH, where Q represents a bifunctional aliphatic chain, branched aliphatic chain, cycloalkanediyl, cycloalkenediyl, phenylene, furandiyl, oxydiethylene, diisocyanates of the general formula OCN-T-NCO and diisothiocyanates SCN-T-NCS, where T represents a divalent aliphatic chain, cyclohexane- 1 ,4-diyl, methylenedi-l,4-phenylene, oxydi-l,4-phenylene, methylenedicyclohexane-l,4-diyl.
• bifunctional reactive systems e.g. dichlorides or chloride-esters of
• Another possibility of activation of hydroxy or amino groups is the use of cyanogen bromide, phosgene, diphosgene, chlorocarbonic esters of aliphatic alcohols, branched aliphatic alcohols, cyclic alcohols, l,l '-carbonyldiimidazole or other derivatives of carbonic acid commonly used in the field.
• the thus modified matrix surface selectively reacts in another step with an aliphatic amino group (J. Drobnik, J. Labsky, H. Kudlvasrova, V. Saudek, F. Svec: Biotechnol. Bioeng. 2, 487, 1982, J. Drobnik, J. Kalal, J. Labsky, V. Saudek, F. Svec: Czech Pat.
• Carboxylic groups can be built-in into polymer carrier using an appropriate polymerizable monomer, such as acrylic, methacrylic acid, maleic anhydride, ⁇ -acrylamidoalkanoic acids, ⁇ -methacrylamidoalkanoic acids or by additional modification of other reactive groups. Modification of polymer surface by the reaction of cyclic anhydrides with amino or hydroxy group of the carrier affords carboxylic groups
• polymer matrix with an appropriate derivative of dicarboxylic acid such as dichloride or active ester.
• suitable alcohols for the active esters are, e.g., 4-nitrophenol, pentachlorophenol, pentafluorophenol, 2,4-dinitrophenol, N- hydroxysuccinimide, N-hydroxyphthalimide, l-hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid and other compounds used in the field.
• excess reagent predominantly one group of dicarboxylic acid reacts; the other can be utilized for the reaction with an aliphatic amino group, e.g. of a saccharide.
• Activation of the carboxylic group is possible in several ways. Its transformation into acid chloride by the action of, e.g., thionyl chloride, phosphorus trichloride, phosphorus pentachloride, oxalyl chloride is easy. A necessary requirement is that the polymer carrier be inert (A. K. ghost et al.: Tetrahedron Lett. 33, 2781, 1992, J. P. Greenstein, M. Winitz: Chemistry of the Aminoacids. J. Wiley, New York 1961)
• Another possible activation of the carboxyl is the transformation of an acid ester to acid hydrazide and further to azide.
• the reaction is performed by the action of hydrazine, most frequently on an acid ester and by subsequent reaction with sodium nitrite or a nitroso compound (J. P. Greenstein, M. Winitz: Chemistry of the Aminoacids. J. Wiley, N.Y, 1961).
• the hydrazide can be also prepared by the carbodiimide-activated reaction of an acid with hydrazine " (J. ' -Drobnik, J. Kalal, J. Labsky, V. Saudek, F. Svec: Czech Pat. 204 190, US 4 245 064, 1979).
• a frequently used activation of the carboxyl group is performed by activation with carbodiimides or analogous compounds, where active ester is an intermediate.
• the technique is perfectly elaborated, offering the reaction proceeding in both aqueous and organic media; the yields of the condensation reaction often range above 80 %.
• the intermediate is given formed by the reaction of the carboxyl with dicyclohexylcarbodiimide (M. K. Dhaon et al: J. Org. Chem. 47, 1962, 1982, J. C. Sheehan et al: J. Am. Chem. Soc. 95, 875, 1973).
• a variety of reactions can be used for activation of polymer matrix with aromatic amines or phenols, which make it possible to build-in an aromatic system suitable for diazotation and azo coupling. Examples of the compounds used are as follows.
• the resulting polymer matrices suitable for azo coupling with diazonium salts have, e.g., the following structure
• D-Mannose and D-galactose or their 2-amino-2-deoxy derivatives often occur in natural materials. These saccharide units are frequently located at the nonreducing ends of saccharide chains and are key saccharides for interactions with active centres of biological systems. Saccharides bonded to polymer matrices have the ⁇ or ⁇ structure; most often they are mixtures of the stereoisomers in dependence on the used reactions. The structure details, however, cannot be determined in the matrix and are not a hindrance in the use of matrices.
• mannosyl-, lactosyl- and galactosylamine can be used, which are easily prepared from mannose, lactose or galactose by the reaction with ammonia, ammonium hydrogencarbonate or ammonium carbonate (L. M Likhosherstov et al: Izv. Akad. Nauk, Ser. Khim. 8, 1461, 2000, R. Duncan et al: J. Controlled Release 10, 51-63, 1989, T. Raderaum, I.D. Manger: US 5,280,113).
• the derivatives appropriate for modification of polymer carriers are, e.g., ⁇ -amino-N- (D-mannopyranosyl)alkanamide or N-( ⁇ -aminoalkyl)-N-(D-mannopyranosyl)alkanamide (J. J. Garcia-Lopez et al: Chem. Eur. J., 5, 1775-1784, 1999, A.Ya. Chernyak et al.: Carbohydr. Res. 223, 303-9, 1992) or analogous derivatives of galactose:
• Mannosylamine, galactosylamine and lactosylamine can also be prepared by reduction of the corresponding azide.
• O-acetylated or O-benzoylated derivatives are prepared, which, after reduction and attachment to polymer matrix, can be easily deacetylated with a methanolic solution of sodium methanolate.
• Methyl 6-amino-6-deoxy-D-mannopyranoside accessible from methyl ⁇ -D-mannopyranoside by tosylation and subsequent reaction with sodium azide and reduction, was also used for the mannosylation reaction (A. L. Cimecioglu et al: Macromolecules, 30, 55-6, 1997, R. Roy et al: Glycoconjugate J. 15, 251-63, 1998).
• a frequently used mannose derivative is 2-aminoethyl ⁇ -D-mannopyranoside, which can be prepared in several ways, e.g. (A.Ya. Chernyak et al: Carbohydr. Res. 223, 303-9, 1992, A.Ya. Chernyak et al: Glycoconjugate J. 8, 82-9, 1991, Patel et al: J. Org. Chem. 2002, 79-86, J. M. Kim, R. Roy: J. Carbohydr. Res. 16, 1281-92, 1997):
• reaction can be extended to halogenated alcohols of the general formula
• the reductive amination of the aldehyde group in a reducing disaccharide can also be used for the preparation of saccharide amines.
• glycosides were prepared, where a carboxyl group is present in the aglykon, e.g. methyl 8-[l-O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-mannopyranosyl)]octanoate (B. M. Pinto et al: Carbohydr. Res. 124, 313-18, 1983, K. Wada et al: J. Carbohydr. Chem. 13, 941-65, 1994).
• the ester can be- transformed into derivatives appropriate for the reaction with activated matrix (carboxylic acid, active ester, hydrazide, azide) (R. U. Lemieux, D. A. Baker: Can. J. Biochem. 55, 507-12, 1977).
• the carboxyl group can be generated at another site of saccharide molecule, e.g. by the reaction of an amino group with cyclic anhydrides of dicarboxylic acids:
• Mannosylamine and galactosylamine can be easily transformed into derivatives that can be used for the reaction with activated matrix, such as (T. Rademacher: US 5 280 113, ID. Manger: EP 0413675, H. Parot Lopez et al: Tetrahedron Lett. 33, 209-13, 1992, D. Vetter: Bioconjugate Chem. 6, 319-22, 1995, J. R. Rasmussen: US 5 283 353):
• N-succinimidyl glycopyranosides are saccharide derivatives, which, in the reaction with the matrix amino group, form a straight-chain spacer (J. M. Kim, R. Roy: J. Carbohydr. Res. 16, 1281-92, 1997, S. Cao, F. D. Topper, R. Roy: Tetrahedron 51, 6679-86, 1985):
• 4-lsothiocyanatophenyl ⁇ -D-mannopyranoside can be prepared from 4-nitrophenyl ⁇ -D- mannopyranoside by reduction of nitro group and consecutive reaction with thiophosgene.
• An analogous derivative was prepared from ⁇ -aminoalkyl ⁇ -D-mannopyranoside (C. R. Broom et al: Methods Enzymol. 28, 212-19, 1972, D.F. Smith et al: Methods Enzymol. 50, 163-75, 1978, S. Kotteer: J. Chem. Soc, Perkin Trans. 1, 2193, 1998)
• lsothiocyanates were derived that contain a spacer between the aromatic ring and saccharide, e.g. (M. Andersson et al: Bioconjugate Chem. 4, 246-9, 1993):
• amidines which can be easily prepared by the reaction of 2- imino-2-methoxy ethyl 1-thioglycopyranosides with amines (Y. C. Lee, C.P. Stowell, M. J. Krantz: Biochemistry 15, 3956, 1976)
• Lactones ⁇ -Galactopyranosyl-(l— 4)-D-glucono-l,5-lactone reacts with an aliphatic amino group under the formation of amide (K. Aoi et al: Macromolecules 28, 5391, 1995). Di- and oligosaccharides that, contain mannose in the nonreducing part react analogously.
• Aminophenyl derivatives of saccharides and disaccharides can be azo-coupled with biological materials, which contains groups (tyrosine) undergoing azo coupling. This fact can be utilized for binding appropriate derivatives to matrices with attached phenols, e.g. (C. P. Stowell, Y. C. Lee: Adv. Carbohydr. Chem. 37, 225-281, 1980, M. Wichek et al: PCT Int. Appl. WO 2000027814 Al, 2000):
• Example 2 75 g (0.5 mol) of triethylene glycol is mixed with sodium methanolate (from 11.5 g sodium and 200 ml methanol) under cooling and methanol is evaporated in vacuum. To the reaction mixture diluted with 300 ml of acetone is dropped 50 g (0.5 mol) of methacryloyl chloride during 30 min at 0 °C under cooling. The mixture is left standing for 24 h, the precipitated sodium chloride is filtered off and volatiles are evaporated in vacuum. The residue contains 57 % triethylene glycol, 26 % of its monomethacryloyl derivative and 5.1 % of the dimethacrylate.
• a mixture of 40 ml of 2-hydroxyethyl methacrylate, and 25 ml of the product mixture containing tetraethylene glycol monomethacrylate (Example 1) and 1 ml of Darocur 1 173 (Ciba) was bubbled with argon for 10 min and poured into a mould for preparation of films of 1.6 mm thickness.
• the polymerization mixture was irradiated with Philips UN lamps of total output 160 W from a distance of 20 cm for 20 min.
• the obtained film was extracted with 30% ethanol in distilled water and subsequently with distilled water and then dried.
• a mixture of 30 ml of 2-hydroxyethyl methacrylate, 0.3 ml ethylene dimethacrylate and 0.6 ml of Darocur 1173 (Ciba) was bubbled with argon for 10 min and poured into a mould for preparation of films of 1.6 mm thickness.
• the polymerization mixture was irradiated with Philips UV lamps of total output 120 W from a distance of 20 cm for 20 min.
• the obtained film was extracted with 30% ethanol and water.
• Example 5 A mixture of 40 ml of 2-hydroxyethyl methacrylate, 20 ml triethylene glycol mo no methacrylate (Example 2) and 1 ml of Darocur 1173 (Ciba) was bubbled with argon for 10 min and poured into a mould for preparation of films of 1.6 mm thickness. The polymerization mixture was irradiated with Philips UN lamps of total output 120 W from a distance of 20 cm for 20 min. The obtained film was extracted with 30% ethanol and water. From the swollen film were cut rings 21 mm in diameter.
• a mixture of 30 ml of 2-hydroxyethyl methacrylate, 5 g 7-methacrylamidoheptanoic acid and 0.2 g Irgacure 2959 (Ciba) was bubbled with argon for 10 min and poured into a mould for preparation of films of 1.6 mm thickness.
• the polymerization mixture was irradiated with Philips UN lamps of total output 120 W from a distance of 20 cm for 20 min.
• the obtained film was extracted with 30% methanol and distilled water.
• Example J " * ' ' * ' A mixture of 40 ml of 2-hydroxyethyl methacrylate, 10 ml 2,3-dihydroxypropyl methacrylate, 5 ml N,N-dimethacrylamide and 0.4 g Irgacure 2959 (Ciba) was bubbled with argon for 10 min and poured into a mould for preparation of films of 1.8 mm thickness. The polymerization mixture was irradiated with Philips UN lamps of total output 120 W from a distance of 20 cm for 25 min. The obtained film was extracted with 25% methanol and water and dried.
• Example 9 A mixture of 30 ml of 3-hydroxypropyl methacrylate, 10 ml 2-methoxyethyl methacrylate, 15 ml of a mixture containing tetraethylene glycol monomethacrylate (Example 1) and 0.26 g 2,2'-azobis(2-methylpropanenitrile) was bubbled with argon for 10 min and poured into a mould for preparation of films of 1.6 mm thickness. The polymerization mixture was heated gradually to 48 - 62 °C for 11 h. The obtained film was extracted with 30% methanol and water and dried before use. .
• Example 9 A mixture of 30 ml of 3-hydroxypropyl methacrylate, 10 ml 2-methoxyethyl methacrylate, 15 ml of a mixture containing tetraethylene glycol monomethacrylate (Example 1) and 0.26 g 2,2'-azobis(2-methylpropanenitrile) was bubbled with argon for 10 min and poured into
• a mixture of 40 ml of 2-hydroxyethyl methacrylate, 20 ml of diethylene glycol monomethacrylate and 0.38 g of 4,4'-azobis(4-cyanopentanoic acid) was bubbled with nitrogen., for .11 .min and poured into a mould for preparation of films of 1.6 mm thickness.
• the polymerization mixture was heated gradually at 50 - 63 °C for 12 h. After taking out From the mould, the film was extracted with 30% methanol and water and dried.
• a mixture of 30 ml of 1 -vinylpyrrolidin-2-one, 20 ml 2-hydroxyethyl acrylate, 15 ml 4- hydroxybutyl methacrylate, 2 ml 2- ⁇ [(vinyloxy)carbonyl]oxy ⁇ ethyl methacrylate, and 1 ml of Darocur 1173 (Ciba) was bubbled with carbon dioxide for 10 min and poured into a mould for preparation of films of 1.8 mm thickness.
• the polymerization mixture was irradiated with Philips UV lamps (120 W) from a distance of 22 cm for 15 min. The obtained film was extracted in a standard way.
• a mixture of 25 ml of 2-hydroxypropyl methacrylate, 25 ml 2-hydroxyethyl methacrylate, 3 ml triethylene glycol dimethacrylate, 10 ml glycerol methacrylate (mixture of isomers), 10 ml of 2-acetoxyethyl methacrylate, and 1 g Irgacure 2959 (Ciba) was bubbled with nitrogen for 15 min and poured into a mould for preparation of films.
• the polymerization mixture was irradiated with Philips UN lamps (100 W) from a distance of 25 cm for 23 min.
• the obtained film was extracted in a standard way.
• Example 12 A mixture of 50 ml of 2-hydroxypropyl methacrylate, 20 ml 2-hydroxyethyl methacrylate, 10 g N-(6-aminohexyl)methacrylamide and 0.7 g Darocur 1173 (Ciba) was bubbled with nitrogen for 15 min and poured into a mould for preparation of films (1.5 mm). The polymerization mixture was irradiated with a UN source (120 W) from a distance of 20 cm. The obtained film was washed in a standard way.
• a UN source 120 W
• a mixture of 20 ml of 2-hydroxyethyl methacrylate, 30 ml poly(ethylene glycol) monomethacrylate (Polyscience, Inc., /. 200), 2 ml diethylene glycol dimethacrylate and 0J ml Darocur 1173 (Ciba) was bubbled with nitrogen for 15 min and poured into a mould for preparation of films (1.6 mm).
• the polymerization mixture was irradiated with a UV source (120 W) from a distance of 25 cm for 20 min.
• the monomer residues after polymerization were removed by extraction of the film with 30% ethanol and water.
• the polymer prepared according to Example 3 (rings 21 mm in diameter, cut in the swollen state, dry weight 5 g) was immersed in a solution of 3 g 4-nitrophenyl chlorocarbonate in 30 ml toluene, 5 ml pyridine and 10 ml acetone for 48 h. The polymer was washed with acetone three times (total 200, ml) and dried.
• Example 17 5 g of the polymer prepared according to Example 9 was immersed in a solution of 3.5 g of 2- [(chlorocarbonyl)oxy]isoindoline-l,3-dione in 50 ml of an acetone-dioxane 1: 1 mixture and 5 ml of triethylamine. After 24 h the solution was removed and the polymer washed successively with dioxane and acetone and dried.
• the polymer prepared according to Example 18 (6 g, rings 11 mm in diameter) was immersed in a solution of 5 g of hexamethylenediamine in 30 ml dioxane. After 24 h the solution was removed and the polymer was washed with a mixture of 30% methanol and 0.5%> sodium hydrogencarbonate.
• Example 22 1 g of the polymer prepared according to Example 14 as a 1.3 mm-thick film was immersed into a solution of 0.5 g of 4-(2-aminoethyl)aniline in 20 ml dioxane. The solution was removed after 48 h. the polymer was extracted for 2 days with a 5 %> solution of sodium hydrogencarbonate in 30% methanol.
• the rings (17 mm in diameter, 1.5 g) prepared according to Example 17 were covered with a solution of 0.35 g of N-( ⁇ -D-mannopyranosyl)glycinamide in 3 ml water and 1.5 ml dimethyl sulfoxide. The reaction was quenched after 2 days by washing with excess distilled water.
• Cultivation supports (thickness 1.3 mm, 2.1 g) prepared according to Example 10 were covered with a solution of 1 g 4-nitrophenyl chlorocarbonate, 10 ml dioxane and 2 ml pyridine. After 2 days, the polymer supports were washed with a mixture of acetone, dioxane and dichloromethane. The polymer was reacted with a solution of 0.4 g of 2-amino-2-deoxy-
• Example 27 25 The polymer supports 2x2 cm prepared according to Example 22 (thickness 1.8 mm, 5.6 g) were moistened with a solution of 2 g thiophosgene in 10 ' ml of acetone. The reaction was quenched after 2 days by washing the film three times with acetone and drying.
• Example 29 To the polymer matrix prepared according to Example 18 (rings 16 mm in diameter, thickness 1.3 mm, 5 g) was added a solution of 1.2 g of methyl 6-amino-6-deoxy- ⁇ -D-mannopyranoside and 0.2 g 4-(dimethylamino)pyridine in 12 ml water and 3 ml dimethyl sulfoxide. The reaction was stopped after 2 days by washing the film with water and drying.
• Example 29 To the polymer matrix prepared according to Example 18 (rings 16 mm in diameter, thickness 1.3 mm, 5 g) was added a solution of 1.2 g of methyl 6-amino-6-deoxy- ⁇ -D-mannopyranoside and 0.2 g 4-(dimethylamino)pyridine in 12 ml water and 3 ml dimethyl sulfoxide. The reaction was stopped after 2 days by washing the film with water and drying.
• Example 29 To the polymer matrix prepared according to Example 18 (rings 16 mm in diameter, thickness
• Example 30 The polymer film prepared according to Example 12 was overlayered with a solution of 1.3 g of 4-isothiocyanatophenyl ⁇ -D-mannopyranoside in 10 ml of acetone - isopropyl alcohol 1 :1. After 2 days the polymer film was washed three times with acetone, twice with methanol, then with water and dried.
• the polymer prepared according to Example 20 (6 g, rings 11 mm in diameter) was covered with a solution of 1.8 g of ⁇ -galactopyranosyl-(l— 4)-D-glucono-l,5-lactone and 0.1 g 4- (dimethylamino)pyridine in 10 ml of dioxane - water 1 :1. The reaction was stopped after two days and the film was washed with dioxane, methanol and water.
• a polymer support prepared according to Example 17 (rings 15 mm in diameter, 5 g, thickness 1.8 mm) was added to a solution of 1.2 g of 2-aminoethyl ⁇ -D-mannopyranoside in a mixture of 5 ml dimethylformamide and 8 ml water. The reaction mixture was left standing for three days and then the support washed with water, 30%> methanol and water.
• Example 12 The film according to Example 12 (3.7 g, ring diameter 18 mm, thickness 1.8 mm) was treated with a solution of 1.1 g of 2,3,4,6-tetra-O-acetyl- ⁇ -D-mannopyranosyl isothiocyanate in 15 ml dichloromethane at laboratory temperature. After 24 h, the rings were washed with dichloromethane, methanol and water.
• Example 36 A polymer film according to Example 20 (ring diameter 19 mm, thickness 1.8 mm, 3.7 g) was immersed in a solution of 1.8 g 2-isocyanatoethyl ⁇ -D-mannopyranoside in a mixture of 10 ml acetone and 5 ml dioxane. After two days, the film was washed with dioxane, acetone and water.
• the film according to Example 10 (4.5 g, ring diameter 18 mm, thickness 1.2 mm) was immersed in a solution of 5-carboxypentyl ⁇ -D-mannopyranoside, 0.3 g N-[3- (dimethylamino)propyl]N-ethyl-carbodiimide and 0.2 g 1-hydroxy benzotriazole in a mixture of 15 ml dimethylformamide and 5 ml water. After 2 days, the solution was removed, the film was washed successively with dioxane, 5% acetic acid, 5% sodium hydrogencarbonate and water.
• a polymer film according to Example 36 (ring diameter 20 mm, thickness 0.9 mm, 3 g) was covered with a solution of 2 g 4-nitrophenyl chlorocarbonate in 10 ml dichloromethane. After two days, the film was washed with 3x50 ml dichloromethane and dried.
• Example 39 A polymer film according to Example 38 (1 g, ring diameter 20 mm, thickness 0.9 mm) was immersed in a solution of 1 g of 3,3'-oxydipropan-l-amine in 10 ml of a mixture dioxane - isopropyl alcohol 1 : 1. After two days the product was washed three times with dioxane.
• the film rings prepared according to Examples 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35 and 36 were used for cultivation of keratinocytes [1-4]. In all cases the cultivation was more successful than on standard films prepared from 2-hydroxyethyl methacrylate. The films from Examples 24, 25 and 28 were better by 50-80 %, while the other exceeded pronouncedly 100 %.

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