CS247431B1 - Polymeric compositions for stimulating adhesion, growth and differentiation of animal cells - Google Patents

Abstract

The solution relates to polymer compositions capable of stimulating adhesion, growth and differentiation of animal cells. The essence of the biological activity of polymer compositions lies in the presence of aldimine binding groups in the polymer matrix of the composition. The polymer matrix consists of synthetic or natural polymers or copolymers. The formation of the aldimine binding group can involve substances containing reactive aldehyde groups and substances containing primary amino groups. Aldehydic groups or primary amino groups can optionally be an integral part of the polymer matrix. Biologically active polymer compositions are used mainly in the form of culture media in the cultivation of animal cells. The cultivation of these cells is carried out under otherwise usual conditions, e.g. in the pH range of 7.2 to 7.4, where the aldimine binding group is completely stable. The polymer compositions are intended mainly for the cultivation of animal cells in laboratories or biotechnology and may also find application in the field of medicine.

Description

(54) Polymer compositions for stimulating the adhesion, growth and differentiation of animal cells

The invention relates to polymer compositions capable of stimulating the adhesion, growth and differentiation of animal cells.

The essence of the biological activity of the polymer compositions lies in the presence of aldimine binding moieties in the polymer matrix of the composition. The polymer matrix consists of synthetic or natural polymers or copolymers. Substances containing reactive aldehyde groups and substances containing primary amino groups may be involved in the formation of the aldimine linkage group. The aldehyde groups or primary amino groups may optionally be an integral part of the polymer matrix.

Biologically active polymer compositions are used in particular in the form of culture supports in the cultivation of animal cells. Culturing of these cells is performed under otherwise usual conditions, e.g. in the range of pH 7.2 to 7.4, where the aldimine binding moiety is completely stable.

The polymer compositions are mainly intended for the cultivation of animal cells in laboratories or biotechnologies and may also find application in the medical field.

The invention relates to polymer compositions for stimulating the adhesion, growth and differentiation of animal cells.

Natural substances are known which allow the cells to attach to the culture medium. These are some high molecular weight tissue components, proteins or glycoproteins such as collagen or elastin, fibronectin and laminin.

Certain synthetic polymers and polymer composites or chemically modified solid culture surfaces in the form of bowls, bottles, flat or capillary membranes are also used for this purpose. More recently, spherical microcarriers are available which are suitable for cell cultivation in laboratories and biotechnologies. The range of materials used so far is very diverse. These include, for example, polylysine, polyHEMA-collagen composite, chemically modified polystyrene, dextran, agarose, cellulose, gelatin and other substances. A disadvantage of said materials is their low biological activity and / or incomplete characterization of the culture surfaces themselves.

A novel solution to this problem is the biologically active polymer composition of the invention, characterized in that it consists of a matrix of synthetic or natural polymer or copolymer, a monofunctional or difunctional or polyfunctional aldehyde, and a physiologically acceptable substance containing at least one primary amino group in its molecule. all reactive aldehyde groups are bonded to the primary amino groups in the form of an aldimine moiety.

The polymer compositions of the invention differ from all known materials used to grow animal cells in that they contain a defined number of aldimine binding moieties. Although the existence of an aldimine binding moiety has long been known, its biological activity in terms of stimulating the adhesion, growth and differentiation of animal cells has not been described anywhere. Only the use of the aldimine bond to immobilize pharmaceuticals, enzymes and some other substances is known. In such systems, the aldimine binding moiety has only the function of a ligand to bind substances which, by their nature and by their nature, exhibit some biological activity.

The following substances, which can be formally divided into three groups, can be used to prepare the biologically active polymer compositions of the present invention:

I. Substances forming the basic polymer matrix.

This group consists of physiologically acceptable synthetic or natural polymers or copolymers. Depending on their nature, they may be hydrophilic or hydrophobic, soluble or insoluble, respectively. three-dimensionally cross-linked. The starting polymeric substances may be completely inert, or may contain either aldehyde or primary amine groups, which further participate in the formation of their own aldimine bonding moiety. Examples of suitable polymeric substances are in particular:

Synthetic polymers and copolymers based on methacrylic or acrylic acid esters or amides, such as glycol esters such as poles (2-hydroxyethyl methacrylate) or polyesters (4-hydroxybutyl acrylate), glycol ether esters such as (5-hydroxy-3-) poles polyhydroxyl esters such as polyglyceryl methacrylate or polysorbityl methacrylate, alkoxyalkyl esters such as poly (2-ethoxyethyl methacrylate), acrylic esters such as polymethyl methacrylate, and acrylic (n) polyesters which may optionally be N-mono or Ν, Ν-disubstituted such as poly (N- (2-hydroxypropyl) methacrylamide) or poly (Ν, Ν- (diethyl) acrylamide).

Synthetic polymers and copolymers based on other vinyl monomers, such as polystyrene, polyvinyl acetate, polyvinylmethyl ether, polyvinyl alcohol, polyvinylbutyral and polyvinylpyrrolidone.

Synthetic polymers and copolymers of ethylenically unsaturated monomers such as polyethylene, polypropylene, polyisobutylene, polyisoprene and polytetrafluoroethylene.

Synthetic polycondensates of, for example, polyamides, polyesters, polyethers and polydimethylsiloxane types.

Similarly, polymer hydrogels based on chitosan, alginate, agar, starch, dextran, agarose and other physiologically acceptable natural polymers or derivatives thereof may be used herein.

Examples of polymeric substances which contain reactive aldehyde groups include, in particular, polymers and copolymers of methacrolein or acrolein, methacrylaldehyde and oxo derivatives of polysaccharides, e.g. starch or cellulose.

Examples of polymeric substances containing primary amino groups include, but are not limited to, polyesters (2-aminoethyl methacrylate), polyesters (vinyl-4-aminobenzoate), 2-aminoethylcellulose, 4-aminobenzoylcellulose, gelatin, collagen or elastin proteins. . and other polyaminoderivatives.

All starting polymeric substances must be carefully free of undesirable impurities so that they do not have a cytotoxic effect. The polyfunctional aldehyde derivatives of polymers or copolymers must first be converted to the corresponding derivatives with primary amino groups, i.e., aldimines, before they come into contact with living cells.

II. Substances containing reactive aldehyde groups.

In addition to the polyfunctional aldehyde derivatives of the polymers or copolymers mentioned in group I, conventional low molecular weight aldehydes can also be used according to the present invention. These may be aliphatic or aromatic. further substituted, e.g., by a hydroxyl group, a keto group, or a carboxyl group. Examples of suitable substances are aliphatic aldehydes such as formic, acetic, butyric, enanthic, stearic and the like, aliphatic dialdehydes such as glyoxal or glutaraldehyde, ketonaldehydes such as methylglyoxal, hydroxyaldehydes such as beta-hydroxybutyral or acetaldol, semialdehydes dicarboxylate acids such as oxalic acid semialdehyde, aromatic aldehydes such as benzoic acid, salicylic acid, cinnamon, phenylacetic acid, and the like. Likewise, some biologically important substances containing a free aldehyde group such as glyceraldehyde or pyridoxal may be used herein.

It is important that all reactive aldehyde groups are further bound to the primary amino groups, i.e. in the form of aldimines, as the free aldehydes are highly cytotoxic.

For simplicity, substances II. groups are hereinafter referred to as aldehydes.

III. Substances containing primary amino groups.

In addition to the polyfunctional amine derivatives of the polymers or copolymers mentioned in group I, low molecular weight substances which contain at least one primary amino group in their molecule can be used according to the present invention. Examples of suitable substances are in particular aliphatic primary amines or diamines such as n-butylamine, ethylenediamine or hexamethylenediamine, amino acids such as 4-aminobutyric or 6-aminocaproic acid, alpha-amino acids such as glycine, lysine and other essential or non-essential amino acids, aminoketones such as aminoacetone, amino sugars such as glucosamine or galactosamine, biogenic amines such as ethanolamine or propanolamine, cysteamine, serotonin, tryptamine, adrenaline, dopamine, histamine, tyramine and others.

In addition to the aforementioned primary amines, other reactive substances may be used according to the present invention, such as the hydroxylamine, hydrazine, semicarbazide and thiosemicarbazide types.

Although the reaction of these compounds with aldehydes does not produce classical aldimines, the resulting binding moiety is chemically very related and also exhibits similar properties.

Said substances may have their amino groups bound, for example with hydrochloride or sulfuric acid, and must therefore be released first by addition of an equivalent of alkali hydroxide or carbonate. This is best done directly in the reaction environment, before creating your own aldimines.

For simplicity, substances III. groups are hereinafter generally referred to as amines.

For practical reasons, it is desirable that aldimine be well anchored in the material that constitutes the actual culture pad. The low stability of the aldimine linkage group in an acidic medium is generally known, but aldimines are very stable at physiological pH values of 7.2 to 7.4. This also enables their use for solved biological applications in the form of polymer compositions according to the present invention.

Since the list of substances in Groups I, II and III is extremely broad and each substance has its specific properties, the preparation and use of the resulting products should be assessed on a case-by-case basis. It should be noted here that the essence of the present invention is not the preparation of known or lesser known aldimines, but their entirely new application in the field of biology.

The examples below serve to illustrate the invention and do not limit the number of possible combinations, interrelationships, and uses of the individual substances.

Example 1

Synthetically, a poly (2-hydroxyethyl methacrylate) polyel-hydrogel hydrogel in a non-crosslinked 5 form, with an average molecular weight of 8.7x10 g / mol, was prepared as a thin film inside standard plastic petri dishes of 0 60 mm. The polymer film was obtained by evaporating a solution of said polyHEMA in pure ethanol, 10 wt% dry matter, in a dust-free environment at room temperature. The film in the dish was then swelled in 4 ml of an aqueous solution of glutaric aldehyde at a concentration of 2.5% by weight over two hours. The excess aldehyde solution was poured and poured into a dish with 5 ml of a 5% by weight aqueous solution of ethylenediamine hydrochloride. and 1 ml of 10% w / w sodium carbonate solution.

The reaction mixture was allowed to stand overnight until the polymer film stained with a reddish brown aldimine. The dishes were then leached with an excess of 0.9 wt. sodium chloride, whose pH was adjusted to 7.4 with phosphate buffer 0.016 M.

Plates with washed polymer films were sterilized by ultraviolet light with a germicidal lamp for 2 hours.

To test the biological properties of the polymer composite, a tissue culture technique was chosen where the test material served as a culture support. The plates were dosed with 8 to 10x10® dissociated embryonic muscle cells, which were obtained using known techniques from ten-day chicken embryos. Three dishes were tested for each type of polymer composition, and polyHEMA alone films served as a negative control. Plates coated with collagen, isolated by acidic extraction of rat tail tendons, served as a positive control here. Muscle cells were cultured under otherwise conventional standard conditions.

During the cultivation, the attachment of the cells to the culture plate was monitored by light microscopy and their further growth and differentiation were performed on the sixth day of cultivation, when the highest density of myotubes in the culture was reached, comparative readings were made. The highest density of myotubes was achieved in collagen control plates, i.e. 11 myotubes / mm. Almost the same results were obtained for the experimental aldimine plates, i.e. 10.4 myotubes / mm. In polyHEMA alone dishes used under otherwise identical conditions, no myotubes were formed, although single mononuclear cells were sporadically attached to the support. The result of this experiment suggests that the aldimine binding moiety clearly has the indicated biological activity.

Example 1 a d 2

The procedure of Example 1 was followed, but other starting materials were used to prepare aldimine polymer films.

To 100 ml of an aqueous solution of polyvinyl alcohol at a concentration of 5% by weight, of a molecular weight

7.2x10 ⁴ g / mol and a degree of hydrolysis of 98.5 mol%, 1.2 ml of a 40 wt.% Glyoxal were added. and the mixture was briefly homogenized. Polymer films were obtained by evaporating 0.5 ml of this mixture in each dish under the above conditions. The polymer films were then treated with 5 ml of a 2% by volume n-butylamine solution for 15 hours. The aldimine films were then washed in a conventional manner from all ballast substances, sterilized and subjected to appropriate tissue culture testing. Here, too, the desired biological activity was achieved, which is absolutely comparable to control samples of collagen alone.

Example 3

The procedure of Example 1 was followed, but the following materials were used:

An aqueous solution of high-quality gelatin, which is typically used to prepare photographic emulsions at a concentration of 15% by weight, was prepared using previous swelling of gelatin at room temperature for 2 hours and melting the swollen particles at 60 ° C with stirring. The warm gelatin solution was poured into the trays, the excess was poured so that inside the trays the remainder of the solution formed a continuous layer which then solidified at room temperature. The gelatin layers were then treated with an aqueous solution of glutaric aldehyde at a concentration of 2.5% by weight. for 5 hours, 4 ml per dish. After the excess aldehyde solution was poured, 5 ml of a 1% by weight aqueous glycine solution was poured into the dishes, the pH of which was adjusted to 7.4 by addition of 1M NaOH solution.

Washing, sterilization and proper testing of biological properties were the same as previously stated. The gelatinized aldimine-coated layers even had a higher biological activity for differentiating myoblasts into myotubes than that of control collagen. Layers prepared from hydrated gelatin alone lack these biological properties.

He attaches

Commercially available regenerated cellulose films, the so-called cellophane, were chemically modified to an oxo derivative with a content of 42.7 mol%. of aldehyde groups determined by oximation method.

The usual periodate method was used to oxidize the cellophane. The water-swollen films were then converted to the aldimine form with a solution of n-butylamine in isopropanol at a concentration of 1% by volume and treated in excess at room temperature for 15 hours. The treated cellophane films were then thoroughly washed with distilled water, equilibrated with saline, pH adjusted to 7.4 with phosphate buffer, and then sterilized with a germicidal lamp as previously described.

Discs cut with a 55 mm circular knife from the material were placed in plastic trays of 0 60 mm prior to sterilization and tested in tissue culture of embryonic muscle cells as shown in Example 1. Cellophane films with aldimine showed significant biological activity in cell attachment, further growth and differentiation. Cellophane films alone did not allow simple attachment of mononuclear cells to their hydrated surface.

Example 5

Conventional 60 mm plastic tissue culture plastic trays, made of polystyrene, were subjected to the following chemical treatment: 5 ml of a solution of n-butyral in amyl alcohol at a concentration of 10% by volume was dispensed into the dish and allowed to diffuse into the surface layer bowls. After 15 hours, the aldehyde solution was discarded from the plates, the plates were rinsed with pure amyl alcohol and dosed with 5 ml of a 2% by weight solution of hexamethylenediamine in amyl alcohol. The duration of action of the solution during which aldimine is formed in the surface layer of the plastic dish is only 4 to 6 hours, since diffusion; the second of the reactants is somewhat facilitated by prior swelling of the surface layer of said material. This is followed by thorough extraction of the dishes with aqueous ethanol at a concentration of 40% by volume and then with an excess of distilled water. The culture dishes thus treated had the desired biological properties, i.e., they enabled attachment, further growth and differentiation of embryonic muscle cells. The original dishes allowed for cell attachment, but in no way differentiated them.

Example6

Following Examples 1 and 2, the following water-soluble polymer materials were used in place of the aqueous polyvinyl alcohol solution: carboxymethyl starch derivative, hydroethyl cellulose derivative, dextrin, sodium alginate, copolymer containing 83.5 mol%. % Of 2-hydroxyethyl methacrylate and 16.5 mol. vinylpyrrolidone units, and poly (N- (2-hydroxypropyl) methacrylamide). Polymer films crosslinked with glyoxal and converted to aldimine, as described in Example 2, exhibited the desired biological activity independent of the type of polymeric material used.

Example 7

The procedure of Example 6 was followed, but instead of the aqueous glyoxal, a corresponding amount of glutaric aldehyde of 25 wt%, th. 2 ml per 100 ml of an aqueous solution of the respective polymer or copolymer. The results were similar and independent of the type of dialdehyde used.

*

Example 8

The procedure of Example 3 was followed, but instead of glycine, the following substances with primary amino groups were used: phenylalanine, 4-amino butyric acid, galactosamine, hydroxylamine, ethanolamine and phenylhydrazine. Regardless of the diversity of amine substances used, in all these cases the respective aldimines showed the desired biological activity.

Claims (5)

SUBJECT OF THE INVENTION 1. A polymer composition for the stimulation of adhesion, growth and differential ⁴ '> animal cells, characterized in that they consist of a matrix of synthetic or natural polymer or copolymer and containing a defined quantity of aldimine binding group of formula -CH = N-. 2. Polymeric composition according to claim 1, characterized in that a substance containing reactive aldehyde groups, such as aliphatic or aromatic aldehydes or dialdehydes, is involved in the formation of the aldimine linkage group. 3. Polymer compositions according to claim 1, characterized in that substances containing primary amine groups, such as primary amines, amino acids, amino sugars, hydroxylamine, hydrazine, semicarbazine or thiosemicarbazide, are involved in the formation of the aldimine linkage group. 4. Polymer compositions according to claims 1 to 3, characterized in that the aldimine bonding moiety is involved in either aldehyde or primary amine groups which are an integral part of the basic polymer matrix. 5. Polymer compositions according to claims 1 to 4, characterized in that they are a substrate in the cultivation of animal cells under conventional culture conditions, e.g. in the range of pH 7.2 to 7.4.