RU2657608C1 - Method for obtaining polysaccharide-containing polymer matrices - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the chemical-pharmaceutical industry, particularly to a method for producing polysaccharide-containing polymer matrices, which comprises mixing, in a volume ratio of 2:1:2, 30 % acrylamide solution in 1 % methylene bisacrylamide solution, a tris buffer with pH=7.7 and a 1–2 % aqueous polysaccharide solution, adding the resulting solution in a dropwise manner to n-hexane, stirring at a rate of 500 rpm for 1–2 minutes, adding 10 % ammonium persulphate solution to start polymerisation, stirring for 30 minutes, adding a sorbitan monooleate emulsifier while continuing to stir for 15 minutes, then distilling off the n-hexane on a rotary evaporator, distributing the resulting matrices on filter paper, drying, holding in a refrigerator for 48 hours at a temperature of 4–8 °C, washing with chloroform, holding in a desiccator at 4 °C for 100–120 h, wherein said polysaccharide solution used is either an aqueous solution of fucoidan obtained by fractional extraction from brown alga Laminaria saccharina, or an aqueous solution of mannan obtained by separation of yeast autolysate Saccharomyces cerevisiae at a temperature of 45–50 °C for 20–24 hours, or a mixture, in volume ratio of 1:1, of said aqueous solution of fucoidan and said aqueous solution of mannan.

EFFECT: invention enables to obtain polysaccharide-containing matrices with a long shelf life, used to include a wide range of biologically active substances.

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Description

The invention relates to the medical and pharmaceutical industries and is intended to produce polysaccharide-containing polymer matrices. The invention can also be used in cosmetics, chemical industry and agriculture.

A known method of producing microcapsules (patent US 33699000, 1968), which uses modified with phthalic anhydride gelatin. The disadvantage of gelatin-based capsules is that they are not suitable for encapsulating hygroscopic substances due to the high moisture content in the capsule shell.

Known technologies for creating soft capsules based on non-gelatin polymers such as starch, cellulose, dextrin, pullulan, gellan, mannan, polyvinyl alcohol and their derivatives, further comprising a gelling hydrocolloid, for example carrageenan, alginate or guar gum (Patent RU 2428971, RU 2005135 Application RU 2010145522). When creating capsules using these methods, additional costs are required for the second gelling agent, selection of the optimal ratios of the two polymers, selection of a complexing agent for imparting gel-forming properties (Ca ²⁺ , Na ⁺ , K ⁺ cations), which leads to more expensive technology and higher cost of the final product.

A known method of producing shells based on chitosan and salts of alginic acid for microcapsules containing phospholipid micelles (patent RU No. 2411077 dated 02.10.2011), in which the cores of microcapsules are sequentially kept in a solution of medium or low viscosity chitosan in acetic acid, in a solution of alkaline earth metal chloride , in a solution of sodium alginate and re-in a solution of alkaline earth metal chloride. The disadvantages of the method include the use of a specific polysaccharide polymer of salts of alginic acid, which gives fragility and softness to the shells of liposomes, which are easily destroyed by mechanical stress. The creation of such a shell does not allow the production of drugs according to the technology for soft gelatin capsules, which leads to the creation of a new production line and economic losses.

The most suitable thickener and gelling agent, which forms elastic reversible gels and has the most suitable properties for encapsulation, is a polysaccharide - agar-agar. In physical and chemical properties, it is similar to a polymer of protein origin - gelatin and therefore can be used as an alternative material for creating a capsule shell. The invention is known for creating capsules having a shell based on agar (Application RU 2011109018, Application RU 2007139819). The disadvantage of these capsules is that their shell, without increasing its density, is easily destroyed by mechanical stresses and, therefore, difficulties arise in the production of capsules, since the washing process from the process oil is difficult and is accompanied by large losses, which is not economically feasible.

A known method of manufacturing microparticles with an improved release profile (patent RU No. 2291686 dated 01/20/2007), in which several stages of formation of microparticles are carried out: a) a composition containing the active substance is added to the organic polymer solution and dispersed in it, b) an emulsion or dispersion , formed in stage a), is added to the external phase and dispersed in it, while the external phase at the time of addition has a temperature from 0 ° C to 20 ° C, and the external phase in the form of an aqueous solution of sludge can be used as the external phase and in the form of an “oil” phase containing an emulsifier and / or protective colloid, and c) the organic solvent is removed by exposing the dispersion or emulsion formed in step b) to a pressure of less than 1000 mbar or introducing an inert gas into the dispersion or emulsion formed in step b) . According to the W / O / W method, the aqueous phase (W1) containing the active substance is first dispersed in an organic polymer solution (O), then the resulting W1 / O emulsion is dispersed in the next aqueous phase (the so-called external phase W2). The polymer coacerves by removing the organic solvent and forms microparticles. An appropriate dispersion method may affect particle size. The formation of microparticles also depends on the possibility of evaporation of the solvent. Therefore, the W / O / W double emulsion method is also referred to as the “technical method of separation by evaporation of the solvent”. After curing the microparticles and removing the solvents, microparticles containing the active substance are obtained. Often such microparticles contain viscosity enhancers, for example gelatin. And according to the S / O / W method, the active substance is not in an aqueous solution, but is presented in the form of a solid (S). The solid is then dispersed directly in the organic phase (O). Finally, the S / O / O method, in which the external phase is not an aqueous phase, but is a non-aqueous phase containing a protective colloid or emulsifier. The disadvantage of this method is the technical complexity of the manufacture of microparticles.

A known method for producing dry liposomal drugs is carried out by mixing in a container of a biologically active substance (BAS), phospholipids, a solvent and a powdery filler until the composition is homogeneous, after which a liposomal preparation is obtained by distilling off the solvent with stirring under reduced pressure (RU 2130771, 1999) . The disadvantages of this method are the limited scope of its application due to the possibility of loss of activity of many biologically active substances, in particular proteins and enzymes, due to the denaturing action of organic solvents.

The closest to the proposed technical essence and the achieved positive effect is the method of producing microspheres (patent RU 96121918 from 02.20.1999), characterized in that they contain a gelled polar core, on which are applied concentrically and alternately n lipid double layers or n aqueous layers in the liquid state and n gelled polar layers, n being an integer equal to or greater than 1, and obtained by delipidation of liposomes, called liposomes with a gelled polar core, which contain, according to enshey least one outer lipid bilayer and at least one inner polar aqueous phase containing a gelified substance. The gelled substance is selected from gelled compounds, polymerizable or not, such as polysaccharides, polypeptides or polyacrylamides. Gelatin, agarose and carrageenans are selected as the gelled polymerizable substance, and the gelled polymerizable substance is selected from polyacrylamide gels. The disadvantage is the use of gelatin, since the main factors limiting the use of gelatin capsules are: susceptibility of the gelatin to microbial contamination, the capsules can break down or change their shape under direct sunlight, at a temperature of more than 40 ° C or at a humidity of more than 75%.

The technical result of the present invention is the development of a more efficient technology for producing polysaccharide-containing matrices, with a long storage time, containing biologically active components used to include a wide range of biologically active substances in the matrix.

To achieve a technical result, the proposed method for producing polymer matrices uses natural polysaccharides (fucoidan and mannam) included in the structure of a polyacrylamide gel.

A method of obtaining a polymer matrix is as follows. The matrix is obtained by mixing in a volume ratio of 2: 1: 2 a 30% solution of acrylamide in a 1% solution of methylenebisacrylamide, Tris buffer with pH = 7.7 and a 1-2% aqueous solution of fucoidan polysaccharide obtained by fractional extraction from brown algae Laminaria saccharina. The resulting solution was added dropwise to n-hexane, stirred at a speed of 500 rpm for 1-2 minutes, a 10% solution of ammonium persulfate was added to start polymerization, and stirred for 30 minutes. To prevent aggregation of the resulting emulsion, the emulsifier sorbitan monooleate is added while stirring for 15 minutes. Then n-hexane is distilled off on a rotary evaporator, the resulting matrices are distributed on filter paper, dried, kept in a refrigerator for 48 hours at a temperature of 4-8 ° C, washed with chloroform, kept in a desiccator at 4 ° C for 100-120 hours.

Implementations of this method are possible when a 1-2% aqueous solution of mannan obtained by separating a Saccharomyces cerevisiae yeast autolysate at a temperature of 45-50 ° C for 20-24 hours or a mixture in volume ratios of 1: 1 is used as a 1-2% aqueous polysaccharide solution 1 1-2% aqueous solution of fucoidan obtained by fractional extraction from brown algae Laminaria saccharina, and 1-2% aqueous solution of mannan obtained by separation of the yeast autolysate Saccharomyces cerevisiae at a temperature of 45-50 ° C for 20-24 hours

This method of obtaining microcapsules is illustrated by specific examples.

Example 1. In a reactor equipped with a stirrer, add 5 ml of a 30% solution of acrylamide in a 1% solution of methylenebisacrylamide, 2.5 ml of Tris buffer with pH = 7.7 and 5 ml of a 1-2% aqueous solution of fucoidan. The resulting solution was added dropwise to n-hexane, stirred at a speed of 500 rpm for 1-2 minutes, a 10% solution of ammonium persulfate was added to start polymerization, and stirred for 30 minutes. To prevent aggregation of the resulting emulsion, the emulsifier sorbitan monooleate is added while stirring for 15 minutes. Then n-hexane is distilled off on a rotary evaporator, the resulting matrices are distributed on filter paper, dried, kept in a refrigerator for 48 hours at a temperature of 4-8 ° C, washed with chloroform, kept in a desiccator at 4 ° C for 100-120 hours.

Example 2. In a reactor equipped with a stirrer, add 5 ml of a 30% solution of acrylamide in 1% solution of methylenebisacrylamide, 2.5 ml of Tris buffer with pH = 7.7 and 5 ml of a 1-2% aqueous mannan solution. The resulting solution was added dropwise to n-hexane, stirred at a speed of 500 rpm for 1-2 minutes, a 10% solution of ammonium persulfate was added to start polymerization, and stirred for 30 minutes. To prevent aggregation of the resulting emulsion, the emulsifier sorbitan monooleate is added while stirring for 15 minutes. Then n-hexane is distilled off on a rotary evaporator, the resulting matrices are distributed on filter paper, dried, kept in a refrigerator for 48 hours at a temperature of 4-8 ° C, washed with chloroform, kept in a desiccator at 4 ° C for 100-120 hours.

Example 3. In a reactor equipped with a stirrer, add 5 ml of a 30% solution of acrylamide in 1% solution of methylene bisacrylamide, 2.5 ml of Tris buffer with pH = 7.7 and 5 ml of a mixture of 1-2% aqueous mannan and 1-2 % aqueous solution of fucoidan in a volume ratio of 1: 1. The resulting solution was added dropwise to n-hexane, stirred at a speed of 500 rpm for 1-2 minutes, a 10% solution of ammonium persulfate was added to start polymerization, and stirred for 30 minutes. To prevent aggregation of the resulting emulsion, the emulsifier sorbitan monooleate is added while stirring for 15 minutes. Then n-hexane is distilled off on a rotary evaporator, the resulting matrices are distributed on filter paper, dried, kept in a refrigerator for 48 hours at a temperature of 4-8 ° C, washed with chloroform, kept in a desiccator at 4 ° C for 100-120 hours.

The obtained polysaccharide-containing polymer matrices showed high stability and great potential for controlled delivery of biologically active substances, since they have high loading efficiency (up to 60% with varying polysaccharide concentrations, for example, bovine serum albumin) and the degree of encapsulation (up to 40%, for example, the same protein) . These microstructural objects can be used in the cosmetic and pharmaceutical industries.

Advantages of using the obtained polymer matrices include:

- polymer matrices can be stored for 3 years without aggregation and loss of activity of the encapsulated substance;

- the use of biocompatible polysaccharides in the preparation of polysaccharide-containing polymer matrices allows the use of microcapsules as drugs that provide pH-sensitive release of the active encapsulated substance;

- polymer matrices based on natural polysaccharides with the encapsulated substance included can be used in biotechnology as containers containing active substances.

The advantage of using fucoidan and mannan in the production of polymer matrices is as follows:

- biocompatibility, the involvement of carbohydrate components in metabolism, ensuring a decrease in allergic reactions to polymer matrices;

- additional provision of the body with minor carbohydrates fucose and mannose;

- fucoidan and mannan have a wound healing, anti-inflammatory effect.

Claims (1)

A method of producing polysaccharide-containing polymer matrices, characterized in that they are mixed in a volume ratio of 2: 1: 2 30% solution of acrylamide in 1% solution of methylenebisacrylamide, Tris buffer with pH = 7.7 and 1-2% aqueous solution of polysaccharide, then the resulting solution added dropwise to n-hexane, stirred at a speed of 500 rpm for 1-2 minutes, to start polymerization add 10% solution of ammonium persulfate, mix 30 minutes, to prevent aggregation of the emulsion, add the emulsifier sorbitan monooleate, while stirring 15 min, then n-hexane is distilled off on a rotary evaporator, the resulting matrices are distributed on filter paper, dried, kept in a refrigerator for 48 hours at a temperature of 4-8 ° С, washed with chloroform, kept in a desiccator at 4 ° С for 100 -120 h, while the indicated polysaccharide aqueous solution is either an aqueous solution of fucoidan obtained by fractional extraction from brown algae Laminaria saccharina, or an aqueous solution of mannan obtained by separation of the Saccharomyces cerevisiae yeast autolysate 5-50 ° C for 20-24 hours, or a mixture in a volume ratio of 1: 1 of the specified aqueous solution of fucoidan and the specified aqueous solution of mannan.