RU2647466C1 - Method for obtaining native protein with prolonged action in the composition of polymeric nanospheres and resorbed microspheres for delivery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, in particular to nanomedicine, which uses biodegradable nanospheres and microspheres to include biologically active proteins in their composition to stabilize their structure. Method provides for the preliminary inclusion of a histone of animal origin in the composition of the matrix of nanospheres with a diameter of not more than 1000 nm, which are obtained on the basis of dextran, and the subsequent inclusion of the histone-containing nanospheres into the resorbable microspheres, which are prepared on the basis of the polylactide-glycolide copolymer, the microspheres are deposited with ethyl alcohol, the deposit is filtered and dried. Extraction of histone from the resorbed microspheres takes place for at least 50 days.

EFFECT: invention ensures preservation of the native structure of the protein due to its inclusion in the composition of polymeric nanospheres, and increases the reliability and simplifies the method of obtaining native protein in the composition of polymeric nanospheres and resorbable microspheres that are intended to prolong the physiological action of native protein used for therapeutic purposes.

1 cl, 3 ex, 2 dwg, 1 tbl

Description

The invention relates to medicine, in particular to nanomedicine, which uses nanotechnology and nanostructure systems in the field of nanodiagnostics, nanotherapy and tissue engineering.

Nanoparticles are defined as dense colloidal particles, the sizes of which lie in the range from 10 to 1000 nm. Nanoparticles can mainly be constructed from macromolecules or ensembles of macromolecules, such as synthetic preformed polymers (e.g., polylactide-glycolide copolymer, polylactide-polyethylene glycol), or from natural macromolecules, such as alginate, chitosan, gelatin, and also from polysaccharides ( dextran). In nanoparticles, the drug or biologically active macromolecular compounds are either dissolved, incorporated, encapsulated, or adsorbed, or chemically bound to the matrix of nanoparticles [1]. Depending on the manufacturing method, the nanoparticles can be obtained in the form of nanospheres or nanocapsules. Nanocapsules is a system in which a drug is enclosed in a capsule cavity surrounded by a specific polymer membrane. Nanospheres are a matrix system in which a drug is physically and uniformly dispersed in a polymer matrix.

Biodegradable micro- and nanoparticles are used to include biologically active macromolecules in their composition to increase their stability, bioavailability and increase the therapeutic index with continuous and controlled release over a long period of time [2-4].

Three possible mechanisms for the release of drugs or biomacromolecules included in micro- and nanoparticles are considered:

- as a result of diffusion through the pores;

- as a result of enzymatic cleavage or degradation of the polymer;

- upon dissociation of a drug or biomacromolecules associated with the polymer.

The release of encapsulated macromolecules as a result of polymer degradation is considered as a mechanism for the controlled release of macromolecules, which depends on the composition of the polymer (homopolymer or heteropolymer), the molecular weight of the polymer, and the ratio of the monomers included in the heteropolymer.

The ability of targeted delivery of drugs to the target is determined by such factors as the size of the nanoparticles, their surface charge, hydrophilicity and the nature of the modification of their surface [5-7].

Based on this, in each case, for a new class of protein molecules intended for encapsulation in nanoparticles, individual design of the technological process of their preparation is required, i.e. the process of formulating, taking into account the peculiarities of their physicochemical properties, the choice of the target of their action and the routes of introduction into the body.

The process of formulating nanospheres with preformed polymers involves the removal of an organic solvent from the formulation system [8]. In this method, the polymer is dissolved in an organic solvent. The drug is dispersed in a solution of the preformed polymer, then the mixture is emulsified in an aqueous solution to obtain an emulsion as oil (O, oil) in water (W, water), i.e. an O / W emulsion using an emulsifying agent. After the formation of the emulsion, the organic solvent is removed either at elevated temperature, or in vacuum, or with prolonged stirring, which leads to the formation of nanoparticles. The method of spontaneous emulsification (organic solvent diffusion method) is considered as a modified version of the organic solvent removal method. In this method, a water-soluble organic solvent, along with a water-insoluble organic solvent, is used as the organic phase. Due to the spontaneous diffusion of a water-soluble organic solvent, turbulence occurs at the interface between the two phases, which leads to the formation of small particles. This technique for producing nanospheres is also known as a nanoprecipitation method.

In fact, the formulation of nanoparticles is based on the use of nanoemulsion systems, which can be considered as matrices for producing nanoparticles.

Microspheres based on a resorbable polylactide-glycolide copolymer are often used to create delivery systems for biologically active macromolecules. However, there are a number of problems associated with the use of these systems. First, organic solvents are used to prepare protein-loaded microspheres, which adversely affect the stability of protein molecules.

Secondly, proteins are released from the microspheres either through the pores or by degradation of the polymer matrix of the microspheres. Thirdly, during the degradation of the polymer matrix, degradation products are generated, which, possibly, leads to a decrease in the pH in the system and chemical degradation of the encapsulated protein. Along with this, the creation of microspheres for prolonged release of protein over an extended period of time (up to several months) requires the development of a method for efficiently encapsulating protein in microspheres.

Optimization of the processes for the preparation of microparticles based on a polylactide-glycolide copolymer loaded with a protein for their subsequent prolonged release requires complete protection of the native protein structure both during its preparation and its use. Denaturation and aggregation of protein molecules not only cancels their therapeutic activity, but also causes side effects, such as immunogenicity and toxicity.

The loading of a protein drug into nanoparticles is achieved in two ways [9]. The first method is based on the inclusion of the drug in the process of obtaining nanoparticles. The second method is based on the adsorption of the drug on the already formed nanoparticles as a result of the process of incubation of the nanoparticles in the drug solution.

In addition to the method of incorporation and adsorption of a drug, methods are known for chemical conjugation of drugs on the surface of nanoparticles [4].

In all known microparticle preparation methods, the starting position is to dissolve the polylactide-glycolide copolymer in an organic solvent, usually in methylene chloride or ethyl acetate (organic phase).

Two different approaches are considered in the process of encapsulating a protein preparation, which differ depending on the physical state of the protein. Either protein solutions (soluble form) are used for encapsulation, or a freeze-dried protein preparation is used, or the protein is converted to an insoluble form and “solid” particles of the protein are obtained (“solid” form of the protein). To obtain "solid" particles containing a protein preparation, protein stabilizers are used, such as salts, sugars, detergents, metal ions.

However, many protein stabilizers limit the duration and continuity of the release of a protein after it is encapsulated in a continuous release system.

Modern strategies for the formulation of protein preparations can be attributed to two categories:

- formulating proteins into solid particles before encapsulating them in order to increase their resistance to organic solvents;

- nano-encapsulation of proteins in polymeric materials that are soluble in water, which avoids contact with organic solvents that are used to dissolve biodegradable polymers in continuous release systems [10].

Known methods for converting proteins into solid particles before microencapsulation in polymer structures for their subsequent continuous release from the system [11]. Protein particles are formed by phase separation of a protein-polyethylene glycol (PEG) co-solution during freezing. Since PEG is soluble in organic solvents, purified protein particles are obtained by sublimation of a joint solution of protein and PEG, followed by removal of the PEG phase. The principle of this method is based on the phase separation of the PEG-protein aqueous mixture induced by the use of “freeze condensation” technique. Protein particles are formed by phase separation of a protein-PEG co-solution during freezing. As a result of the subsequent lyophilization process, spherical microdomains of a protein dispersed in the continuous “solid phase” of PEG can be formed under specific conditions. After the PEG is dissolved in methylene chloride, a suspension of micronized protein particles is obtained. The resulting protein particles are added to a solution of a polylactide-glycolide copolymer in an organic solvent to form a suspension of a protein-in-solution of a polylactide-glycolide copolymer and emulsified in an aqueous continuous phase containing polyvinyl alcohol as an emulsifier to form complex microspheres. This process is known as S / O / W (particulate matter - in oil - in water) microencapsulation.

However, the protein particles during encapsulation are surrounded by a hydrophobic polymer of the matrix of microspheres (for example, a polylactide-glycolide copolymer). Therefore, the disadvantages of this method include both the presence of organic solvents and a hydrophobic polymer matrix.

It is known that dextran microspheres are obtained using emulsification in a water-in-water system [12]. This technique is based on the inability to mix water-soluble polymers in aqueous solutions. The polymers remain in solution, but are stratified into two aqueous phases when the concentration exceeds a certain value. When aqueous solutions of PEG and dextran are mixed, a biphasic system is obtained depending on the molecular weight and concentration of both polymers. After the emulsion is prepared, the dextran particles are chemically crosslinked (methacrylate residues are introduced into the dextran first). In this case, microparticles acquire the properties of hydrogels. Crosslinked dextran particles are separated by centrifugation, with an average particle size ranging from 2.5 to 25 microns. The release of protein from such microparticles is carried out using endo-dextranase previously included in the microspheres. Moreover, the release of protein from the microspheres is linearly related to the amount of dextranase incorporated.

Thus, dextran microspheres, which undergo enzymatic degradation, have some disadvantages. First, the regulation of protein release is determined by the change in the number of crosslinks with methacrylate. Secondly, it is necessary to administer the target protein together with dextranase.

A known method for loading proteins into polysaccharide particles in the absence of hydrophobic-hydrophilic surface tension. This method is based on the separation of two aqueous phases [13]. In this method, to prevent the fusion of the polysaccharide-dispersion phases and the formation of a blocking phase, the third component is introduced into the dextran-PEG double aqueous phase system, using sodium alginate [14]. The anionic polysaccharide sodium alginate forms a diffusion charged layer around dispersed dextran droplets, this keeps them from merging with each other. As a result, the two-phase system becomes stable in the liquid-liquid emulsion.

Thus, proteins that are preferably separated into the dextran-dispersion phase when two aqueous phases are separated (see 11), in this case, they are preliminarily transferred to the phase of dense glassy (glassy, non-crystalline or glassy) nanoparticles that are resistant to organic solvents during lyophilization. Such dextran nanoparticles having a diameter of 200-500 nm are incorporated into microspheres based on a polylactide-glycolide copolymer using the S / O / W microencapsulation process.

The “packing” of proteins into dextran vitreous nanoparticles before microencapsulation protects the proteins from degradation, reduction of biological activity and adsorption on polylactide-glycolide copolymer both during their formation into microspheres and during their continuous release from the polymer system for a long period of time.

However, this system has some disadvantages. For example, the need to use relatively concentrated aqueous solutions of two hydrophilic polymers (polysaccharide and PEG solution) and the use of a third water-soluble polyelectrolyte polymer that carries a negative charge both to form a water-water emulsion and to stabilize the dispersion polysaccharide phase from fusion. In addition, some proteins can interact intensively with polyelectrolytes due to ionic interactions.

A known method of preparing polysaccharide vitreous microparticles that have a diameter of less than 10 microns, as well as nanoparticles that have a diameter of 500 nm and contain degradable agents such as proteins, peptides, genetic engineering producers, vaccines, antibodies, viruses, liposomes. The method is based on the principle of emulsification in the water-water system at low temperature and phase separation induced by freezing [15, 16].

This method provides the preparation of polysaccharide particles containing structurally unstable agents, such as proteins without the use of polyelectrolytes. However, without the formation of a charged diffusion layer around the dispersion phase (see 13-14), the dispersion droplets merge, and the formation of two phases is blocked immediately after the termination of mechanical stresses (for example, mixing). To avoid the effect that leads to blocking the formation of phases, use technological methods that stabilize water droplets in an aqueous continuous phase without the use of polyelectrolytes. Two methods are used to stabilize dispersed water droplets from aggregation and the merging of two phases, which are based on carrying out processes at a low temperature: low temperature in the preparation of an aqueous emulsion and freezing, inducing biphasic water separation. The disadvantages of this method include the ability of proteins dissolved in a PEG solution to form particles upon freezing-drying. In other words, freeze-induced phase separation can also occur for protein-PEG solutions (see 11). Therefore, proteins that form their own particles along with dextran particles will co-precipitate from the PEG solution while continuing to freeze.

-аминогруппами лизиновых остатков в молекуле гистона и активными группами в цепях полисахаридов. Количество ковалентно связанного гистона с поверхностью микросфер составляет от 60 до 200 мкг белка на 1,0 г наносфер.A known method of preparing polymer microspheres, which are obtained on the basis of crystallized dextran by emulsification of an aqueous solution of dextrans with a molecular weight of 500 kDa and 10 kDa in an organic emulsion medium, which is used as a vegetable oil [17]. Such an emulsion of two immiscible liquids is subjected to ultrasonic treatment. Then, the resulting suspension is introduced into acetone to crystallize the polysaccharide in an emulsion medium. The formed microspheres are separated from the suspension by centrifugation. Finally, the precipitate of the microspheres is dried at room temperature. Samples of microspheres from crystallized dextran according to electron microscopy have a diameter of not more than 1000 nm. The known method involves the covalent binding of positively charged proteins, which use histones, with the surface of dextran microspheres. Covalent binding is due to the reaction between

-amino groups of lysine residues in the histone molecule and active groups in polysaccharide chains. The amount of covalently bound histone with the surface of the microspheres is from 60 to 200 μg of protein per 1.0 g of nanospheres.

The disadvantage of this method is the surface modification of dextran microspheres by positively charged histones, which prevents the creation of microparticles for prolonged circulation after their introduction in vivo [17, 18].

It is known that the adsorption of certain plasma proteins, such as IgG, albumin, complement components (the common name of these proteins is “opsonins”), as well as histones [19, 20], on the surface of microparticles and phagocytosis of microparticles are interrelated processes. Opsonins act as a “bridge” between microparticles and phagocytes. When microparticles are administered intravenously, they are easily recognized by the body’s immune system and then removed from circulation (immune clearance).

Therefore, the study of the process of opsonization of nanocarriers, along with the establishment of their distribution in various organs after in vivo administration, as well as subsequent excretion (clearance) from the body, is equally important for the development of nanomedical technologies.

A known method of preparing a composition for the controlled release of recombinant human insulin during oral administration, which is the closest in technical essence to the proposed invention and selected as a prototype [21]. The known composition includes microparticles, which are preliminarily prepared on the basis of crystallized dextran and recombinant human insulin, which is added to an aqueous suspension of microparticles, moreover, insulin is adsorbed on the surface of microparticles, which are obtained on the basis of an aqueous solution of dextran of 55-65% concentration with a molecular weight of 20 up to 75 kDa, then the solution is kept for 3 hours at a temperature of 60 ° C, and after crystallization of dextran, microparticles are formed as a result of water evaporation, which precipitate from the solution by centrifugation and dried at a constant temperature, the microparticles having a diameter of more than 500 nm and not more than 5000 nm, then an aqueous suspension of microparticles is prepared and an equal volume of insulin is added to the resulting suspension, which is dissolved to a concentration of 15-30% in distilled water, the suspension is then centrifuged again and dense microparticles containing insulin are obtained, the insulin being adsorbed on the surface and / or in the pores of the dextran matrix.

A disadvantage of the known method of preparing a composition for the controlled release of insulin, in which insulin is adsorbed on the surface of a dextran matrix, is the availability of the protein to the action of proteolytic enzymes, in particular, during its release from the composition upon oral administration to animals. Therefore, the action time of insulin is limited to one day, as shown in the experiment.

In addition, microparticles obtained on the basis of crystallization of dextran represent a population of microparticles heterogeneous in size, the diameter of which lies in the range of more than 500 nm and not more than 5000 nm.

It should be noted that microparticles with a diameter of 10 to 1000 nm are currently defined as dense colloidal nanoparticles or nanospheres [1]. Therefore, it can be assumed that in this population of a microparticle heterogeneous in size, there are both nanoparticles or nanospheres, and microparticles or microspheres.

The present invention is devoid of these disadvantages due to the preliminary inclusion of a biologically active protein, which is used histone of animal origin, in the structure of the matrix of dextran nanospheres in the process of their preparation. Such histone-containing nanospheres are intended for their subsequent incorporation into resorbable microspheres based on a polylactide-glycolide copolymer for prolonged release of the native histone.

In the scientific and technical literature, protein release profiles from various microparticles are classified into four categories: A, B, C, D. The separation is based on the size of the initial burst release, the duration of protein release, and the kinetics of the steady state release after burst release, respectively, in each category [ 22]. Burst release is considered the initial release during the first 24 hours. In Category A, the initial burst release (with a yield of more than 30%) is followed by a minor incremental release. In category B, a low initial burst release (less than 30%) is followed by a minor incremental release. If the formulated protein, when released, has an initial burst release of less than 30%, but the total protein release is more than 60%, then this type of release is categorized as Category C. Category D is considered as an ideal low burst release protein release profile, followed by steady state of release of all protein loaded into the system.

Incomplete release of a bioactive protein from microspheres based on a resorbable polylactide-glycolide copolymer is associated with one of the following phenomena: covalent or non-covalent aggregation, hydrolysis, or nonspecific adsorption on a polymer matrix. The instability of the protein during the release process may be due to a local decrease in pH inside the microparticles due to the delay of the acidic products of polymer degradation.

To clarify the properties of proteins, in particular the histones used in the claimed invention, the applicant considers it appropriate to give their characteristics in the appendix to this description (see Appendix).

The technical result of the claimed invention is the preservation of the native structure of the protein, previously included in the structure of the matrix of nanospheres with a diameter of not more than 1000 nm, prepared on the basis of dextran, increasing the reliability and simplification of the method of incorporating protein and cheapening the method of producing nanospheres intended for subsequent inclusion in resorbable polymer microspheres, which obtained on the basis of a polylactide-glycolide copolymer, which ensures prolonged histone release as a result of resorption microspheres for at least 50 days and has several advantages, which are mainly due to the fact that:

1) the physical incorporation of protein into the nanospheres is a less complex process than the chemical attachment of nanospheres to the surface;

2) obtaining nanospheres with proteins included in their composition is cheaper and can have an industrial scale in comparison with the chemical attachment of a protein to the surface of nanospheres;

3) the encapsulation of nanospheres with the proteins included in their composition in microspheres with the prolonged release of proteins from their composition ensures their effectiveness and specificity of action due to:

- protection of proteins from the action of proteolytic enzymes and hydrolytic degradation when interacting with biological components in vivo;

- eliminating the potential toxic effects of proteins when directly introduced into the body in various ways;

- increase the biological half-life of the protein in the circulation system and the possibility of reducing therapeutic doses of the protein;

- an increase in the therapeutic index (ratio between LD 50 and ED 50, where LD 50, semi-lethal dose and ED, 50% effective dose), due to the continuous release of the protein drug over a long period of time;

- the advantages of using continuous delivery of protein drugs for therapeutic purposes, which consist in maintaining its concentration in serum within the “therapeutic window” (therapeutic window, the time period within which the concentration of the drug is below the toxic value and above the therapeutic value).

The technical result is achieved by the fact that in the known method for the preparation and use of an oral composition containing recombinant human insulin, which includes known and common features of the claimed new method, where the composition contains an aqueous suspension of microparticles with a diameter of more than 500 nm and not more than 5000 nm, which are obtained on the basis of crystallized dextran and an aqueous solution of insulin, which is introduced into a previously prepared aqueous suspension of microparticles, where insulin is adsorbed on the surface of dex trans matrix, the proposed method uses a protein of animal origin, and the protein used is freeze-dried histone, which is dissolved to a 1.0% concentration in distilled water, then an aqueous solution of histone is added in an equal volume to an aqueous solution of dextran 6% concentration with a molecular weight of 50 to 70 kDa, then the solution containing dextran and histone is kept for 30 ± 5 min at a temperature of 6 ± 2 ° C, then this solution is added to an organic liquid, which is used as a plant oil, in an amount of 3.5 volumes from the volume of the initial solution, and a mixture of two immiscible liquids is obtained, which is stirred and re-aged for 30 ± 5 min at a temperature of 6 ± 2 ° C and a suspension is obtained, then the suspension is subjected to ultrasonic treatment for 45 ± 5 s at a radiation power of 100 W, then the resulting suspension is centrifuged, and centrifugation is carried out for 5 ± 1 min at 2000 g, then the precipitate is dried at room temperature and nanospheres with a diameter of not more than 1000 nm containing histone are obtained, after which prepare a primary suspension of nanospheres in a pre-prepared polymer solution, which is used as a resorbable polylactide-glycolide copolymer with a molecular weight of from 50 to 80 kDa with a molar ratio of monomers equal to 75:25, which is dissolved to a 10% concentration in a hydrophobic organic solvent, the methylene chloride being used is stirred for 60 ± 5 min at a temperature of 6 ± 2 ° C; moreover, nanospheres with histone are added to the polymer solution at a weight ratio of nanospheres to polymer, SG 1:50 then added to a solution of ethyl alcohol in an amount of about 10 volumes relative to the volume of the starting solution, then the mixture was stirred to give a secondary slurry which is subjected to dispersion with stirring for 10 min. at room temperature for uniform distribution of the polymer in the organic solvent and the formation of microspheres, then the microspheres are precipitated from the suspension by adding chilled ethyl alcohol, which is added in a 100-fold volume relative to the volume of the suspension, then the precipitate of the microspheres is placed on a fine-porous glass filter and filtered, then the precipitate was collected and dried at room temperature for 8 ± 2 h, and the resulting resorbable microspheres containing nanospheres with histone containing m protein per 1.0 mg dry weight of microspheres from 2.0 to 4.0 μg is introduced into a model incubation medium, which is used as a phosphate-buffered saline solution, pH 7.2 and incubated at + 21 ° C for 2 months, while monitoring every day for at least 50 days the concentration of protein in the incubation medium, after which the resulting native proteins as part of the resorbable microspheres are used as the basis for prolonging the physiological action of proteins.

Thus, the preliminary inclusion of protein in the matrix of nanospheres and the subsequent inclusion of nanospheres in the composition of resorbable microspheres ensures the preservation of the native protein structure due to the resistance of the protein to the action of proteolytic enzymes, as well as to organic solvents and the acid environment in the composition of the microspheres during prolonged release of protein .

The claimed method was tested in laboratory conditions at the laboratory base of St. Petersburg State University. The results of the studies are illustrated by the following examples and drawings.

In FIG. 1 presents the results of electron microscopy of nanospheres obtained on the basis of dextran (× 1000). Adsorption on the surface of the glass. It was shown that nanospheres have an average diameter of not more than 1000 nm and form a uniform layer on the glass surface. The layer of nanospheres on the glass surface is resistant to the action of the incubation medium during long-term storage.

In FIG. Figure 2 shows a graph of the release of histone H1 from resorbable microspheres prepared on the basis of a polylactide-glycolide copolymer containing dextran nanospheres with histone H1 included in their composition in a model incubation medium depending on the time from the start of incubation in phosphate-buffered saline for 50- that day. The abscissa shows the incubation time in hours.

The ordinate is the amount of protein released as a percentage of the initial amount of protein in the microspheres.

The graph shows that during the first 2 days the amount of histone H1 released is 20-22% of the amount of the original protein. After 120 hours, the protein yield was 43%, after 240 hours - 79%, after 480 hours - 84% and after 720 hours - 92%. Full release of histone H1 occurs within 50 days (1200 h) and is 97% of the start of incubation.

Example 1. In this series of experiments implementing the present invention, a method for producing a biologically active protein in the composition of polymer nanospheres involves the use of a stable polymer suspension of two immiscible liquids (aqueous solution - organic liquid), which are used as an aqueous solution of dextran containing a protein preparation (dispersion phase) and organic liquid, which is used as a vegetable oil (continuous phase).

In the particular case of the method is as follows. As a model protein using histone H1. Obtaining histone H1 is as follows. Preliminarily, a preparation of total histone sulfate from calf thymus tissue is obtained in a known manner [23]. Then, histone H1 is separated from other histone classes from an aqueous solution of total histone in a known manner [24].

An aqueous solution of dextran and an aqueous solution of protein are prepared, and freeze-dried histone is used, which is dissolved to a 1.0% concentration in distilled water, then an aqueous solution of histone is added in an equal volume to an aqueous solution of dextran of 6% concentration with a molecular weight of 50 to 70 kDa, then a solution containing dextran and histone is incubated for 30 ± 5 min at a temperature of 6 ± 2 ° C, then this solution is added to an organic liquid, which is used as a vegetable oil, in an amount of 3.5 volumes from the volume of the initial solution, and get a mixture of two immiscible liquids, which is mixed and re-aged for 30 ± 5 min at a temperature of 6 ± 2 ° C and a suspension is obtained, then the suspension is subjected to ultrasonic treatment for 45 ± 5 s with a radiation power of 100 W , then the resulting suspension is centrifuged, and centrifugation is carried out for 5 ± 1 min at 2000 g, then the precipitate is dried at room temperature and nanospheres with a diameter of not more than 1000 nm are obtained containing histone H1 in the dextran matrix.

The determination of the amount of protein included in the nanosphere is carried out using amino acid analysis. Dextran nanospheres containing histone H1 included in the matrix are dried to constant weight at 50 ° C. A histone H1 preparation is used as a standard. Samples hydrolyze 6 N. hydrochloric acid in sealed tubes for 24 hours at 110 ° C.

After drying the samples to constant weight, an amino acid analysis is performed and the amount of protein is calculated.

The amount of protein is estimated based on the content of hydrolysates of dehydration-resistant amino acids, such as aspartic acid, glycine, glutamic acid.

Samples of nanospheres that are prepared based on dextran are analyzed using electron microscopy when applied to a glass surface. For this, standard glass samples are treated with ethanol-ether mixture (in equal volumes) for 1 h and kept at 500 ° C for 1 h. The glass surface is covered with an aqueous suspension of nanospheres and dried at room temperature.

The results of scanning electron microscopy of nanospheres obtained on the basis of dextran are presented in FIG. 1. It is shown that nanospheres have a diameter of not more than 1000 nm.

Example 2. In this series of experiments implementing the claimed invention, a method was used to incorporate nanospheres containing proteins into the matrix of resorbable microspheres based on a polylactide-glycolide copolymer, where the nanospheres with the protein represent the “solid” form of the protein.

In the particular case of the method is as follows. Nanospheres with histone H1 in their composition are prepared as described in Example 1. An initial suspension of nanospheres in a pre-prepared polymer solution is prepared, using a resorbable polylactide-glycolide copolymer with a molecular weight of 50 to 80 kDa with a molar ratio of monomers of 75 : 25, which is dissolved to a 10% concentration in a hydrophobic organic solvent, which is used as methylene chloride, is stirred for 60 ± 5 minutes at a temperature of 6 ± 2 ° C, and in the polymer solution add the nanospheres with histone are added at a weight ratio of nanospheres to polymer of 1:50, then ethanol is added to the solution in an amount of 10 volumes relative to the volume of the initial solution, then the mixture is stirred and a secondary suspension is obtained, which is dispersed under stirring for 10 minutes at room temperature for uniform distribution of the polymer in the organic solvent and the formation of microspheres, then the microspheres are precipitated from the suspension by adding chilled ethyl alcohol, which is added t in a 100-fold volume relative to the volume of the suspension, then the precipitate of microspheres is placed on a fine-porous glass filter and filtered off, then the precipitate is collected and dried at room temperature for 8 ± 2 hours, and the resulting resorbable microspheres containing nanospheres with histone with a protein content of 1.0 mg dry weight of microspheres from 2.0 to 4.0 μg, and the release of histone from microspheres as a result of their resorption occurs for at least 50 days, as described below in example 3.

Example 3. In this series of experiments, the rate of release of protein from resorbable microspheres is estimated by the amount of protein in the incubation medium depending on the time of incubation.

In model experiments, the rate of protein release from microspheres prepared on the basis of a resorbable polylactide-glycolide copolymer containing nanospheres with histone H1, depending on the time of in vitro incubation, is estimated.

In the first series of experiments: the rate of release of protein from nanospheres prepared on the basis of dextran is evaluated. The rate of release of protein from nanospheres is estimated by the amount of protein in the model medium during incubation of nanospheres in phosphate-buffered saline, pH 7.2 and a temperature of + 21 ° C, while the protein concentration is determined in the incubation medium for the first 5 hours and up to 120 hours

In a particular particular case, nanosphere samples with a diameter of not more than 1000 nm are used, containing from 110 to 130 ng of protein per 1.0 μg of dry weight of nanospheres. A 0.98 g sample of nanospheres is placed in a 65 ml incubation medium and incubated at + 21 ° C with stirring.

To analyze the amount of histone H1 released from the model incubation medium, aliquots of the medium are sequentially taken during the first 5 hours (every hour), from 10 to 24 hours (every 5 hours) and up to 120 hours from the start of incubation and concentration is determined protein in aliquots of the incubation medium.

The experimental results show that the main amount of histone H1 is released in the first 10 hours of incubation and is 40%, then after 24 hours the release of histone H1 from the system reaches 70%. The remaining 30% of the protein is released gradually over the next 4 days, i.e. complete release is achieved after 120 hours

Based on the data obtained, it can be concluded that the biodegradation of nanospheres prepared on the basis of dextran mainly proceeds during the first days from the start of incubation.

In the second series of experiments, the rate of resorption of microspheres prepared on the basis of a resorbable polylactide-glycolide copolymer with nanospheres encapsulated in their matrix containing histone H1 is estimated.

The rate of resorption of the microspheres is estimated by the release of protein into the model medium upon incubation of the microspheres in phosphate-buffered saline, pH 7.2 and a temperature of + 21 ° C, and is observed during the first day and up to 50 days of incubation.

A sample of microspheres is placed in an incubation medium, using phosphate-saline buffer solution, pH 7.2, and incubated at a temperature of + 21 ° C with constant stirring on an orbital shaker at a speed of 30 rpm. Then determine the amount of protein in the incubation medium for 50 days from the start of incubation.

In the particular case of using a sample of microspheres containing in its composition from 2 to 4 μg of protein per 1.0 mg of dry weight microspheres. A sample of microspheres in an amount of 1.54 g is placed in an incubation medium with a volume of 180 ml and incubated at + 21 ° C with stirring. Determine the concentration of protein in aliquots of the medium, which are selected from the incubation medium (volume 5.0-10.0 ml) during the first day after 1 hour, 5 hours, 12 hours and 24 hours and from 2 days to 50 days (after 2, 5, 10, 30 and 50 days) from the start of incubation. Based on the data on determination of protein concentration in the selected samples (in μg / ml), the total amount of histone H1 released during each specific period from the beginning of incubation is calculated (in absolute values (μg) and in percentage terms) (Table 1).

Table 1 shows the data on the amount of histone H1 released from the microspheres during the first hours, first days, and up to 50 days from the start of incubation. It can be seen from the above data that the amount of protein in the medium increases with the incubation time and is characterized as a continuous process of the protein leaving the system.

The graph (Fig. 2) shows the release profile of histone H1 from microspheres containing nanospheres with histone H1 to the incubation model versus time from the start of incubation in phosphate-buffered saline for 50 days.

Protein release from microspheres based on a resorbable polylactide-glycolide copolymer is usually characterized by an initial diffusion-controlled phase. The process of protein exit from the system in the first 24 hours is determined by the release of adsorbed protein on the surface of the microspheres. The initial release of positively charged histone H1 from microspheres prepared from a resorbable polylactide-glycolide copolymer can be controlled by the electrostatic interaction between the NH ₂ groups of histone H1 and the free carboxyl groups of the heteropolymer chains. This fact is confirmed by an increase in the period of primary release of histone H1 from the system up to 2 days. During the first 2 days, the amount of histone H1 released remains at the same level and amounts to 20% and 22% in the first 24 hours and 48 hours, respectively.

The subsequent release of histone H1 from the microspheres can be characterized as a continuous and uniform release of protein from the incubation system as a result of resorption of the microspheres for 50 days. After 120 hours, the protein yield was 43%, after 240 hours - 79%, after 480 hours - 84% and after 720 hours - 92%. Full release of histone H1 occurs within 50 days (1200 h) and is 97% of the start of incubation.

The presented invention ensures the preservation of the native structure of the protein due to its inclusion in the composition of polymer nanospheres and increases reliability, and simplifies the method of producing the native protein in the composition of polymer nanospheres and resorbable microspheres, which are designed to prolong the physiological effect of the native protein used for therapeutic purposes.

The claimed invention can be used to deliver protein drugs to target cells and body tissues and has advantages when used in various fields of nanomedicine, for example, when creating vaccines for immunization against a number of diseases; in bioregenerative engineering to stimulate tissue regeneration; in the field of nanodiagnostics based on the use of molecular detectors, biosensors and fluorescent nanoparticles.

You can consider other numerous possibilities of using microencapsulation of protein drugs, for example, for their local delivery in areas that, in certain situations, may be inaccessible for blood flow in the body. Moreover, proteins with unfavorable pharmacokinetics, for example, with a very short half-life or unable to reach the corresponding targets, also require the use of continuous delivery methods for their further therapeutic use.

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Claims (1)

A method of obtaining a native protein, as the basis for prolonging the action, in the composition of polymer nanospheres and resorbable microspheres, which consists in preparing an aqueous solution of dextran and an aqueous solution of protein, obtaining a suspension, centrifuging the suspension, drying the precipitate at room temperature and obtaining nanospheres containing a protein, characterized in that they use a protein of animal origin, and freeze-dried histone is used as a protein, which is dissolved to a 1.0% concentration in distilled water, s thus, an aqueous solution of histone is added in an equal volume to an aqueous solution of dextran of 6% concentration with a molecular weight of 50 to 70 kDa, then the solution containing dextran and histone is kept for 30 ± 5 min at a temperature of 6 ± 2 ° C, then this solution is added to the organic liquid, which is used as vegetable oil, in an amount of 3.5 volumes of the volume of the initial solution, and a mixture of two immiscible liquids is obtained, which is mixed and re-aged for 30 ± 5 min at a temperature of 6 ± 2 ° C and get a suspension, s the suspension is then subjected to ultrasonic treatment for 45 ± 5 s at a radiation power of 100 W, then the resulting suspension is centrifuged, and centrifugation is carried out for 5 ± 1 min at 2000 g, then the precipitate is dried at room temperature and nanospheres with a diameter of not more than 1000 nm are obtained containing histone, after which a primary suspension of nanospheres in a pre-prepared polymer solution is prepared, using a resorbable polylactide-glycolide copolymer with a molecular weight of from 50 to 80 kDa at molar s the ratio of monomers equal to 75:25, which is dissolved to a 10% concentration in a hydrophobic organic solvent, which is used as methylene chloride, stirred for 60 ± 5 min at a temperature of 6 ± 2 ° C, and nanospheres with histone are added to the polymer solution when the weight ratio of nanospheres and polymer is 1:50, then ethyl alcohol is added to the solution in an amount of 10 volumes relative to the volume of the initial solution, then the mixture is stirred and a secondary suspension is obtained, which is subjected to dispersion with stirring for 10 min at room temperature for uniform distribution of the polymer in the organic solvent and the formation of microspheres, then the microspheres are precipitated from the suspension by adding chilled ethanol, which is added in a 100-fold volume relative to the volume of the suspension, then the precipitate of the microspheres is placed on a finely porous glass filter and filter, then the precipitate is collected and dried at room temperature for 8 ± 2 hours, and the resulting resorbable microspheres containing nanospheres with a histone with a protein content of 1.0 mg dry weight of microspheres from 2.0 to 4.0 μg is introduced into a model incubation medium, which is used as a phosphate-buffered saline solution, pH 7.2 and incubated at + 21 ° C for 2 months, while monitoring every day for at least 50 days the concentration of protein in the incubation medium, after which the resulting native proteins as part of the resorbable microspheres are used as the basis for prolonging the physiological action of proteins.

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