RU2722822C1 - Method of producing polymer nanospheres for targeted delivery to target tissue - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to production of biodegradable nanospheres for inclusion of biologically active proteins in their composition for stabilization of their structure and directed delivery to target tissue with intranasal introduction. Method involves preparation of an aqueous solution of dextran and aqueous solution of protein, mixing them in an equal volume, adding to the obtained solution vegetable oil in amount of 3.5 volume of the volume of the initial solution, obtaining a mixture of two immiscible liquids, ultrasonic treatment and centrifugation of the mixture, drying the obtained nanosphere with diameter of not more than 200 nm. Protein used is a sublimation-dried total histone of animal origin, which is a complex of cationic proteins which is dissolved to 0.1 % concentration in distilled water. Nanospheres thus obtained are modified by a complex of core histones which are covalently bound to the surface of nanosphere, and nanospheres containing not less than 200 mcg of core histones per 1.0 g of nanosphere are obtained.

EFFECT: invention provides preservation of native structure of protein due to its inclusion in composition of resorbable nanospheres intended for intranasal introduction, and modification of surface of nanospheres with positively charged proteins increases degree of absorption of nanospheres by olfactory mucous membrane.

1 cl, 1 dwg, 3 ex

Description

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

One of the essential features of drug therapy for neurological diseases is associated with the presence of a blood-brain barrier (BBB) in the brain structures, which separates circulation from the interstitial space and prevents the entry of a number of polar compounds directly into the brain parenchyma [1].

BBB permeability is determined by endothelial cells of brain capillaries, which have epithelial-like highly resistant tight junctions, which excludes the paracellular pathways of dissolved substances fluctuations through the BBB. Therefore, circulating molecules gain access to the interstitial fluid of the brain only by: a) lipid-mediated transport, b) transport mediated by metabolite carriers, c) transport mediated by receptors for peptides and proteins [2, 3]. The BBB peptide receptor system includes receptors for insulin, insulin-like growth factors, transferrin, lectins and cationic proteins. It is believed that cationic proteins, binding to the anionic sections of the endothelium of the brain capillaries, undergo absorption-mediated transcytosis through the BBB [4]. These data led to the strategy of creating transport systems based on carrier proteins, the receptors for which mediate protein transcytosis through the BBB.

Strategies based on the use of the endogenous transport pathway - transcytosis, are physiological. In addition, there are invasive and pharmacological strategies for delivering drugs to brain tissue. Invasive strategies include intraventricular infusion of drugs, intracerebral implants and destabilization of the BBB, pharmacological - the use of liposomes and / or polymer nanoparticles. The search for vehicles for the transport of a number of polar compounds, characterized by low permeability through the BBB, continues.

Currently, intranasal delivery of biological drugs blocked by the BBB is widely used as an alternative to invasive methods of delivery to the central nervous system (CNS) [5-7].

It has now been established that after intranasal administration, certain medicinal substances based on protein molecules can be transported from the nasal cavity to the central nervous system, bypassing the BBB. A number of studies have shown the entry of protein molecules into the brain after their intranasal administration, however, compared with their intravenous administration, the bioavailability of these molecules is low, usually below 1% [5-7].

Protective "barriers" present in the nasal mucosa affect the low efficiency of the delivery of protein drug substance to the central nervous system. Mucociliary clearance mechanisms quickly remove medicinal substances from their delivery sites, reducing contact with nasal epithelium and delivery to the central nervous system. The mucous membrane of the nasal cavity plays the role of an enzymatic barrier to drugs. Weak permeability of the mucous membrane limits the efficiency of drug delivery based on protein molecules into the brain parenchyma in therapeutically relevant amounts.

The olfactory system among the components of the nasal cavity is of interest not only as an olfactory analyzer, but also because of the ability of the olfactory neurons to provide a "portal" for direct delivery of drugs to the brain parenchyma.

Currently, transport through the olfactory mucosa is widely studied as a promising therapeutic approach for delivering drugs from the nasal cavity directly to the brain parenchyma for the treatment of central nervous system diseases, taking into account the ability to simultaneously bypass the BBB [8].

The olfactory pathway is divided into neuronal and epithelial routes. The neuronal route is characterized by the internalization of drugs into olfactory sensory neurons through endocytosis or macropinocytosis and their intracellular axonal transport along neurons into the olfactory bulbs [8]. From olfactory bulbs, medicinal substances enter the central nervous system via neuronal pathways that are directed to specific areas of brain tissue (for example, the olfactory tract, pyriform cortex, hypothalamus, etc.). The pathway of the olfactory analyzer is represented by a chain of consecutive neurons. At present, axonal transport of protein molecules is being intensively studied [8]. The results of the studies showed that axonal transport takes place over a long period of time for the drug to enter the central nervous system.

It is known that the flow of substances through the neuronal route (intracellular, axonal transport) into the olfactory bulbs proceeds slowly, and the substances are detected in the brain parenchyma 24 hours after intranasal administration, and their amount accumulates gradually.

The delivery of drugs to the brain parenchyma through the olfactory epithelium is more effective than axonal transport, as shown for insulin and nerve growth factor [9]. In this case, the drug substance enters the central nervous system as a result of diffusion through the compartment, which is located near the axon. Axons are surrounded by a continuous layer of olfactory tight cells that cover the axons all the way from the olfactory epithelium to the olfactory bulbs. This layer of lightening cells is additionally covered with a layer of fibroblasts, which ultimately forms a membrane (compartment) near the nerve [9, 10]. Moreover, translocation inside the compartment proceeds much faster than intracellular axonal transport into the olfactory bulbs.

As a result, the olfactory sensory neuron is capable of endocytosis of some proteins from the nasal ducts and their subsequent transport along the axon (anterograde axonal transport - directed along the axon to its terminals) in the direction of the olfactory bulbs. The epithelial pathway is considered as a rapid transport of substances, which takes several minutes with intranasal administration to reach the olfactory bulbs and other areas of the brain parenchyma [8]. Various neurotherapeutic agents, including drug molecules, proteins, peptides, hormones and cells, such as stem cells, can be delivered this way.

Improving the approaches to formulating protein drugs to bypass “barriers” are aimed at increasing their effective delivery to the central nervous system. Encapsulating proteins in nanoparticles not only protects them from degradation, but, importantly, encapsulation improves their absorption by the olfactory mucosa and access to the central nervous system [7] .

Systems based on macro- and nanoparticles are widely used to increase the degree of absorption of drugs. In addition, the possibility of modifying the surface of polymer nanoparticles by the inclusion of peptide and protein molecules that are ligands of cell surface receptors has been shown, which can significantly increase the degree of their absorption.

It is known that microparticles based on chitosan and its derivatives are widely used for intranasal delivery of drugs to the brain parenchyma. Chitosan is a natural polysaccharide and is widely used in various fields of biomedicine due to properties such as biocompatibility and biodegradability. The mucoadhesive properties of chitosan are associated with the presence of positively charged amino groups that can interact with negatively charged molecules of the nasal mucosa, such as sialic and sulfonic acids. The ability of chitosan to increase the permeability of drugs through the epithelium of the mucous membrane is due to the reversible opening of tight contacts [11, 12].

It is known that biodegradable 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 [13, 14].

The ability of targeted delivery of drugs to the target tissue is determined by such factors as the size of the nanoparticles, their surface charge, hydrophilicity and the nature of the modification of their surface [15].

It is believed that nanoparticles after intranasal administration can reach the brain parenchyma using three main routes: a) transport through the nasal respiratory epithelium into the systemic circulation; b) direct transport of paracellular or transcellular via the olfactory neurons (“olfactory neuronal pathway”) or through the olfactory epithelium (“olfactory epithelial pathway”); c) via via trigeminal nerves located in the respiratory and olfactory epithelium [16].

It is known that nanoparticles are absorbed by cells through endocytosis. There are four main mechanisms of endocytosis: clathrin-mediated endocytosis, caveolin-mediated endocytosis, endocytosis independent of clathrin and caveolin and macropinocytosis [17-19]. Internalization of nanoparticles with a diameter less than or equal to 200 nm is determined by clathrin-mediated endocytosis [20, 21].

Currently, information is being accumulated regarding factors such as surface characteristics and the size of nanoparticles that affect their transport from the nasal cavity to the central nervous system.

Data are known regarding the supply of polystyrene (PS) nanoparticles with various physicochemical surface characteristics and various diameters to the murine olfactory epithelium after intranasal administration [16, 22, 23]. PS nanoparticles with a diameter of 100 nm and 200 nm were obtained with characteristic features of their surface, which varied from negatively charged and hydrophobic PS nanoparticles, neutral and hydrophilic, coated with polysorbate 80 (P80-PS) and positively charged and hydrophilic, coated with chitosan (C-PS ) nanoparticles [22, 24].

It was shown that coating the surface of nanoparticles with chitosan or polysorbate 80 effectively increased the interaction of nanoparticles with nasal olfactory mucosal epithelium. The positive effect of nanoparticles is explained by their penetrating and mucodiffusion properties. Along with these data, it was found that none of the studied types of nanoparticles was found in olfactory bulbs.

It was established that C-PS nanoparticles remained in the mucosal layer for a long period of time due to the electrostatic interaction of chitosan containing positively charged amino groups [11, 12, 25] with negatively charged sialic acid of the mucosal layer glycoproteins [26, 27].

Based on this, it is believed that this type of formulation can be used for the delivery of drugs paracellularly or transcellularly through the olfactory epithelium after their release from nanoparticles in the nasal cavity, which will enter the olfactory bulbs, and from them to the central nervous system via neuronal pathways [9 , 10].

These results were confirmed by studies of the mechanism of transport of nanoparticles coated with chitosan (C-PS) and polysorbate 80 (P80-PS) with a diameter of 100 nm and 200 nm through a remote olfactory pig epithelium, which was used as an in vitro model [16]. In this study, no transport of nanoparticles through the extracted olfactory epithelium was detected. Moreover, chitosan-coated nanoparticles were found in the mucosal layer located above the epithelial cell layer of the olfactory tissue. At the same time, polysorbate 80 coated nanoparticles penetrated the epithelial cell layer, while polystyrene nanoparticles had a low degree of interaction with olfactory tissue.

It is known that a key mechanism for increasing nasal absorption of nanoparticles is an increase in their transmucosal transport, which can be provided by modifying the surface of nanoparticles with bioactive peptides, which are known as cell-penetrating peptides (CPPs) [6, 28]. CPPs are positively charged oligopeptides containing amino acid residues of arginine and lysine, which are capable of electrostatic interaction with biological membranes and increase the efficiency of absorption of biomacromolecules by cells through endocytosis.

Known studies on the delivery of drugs from the nasal cavity to the central nervous system, in which it was shown that peptides CPPs significantly increase the penetration of certain nanoparticles through the olfactory epithelium [6].

Among them, the arginine-enriched Tat protein peptide (Tat protein, Transactivator of transcription, human immunodeficiency virus protein) is most often used to increase the penetration of nanosystems through the olfactory epithelium [6, 29]. The Tat protein consists of 86 amino acid residues and contains a domain enriched in arginine. A peptide sequence containing amino acid residues 49-57 of the Tat protein is necessary for cell internalization. The last sequence is an arginine-enriched domain (it contains two lysines and six arginines) [30]. The minimal sequence, which includes amino acid residues 47–57, is necessary and sufficient for translocation through the cell membrane and is known as the “protein transduction domain” [30].

The current model for Tat peptide-mediated (amino acid residues 47-57 Tat protein) protein transduction is a multi-stage process [31]. The first step involves the interaction of the Tat peptide with proteoglycans on the cell surface. The highly charged amino acid residues of arginine in the Tat peptide are necessary for transduction. The guanidine groups of arginine are able to form both electrostatic and hydrogen bonds with charged acceptors on the cell surface (anionic regions of proteins, membrane phosphosolipids, sulfonated glucosaminoglycans) [32]. The next step is associated with the stimulation of macropinocytosis. Some proteoglycans can act as receptors for Tat peptide and induce macropinocytosis of Tat peptide and its associated “load” (for example, protein or drug substance) [33]. The final step in the transduction of Tat peptide is the exit from the pinocytotic vesicles of the Tat peptide and the associated “load” into the cytoplasm.

It is known that the number of amino acid residues in their composition is the determining factor in the entry of polyarginin polymers into cells [34]. Arginine polymers containing less than five amino acid residues enter cells less efficiently than polymers containing 6 to 15 amino acid residues of arginine. However, arginine polymers containing more than 15 amino acid residues are less effective (in molar ratio) compared to peptides containing less than 15 amino acid residues. In addition, the cytotoxicity of arginine polymers increases with increasing peptide size [34].

It is suggested that from 5 to 15 amino acid residues of arginine in the polymer are necessary for adaptation of the biologically active conformation of the peptide and / or the formation of the required number of ionic interactions with specific receptors or phospholipids in order to induce the process of transport into cells [34].

It is known that surface modification of polymer nanoparticles by the inclusion of protein molecules that are ligands of cell surface receptors has led to the development of new drugs that improve intracellular delivery, bioavailability, the ability to carry out targeted action and reduce side effects.

Protein agglutinins (lectins) are specific protein molecules that recognize sugars and therefore are able to bind to glycosylated membrane components and are used to study cell surfaces [35]. Data on the affinity ratios of certain lectins for N-acetyl-D-glucosamine and sialic acid in olfactory mucosa compartments led to the development of studies on the use of lectins to modify the surface of nanoparticles for drug delivery from the nasal cavity to the brain parenchyma. For example, polyethylene glycol polylactide (PEG-PLA) nanoparticles with a diameter of 85-90 nm were obtained, with a fluorescent marker coumarin included in their composition, which were conjugated with agglutinin from wheat seedlings (wheat germ agglutinin, WGA) [36]. In this study, it was shown that after intranasal administration of WGA nanoparticles to rats, the coumarin content in the olfactory bulbs, olfactory tract, large brain, and cerebellum were 2 times higher than lectin-modified nanoparticles.

In another study, it was found that labeled WGA nanoparticles were detected in the olfactory bulbs after 5 minutes and the fluorescent signal reached its maximum value after 30 minutes and disappeared 2 hours after intranasal administration [37]. This suggests that the fast transport of WGA nanoparticles to the olfactory bulbs is via the olfactory epithelium (olfactory route). Along with this, it is known that plant lectins stimulate immune responses after intranasal administration to animals [38].

It is known that natural positively charged proteins of animals and humans have a high ability to penetrate through cell membranes. In addition, it was shown that cationic proteins with a low molecular weight (5.2-15.1 kDa), which contain up to 31 positively charged amino acid residues, are characterized by the ability to deliver macromolecules and have a pronounced penetrating ability, which is not inhibited due to the high value charge of a protein molecule [39]. The high efficiency of positively charged proteins compared to cationic peptides is determined by the difference in the density of positive charges (due to the dispersion of positively charged amino acids throughout the amino acid sequence), protein structure, method of folding the polypeptide chain or surface area.

The identification of proteins of this class with different structures and molecular weights may present new possibilities for the delivery of macromolecular “load” into mammalian cells [39].

It is possible that cationic proteins of cell nuclei - histones can represent a new class of protein molecules that are ligands of cell surface receptors.

Five classes of histones are distinguished, which are characterized and studied in detail [40]. The histone classes have the following notation: histone H1 (rich in lysine), histone H2B (moderately rich in lysine), histone H2A (rich in arginine and lysine), histone H3 (rich in arginine), histone H4 (rich in arginine and glycine). Histones are heterogeneous in molecular weight. The highest molecular weight is histone H1 - 21.5 kDa, the next largest histones are H3, H2A, H2B, H4 - 15.32, 14.0, 13.77, 11.28 kDa, respectively.

It is known that in the polypeptide chain of cationic proteins, such as arginine-rich histones, there are regions containing six or more amino acid residues of arginine that are in close proximity to each other [40]. It is known that histones along with clusters of cationic amino acids (polycationic domains) contain heparin-binding domains. These domains bind to cell surface proteoglycans containing sulfonated glucosaminoglycans, such as chondroitin sulfate and heparan sulfate [41].

In the cell nuclei, histones are involved in the formation of nucleosomes. Each nucleosome incorporates a compact (core) particle onto which a 146 bp DNA fragment is “wound” The core particle represents the histone octamer, which consists of four dimeric complexes - 2 (Н3-Н4) and 2 (Н2А-Н2В). Histones in the composition of the core particle are called core histones [40].

At present, there is a sufficient amount of data that suggests that the biological role of histones is not limited to their participation in DNA compaction. Histones were also found in the extracellular space [42].

Due to the ability of histones to enter intercellular media, they can exhibit biological activity, causing various biochemical and functional changes in the cells. Therefore, exogenous histones attract the attention of researchers in terms of the practical implementation of the beneficial properties of histones in biotechnology and biomedicine.

Exogenous histones are known as antimicrobial and antiviral agents (43, 44).

In addition, exogenous histones are known as bioactive agents, which, due to their high positive charge, are able to significantly increase the permeability of cell membranes for other substances in very small concentrations, including polymer compounds and penetrate through them through endocytosis.

It was shown [45] that the relative rate of histone entry into tumor cells depends on the content of arginine in their composition. So when culturing cells in the presence of various classes of histones, the absorption by the cells of arginine-rich histone and total histone is 10 times higher than that of lysine-rich histone. Along with this, it was found [46] that, during the cultivation of tumor cells, the uptake of added [ ¹³¹ I] albumin by cells increases 15 and 12 times in the presence of arginine-rich histone or total histone, respectively. However, in the presence of a lysine-rich histone at the same protein concentration, the absorption of albumin by the cells increases only 1.5 times.

Along with these data, it was shown that histone molecules are capable of direct translocation through cell membranes and are able to mediate the translocation of related molecules [47, 48]. Thus, the cultivation of cells of different origin in the presence of a protein complex of five fractions of nucleosomal histones and individual fractions of histones led to their direct translocation through cell membranes and into the cell nucleus. Penetration of a protein complex of five histone fractions into the cell was significantly higher compared to individual histone fractions. Moreover, histone fractions differ in their ability to penetrate the membranes of intact cells, the most effective are arginine-rich histones.

It is likely that histone translocation into cells has the characteristic features of translocation of the minimal domain of the human immunodeficiency virus protein (Tat protein, Transactivator of transcription).

Studies are known in which it is shown that exogenous histones pass through the endothelium of brain capillaries, i.e. through the BBB, and as transport systems facilitate the passage of covalently bound substances that do not independently pass normally through the BBB [42, 49, 50].

A study is known in which the penetration into the central nervous system of the drug of total histone with intranasal administration to mice was established [51].

It was shown that after intranasal administration of [ ¹²⁵ I] histone in the first 15 minutes, the content of radioactive histone in the olfactory bulb was almost 2 times higher than in the blood and 40 times higher than in the cerebral cortex. The pattern of the distribution of the radioactive label during intranasal administration of histone suggests two independent channels of its entry into various tissues of the animal. The entry of [ ¹²⁵ I] histone into the blood can be carried out through the capillary network of the respiratory epithelium of the nasal cavity, which is extremely developed. Another part of the radioactive histone can be directly transported to the olfactory bulbs using axoplasmic transport along the fibers of the olfactory neurons. These data are confirmed by studies on the effect of histone on intraspecific aggression in male mice with intranasal and intraperitoneal administration of the drug [52].

Thus, the data presented make it possible to consider histones as potential carrier proteins, which at low concentrations can be used as transport systems for the delivery of a number of drugs that do not penetrate cell membranes and tissue barriers to tissues and organs.

A known method of preparing polymer microspheres with a diameter of not more than 1000 nm based on crystallized dextran coated with different types of histones [53, 54]. The amount of covalently bound histone with the surface of the microspheres ranged from 160 to 200 μg of protein per 1.0 g of nanospheres. Histone-coated microspheres were used to modify surfaces intended for cell culture. Modification of the surface of nanospheres by exogenous histones facilitated their electrostatic interaction with cells [42, 54]. In addition, it is known that histones contain heparin binding domains capable of interacting with cell proteoglycans and inducing intracellular signals accompanied by cytoskeleton reorganization [41].

A known method of producing a native protein prolonging action in the composition of polymer nanospheres and resorbable microspheres for delivery, which is the closest in technical essence of the proposed invention and is selected as a prototype [55].

The known composition includes nanospheres, which are preliminarily prepared on the basis of dextran and an aqueous protein solution, which is used as an animal protein, which is a freeze-dried histone, which is added in equal volume to an aqueous solution of dextran of 6% concentration with a molecular weight of from 50 to 70 kDa, then this solution is added to the organic liquid, which is a vegetable oil, in an amount of 3.5 volumes from the volume of the initial solution, and a suspension is obtained, then the suspension is subjected to ultrasonic treatment, then the resulting suspension is centrifuged, then the precipitate is dried and nanospheres with a diameter of not more than 1000 nm containing histone, after which a primary suspension of nanospheres in a pre-prepared polymer solution is prepared, which is 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 up to 10%concentration in a hydrophobic organic solvent, which is methylene chloride, then nanospheres with histone are added to the polymer solution at a weight ratio of nanospheres and polymer equal to 1:50, then ethyl alcohol is added to the solution in an amount of 10 volumes relative to the volume of the initial solution, and a secondary the suspension is dispersed, then the microspheres are precipitated from the suspension by adding 100 times the volume of chilled ethyl alcohol, then the precipitate is collected and dried to obtain a native protein in the composition of resorbable microspheres suitable for prolonged release.

A disadvantage of the known method of preparing microspheres based on a polylactide-glycolide copolymer containing nanospheres with a protein included in their composition, which used histone HI of animal origin, is the large size of these structures whose diameter exceeds 1000 nm. In addition, microspheres prepared on the basis of a polylactide-glycolide copolymer are characterized as structures that carry negative charges on their surface.

The alleged invention is devoid of these drawbacks due to the production of nanospheres with a diameter of not more than 200 nm based on dextran and the inclusion in the matrix structure of a positively charged protein complex, which is used as the total histone of animal origin, and the coating of such nanospheres with a protein complex, which is used core histones to obtain hydrophilic nanospheres carrying positive charges on their surface. Modification of the surface of nanospheres by core histones allows increasing the degree of their absorption due to interaction with proteoglycans of the cell surface, which can act as receptors for arginine-rich peptides that are present in the structure of histone molecules. Moreover, the total histone is used as a protein for inclusion in the nanosphere matrix, which is a carrier protein for transporting a number of drugs that do not penetrate cell membranes and tissue barriers into tissues and organs.

The technical result of the claimed invention is the preservation of the native structure of the protein included in the structure of the matrix of dextran nanospheres with a diameter of not more than 200 nm in the process of their preparation, increasing the reliability and simplification of the method of inclusion of the protein and increasing the degree of absorption of nanospheres coated with positively charged proteins and has several advantages that mainly by the fact that:

1) obtaining nanospheres with the protein included in their composition and subsequent coating of such nanospheres with a positively charged protein ensures their effectiveness and specificity of action by protecting proteins from the action of proteolytic enzymes and hydrolytic degradation when interacting with biological components in the nasal cavity, eliminating the potential toxic effects of proteins with their intranasal use, increasing the biological half-life of the protein in the nasal cavity and the possibility of reducing therapeutic doses of the protein;

2) increasing the mucoadhesive properties of dextran nanospheres with a diameter of not more than 200 nm coated with histones, i.e. the degree of their absorption due to the electrostatic interaction of positively charged amino groups of histone molecules with negatively charged sialic acid of glycoproteins that are present in the mucosal layer of the olfactory mucosa, the association of nanospheres with the mucosal layer of the mucous membrane for an extended period of time, and the release of protein from nanospheres, which can occur in nasal cavity.

At the same time, it is assumed that the release of protein during the resorption of nanospheres can occur in the mucosal layer, from which the protein will be absorbed paracellularly or transcellularly into the underlying epithelium of the mucous membrane. Further protein intake can be carried out along the olfactory epithelial route into the olfactory bulbs and from them using neuronal pathways to the central nervous system [22, 23]. This is confirmed by the results of studies on the penetration into the central nervous system of the drug total histone with intranasal administration [51]. It has been shown that [ ¹²⁵ I] histone is directly transported to the olfactory bulbs using axoplasmic transport along the fibers of the olfactory neurons.

The technical result is achieved by the fact that in the known method for the preparation and use of native protein in the composition of polymer nanospheres and resorbable microspheres, suitable for sustained release upon delivery, which includes known and general features of the claimed method, where the composition contains a protein, which is used histone H1 of animal origin, which is previously included in dextran nanospheres with a diameter of not more than 1000 nm, followed by the inclusion of such nanospheres in the composition of resorbable microspheres, which are obtained on the basis of a polylactide-glycolide copolymer, in the proposed method, freeze-dried total histone of animal origin is used as a protein, which is a complex of cationic proteins, which is dissolved to 0.1% concentration in distilled water, then an aqueous solution of the total 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 half the learned solution is added to an organic liquid, to which Span-80 nonionic detergent is preliminarily added to a final concentration of 0.5%, moreover, vegetable oil is used as an organic liquid 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 mixed and subjected to ultrasound treatment, then the resulting suspension is centrifuged for 5 ± 1 min at 2000g, then the precipitate is dried at room temperature and get resorbable nanospheres with a diameter of not more than 200 nm containing a total histone in the matrix, and the protein content is from 5 , 0 to 8.0 mg per 1.0 g of nanospheres, after which such nanospheres are modified with a protein, which is used by core histones, which are a complex of cationic proteins that covalently bind to the surface of nanospheres, and the nanospheres are preliminarily activated by adding to an aqueous suspension of crosslinking nanospheres agent, which is used in the amount of 100 mg of bromine cyan per 1.0 g of nanospheres, at a temperature of not more than 4 ° C and an incubation time of not more than 2 minutes, then the activated nanospheres are precipitated by centrifugation, the precipitate is washed with distilled water and centrifuged again, after which suspension of nanospheres in a solution of core histones is carried out at a weight ratio of protein to nanospheres of 1: 100, and the covalent binding reaction is carried out at pH 7.5-8.0, temperature no more than 4 ° C, incubation time no more than 2 hours, then nanospheres with covalently bound histones are precipitated by centrifugation and dried, and nanospheres containing at least 200 μg core histones per 1.0 g of nanospheres are obtained, with obtaining resorbable nanospheres with a diameter of not more than 200 nm containing a native protein in the nanosphere matrix and a protein immobilized on the surface nanospheres, and such nanospheres are used as the basis for targeted delivery of the native protein to the target tissue with intranasal m introduction.

Thus, the preliminary incorporation of a protein, which is a complex of total histone, into the matrix of resorbable dextran nanospheres with a diameter of not more than 200 nm, intended for intranasal administration, ensures the preservation of the native protein structure due to resistance to the action of proteolytic enzymes, and surface modification of nanospheres by positively charged proteins representing a complex of core histones, increases the degree of absorption of nanospheres by the olfactory mucosa. The release of protein from resorbable nanospheres in the nasal cavity involves direct transport of total histone via axoplasmic transport through the fibers of the olfactory neurons to the target tissue.

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.

A brief description of the figures.

Figure.

In FIG. The results of electron microscopy of nanospheres based on dextran (x1000) are presented. Adsorption on the surface of the glass. It is shown that nanospheres have an average diameter of not more than 200 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.

Example 1

In this series of experiments implementing the invention, the 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 an organic liquid , which use vegetable oil (continuous phase).

The total histone and core histones are used as a model protein. Total histone and core histone preparations containing histones H2A, H2B, H3, and H4 are obtained in a known manner [56].

In the particular case of the method is as follows. An aqueous solution of dextran and an aqueous solution of protein are prepared, using freeze-dried total histone, which is dissolved to 0.1% concentration in distilled water, then an aqueous solution of total histone is added in an equal volume to an aqueous solution of dextran of 6% concentration with molecular weighing from 50 to 70 kDa, then the solution containing dextran and total histone is incubated for 30 ± 5 min at a temperature of 6 ± 2 ° C, then this solution is added to an organic liquid into which the non-ionic detergent Span-80 is preliminarily added to the final a concentration of 0.5%, moreover, vegetable oil in the amount of 3.5 volumes of the volume of the initial solution is used as the organic liquid, 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 a suspension is obtained, then the suspension is subjected to ultrasonic treatment, then the resulting suspension is centrifuged for 5 ± 1 min and 2000g, then the precipitate is dried at room temperature and get resorbable nanospheres with a diameter of not more than 200 nm containing the total histone in the matrix, and the protein content is from 5.0 to 8.0 mg per 1.0 g of nanospheres.

The determination of the amount of protein incorporated in the nanospheres is carried out using amino acid analysis [57]. Dextran nanospheres containing the total histone included in the matrix are dried to constant weight at 50 ° C. As a standard, a total histone preparation is used. Samples hydrolyze 6 N. hydrochloric acid 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.

The results of scanning electron microscopy of nanospheres obtained on the basis of dextran are presented in FIG. It is shown that nanospheres have a diameter of not more than 200 nm.

Example 2

In this series of experiments, the chemical binding of core histones to nanospheres with a diameter of not more than 200 nm is carried out as follows.

An aqueous solution of dextran of 6% concentration with a molecular weight of 50 to 70 kDa is prepared, then the solution containing dextran is kept for 30 ± 5 min at a temperature of 6 ± 2 ° C, then this solution is added to an organic liquid, into which add non-ionic detergent Span-80 to a final concentration of 0.5%, and vegetable oil in the amount of 3.5 volumes of the volume of the initial solution is used as the organic liquid, 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 a suspension is obtained, then the suspension is subjected to ultrasonic treatment, then the resulting suspension is centrifuged for 5 ± 1 min at 2000g, then the precipitate is dried at room temperature and resorbable nanospheres with a diameter of not more than 200 nm are obtained.

A 100 mg sample of nanospheres is suspended in 1.0 ml of distilled water and placed in an ice bath. Prior to immobilization of core histones on the surface of nanospheres, the nanospheres are preliminarily activated with a crosslinking agent, which is used as bromine cyan. For this, 10 mg of bromine cyanide is added to the suspension of nanospheres with vigorous stirring and incubated for two minutes. The activated nanospheres are washed with acetone six times (6 × 12 ml) and twice with distilled water (2 × 12 ml) by centrifugation. After the last centrifugation, 1.0 ml of a 0.1% solution of core histones in phosphate-saline buffer pH 7.5 is added to the activated nanospheres. The resulting secondary suspension of nanospheres at a weight ratio of protein to nanospheres of 1: 100 is incubated at 4 ° C for 2 hours to bind histone proteins to activated nanospheres. Bound protein nanospheres are precipitated from the suspension by repeated centrifugation and washed with distilled water six times (6 × 12 ml), frozen and freeze-dried.

Determination of the amount of protein immobilized on the surface of nanospheres is carried out using amino acid analysis (57). Nanospheres (50 mg), covalently bound to core histones, are dried to constant weight at 50 ° C. A core histone preparation (10 mg) is used as a standard. Both 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 hydrolysis-resistant amino acids, such as aspartic acid, glycine, glutamic acid.

The number of covalently bound core histones with the surface of nanospheres with a diameter of not more than 200 nm is 220 μg of protein per 1.0 g of nanospheres.

Due to interprotein interactions, cortical histones form a protein complex, on the surface of which amino acid sequences are exhibited, which have the highest affinity for the cell surface adhesion receptors. Therefore, the use of cross-linked conjugates of core histones, preserving the originally formed protein-protein interactions, is preferable.

Example 3

A method of producing polymer nanospheres containing a complex of cationic proteins in the matrix and on the surface of nanospheres, for targeted delivery to the target tissue, where the protein is freeze-dried total animal histone, which is a complex of cationic proteins, which is dissolved up to 0.1% concentration in distilled water, then the total histone aqueous solution is added in an equal volume to a 6% concentration aqueous dextran solution with a molecular weight of 50 to 70 kDa, then the resulting solution is added to an organic liquid into which the non-ionic detergent Span is added -80 to a final concentration of 0.5%, and vegetable oil in the amount of 3.5 volumes of the volume of the initial solution is used as the organic liquid, and a mixture of two immiscible liquids is obtained, which is mixed and subjected to ultrasonic treatment, then the resulting suspension is centrifuged for 5 ± 1 min at 2000g, then wasp the dock is dried at room temperature and get resorbable nanospheres with a diameter of not more than 200 nm containing the total histone in the matrix, and the protein content is from 5.0 to 8.0 mg per 1.0 g of nanospheres, after which such nanospheres are modified with protein, the quality of which is used by core histones, which are a complex of cationic proteins that covalently bind to the surface of nanospheres, and the nanospheres are preliminarily activated by adding a crosslinking agent to the aqueous suspension of nanospheres, which use bromine in an amount of 100 mg per 1.0 g of nanospheres , at a temperature of not more than 4 ° C and an incubation time of not more than 2 minutes, then the activated nanospheres are precipitated by centrifugation, the precipitate is washed with distilled water and centrifuged again, after which nanospheres are suspended in a solution of core histones at a weight ratio of protein to nanospheres of 1: 100 and the covalent binding reaction is carried out at pH 7.5-8.0, temperature no more than 4 ° С, incubation time no more than 2 hours, then nanospheres with covalently bound histones are precipitated by centrifugation and dried, and nanospheres containing at least 200 μg core histones per 1 are obtained, 0 g of nanospheres, to produce resorbable nanospheres with a diameter of not more than 200 nm, containing a native protein in the matrix of nanospheres and a protein immobilized on the surface of nanospheres, and such nanospheres are used as the basis for targeted delivery of the native protein to the target tissue during intranasal administration.

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Claims (1)

The method of producing polymer nanospheres for targeted delivery to the target tissue, which consists in preparing an aqueous solution of dextran and an aqueous solution of protein, adding to the resulting solution an organic liquid, which is used as a vegetable oil, obtaining a mixture of two immiscible liquids, ultrasonic processing and centrifugation of the mixture, drying obtained precipitate of nanospheres, characterized in that the protein used is freeze-dried total histone of animal origin, which is a complex of cationic proteins, which is dissolved to 0.1% concentration in distilled water, then the aqueous solution of the total histone is added in an equal volume to aqueous a solution of dextran of 6% concentration with a molecular weight of from 50 to 70 kDa, then the resulting solution is added to an organic liquid to which Span-80 non-ionic detergent is added previously to a final concentration of 0.5%, and I use organic liquid t vegetable oil in an amount of 3.5 volume of the volume of the initial solution and get a mixture of two immiscible liquids, which are mixed and subjected to ultrasonic treatment, then the resulting suspension is centrifuged for 5 ± 1 min at 2000g, then the precipitate is dried at room temperature and get resorbable nanospheres with a diameter of not more than 200 nm, containing the total histone in the matrix, and the protein content is from 5.0 to 8.0 mg per 1.0 g of nanospheres, after which such nanospheres are modified with a protein, which uses core histones, which are a complex cationic proteins that covalently bind to the surface of nanospheres, and the nanospheres are preliminarily activated by adding a crosslinking agent to the aqueous suspension of nanospheres, which use bromine in an amount of 100 mg per 1.0 g of nanospheres, at a temperature of not more than 4 ° C and incubation time no more than 2 min, then activated nanospheres precipitate and centrifugation, the precipitate is washed with distilled water and centrifuged again, after which nanospheres are suspended in a solution of core histones at a weight ratio of protein to nanospheres equal to 1: 100, and the covalent binding reaction is carried out at a pH of 7.5-8.0, at a temperature of not more 4 ° C, incubation time of not more than 2 hours, then nanospheres with covalently bound histones are precipitated by centrifugation and dried, and nanospheres containing at least 200 μg core histones per 1.0 g of nanospheres are obtained, with obtaining resorbable nanospheres with a diameter of not more than 200 nm containing native protein in the matrix of nanospheres and a protein immobilized on the surface of nanospheres, and such nanospheres are used as the basis for targeted delivery of the native protein to the target tissue during intranasal administration.

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