RU2753018C1 - Nanostructured composition for oral delivery of insulin and method for production thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely to a nanostructured composition for oral delivery of insulin, containing nanoparticles with a core-shell structure, wherein the core is made in form of gold nanoparticles sequentially coated with chitosan and insulin, characterised by the fact that the core of the composition is additionally coated with a chitosan shell, wherein the size of the formed gold nanoparticles constitute 3 to 15 nm, gold nanoparticles-chitosan 130 to 143 nm, gold nanoparticles-chitosan-insulin 148 to 160 nm, gold nanoparticles-chitosan-insulin-chitosan 175 to 190 nm, and also relates to a method for producing a nanostructured composition for oral delivery of insulin, including forming cors and producing shells of nanoparticles included in the composition, characterised by the fact that the cores of nanoparticles are formed by complete reduction of trivalent gold Au³⁺ to a zerovalence state under UV irradiation from a dopant of chloroauric acid HAuCl₄ in an aqueous acetic acid solution of chitosan at the following ratio of components, % wt.: chitosan 3, acetic acid 1.5, HAuCl₄ 4.5 relative to the weight of dry polysaccharide, water the rest, followed by applying an insulin layer to the surface of the obtained nanospheres of gold stabilised with a chitosan layer, by introducing an insulin solution directly into the nanosphere dispersion, wherein a ratio of the weight of chitosan-coated gold nanospheres to the weight of insulin of 3:1 is used, and the shells of nanoparticles of the insulin-chitosan composition are obtained by adding a "gold nanospheres-chitosan-insulin" dispersion to the excess chitosan solution.

EFFECT: present invention ensures development of a nanostructured composition for oral delivery of insulin with increased time of effect of insulin, prolonged reduction in blood glucose level, increased safety and biocompatibility, reduced toxicity.

5 cl, 2 dwg, 1 tbl

Description

The proposed group of inventions relates to medicine, pharmacology and biotechnology, and concerns a nanostructured insulin-chitosan composition for an oral insulin delivery system and a method for its manufacture, which can be used, for example, for the treatment of diabetes mellitus.

Diabetes is a serious chronic disease that has increased significantly in recent years. Insulin is a protein with a high molecular weight and is the most used and effective drug for the treatment of diabetes. The insulin molecule was discovered in 1922, since then peptides and proteins have been used as biopharmaceuticals due to their advantages, such as higher potency, specificity and efficacy compared to conventional drugs, but oral protein formulations are still under development. The main problem is that the digestive system metabolizes insulin before it can reach the bloodstream. There are several barriers along the gastrointestinal tract that must be overcome in order to increase the bioavailability of insulin. For example, gastric pH, enzymatic activity, the presence of mucus, and poor intestinal permeability are major concerns for protein activity and efficiency (Hosseininasab S. et al. Retracted: Synthesis, Characterization, and In vitro Studies of PLGA - PEG Nanoparticles for Oral Insulin Delivery // Chem Biol Drug Des. 2014. Vol. 84, No. 3. P. 307-315).

One of the reasons for poor diabetes control is the need for daily injections in patients. In principle, the subcutaneous administration of insulin is not physiological, because it is carried out in a place that is not the most suitable from an anatomical and physiological point of view. In the natural physiology of mammals, insulin produced by the pancreas is delivered to the portal vein system and transported to the liver. When insulin is injected subcutaneously, it first passes through the large and small circles of blood circulation, and to achieve the physiological concentration of insulin in the liver, a much higher level of insulin in the peripheral blood is required than in a healthy person, which leads to peripheral hyperinsulinemia. Oral delivery, on the other hand, mimics the endogenous pathway of insulin after secretion, as it undergoes a first pass to the liver rather than to the systemic circulation. In addition, oral delivery of insulin will provide the following advantages over subcutaneous administration of insulin: reduced pain, reduced likelihood of skin infections and irritation; reducing anxiety and injury associated with injections; preventing the development of diabetes (treatment with low-dose oral insulin suggests this); improving diabetes control through continuous absorption and availability of insulin over many hours. Despite a number of studies on overcoming barriers to oral delivery of insulin, so far none of them have gone beyond the laboratory bench and have not received commercial application due to the use of complex and science-intensive drugs due to the instability of proteins in the gastrointestinal tract and the risk of obtaining side effects.

In addressing the problem of low stability of insulin in the gastrointestinal tract and low intestinal permeability, encapsulation of insulin in polymer-based nanoparticles appears to be a good strategy for improving the oral bioavailability of insulin. In recent years, various strategies and polymers have been used to encapsulate insulin and deliver it orally: chitosan, dextran, alginate, poly (γ-glutamic acid), hyaluronic acid, polylactide, polylactide glygolide (PLGA), polycaprolactone (PCL), acrylic polymers, polyallylamine. There have been promising studies and positive results, but as yet there is no polymer-based nanoparticle system for oral administration of insulin on the market. There are also no long-term toxicity studies of the developed carriers. (Fonte P. et al. Polymer-based nanoparticles for oral insulin delivery: Revisited approaches // Biotechnology Advances. 2015. Vol. 33, No. 6. P. 1342-1354).

Currently, chitosan-based nanoparticles are considered a promising means of oral insulin delivery. The advantages of chitosan-based nanoparticles include the following. Chitosan is a non-toxic and biocompatible polysaccharide derived from crustacean and insect shells and has been demonstrated to be safe in both animal and human models. Chitosan, available as an oral tablet, can reduce the intake of fat and cholesterol in the diet. In addition to protecting insulin from acid denaturation and enzymatic degradation, chitosnan nanoparticles have mucoadhesive properties that can prolong their residence time in the small intestine. Moreover, chitosan may mediate the opening of tight junctions between epithelial cells, thereby facilitating paracellular insulin transport.

A known method for the synthesis of modified particles of chitosan for oral delivery of insulin according to US patent 9828445, IPC A61K 47/36, C08B 37/08, C08F 251/00, A61K 38/28, A61K 47/32, publ. November 28, 2017, including the amidation of chitosan with a fatty acid modified with a fatty acid and / or amino acid. This is a complex multistage process of chemical modification of a fatty acid or amino acid using a catalyst and subsequent crosslinking to obtain modified chitosan particles. Insulin is loaded onto modified chitosan particles by mixing chitosan particles with insulin to form a mixture, the mixture is incubated at a temperature and time sufficient for complete adsorption of insulin on the particles, then the mixture is centrifuged to isolate the particles. In addition to the complexity of the reaction, the disadvantage is the use of substances toxic to nerve tissues - alkylacrylamides, since N-isopropylacrylamide (NIPAm) is grafted onto the amidated chitosan using this method. In addition, the resulting particles, when delivered orally, will not be sufficiently protected from the action of gastric acid enzymes.

A known method of producing nanoparticles loaded with insulin by self-gelation based on chitosan and water-soluble snail mucin as natural polymers. For this, the ionic interaction of mucins with chitosan at various concentrations was used with the use of pluronics and polyvinyl alcohol as a solid surfactant. This composition was chosen to provide the necessary dynamics for the formation of nanoparticles while maintaining surface properties that would facilitate the encapsulation of insulin. (Mumuni M.A. et al. Insulin-loaded mucoadhesive nanoparticles based on mucin-chitosan complexes for oral delivery and diabetes treatment // Carbohydrate Polymers. 2020. Vol. 229. P. 115506). The mixture of chitosan and mucin is stirred using homogenization, then 5 ml of insulin solution is added to the mixture and gently stirred with a magnetic stirrer for 3 minutes at 200 rpm. Thereafter, an aqueous phase containing a mixture of Poloxamer-188 and polyvinyl alcohol is added to the phase containing insulin and chitosan, and homogenized for 5 minutes at 12,000 rpm. The drug is additionally subjected to ultrasonic treatment at 80 W for 2 minutes in an ice bath. Then, aliquots of nanoparticles are washed twice with deionized water by centrifugation (10,000 rpm, 10 ° C, 60 min) and then lyophilized to obtain nanoparticles with insulin. The disadvantage of the analogue, in addition to the complexity of the preparation of the drug, is that ultrasound destroys chitosan to an oligomer. The latter does not possess mucoadhesive properties; as a result, the delivery of insulin into the bloodstream will be limited. The method is also energy intensive.

Known nanostructured insulin-chitosan composition of nanoparticles for oral delivery of insulin and a method for its manufacture WO 2019148810, class. A61K 38/28, A61K 9/51, A61K 47/36, A61P 3/10, publ. 08.08.2019. The nanostructured insulin-chitosan composition of nanoparticles for oral delivery of insulin has a core-shell structure, said core is a nanocomposite core consisting of a quaternary ammonium salt of chitosan and insulin, said shell is hyaluronic acid or thiolated hyaluronic acid applied to the surface of said core. A method for manufacturing a prototype consists of manufacturing said cores using FNC technology and manufacturing said shells by applying hyaluronic acid to the surface of said nuclei. The disadvantage of the prototype is that special techniques are required to obtain hyaluronic acid of high molecular weight, which is capable of providing protective functions. In addition, hyaluronic acid is rapidly degraded by a whole family of enzymes, and its degradation products, in contrast to the high molecular weight polysaccharide, are capable of inducing an inflammatory response in macrophages and dendritic cells.

Closest to the proposed invention is a nanostructured composition for oral delivery of insulin, known from the source Hrushikesh M Joshi. et al., Chitosan Reduced Gold Nanoparticles as Novel Carriers for Transmucosal Delivery of Insulin. Pharmaceutical Research, 2007, Vol. 24, No. 8, pp. 1415-1426, taken as the closest analogue (prototype). The nanostructured composition for oral delivery of insulin consists of spherical gold nanoparticles sequentially coated with chitosan and insulin. The disadvantage of the prototype is the lack of a shell that protects the insulin layer from the destructive action of the gastrointestinal tract when administered orally. Also, the method for manufacturing gold nanoparticles of the prototype does not provide a narrow nanoparticle size distribution, which is important for the transport of nanoparticles in the gastrointestinal tract. Moreover, a significant part of the fractions (up to 40%) of the prototype composition contains gold nanoparticles with a size of 30-50 nm, which will lead to the formation of final multilayer insulin-containing structures with dimensions that prevent penetration through the gap junctions of the gastrointestinal tract. Thus, only part of the insulin-containing structures will enter the bloodstream (internal environment).

The task to be solved by the group of inventions is to obtain a nanostructured composition for oral delivery of insulin, which ensures the flow of insulin into the bloodstream (internal environment) from the cavity of the gastrointestinal tract with subsequent prolonged release of insulin from the preparation into the blood.

The technical result from the use of the proposed group of inventions is an increase in the time of insulin exposure, a prolonged decrease in blood glucose, an increase in safety and biocompatibility, and a decrease in toxicity.

The task is achieved by the fact that in a nanostructured composition for oral delivery of insulin containing nanoparticles with a core-shell structure, in which the core is made in the form of gold nanoparticles, sequentially coated with chitosan and insulin, the core of the composition is additionally coated with a shell of chitosan, while the size of the formed nanoparticles gold nanoparticles are 3-15 nm, gold nanoparticles-chitosan - 130-143 nm, gold nanoparticles-chitosan-insulin - 148-160 nm, gold nanoparticles-chitosan-insulin-chitosan - 175-190 nm.

This task is also achieved by the fact that in the method of manufacturing a nanostructured composition for oral delivery of insulin, including the formation of nuclei and the production of shells of nanoparticles included in the composition, the nuclei of nanoparticles are formed by the complete reduction of trivalent gold Au ³⁺ to a zero-valence state under UV irradiation from the dopant gold chloride. acid HAuCl ₄ in an aqueous acetic acid solution of chitosan at the following ratio of components, wt%: chitosan - 3, acetic acid - 1.5, HAuCl ₄ - 4.5 from the mass of dry polysaccharide, water - the rest, after which on the surface of the resulting nanospheres of gold stabilized with a layer of chitosan, a layer of insulin is applied by introducing an insulin solution directly into the dispersion of nanospheres, and the ratio of the mass of gold nanospheres coated with chitosan to the mass of insulin is 3: 1, and the shells of nanoparticles of the insulin-chitosan composition are obtained by adding a dispersion of nanospheres from gold-chitosan-insulin "in excess of races chitosan; chitosan with a molecular weight of 100,000 to 250,000 and a degree of deacetylation of 78-82% is used as a highly effective stabilizer; the process of nucleation and growth of gold nanospheres is monitored spectrophotometrically, according to the absorption band corresponding to the plasmon resonance of gold nanoparticles at λ = 520-550 nm until the absorption maximum stops changing; the concentration of chitosan, corresponding to its complete adsorption by the surfaces of gold nanospheres, is determined by thin layer chromatography, and the absence of unadsorbed chitosan in the solution is additionally monitored by high performance liquid chromatography.

FIG. 1 schematically shows the structure of nanoparticles of the proposed nanostructured composition for oral delivery of insulin.

FIG. Figure 2 shows TEM images of gold nanospheres (a) and their size distributions according to TEM (histogram) and PMU (curve) (b) data, where TEM is high-resolution transmission electron microscopy and PMU is low-angle X-ray scattering.

The nanostructured composition for oral delivery of insulin contains gold nanospheres 1 with a size of 3-15 nm, stabilized with a layer of chitosan 2, coated with a layer of insulin 3 and a polymolecular protective shell 4 of chitosan.

A method of manufacturing a nanostructured composition for oral insulin delivery is carried out as follows.

A stable aqueous dispersion of gold nanospheres 1, each of which is stabilized by a layer of chitosan 2, is obtained by UV reduction of Au ³⁺ from the dopant hydrochloric acid HAuCl ₄ to a zero valence state in chitosan solutions doped with hydrochloric acid. The development of a method for producing gold nanospheres was based on the results of the work of G.B. Shulpin. with co-authors on a detailed study of the photolysis of gold-containing compounds in aqueous solutions [Shulpin, GB. Photochemical chlorination of saturated and aromatic hydrocarbons / G.B. Shulpin, P. Lederer, Yu.V. Hepety // ZhOKh - 1987. - T. 57. - No. 3 - S. 619.]. The recovery mechanism of chloroauric acid is represented by the following scheme:

The reduction of Au ³⁺ from the dopant chloroauric acid (HAuCl ₄ ) was carried out directly in an aqueous acetic acid solution of chitosan at the following ratio of components, wt%:

chitosan - 3,

acetic acid - 1.5,

HAuCl ₄ - 4.5 by weight of dry polysaccharide,

water is the rest.

For this, for example, 0.135 g of HAuCl ₄ was introduced into 100 g of a chitosan solution containing 3 g of polysaccharide, 1.5 g of acetic acid and 95.5 g of distilled water. Chitosan with a molecular weight of 100,000 to 250,000 and a degree of deacetylation of 78-82% was used as a highly effective stabilizer.

As a result, nanospheres of gold 1 with a size of 3-15 nm were obtained, stabilized by a layer of chitosan 2.

The process of nucleation and growth of nanospheres from gold 1 was monitored spectrophotometrically using the absorption band corresponding to the plasmon resonance of gold nanoparticles at λ = 520-550 nm until the absorption maximum stops changing.

The concentration of chitosan at which it is completely adsorbed on gold 1 nanospheres and stabilizes them was determined by thin layer chromatography. As a stationary phase, we used, for example, an aluminum plate of the Silifol UV-254 brand with a layer of silica gel applied to it. In this case, an acetic acid solution served as an eluent, and iodine vapor was used as a developer. To prove the formation of the system "gold nanospheres - chitosan", in which there is no unadsorbed chitosan, the method of high performance liquid chromatography was used, for example, on a Shimadzu CTO-20A / 20AC instrument with the LC-Solutions-GPC software module, the eluent was acetic acid solution. The resulting system of "gold nanospheres-chitosan" (aqueous dispersion) remained stable for 7 days.

Then, a layer of insulin 3 was applied to the surface of gold nanospheres 1 stabilized with a layer of chitosan 2 by injecting an insulin solution directly into the dispersion of nanospheres, and the ratio of the mass of gold nanospheres coated with chitosan to the mass of insulin was chosen 3: 1.

To determine the concentration of insulin corresponding to its complete adsorption on nanospheres of gold 1, stabilized by a layer of chitosan 2, it was preliminarily carried out in the range of ratios from 1: 1 to 40: 1 by weight, respectively. Insulin binding was monitored by polyacrylamide gel electrophoresis. The study was carried out in a vertical electrophoresis chamber with a VE-10 casting device and an Elf-4 power source. The components of the gel were visualized with an alcohol solution of iodine. The optimal ratio of chitosan-stabilized gold nanospheres to insulin was 3: 1 by weight of dry components, respectively.

To protect the insulin layer 3 of the nucleus from the destructive action of the enzymes of housing and communal services, when administered orally, the outer shell 4 of chitosan was formed. For this purpose, a dispersion of "gold nanospheres-chitosan-insulin" was added to an excess of chitosan solution at the following ratio of components:

"Gold-chitosan-insulin nanospheres" / chitosan = 1/3 by weight. For this, for example, 100 g of a solution of "gold nanospheres-chitosan-insulin" containing 3 wt.% Chitosan, 1.5 wt.% Acetic acid, 4.5% by weight of the dry polysaccharide HAuCl ₄ , the rest is water, was added in 300 g of aqueous acetic acid solution of chitosan containing 3 wt.% chitosan, 1.5 wt.% acetic acid, the rest is water (9 g of chitosan, 4.5 g of acetic acid, 286.5 g of water).

The dimensional characteristics of the developed nanostructured insulin-chitosan composition at various stages of the process were determined by dynamic light scattering on a Photocor Complex spectrometer. Dimensions (diameters) of the formed nanoparticles:

gold nanospheres 3-15 nm,

gold-chitosan nanospheres 130-143 nm,

nanospheres of gold-chitosan-insulin 148-160 nm,

nanospheres of gold-chitosan-insulin-chitosan 175-190 nm

Statistical studies performed for various replications showed that the sizes of nanoparticles of the developed nanostructured insulin-chitosan composition for oral insulin delivery range from 175-190 nm.

The study of the hypoglycemic effect of the manufactured nanostructured insulin-chitosan composition during oral administration was carried out according to blood parameters in experimental animals (rats), which were divided into 4 groups: 1) intact animals, 2) animals of the control group, in which the pathological process was simulated, 3) experimental animals. groups in which the pathological process was modeled and the developed nanostructured insulin-chitosan composition was administered orally; 4) animals of the experimental group in which the pathological process was modeled and insulin was injected. Diabetes mellitus in animals of the control and experimental groups was caused by the introduction of alloxan at a dose of 100 mg / kg of experimental animals. This led to the development of persistent type I diabetes mellitus (insulin dependent). The results of the study of the hypoglycemic effect of the developed nanostructured insulin-chitosan composition for oral administration are presented in Table 1. The insulin content in the specified composition is 50 IU.

Table 1

Group Time after injection Blood glucose level, mmol / l (avg value ± SOS) Intact - 6.98 ± 0.01 Control - 29.1 ± 2.40 Developed nanostructured insulin-chitosan composition, oral 1 hour 11.00 ± 0.78 2 hours 5.25 ± 2.05 3 hours 6.57 ± 0.62 6 o'clock 12.85 ± 3.32 24 hours 21.80 ± 1.79 Injectable insulin, intramuscularly in the thigh 1 hour 7.11 ± 0.72 2 hours 7.97 ± 0.66 3 hours 17.28 ± 1.06 6 o'clock 24.57 ± 0.88 24 hours 29.45 ± 0.46

As follows from table 1, oral administration of the developed nanostructured insulin-chitosan composition provides a decrease in blood glucose at the level of injection of insulin for 3 hours. The effect develops somewhat more slowly than with injection (1 hour), reaching comparable results after 2 hours. However, in the first case, a more pronounced prolonged effect is observed up to 6 hours, compared with injection.

Chitosan is non-toxic, safe and biocompatible, has high mucoadhesive properties and is able to push apart the gap junctions of the gastrointestinal tract walls.

Thus, the obtained nanostructured composition for oral delivery of insulin ensures the flow of insulin into the bloodstream from the cavity of the gastrointestinal tract, has a prolonged effect of insulin, leads to a prolonged decrease in blood glucose in comparison with the injection method of insulin administration, and also has all the advantages of the agent for oral administration (physiological, painless) and consists of non-toxic substances.

Claims (10)

1. Nanostructured composition for oral delivery of insulin, containing nanoparticles with a core-shell structure, in which the core is made in the form of gold nanoparticles, sequentially coated with chitosan and insulin, characterized in that the core of the composition is additionally coated with a shell of chitosan, while the size of the formed gold nanoparticles are 3-15 nm, gold nanoparticles-chitosan - 130-143 nm, gold nanoparticles-chitosan-insulin - 148-160 nm, gold nanoparticles-chitosan-insulin-chitosan - 175-190 nm. 2. A method of manufacturing a nanostructured composition according to claim 1 for oral delivery of insulin, including the formation of nuclei and the manufacture of shells of nanoparticles included in the composition, characterized in that the nanoparticle nuclei are formed by the complete reduction of trivalent gold Au ³⁺ to a zero valence state under UV irradiation from dopant chloroauric acid HAuCl ₄ in an aqueous acetic acid solution of chitosan at the following ratio of components, wt%: chitosan - 3, acetic acid - 1.5, HAuCl ₄ - 4.5 by weight of dry polysaccharide, water - the rest, After that, a layer of insulin is applied to the surface of the obtained nanospheres of gold stabilized with a layer of chitosan, by injecting an insulin solution directly into the dispersion of nanospheres, and using the ratio of the mass of gold nanospheres coated with chitosan to the mass of insulin 3: 1, and the shells of nanoparticles of the insulin-chitosan composition is obtained by adding a dispersion of "gold nanospheres-chitosan-insulin" to an excess of chitosan solution. 3. A method according to claim 2, characterized in that chitosan with a molecular weight of 100,000 to 250,000 and a degree of deacetylation of 78-82% is used as a highly effective stabilizer. 4. The method according to claim. 2, characterized in that the process of nucleation and growth of gold nanospheres is controlled spectrophotometrically, according to the absorption band corresponding to the plasmon resonance of gold nanoparticles at γ = 520-550 nm until the absorption maximum stops changing. 5. The method according to claim 2, characterized in that the concentration of chitosan corresponding to its complete adsorption by the surfaces of gold nanospheres is determined by thin layer chromatography, and the absence of non-adsorbed chitosan in the solution is additionally monitored by high performance liquid chromatography.

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