RU2504582C1 - Method of stabilising nanoparticles of biogenic elements with enzymes - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves synthesis of nanoparticles through a redox reaction while adding an enzyme for stabilising the formed nanoparticles directly into the reaction. The enzyme solution - chymotrypsin, concentration of which is selected from 0.02%, is mixed with a precursor solution of 0.013 M selenous acid or silver nitrate - 1%, 0.01%, 0.001%; the solutions are stirred and held at room temperature of 20°C or low temperature of 0°C for 30-60 minutes. A reducing agent - ascorbic acid or sodium borohydride - is then added to the reaction flask and the solutions are stirred and settled to complete the reaction.

EFFECT: invention enables to obtain stabilised nanoparticles of biogenic elements in form of molecular solutions that are resistant to precipitation over time at different pH, with a controlled diameter and high output.

1 tbl, 2 ex

Description

The invention relates to nanochemistry, nanotechnology and biotechnology, and in particular, to the production of nanocomplexes of the nanoparticle-enzyme type, which act as enzymes with proteolytic activity, conformational stability with temperature and pH, as well as additional properties, depending on the specifics of the nanoparticle. Such nanocomplexes can be used in clinical medicine to develop methods for treating wounds, as vehicles for drug delivery and as a means for immunoassay.

It is known to use as stabilizers nanoparticles of polymers of various structures and bovine serum albumin (BSA), a stabilizer of selenium nanoparticles (J. Zhang, H. Wang, Y. Bao, L. Zhang, BioF actors 15 (2001) 27-30, Nano red elemental selenium has no size effect in the induction of seleno-enzymes in both cultured cells and mice, Zhang J, Spalholz J. Toxicity of Selenium Compounds and Nano-Selenium Particles, Handbook of Systems Toxicology. 2010, Chapter 44: 787-802). However, the use of polymers and BSA is limited. In particular, BSA is applicable to stabilize hydrophobic particles such as selenium. In addition, polymers and BSA, having a sufficiently large molecular weight (M _BSA = 65000) forms large nanocomplexes, which is an undesirable factor.

A known method of stabilizing selenium nanoparticles with bovine serum albumin (Huang B, Zhang J, Hou J, Chen C. Free radical scavenging efficiency of Nano-Se in vitro. Free Radic. Biol. Med. 2003, 35 (7), 805-813) according to which 4 ml of a 25 mM glutathione solution containing 200, 20 and 2 mg of BSA for the preparation of small, medium and large selenium nanoparticles, respectively, was added to 1 ml of a 25-mM sodium selenite solution. At the same time, the pH of the medium was adjusted to 7.2 with 1.0 M sodium hydroxide. Then the solution was dialyzed and lyophilized. This method is the closest in essence and the achieved result.

The described method is not universal for nanoparticles, since glutathione is used as a reducing agent, which reduces selenium from selenic acid in a neutral medium, and this method is not applicable for the preparation of metal nanoparticles. It should also be noted that using this method, nanoparticles were synthesized in order to use the ability of selenium to bind free radicals in living organisms. This is a private task, and the resulting product is not suitable for use in other areas.

The technical task and the positive result of the claimed invention is the development of a method for stabilizing nanoparticles of nutrients in a wide pH range, the result of which is to obtain stable molecular, time-stable solutions suitable for further processing, as well as ready for use in a wide range of tasks, and not possessing the disadvantages of the claimed prototype.

The essence of the invention lies in the development of a method for the synthesis of highly efficient nanocomplexes based on nanoparticles of biogenic elements (selenium and silver, etc.) and enzymes (chymotrypsin). Enzymes stabilize nanoparticles of both metals and non-metals, are stable and stable in solution and meet the requirements of such derivatives in medicine and bionanotechnology. The presence of both charged groups and hydrophobic fragments in the enzymes makes them universal stabilizers of nanoparticles and ensures the formation of molecular solutions of any nanoparticles, regardless of the reducing agent used, since the enzyme can be stable both in the acidic and alkaline regions. The combination of properties of the enzymes used as stabilizers, allows to eliminate the disadvantages of both polymer and other stabilizers.

The specified task and result in the invention is achieved by the implementation of a redox reaction with the introduction of a stabilizer - an enzyme, the resulting nanoparticles directly into the reaction. To a solution of chymotrypsin, the concentration of which can vary from 0.01 to 0.1%, a solution of a precursor is added - selenic acid 0.013M concentration or silver nitrate - 1%, 0.01%, 0.001%, the solutions are mixed and kept at room temperature 20 ° C or lowered 0 ° C temperature for 30-60 minutes. Then, a reducing agent is introduced into the reaction flask: ascorbic acid or sodium borohydride, the solutions are mixed and left to complete the reaction; the reaction of obtaining nanoparticles is carried out in the presence of an enzyme and the nanoparticles of biogenic elements formed are stabilized almost at the time of their formation, while stabilized nanoparticles of biogenic elements can be obtained in the form of molecular solutions stable in time to precipitate at different pH of the medium, of various controlled diameters with a yield of 75-97%.

Distinctive features of the proposed method are the above; the proposed method for stabilizing nanoparticles with enzymes has obvious advantages over the prototype.

The analysis of the prior art did not allow to find published solutions that would use the whole set of essential features of the claimed method. This indicates the conformity of the method of the invention under the conditions of patentability as "novelty" and "inventive step".

In accordance with the proposed technical solution, it is possible to obtain stabilized nanoparticles of biogenic elements in the form of molecular solutions stable in time to precipitate at different pH of the medium, of various controlled diameters with a yield of 75-97%. The size of the nanocomplex is controlled by the introduction of the amount of reducing agent and the pH of the medium.

In identifying the materiality of the novelty of the features, the following was obtained.

The enzyme is a good stabilizer of various in nature nanoparticles of biogenic elements (metal and non-metal).

The reaction of obtaining nanoparticles proceeds in the presence of an enzyme and the resulting nanoparticles of nutrients stabilize (interact with the enzyme) almost at the time of formation.

By varying the concentration of the stabilizing enzyme, a different degree of stability of the resulting nanocomplex over time is achieved.

By changing the ratio of nanoparticle: enzyme, nanocomplexes can be obtained in a wide pH range.

The resulting nanocomplex allows the use of the properties of nanoparticles (antimicrobial, antioxidant, anti-inflammatory, anticarcinogenic, detoxifying) and the properties of the enzyme (proteolytic activity).

To prove the conformity of the claimed solution to the condition of patentability "industrial applicability" and to better understand the essence of the claimed invention, examples of specific performance are given.

Example 1. The synthesis of selenium nanoparticles stabilized by chymotrypsin in the ratio of selenium: chymotrypsin 0.01: 0.01 mass%. 0.5 ml of a 0.2% solution of XT is placed in a 30 ml flat-bottomed flask, 7.5 ml of distilled water are added, then 1 ml of 0.013 M selenic acid (H ₂ SeO ₃ ) is added with stirring on a magnetic stirrer and after stirring for Ascorbic acid (C ₆ H ₈ O ₆ ) 1 ml of 0.025 M is added as a reducing agent for 10 min. Stirring is continued for another 5 minutes and then left to complete the reaction (24 hours) at room temperature. The resulting molecular solution has a characteristic reddish-orange color, the pH of the solution is 3.2. The solution is stable up to 4 years.

Example 2. The synthesis of silver nanoparticles stabilized by chymotrypsin in the ratio of silver: chymotrypsin 0.1: 0.1. 0.2114 g of sodium borohydride (NaBH ₄ ) was placed in a 30 ml flat-bottomed flask, a flask was placed in ice, and 10 ml of distilled water was added. Dissolved NaBH _{4 is} aged in ice for 30-40 minutes. At the same time as NaBH _{4, a} 50 ml flask was placed in ice, into which 5 ml of a 0.2% solution of XT and 1 ml of a 1% solution of silver nitrate (AgNO ₃ ) were introduced. The mixture is mixed in a circular motion of the flask in a mold with ice and remains in it for 30-40 minutes. After the temperature is equalized, 4 ml of the prepared solution is introduced into the flask with XT and silver nitrate. A violent reaction begins, the reaction flask remains in ice for 1 hour, and then placed in the refrigerator for 18-24 hours. As a result of the reaction, a black solution is obtained whose pH is 10.4. The solution is stable from 1 month to 1 year.

The data of specific synthesis examples are summarized in the table:

XT Precursor Conditions Reducing agent Selenium: chymotrypsin 0.5 ml H ₂ SeO ₃ Room by that. C ₆ H ₈ O ₆ 0.01: 0.01 0.2% 1 ml, 0.013 M 1 ml, 0.025 M 0.5 ml H ₂ SeO ₃ Room by that. C ₆ H ₈ O ₆ 0.02: 0.01 0.2% 2 ml 2 ml 0.5 ml H ₂ SeO ₃ Room by that. C ₆ H ₈ O ₆ 0.015: 0.01 0.2% 1,5 ml 1,5 ml 5 ml H ₂ SeO ₃ Room by that. C ₆ H ₈ O ₆ 0.01: 0.1 0.2% 1 ml 1 ml 5 ml AgNO ₃ Cool. NaBH ₄ 0.1: 0.1 0.2% 1 ml 1% 4 ml 5 ml AgNO ₃ Chill out. NaBH ₄ 0.01: 0.1 0.2% 1 ml 0.1% 0.4 ml 5 ml AgNO ₃ Chill out. NaBH ₄ 0.001: 0.1 0.2% 1 ml 0.01% 0.04 ml 0.5 ml AgNO ₃ Chill out. NaBH ₄ 0.01: 0.01 0.2% 1ml 0.1% 4 ml

Thus, as described above, the effectiveness of the method lies in the fact that stabilization of any nanoparticles is carried out in a wide pH range, the result of which is to obtain stable molecular, time-stable solutions, suitable both for further processing and ready for use in a wide range of tasks .

Claims (1)

A method of stabilizing nanoparticles of nutrients by enzymes, including the synthesis of nanoparticles through a redox reaction with the introduction of a stabilizer-enzyme, the resulting nanoparticles directly into the reaction, characterized in that the solution of the enzyme is chymotrypsin, the concentration of which is selected from 0.02%, add a solution of the precursor - selenic acid 0.013 M concentration or silver nitrate - 1%, 0.01%, 0.001%; the solutions are stirred and kept at room temperature 20 ° C or lower 0 ° C for 30-60 minutes, then a reducing agent is introduced into the reaction flask: ascorbic acid or sodium borohydride, the solutions are mixed and left to complete the reaction; in this case, the reaction of obtaining nanoparticles is carried out in the presence of an enzyme and the resulting nanoparticles of biogenic elements are stabilized at the time of formation, while stabilized nanoparticles of biogenic elements are obtained in the form of molecular solutions that are stable in time against precipitation at different pH of the medium, of various controlled diameters with a yield of 75-97%.