RU-2861486-C1 - METHOD FOR OBTAINING NIOSOMAL FORM OF OLIGODEOXYNUCLEOTIDES

Abstract

FIELD: medicine; biotechnology. SUBSTANCE: invention relates to a method for obtaining a niosomal form of oligodeoxynucleotides by the reverse-phase evaporation method. Sorbitan monostearate, cholesterol, and dicetyl phosphate are dissolved with stirring in dichloromethane. A solution containing 0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 8.0, and 50 mcM oligodeoxynucleotide is added to the mixture. The mixture is subjected to ultrasonic disintegrator treatment for 5 minutes, with an amplitude of 7.5 mcm, a frequency of 20 kHz. The emulsion is transferred to a round-bottom flask and dichloromethane is distilled off on a rotary evaporator. Then 20% of the initial volume of the aqueous phase is added to the mixture, and distillation is continued. 10 ml of a buffer solution containing 0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 8.0, is added to the preparation, after which it is centrifuged for 30 minutes at 12000 rpm. The supernatant is removed, 2 ml of a buffer solution containing 0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 8.0, is added to the residue, and transferred to a clean vessel. EFFECT: provision of a method for obtaining niosomes based on non-ionic surfactants with encapsulated oligodeoxynucleotide, with a high efficiency of active substance inclusion. 1 cl, 4 ex

Inventors

• Zhirov Andrej Mikhajlovich
• Dementeva Elena Nikolaevna
• Lobach Irina Vladimirovna
• Kovalev Dmitrij Anatolevich
• KULICHENKO ALEKSANDR NIKOLAEVICH

Dates

Publication Date

20260505

Application Date

20251020

Claims (1)

1. A method for obtaining a niosomal form of oligodeoxynucleotides, including obtaining by reverse-phase distillation, characterized in that 0.490 g of sorbitan monostearate, 0.251 g of cholesterol and 0.052 g of dicetyl phosphate are dissolved with stirring in 50 ml of dichloromethane and mixed with 10 ml of a solution containing 0.05 M tris (hydroxymethyl) aminomethane, 0.15 M NaCl, pH 8.0, and 50 μM oligodeoxynucleotide, then the mixture is emulsified in an ultrasonic disintegrator for 5 min, at an amplitude of 7.5 μm, a frequency of 20 kHz, the emulsion is transferred to a round-bottomed flask and dichloromethane is distilled off on a rotary evaporator for 20 min at a pressure of 0.35 bar, a temperature of 26±1 °C and 150 rpm, then 25 min at a pressure of 0.35 bar, a temperature of 55±1°C, 200 rpm, then 2 ml of type I water are added to the mixture and distillation is continued for 45 min at a pressure of 0.35 bar, a temperature of 26±1°C and 140 rpm, 10 ml of a solution containing 0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 8.0 are added to the preparation, after which it is centrifuged for 30 min at 12,000 rpm, the supernatant is removed, 2 ml of a solution containing 0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 8.0 are added to the sediment, and it is transferred to a clean container.

Description

Field of technology This invention relates to medicine and biotechnology, specifically to a method for microencapsulating oligodeoxynucleotides in niosomes. It can be used in medical and veterinary practice for the development and use of vaccines in niosomes, which reduces the degree of their inactivation and optimizes their action through slow release from microvesicles based on nonionic surfactants. State of the art DNA and RNA oligonucleotides are increasingly being used as components of prophylactic and therapeutic drugs. CpG oligodeoxynucleotides are well-known and widely used vaccine adjuvants, and small interfering RNAs are being explored as a promising platform for the treatment of genetic and infectious diseases. The ability to program structures and their high biocompatibility make DNA oligonucleotides potential vaccine carriers [1, 2]. The rapid degradation of oligodeoxynucleotides by nucleases is a serious problem when used in vivo. A sufficient shelf life of DNA/RNA oligonucleotides after administration is a prerequisite for good therapeutic efficacy. Numerous strategies exist for enhancing the biostability of DNA/RNA oligonucleotides, employing modifications of the DNA backbone, such as phosphorothioate and triazole modification of the phosphodiester bond, the introduction of non-canonical secondary structures, and encapsulation in lipid vesicles. The present invention proposes an approach to increasing the stability of oligodeoxynucleotides under conditions close to in vivo conditions (20% fetal bovine serum in 0.01 M phosphate-buffered saline, pH 7.4, temperature (37±1)°C) by encapsulation in Span60-based niosomes. A known method for producing niosomal nucleic acid preparations involves mixing Span20 and cholesterol in a molar ratio of 2.5:2.5 mM in a methanol:chloroform solvent mixture (1:1 v/v). Flexible cationic lipid A (PCL-A) in a methanol:chloroform solvent mixture (1:2 v/v) with different PCL-A concentrations (0.5, 1, 1.5, 2, and 2.5 mM) was then added to the mixture. The solvents were evaporated under a stream of nitrogen to obtain a thin film. The film was left in a desiccator overnight, then hydrated in Tris-buffered saline with a pH of 7.4 at 60°C and stirred for 30 s. The mixture was then sonicated for 30 min. The size of niosomes was 158.6-1743 nm, zeta potential from -21.77 to 62.2 mV [3]. A system for targeted delivery of oligonucleotides in cancer therapy based on niosomes is known, according to which a mixture of 300 mg Tween 80, 300 mg Span 80 and 80 mg cholesterol is dissolved in chloroform in a round-bottomed flask, after which the solvent mixture is distilled off on a rotary evaporator at 60 °C and reduced pressure. Then, an oligonucleotide solution containing 20 mg/ml bovine serum albumin is slowly added to the surfactant film, after which the mixture is treated with ultrasound for 20 min. The size of niosomes ranged from 126.9 ± 3.5 nm to 148.53 ± 0.5 nm, the zeta potential from -18.03 ± 0.83 mV to -36 ± 2.26 mV [4]. The closest invention to the described method in technical essence is a method for producing a niosomal form of cefotaxime by reverse-phase distillation. A chloroform solution of sorbitan monostearate, cholesterol, polyethyleneglycol-4000, and dicetyl phosphate in a molar ratio of 60:34:5:1, respectively, is mixed with 0.01 M phosphate-buffered saline, pH 7.40, containing cefotaxime, in a ratio of 5:1 by volume and emulsified using an ultrasonic disintegrator for 5 minutes. Chloroform is then removed by distillation at a pressure of 0.175 bar and a temperature of (26±2)°C using a rotary evaporator, the gel is hydrated for 25 minutes at (55±1)°C, after which 20% of the initial volume of the aqueous phase is added to the resulting surfactant-lipid gel and the niosome dispersion is maintained at a pressure of 0.175 bar and a temperature of (26±2)°C for 45 minutes. The efficiency of antibiotic incorporation was 63.7±1.2% [5]. The method under consideration has a number of disadvantages, the main ones being the lack of standard conditions for reversed-phase distillation and low inclusion efficiency for hydrophilic compounds such as cephalosporin antibiotics. The aim of the invention is to develop a method for obtaining a niosomal form of oligodeoxynucleotides based on non-ionic surfactants. Disclosure of invention The technical result of the proposed invention is achieved by optimizing the niosome construction technology, composition, and ratio of structure-forming components. The claimed method possesses the following distinctive features compared to the prior art: - The use of dichloromethane as an organic solvent and three-stage distillation allows for the effective, reproducible and most complete removal of the solvent from the finished product, without uncontrolled boiling of the mixture and at a lower distillation pressure. - Washing the niosomal preparation with a buffer solution containing 0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 8.0, rem