RU2320323C1 - System for delivery of bioactive substances by using niosomes - Google Patents

Abstract

FIELD: biology, medicine.

SUBSTANCE: invention relates to niosomes prepared on the base of modified silicone emulsifiers namely dimeticone copolyols forming vesicules-niosomes with included biologically active substances therein. Said biologically active substances represent extract from stem cells of porcine placenta, avocado oil, and cyclometicone which are included in gel composition.

EFFECT: gel of increased stability.

3 ex, 3 tbl

Description

The invention relates to biology and medicine and can be used to deliver biologically active substances (BAS) in the tissue of a macroorganism in pharmacology and cosmetology. The inclusion of biologically active substances in niosomes ensures their effective metered delivery to tissue cells. This is due to the ability of a nonionic surfactant (SAS) to form a niosome, a structure similar to the cytoplasmic membrane of a cell.

Niosomes are vesicles consisting of a shell in the form of a water-insoluble double layer of a nonionic emulsifier (surfactant), which is a group of dimethicone copolyols that are esters of a polyethylene glycol and a polydimethylsiloxane base and a biologically active substance enclosed inside a capsule. Thus, dimethicone copolyols are a hybrid of silicon and carbon. Unlike liposomes made on the basis of phospholipids, niosomes have a number of advantages for the delivery of biologically active substances. The presence of the covalent Si – O bond in the hydrophobic part of the polydimethylsiloxane emulsifier base molecule, which has great elasticity and reactivity, allows targeted delivery of a wide range of biologically active substances with the help of reactive sites and their targeted release from the vesicles. These properties allow the use of niosomes for interstitial delivery of biologically active substances (antibiotics, vitamins, plant and animal extracts).

Known interstitial method of delivery of biologically active substances using liposomes (RF Patent No. 2234311, publ. 08/20/2004. Bull. No. 23). The delivery of ingredients is carried out due to the fact that the components of the bilayer membrane of liposomes are actively involved in metabolic processes, interacting with the cells of the macroorganism, and cause changes in the functional state of individual organs and systems.

The study of the biological and functional activity of liposomes has opened up the possibility of using their transport function, which consists in delivering biologically active substances into the skin cell (RF Patent No. 2241440, publ. 10.12.2004. Bull. No. 34).

However, the disadvantage of liposomes is the content of amphiphilic phospholipids in their structure, which have a high ability to oxidize (rancid), as well as the presence of residual carcinogenic solvents (chloroform, methanol) used in the manufacture of liposomes, and significant expenditures of mechanical energy for the formation of a vesicle.

Closest to the proposed invention relates to a method for the delivery of biologically active substances using siloxane emulsifiers (US patent - US Patent 5364633. Silicone vesicles and entrapment, 15/11/1994), the essence of which is the inclusion of water-soluble biologically active substances in vesicles formed from siloxane surface active substance, preliminary dissolution of the BAS and its involvement in the vesicle with the addition of a siloxane surfactant, easy mixing of the mixture, further removal of excess water and BAS.

The disadvantage of the prototype is the lack of reproducibility of the vesicles, accompanied by the formation of micelles (inferior vesicles), and, as a result, the insufficient inclusion of biologically active substances in the vesicles. The lack of reproducibility of vesicles of a given size is accompanied by an increase in the consumption of siloxane emulsifier, which can lead to possible irritation of the skin and the cost of the target product.

The aim of the invention is to develop a system for the effective delivery of biologically active substances in tissue. This goal is achieved by the fact that for the delivery of biologically active substances in tissues, modified silicone non-ionic emulsifiers are used, which are copolyols dimethicone capable of forming niosome vesicles.

In relation to the prototype, the claimed invention is distinguished by the fact that the obtained niosomes have increased stability due to the presence of hydrophilic residues of polyethylene glycol (PEG) and the presence of reaction sites in dimethicone (polydimethylsiloxane part of the surfactant molecule). Niosomes obtained from dimethicone copolyols are complete multilamellar vesicles with good reproducibility and are an excellent tool for delivering a wide range of biologically active substances. The presence of a polydimethylsiloxane base allows one to obtain vesicles capable of withstanding a wide range of solvents such as ethyl alcohol, isopropyl palmitate, etc. Thus, unlike other vesicles, niosomes can be used in alcohol-containing formulations.

For delivery of niosomes to tissue cells, a gel can be used as a carrier, the spatial structure of which allows niosomes to be kept in suspension, while maintaining their structural integrity.

The possibility of carrying out the claimed invention is confirmed by examples of specific performance.

Example 1

As a copolyol dimethicone for the formation of niosomes, a surface-active compound containing 18 polyoxyethylene groups in polyethylene glycol (PEG-18 dimethicone) was used.

Due to the fact that biologically active substances occupy a small volume relative to other components of the gel, its incorporation into niosomes is performed by adding 2% PEG-12 dimethicone to a biologically active substance solution in a separate container. The process is carried out at room temperature and intensive mechanical shaking for 5 minutes. After the inclusion of biologically active substances in niosomes, a suspension of niosomes is introduced to other components of the gel at room temperature. A feature of the inclusion of the lipid phase of the gel, containing 5% avocado oil and 5% cyclomethicone, in niosomes is the preliminary dissolution of 2% PEG-18 dimethicone in it, mixing and subsequent introduction of the mixture into the aqueous phase. The final stage of the formation of vesicles occurs with intensive mechanical stirring of the mixture.

As a delivered biologically active substance, pig placenta stem cell extract is used to accelerate the proliferation of keratinocytes (human skin cells) - Decision on the grant of a patent of the Russian Federation dated April 24, 2007 according to application No. 2006105864/15.

The process of preparing a stem cell extract consists in washing pork placenta tissue in phosphate buffer or Hanks solution, followed by grinding it into pieces of 2-3 cm ² , pour the pieces into 150 ml of a 25% trypsin solution, trypsinization on a magnetic stirrer for 3 hours at 20 ° C, then the biomass of regional stem cells is grown in a monolayer on medium 199 followed by water-salt extraction.

The resulting water-salt extract of stem cells with 5, 15 and 30% protein content was used for inclusion in niosomes.

The phases of preparation and formulation of the gel are presented in table 1.

Phase A is prepared at room temperature by mechanically mixing the components in a mixer. Phases B and C containing suspensions of niosomes are made separately according to the technology described above and are introduced into phase A with stirring. To stabilize the concentration of hydrogen ions (pH) to 6.6-7.0 and the formation of the structure of the gel contribute phase G.

The prepared gel is applied to previously cleaned skin 1 time per day. Every day before re-applying the gel, the degree of keratinization is evaluated. The maximum increase in the degree of keratinization is 10% of the initial values and occurs on day 5 from the application of the gel.

Example 2. It is carried out analogously to example 1, only as a copolyol dimethicone for the formation of niosomes, a surface-active compound containing 12 polyoxyethylene groups in polyethylene glycol (PEG-12 dimethicone) was used, as well as a suspension of niosomes introduced in an amount of 5%, of which 15 % (protein) stem cell extract. The maximum increase in the degree of keratinization is 30% of the initial values and occurs on day 5 from the application of the gel.

Example 3. It is carried out analogously to example 1, only as a copolyol dimethicone for the formation of niosomes, a surface-active compound containing 20 polyoxyethylene groups in polyethylene glycol (PEG-20 dimethicone) was used, as well as a suspension of niosomes introduced in an amount of 8%, of which 30 % (protein) stem cell extract. The maximum increase in the degree of keratinization is 30% of the initial values and occurs on day 5 from the application of the gel.

Thus, the optimal delivery system is a suspension of niosomes in an amount of 5% (by weight of the gel) with a content of 15% (by protein) of stem cell extract (example 2). A further increase in the percentage of niosomes and stem cell extract does not lead to an increase in the degree of keratinization.

The designed gel was clinically tested in the regional dermatovenerological dispensary and at the Department of Dermatology and Cosmetology of the Stavropol State Medical Academy (protocol No. 11 of November 25, 2005). As a result of the studies, the optimal percentage ratio of niosomes and stem cell extract was selected, which allows to obtain the maximum increase in the degree of keratinization (table 2).

Table 1. Stages of preparation and formulation of a gel with stem cell extract No. p / p Name of ingredient Content in% (by weight) Phase A one Distilled water up to 100 2 Preservative 0.05 3 Gelling agent 2,5 four Perfume 0.15 5 Hyaluronic acid 0.1 Phase B 6 Niosome suspension containing stem cell extract 3.0-8.0 Phase B 7 Nyosome suspension containing lipid fraction (5% avocado oil, 5% cyclomethicone) 12.0 Phase G 8 Triethanolamine 2.0

Table 2. The percentage of niosomes and stem cell extract Examples one 2 3 Niosome Suspension 3 5 8 (wt.% application to the gel) Stem cell extract 5 fifteen thirty (wt.% protein) The degree of keratinization (% increase from the initial value) 10 thirty thirty

Clinical Testing Protocol No.11 of November 25, 2005 Stavropol State Medical Academy Department of Dermato-Venereology and Cosmetology Niosome gel Applicant: Bazikhov Igor Aleksandrovich Stavropol, Oktyabrskaya Ave. 34, apt. 4. Safety indicators: SanPiN 1.2.681-97 "Hygienic requirements for the production and safety of perfumes and cosmetics" tab. Number 3 TEST RESULTS No. p / p Name of indicator * Gel with biologically active substances ** (control) Gel with biologically active substances ** included in niosomes The content of nios in the gel,% 3% 5% 8% one The degree of keratinization (% increase from the initial value) 6 10 thirty thirty 2 The degree of moisture (% increase from the original value) 2 5 thirty thirty 3 The content of stem cell extract (wt.% Protein) fifteen 5 fifteen thirty four Kind of dimethacon copolyol used for nios preparation - PEG-18 Dimethicone PEG-12 Dimethicone PEG-20 Dimethicone 5 Drip test
mild hyperemia
swelling
units vesicles, rashes lack of
lack of
lack of lack of
lack of
lack of lack of
lack of
lack of lack of
lack of
lack of 6 Compressor test
mild hyperemia
swelling
units vesicles, rashes lack of
lack of
lack of lack of
lack of
lack of lack of
lack of
lack of lack of
lack of
lack of * The gel was tested on 27 test agents for 5 days ** As a BAS, stem cell extract from pig placenta, cyclomethicone and Avocado oil were used

Claims (1)

The delivery system of biologically active substances, consisting of surfactants, which are modified silicone emulsifiers - dimethicone copolyols, forming vesicles - niosomes, biologically active substances included in the vesicles, which are stem cell extracts from pig placenta, avocado oil and cyclomethicone, which included in the composition of the gel, containing, wt.%:  suspensions of niosomes containing stem cell extract 3.0-8.0  suspensions of niosomes containing avocado oil and cyclomethicone 12.0  triethanolamine 2.0  gelling agent 2,5  hyaluronic acid 0.1  perfume 0.15  preservative 0.05  distilled water up to 100