RU2311449C2 - Method for preparing liposomal composition - Google Patents

Abstract

FIELD: food processing industry, veterinary science, pharmacology, biology, medicine and cosmetic industry.

SUBSTANCE: method involves forming a mixture of lipids with lipophilic and hydrophilic supplements, small-dispersed powder and dispersing process. On preliminary step in preparing a lipid phase it is treated with amino-containing compounds for charge redistribution and lipids are subjected for partial hydration also. Then supplements are added in necessary amounts, and liposomes are formed on the interface water/water-insoluble particles biphase system. Invention provides preparing the composition showing high degree of stability and incorporating capacity, and to reduce energy consumption also.

EFFECT: improved preparing method.

3 cl, 6 ex

Description

The invention relates to biotechnology and can be used in biology, pharmacology, medicine, the cosmetic industry, veterinary medicine and the food industry.

Liposomes or phospholipid vesicles (FLV) are used in biology, pharmacology, medicine, and the cosmetic industry to create systems for the targeted transport of biologically active substances in order to increase the therapeutic activity of finished medicinal, cosmetic, and veterinary drugs.

Preparations based on phospholipid vesicles (FLV) appeared relatively recently and in a short time became widespread, first in biology, and then in medicine and cosmetics. The effectiveness of exposure to FLV is due to their properties, in particular the property of tropism, which ensures their rapid penetration into tissues and transdermal delivery of substances useful to the body, including phospholipids. Imbalance of phospholipids leads to various pathological changes in the cell membranes and to the violation of the epidermal barrier. The use of drugs based on liposomes (LP) provides a quick restoration of the epidermal barrier, membranes of living cells and helps eliminate phospholipid syndrome. Liposomal transport provides almost complete utilization of the included substances and the ability to reduce the concentration of the active principle, which is especially important when working with therapeutic substances that can have side effects, as well as economically justified.

Depending on the production method, phospholipid vesicles can have larger or smaller sizes, different layers (one or more lipid bilayers). In cases where it is necessary to enclose high-molecular substances in a liposome (for example, when solving genetic engineering problems when incorporating DNA molecules), large liposomes, including multilayer ones, are used. For effective action on the skin and mucous membranes, small single-layer vesicles are usually preferable.

Of the previously developed methods for producing liposomes, a method for producing a liposome composition is known (RF patent No. 20144070, A61K 9/127, 1991).

The disadvantage of this method is that it does not provide liposomes having small sizes. In addition, the known method is characterized by considerable complexity.

There is also a known method for producing a liposomal composition (European Patent No. 0130577, MKI ⁷ A61K 9/50, 7/48, 1989), comprising mixing the components of the liposome membrane with a water-soluble non-volatile physiologically acceptable solvent and dispersing the resulting mixture in an aqueous medium using a conventional mixer.

The liposomes obtained by this method have a relatively large size of 2500 nm, which is the main disadvantage of this method.

A known method of producing organosilicon microcapsules for the creation of medical, cosmetic preparations, comprising mixing lipids with water-soluble additives and organosilicon compounds, and the dispersion is carried out on a rotary-cavitation unit (RF patent No. 2173140 MKI ⁷ A61K 9/127, 9/50, BI No. 25, 2001).

The disadvantage of this method is that special equipment is required (RKU - rotary cavitation unit), where liposomes are formed under the influence of ultrasonic and hydroacoustic vibrations, and water softening is achieved using Trilon B, however, liposome aggregation is slow during storage and packaging , which violates the stability of the finished form.

Closest to the technical nature of the present invention is a method for producing a liposomal composition, comprising forming a mixture containing at least one component of the liposomal membrane, water and an additive, dispersing the mixture, in which either submicro powder of a substance insoluble in water and in water is used as an additive organic solvent, or water-swellable polymers, or a liquid not miscible with water and other components of the mixture (RF patent No. 2104691 MKI ⁷ A61K 9/127, 9/50, 9/66, BI No. 5, 1998).

The disadvantages of this method is the following:

- for stable reproducibility of the method on an industrial scale, special equipment is needed (mixing devices with powerful engines or frame mixers, flow-through mixing mode);

- strict selection of all lipid components in order to avoid their aggregation at the initial stage when they are introduced into the Sacap polymer solution having an acidic medium or when interacting with other active submicroscopic powders in an acidic medium when preparing a liposome composition;

- the use of distilled or highly purified water only, since the system is very sensitive to the presence of an insignificant salt content, even those contained in water-alcohol extracts;

- the impossibility of increasing the load of lipid components, since with an increase in their concentration over 3% there is a rapid aggregation of liposomes, which limits the range of incorporated active components and the variety of finished products.

Due to the fact that to solve the problems of transdermal delivery of biologically active substances, single-layer small vesicles capable of penetrating through the intercellular space are needed, a more universal method is needed. The method should provide regulation of the conditions of the equipment operating modes (input rate of the lipid phase, diameter of the inlet for the introduction of the lipid mixture, power and speed of the mixer, etc.), i.e. ensure the universality of work and guarantee the stability of liposomes over the entire shelf life, change the size and properties of liposomes depending on the purpose of the finished form of the drug.

The objective of the invention is to provide a method for producing a liposomal composition with particles of certain sizes from 40-100 nm to 200-3500 nm with a high degree of stability and incorporating ability, as well as reducing energy consumption by using simple mixing devices and reducing the concentration of active components.

As a result of using the present invention, it becomes possible to obtain a more perfect basis for the preparation of medical, cosmetic, pharmacological, veterinary and other drugs. Certain sized liposomes containing biologically active substances (BAS) and / or medicinal substances and possessing aggregative stability in media with a pH range from 5.1 to 9.0 and a salt content in the aqueous phase of up to 0.1% (mainly Ca ⁺⁺ and Mg ⁺⁺ ), when stored for 2 years, they appear to be the most versatile carriers for the creation of medical, cosmetic and veterinary preparations.

The technical result is achieved by the fact that in the proposed method for producing a liposomal composition, including the formation of a mixture of lipids with lipophilic and hydrophilic additives, finely dispersed powder structure-forming agent and dispersion, the lipids are treated with amine-containing compounds in advance to prepare the fat phase to redistribute the charge, as well as partial lipid hydration , then additives are added in the required amounts, and liposomes are formed at the interface of the two-phase systems Water / water-insoluble particles.

When using the method, lipophilic and / or hydrophilic active components of plant origin are introduced as additives: oil extracts (fir, burdock, flax, chamomile), oils (olives, jojoba, cocoa), lecithin (egg, soy), essential oils (rosemary, fennel, Chinese magnolia vine, wormwood, cedar, pine), dry extracts (fir, golden root, henna, etc.) and / or animal origin (ASD - Dorogov's antiseptic stimulator, lanolin, etc.), and / or collagen of marine origin - fractions from algae, plankton, fish oils and fish ir s, etc.).

In the method, polymers of the type Carbomer and / or aristoflex, and / or collagen, and / or polymers of cellulose and / or chitosan and / or gelatin, dicalcium phosphate, and / or aerosil, and / or chalk, and / are used as a fine powder or inert clay.

In the method, lipophilic or hydrophilic drug substances (tocopherol acetate, diclofenac, demidrol, urea, analgin, peromecaine, darinate, etc.) are introduced as additives.

In the method, lipophilic or hydrophilic veterinary preparations (methylene blue, sulfur, permethrin, ASD, antibiotics, etc.) are introduced as additives.

Lecithin is plasticized with olive oil or jojoba oil and an amine-containing substance (for example, triethanolamine, carbomide, ASD) and an antioxidant complex based on vitamins P, E, A, D are introduced and placed in a mixing tank. As a dispersing device use any mixer-homogenizer, which provides mixing of the mixture of the aqueous phase with particles of fine powder and lipids. In the process of dispersing lipids in an aqueous phase saturated with small particles of a water-insoluble powder of the structure-forming agent - polymer, small single-layer vesicles are formed at the interface between the solid and liquid phases, and an increase in the content of powder particles increases the area for liposome formation (increases the saturation of the suspension with liposomes).

The proposed method allows the use of lipids, excluding the presence of organic solvents, due to the plasticization of lecithin with olive oil, polyethylsilaxane PES and the introduction of amine-containing compounds. In the proposed method, conventional homogenizing mixers are used to obtain small single-layer vesicles, by adjusting the speed of the mixer, MOBs of the required sizes are formed (from 40-60 nm to 100-250 nm), and the number of liposomes is increased by the number of fine particles and the rate of lipid mixture entering the unit .

Amine-containing substances are added in acceptable amounts as a stabilizer of the lipid phase, a homogenizer is used as a dispersing device, and a standardized polymer of the Carbamer type is used as fine particles. In certain cases, it is possible to use microscopic particles of water-insoluble chemical elements (for example, ZnO, S) or other targeted additives.

Using a mixture of lecithin / olive oil / amine-containing compounds allows you to get a uniform thin lipid film at the interface between solid and liquid phases (water / water insoluble particles), which is very important for the formation of membrane structures.

The proposed method allows you to create a lipid mixture, regulate the mode of its introduction and as a result receive stable liposomal suspensions and exclude the use of alcohol and other organic solvents.

The method for producing the liposomal composition makes it possible to produce medical and cosmetic preparations according to a universal technological scheme that can be easily scaled in any production conditions (from laboratory to large-workshop) on any industrial and semi-industrial equipment, including the preparation of ingredients, and the dispersion process itself, and the presence of a certain number in volume dispersing microparticles provides the creation of liposomal suspensions of different saturations.

Specific examples of the invention are presented below.

Example 1

Preliminarily, at the stage of preparation of the fat phase, all oil components (lecithin, olive oil) are mixed and the lipid mixture is treated with a solution of urea (urea) to redistribute the lipid charge and partially hydrate.

Prepare aqueous solutions of dry extracts of succession and lavender.

Prepared hydrated standardized polymer powder - carbomer 2001 is loaded into the homogenizer mixer and it is thoroughly mixed in the required volume of water at room temperature (polymer concentration 0.40%).

Then, the process of forming a liposomal suspension is carried out: a lipid mixture of lecithin, olive oil and urea in a ratio of 1: 2: 0.5, respectively, and aqueous solutions of dry extracts of medicinal herbs - extracts of a series and lavender (additives) are introduced into the reactor at the maximum intensity of the homogenizer ( 4 thousand rpm).

As a result, liposomes are formed at the interface of the two-phase water / water-insoluble particles (solid and liquid phases), which have an average size of 80 to 100 nm. After the final neutralization of the total mass with an alkaline solution (sodium hydroxide or triethanolamine), the liposomal substance can be used at various pH values from 5.5 to 7.0, depending on the purpose of the cosmetic product. The tool can be used in children's and medical cosmetics for the prevention of skin diseases.

Example 2

Previously, at the stage of preparing the fat phase, mixing all the oil components (lecithin, olive oil, vitamin complex), the lipid mixture is treated with a solution of triethanolamine to redistribute the lipid charge and partially hydrate.

Aqueous solutions of anestezin and diclofenac are prepared.

Prepared hydrated polymer powder - carbomer 98 is loaded into the homogenizer, and it is thoroughly mixed in volume with distilled water at a temperature of 32 ° C, the polymer concentration is 0.5%, then the pre-treated fat phase (lecithin, olive oil, vitamin complex) is introduced, triethanolamine solution, solutions of diclofenac, anastesin (additives) in a ratio of 1: 4: 0.5: 0.01: 0.003: 0.1, respectively, with vigorous stirring (rpm 3.5-4.0 thousand).

In the process, a liposomal suspension is formed with an average particle size of 50-100 nm. In this case, the pH of the medium ranges from 5.6-5.8. When the obtained substance is neutralized, the polymer thickens and a plastic gel mass is formed - a new dosage form for the treatment and prevention of inflammatory diseases in the joints. The recipe is reproduced using a laboratory homogenizer or a household mixer with a mixer speed of 1.5-2 thousand rpm and can be used to obtain ointments according to the prescriptions in the prescription department with a minimum volume of the finished composition of 100 ml.

Example 3

Preliminarily, at the stage of preparation of the fat phase, the fat phase is prepared by mixing all the oil components (lecithin, olive oil) and treated with a solution of ASD (Dorogov's antiseptic stimulant) and aqueous-alcoholic extract of burdock for redistribution of lipid charge and partial hydration.

An aqueous solution of dry yarrow extract is prepared.

The prepared hydrated standardized polymer powder, Aristoflex, is loaded into the homogenizer, and it is thoroughly mixed in a volume of water at room temperature (polymer concentration 0.15%), then the fatty phase from lecithin, olive oil, pre-treated with a solution of ASD, water-alcohol extract is introduced burdock and yarrow extract in a ratio of 0.1: 6: 0.36: 0.1: 1, respectively, with a maximum intensity of the homogenizer.

As a result, at the interface between the two-phase system water / water-insoluble particles form liposomes, which have an average size of 60 to 120 nm. After the final neutralization with an alkaline solution (sodium hydroxide or triethanolamine), a creamy mass is formed for the cosmetic milk from the resulting suspension.

Milk is used for problem skin, has a moisturizing, cleansing and regenerating effect. An example demonstrates the possibility of obtaining liquid and semi-liquid forms.

Example 4

Preliminarily, at the stage of preparation of the fat phase, the fat phase is prepared by mixing all the oil components (lecithin, fish oil, essential oils of rosemary, pine, anise) and treated with a solution of triethanolamine to redistribute the lipid charge and partially hydrate.

An aqueous solution of hyaluronic acid is prepared.

The prepared hydrated polymer powder, hyaluronic acid solution and carboxymethyl cellulose as additives are loaded into the homogenizer and mixed thoroughly in a volume of water at room temperature (total polymer concentration of 0.90% in different ratios), then the pre-treated fat phase from lecithin is introduced, fish oil, a solution of triethanolamine, essential oils of rosemary, pine, anise in a ratio of 2: 2: 0.26: 0.4: 0.1: 0.03, respectively, with a maximum intensity of the homogenizer. Moreover, liposomes form particles at the interface of a two-phase water / water-insoluble system, which have an average size of 80 to 150 nm. After the final neutralization with an alkaline solution (sodium hydroxide or triethanolamine), the substance can be used at a pH of 5.5 to 7.0, depending on the purpose of the product. By adjusting the percentage of essential oils (EM), you can get an effective prophylactic or food supplement in the form of a gel sauce.

Example 5

Preliminarily, at the stage of preparation of the fat phase, the fat phase is prepared by mixing all the oil components (lecithin, burdock oil, vitamin complex, lanolin), and is treated with an ASD solution for redistribution of lipid charge and partial hydration.

Prepared hydrated fine powder - Aristoflex, is loaded into the homogenizing mixer and it is thoroughly mixed in a volume of water at a temperature of 32 ° С, polymer concentration is 0.9%, then a lipid mixture of lecithin, burdock oil, vitamin complex, lanolin and ASD solution are introduced in the ratio 1: 2: 0.3: 0.1: 1.0, respectively, with vigorous stirring.

In the process, a liposomal suspension with an average particle size of 80-150 nm is formed at the interface between the solid and liquid phases. In this case, the pH of the medium ranges from 5.6-6.8. When neutralizing the obtained substance with triethanolamine, the polymer thickens and a plastic gel mass is formed - a new form of the veterinary drug for the treatment and prevention of allergic diseases of animals.

The tool has a general tonic effect, moisturizes the skin, stimulates blood circulation in the capillaries, improves metabolic processes, skin turgor, strengthens the immune system, accelerates cell renewal processes.

Example 6

Preliminarily, at the stage of preparation of the fat phase, the fat phase is prepared by mixing all the oil components (lecithin and essential oils of peppermint and rosemary), and is treated with a solution of sodium hydroxide to redistribute the lipid charge and partially hydrate.

An aqueous solution of a dry extract of stevia herb is prepared.

Prepared hydrated toothpaste abrasive powder (aerosil) and carboxymethyl cellulose with glycerin are loaded into a homogenizer and mixed thoroughly in a volume of water at room temperature, then a lipid mixture of lecithin, sodium hydroxide, peppermint and rosemary essential oils is added in a ratio of 20: 1 : 1: 25: 1: 0.4: 0.3: 0.1, respectively, at the maximum intensity of the homogenizer.

As a result, liposomes are formed at the interface between solid and liquid phases, which have an average size of 80 to 150 nm. After the final neutralization with an alkaline solution (sodium hydroxide or triethanolamine), the substance can be used at a pH of 5.5 to 7.0, depending on the purpose of the product. The liposomal composition is used in the preparation of toothpaste without surfactants for hypersensitive gums.

The method of obtaining the liposomal composition allows to increase the load on the lipid components and significantly expand the range of incorporated active substances. The method is time-saving, universal, allows you to get a series of tools on one industrial installation, is easily adapted under various conditions of technical equipment and laboratory support (from innovative devices to simple mixing plants).

Claims (3)

1. A method of obtaining a liposomal composition, comprising the formation of a mixture of lipids with lipophilic and hydrophilic additives, fine powder and dispersion, characterized in that the lipids are treated with amino-containing compounds at the stage of preparation of the fat phase to redistribute the charge, as well as partial lipid hydration, and then introduced into the required amounts of additives, and liposomes form at the interface of the two-phase system water / water-insoluble particles. 2. The method according to claim 1, characterized in that the lipophilic and / or hydrophilic active components of a plant and / or animal and / or marine origin are introduced as additives. 3. The method according to claim 1, characterized in that the lipophilic or hydrophilic drug substances are administered as additives.