KR20190009931A - Biomembrane, closed structure with biomembrane characteristics or cellular compartment derived from natural sources and/or self-assembly techniques, preparation method and applications thereof - Google Patents

Abstract

Provided are a biomembrane, a closed structure or a cellular compartment with characteristics of the biomembrane, and a preparation method thereof. The method comprises: (1) a step of obtaining biological cells from natural tissues or natural biological species; (2) a step of culturing the cells in the step (1) in a large volume in an adequate environment; and (3) a step of obtaining various biomembranes or a mixture belonging to the present invention through separation and purification of different methods after obtaining a lysed solution of the cells in the step (2). Methods for obtaining the biomembrane in vitro include a differential centrifugation method, a dense gradient centrifugation method, and a two-phase extraction method. A procedure for extracting the biomembrane and the closure structure or the cellular compartment with the characteristics of the biomembrane is performed by using the methods solely or in a pair of two or three. The membrane as the natural biomembrane and the closed structure and the cellular compartment with the characteristics of the biomembrane are used to package active ingredients in various fields.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a closure structure or a cell compartment having a biological membrane and a biological membrane obtained by natural and / or self-assembling techniques, and a production method and an application thereof (BIOMEMBRANE, CLOSED STRUCTURE WITH BIOMEMBRANE CHARACTERISTICS OR CELLULAR COMPARTMENT DERIVED FROM NATURAL SOURCES AND / OR SELF-ASSEMBLY TECHNIQUES , PREPARATION METHOD AND APPLICATIONS THEREOF}

The present invention belongs to the field of biomedical and polymeric materials, and specifically relates to closure structures or cell compartments having biomembrane and biomembrane properties obtained by naturally derived and / or self-assembled techniques, and a production method and application thereof.

Biomembranes or biological membranes are the collective term for all membrane structures in contact with cells, cell organs, and the environment. They serve as an important part of cellular communication, . In vivo, all but the virus has a biomembrane. Eukaryotic cells have a membrane system that separates various cell organs in addition to the plasma membrane (also called a cell membrane), and includes a nuclear membrane, a mitochondrion membrane, an endoplasmic reticulum membrane, an endosomal membrane, a Golgi membrane, a chloroplast membrane, a vacuole, .

The biomembrane morphologically has a layered structure of lipid bilayer, mainly composed of lipids, and a small amount of proteins and saccharides. This lipid bilayer structure of the biomembrane has the following performance. It controls and controls the entry and exit of substances, the packaging and transport of different intercellular organelles within cells, the formation of specific transport routes and cellular compartments in some reagents and signaling materials to provide storage space for substances give.

For the sake of research convenience, up to now, various membrane structures such as a monolayer membrane, a cumulative membrane, a lipid membrane, a bilayer membrane and a lipid membrane composed of single or multiple lipids have been proposed . It is also possible to construct reconstructed membranes by inserting proteins. These membranes are called "membranes". Artificial membranes are already widely used in practice, for example, they are used to efficiently separate and concentrate substances from solutions such as seawater to provide dialysis membranes and clinical diagnosis and treatment of renal disease patients. In recent years, lipid-carrier drugs have been produced using the feature that lipids can be fused with cell membranes, which is another relatively large development of the membranes.

However, membranes such as lipids have weaknesses such as oxidation, leakage, and poor shelf life. It is biodegraded by enzymatic materials in the body and can not reach target tissues because it is consumed by macrophages. In addition, by adding an artificial synthetic material, the artificial membrane is embedded in a human body in the form of a polymer material, and inevitably causes a rejection reaction in the body.

During the long evolutionary process of evolution from simple living organisms to complex living organisms, the emergence of biomembranes can be a breakthrough, and the biomembrane has evolved over a billion years, Has a mechanism of functional action, which itself has the two greatest properties, namely the fluidity of the membrane and the asymmetry of the membrane. The fluidity of membrane means that the biomembrane is in motion and the lipid molecules in the membrane constantly change its position, which is an important condition for the biomembrane to have functions such as endocytosis, exocytosis, mass transfer and cell fusion. However, due to the continuous deformation of the membrane due to the fluidity of such membranes, instability and other factors of the relatively simple membrane structure are caused by the components, stability and storage stability when the membrane containing the lipid body is used as a carrier are inferior, . The biomembrane is divided into myoclonus and noncellular two layers. The components and structure of the inner and outer layers of the biomembrane are very different from each other. The difference is called asymmetry. By this asymmetry, It has an important influence on the intermolecular discrimination, and this asymmetry can not be found in the synovial membrane.

 Life is the highest form of existence of matter, and the most basic feature of life is the ability to realize self-control, self-reproduction, and self-assembly through metabolism. Self-assembly is a component of a system, which refers to a phenomenon in which humans are organized and organized into a regular structure under a situation where they are not subjected to external forces. The self - assembly is the basis for forming various complex biological structures and has a close relationship with life phenomena. Biomembrane is also a natural model of self - assembly research provided by living things to our humans.

An object of the present invention: In order to overcome the ingongmak drawback of that present in the application so far, the first object of the present invention provides a closure structure or a cell compartment having a biological membranes and membranes properties obtained by naturally occurring and / or self-set technology And a second object of the present invention is to provide a closure structure or a cell compartment having a biomembrane and biomembrane properties obtained by naturally derived and / or self-assembly techniques, and a third object is to provide a closure structure Or application of cell compartments. Closure structures or cell compartments having biomembrane and biological membrane properties obtained by the above method are used in biopharmaceutical carrier technology, in particular, as genetically modified carriers and drug carriers, and are also used in research and product development of cosmetic additives and cosmetic active ingredient carriers It is also used in the research and product development of vaccines and immunomodulators, and in the research and product development of polymer materials.

The present invention provides a biomembrane or a biomembrane fragment on the one hand, which is derived from a natural natural species and has a morphological characteristic double-layer structure, its constituent being mainly lipid and protein, Of the saccharide is bound onto the lipid or protein by a covalent bond.

Preferably, such biomembrane encompasses closure structures or cell compartments having biomembrane properties, or closure structures or cell compartments having biomembrane properties of the biomembranes.

Preferably, the biological origin is a kind of natural plant, animal or microorganism.

Preferably, the diameter of the closure structure or cell compartment having the biological membrane and the biological membrane property is 10 nm to several tens of 탆.

Preferably, the shape of the closure structure or the cell compartment having the biological membrane and biomembrane property includes various types of structures such as a spherical shape, a cyst shape, a rod shape, a spiral shape, a single layer, or a plurality of layers.

Preferably, the biological membrane includes one or more of a plasma membrane, a nuclear membrane, a mitochondrion membrane, an endoplasmic reticulum membrane, an endosomal membrane, a Golgi apparatus membrane, a chloroplast membrane, a vacuole membrane and a peroxisome membrane.

Preferably, the electrocellular compartment generally refers to a cell organelle. More preferably, the electrocellular compartment is one or more of mitochondrial, chloroplast, peroxisome, endoplasmic reticulum, nucleus, Golgi, and cysts and microtubules.

In order to realize the second object, the present invention provides a method for obtaining a biological membrane of the present invention.

The closure structure or the cell compartment having the biomembrane and biomembrane properties described in the above technical means is produced by the following procedure.

1) Biological cells are obtained.

2) The cells described in Procedure 1) are cultured in a large volume in an appropriate environment.

3) The cell lysate obtained in step 2) is obtained, and the cell lysate obtained by fractionation centrifugation, density gradient centrifugation and biphasic extraction can be used alone or in combination with two or three together. And cell membrane compartments with biomembrane and biomembrane properties are extracted.

Preferably, the extraction by the electroporation centrifugation method is performed by the following procedure.

1. Centrifuge the cell lysate at 15,000-30,000 × g for 10-30 min at 1-6 ° C, discard the precipitate, and collect the supernatant.

2. The supernatant is centrifuged at 100 to 200,000 g for 30 to 90 minutes at an ultrahigh speed under the condition of 1-6 DEG C and the supernatant is thrown and the precipitate is collected to extract the closure structure or cell compartment having the required biological membrane and biological membrane properties , And the precipitate is resuspended in 15-30% glycerin in PBS / physiological saline and stored.

Preferably, the extraction by the density gradient centrifugation method is performed by the following procedure.

3. The cell lysate precipitate obtained in the above procedure 2 was resuspended and put into a sucrose solution having a different concentration in the re-suspension, subjected to ultra-high speed centrifugation under the conditions of 150,000-300,000 × g, 60-90 min, 1-6 ° C., Collect the required layers.

4. Centrifuge the collected liquid at 100,000-200,000 × g for 30-90 min at 1-6 ℃, throw the supernatant, collect the precipitate, and collect closure or cell compartments with the required biomembrane and biofilm properties. And the precipitate is resuspended in 15-30% glycerin in PBS / physiological saline and stored.

Preferably, the mass percent-percent concentration range of the sucrose solution of Electrical Procedure 3 is 10-70%, more preferably the unconstrained mass percent concentration of the sucrose solution of Electrical Procedure 3 is 10%, 20%, 30%, 40% %, 45%, 50%, 55%, 60%, 65%, 70%.

Preferably, the electric two-phase extraction method has the following characteristics.

5. Prepare an aqueous two-phase mixture of glucan / polyethylene glycol, mix homogeneously in a separating funnel, separate the layers by standing at 4 ° C and overnight, carefully separate upper and lower layers to obtain fresh top and bottom images .

6. Resuspend the cell lysate precipitate obtained in Procedure 5, add it in a water two-phase mixture, and slowly transfer it 30-40 times to the bottom.

7. The upper and lower phases were continuously placed in a two-phase system under the conditions of 2,000-4,000 × g, 5-10 min, and 4 ° C., separated 3 times, merged with the upper phase, diluted 5-fold and then diluted 60,000-100.000 × g, Centrifuging under the condition of 30-90 min at 4 ° C and collecting the precipitate to extract the closure structure or cell compartment having the required biological membrane and biological membrane properties and resuspending the precipitate with 15-30% glycerin in PBS / physiological saline Preservation.

More preferably, the electrical two phase is a two phase mixture of glucan / polyethylene glycol.

More preferably, the electric two-phase includes an aqueous two-phase, two-phase organic, an aqueous solution and an organic phase solution, and the electric solvent includes water, acetonitrile, acetone, tetrahydrofuran, methanol, ethanols, That is the component.

The closure structure and the cell compartment having the biomembrane and biomembrane properties by the self-assembly include the manufacturing method described in the above technical means, and the closure structure and the cell compartment material having the biomembrane and bio- After adhering to the container wall in the form of a dry film, water or buffer is poured slowly, and light or hard vibration is applied to obtain a closure structure and cell compartment having biomembrane and biological membrane properties required by the self-assembly.

Preferably, the material obtained by the electrical procedure 3) is dissolved in chloroform (a similar organic solvent, such as any other organic solvent), put into a container, adhered to the surface of the container by vacuum evaporation, After deposition, PBS buffer was added, and the mixture was slowly shaken for 0.5 ~ 3h. The supernatant was centrifuged at 100,000-200,000 × g for 30-90min at 1-6 ℃, and the precipitate was collected by collecting the supernatant. Resulting in closure structures and cell compartments.

In order to achieve the third object, the present invention uses the following technical means.

In applications of closure structures or cell compartments having biomembrane and biomembrane properties as described in any of the above technical means, this application may be applied to the membrane internal package, surface adsorption, surface adsorption Cross-linking, intermembrane insertion, and adding a target to the in-film package.

Preferably, the electroactive component encompasses a vaccine or immunomodulating active ingredient, a cosmetic or cosmetic active ingredient, a drug active ingredient, a genetic material and a cell or tissue.

The closure structure or cell compartment having biomembrane and biomembrane properties of the present invention is used in biomedical carrier technology, particularly in research and product development of genetically modified, drug carrier and cosmetic active ingredient carriers, and researches and products of vaccines and immunomodulators It is also used for development, research and product development of polymer materials and so on. The present invention is to provide a method of using a biodegradable polymer membrane obtained by naturally occurring and / or magnetic aggregation for the first time as a carrier and / or an immunomodulator in chemical, pharmaceutical and cosmetic fields, and to provide various polymer materials, medicines, Cosmetics raw materials and intermediates.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a comparative view of the solubility of quercumin packaged in biological membranes.
Fig. 2 is a comparative chart of cytotoxicity of the coumarin packaged in biological membranes.
Figure 3 is the fluorescence intensity of the green color observed after fluorescence microscopy after 48 h of cell transfection, indicating transfection success.
Fig. 4 is a graph showing the effect of effectively transfection efficiency of a biological membrane as a transfection reagent.
5 is a long-term whitening effect of phenylethylresorcinol incorporated into a biological membrane.
Fig. 6 is an effect of remarkably increasing the cell uptake rate after packaging the quercumin with the biomembrane as a carrier.
FIG. 7 is an effect of significantly suppressing cell melanin after packaging of a coumarin with a biomembrane as a carrier.

Terms and Interpretation

Regarding the meaning interpretation of the terminology of the present invention, unless otherwise specified in the present invention, the meaning of the patent term is interpreted in the ordinary definition of the present field.

Biomembrane: The "biological membrane" described in the present invention differs from the conventional "seminal membrane" in that the biological membrane of the present invention can be directly or indirectly obtained from the natural organism or any specific tissue of the organism by the method of the present invention It is a point. An organism described herein refers to an object having an existing life characteristic, including plants, animals, microorganisms, algae, and even viruses. Some organism tissues include any organs of the animal, or tissues of any part of the plant, and may be tissues such as blood of an animal or human.

As used herein, the term " biological membrane " refers to a lipid bilayer structure morphologically composed mainly of lipids and proteins. In addition, small amounts of saccharides are bound to lipids or proteins by covalent bonds. As used herein, the term " biological membrane " refers to a closure structure or cell compartment having a biomembrane property, or a closure structure or cell compartment having biomembrane properties of the biological membrane.

Such closure structures or cell compartments having biomembrane and biomembrane properties may be fine particles having a particle size of 1-10 nm or 10 nm or more.

The term " closure structure " or " cell compartment " having biocompatibility and biological membrane properties encompasses various types of structures such as spherical, cystic, rod-shaped, spiral single layer or plural layers and multiple chambers.

Biomembranes are classified into types, a plasma membrane, a nuclear membrane, a mitochondrial membrane, an endocardial membrane, an endosomal membrane, a Golgi apparatus membrane, a chloroplast membrane, a vacuole, and a mixture or mixture thereof.

The term " cell compartment " as used herein refers to a general cell organelle, and the cell compartment is composed of mitochondria, chloroplast, peroxisome, endosome, endoplasmic reticulum, nucleus, Golgi apparatus, cysts and microtubes.

The term "closed structure" as used herein refers to a structure in which a component constituting a membrane under certain "conditions" has characteristics of a film to be automatically assembled or a film to be clogged or a film, and when the "condition" The component is decomposed. The components constituting such membranes, or the minimum units derived from natural life forms or organisms, are obtained by the method of the present invention.

Cell compartments: Cells are the least independent members of living organisms. Cells are encompassed by several cell organs, all of which have a variety of independent functions, all of which are cleaved by membranes, Membranes include nuclear membranes, mitochondria membranes, endoplasmic reticulum membranes, endosomal membranes, Golgi body membranes, chloroplast membranes, vacuoles, and peroxisome membranes. Herein, the cell compartment can be abbreviated as " biological membrane " in the sense of the present invention.

Acquisition of biomembranes or cell compartments

Conventional techniques can extract simple biological membranes, and what is generally obtained is a mixture of various biological membranes or biomembrane fragments, and at the same time, some cell lysates, such as nucleic acid molecules, fatty molecules, oily molecules, , These impurities affect the properties of the membrane in vitro packaging. Mixtures with these membranes can achieve some functionality, such as a secondary package, but are still difficult to use in real products and are still in the lab. Although the biological membrane obtained by such an in vitro extraction method can realize a function similar to a membrane in an organism, there is still a great difficulty. In particular, it takes a long time for a cell compartment containing a biological membrane or a membrane obtained by an in vitro extraction method to actually operate in a specific product. The present invention is based on the finding that, in response to such a technical problem, a cell compartment containing a biomembrane or a membrane is extracted from a tissue derived from a natural organism to obtain a precisely fragmented pure structure or component, or a fragment of the membrane, The film formed by direct extraction and / or secondary processing has a function similar to that of a membrane inside some biofilm, and has some characteristics of the membrane reserved. Thus, the membrane is practically used in many specific fields .

How to make biomembrane

The method of the present invention can obtain the desired membrane component or membrane fragments or complete membranes on one side, removing some adverse components or components that adversely affect the extracorporeal operation. The biomembrane obtained by the present invention has a lipid bilayer structure in the form of lipids and proteins. In addition, a small amount of saccharides is bound to lipids or proteins through covalent bonds. Other components are effectively removed Therefore, the obtained biological membrane has a more effective action and utility value.

In some specific methods, the closure structure having a biomembrane and biomembrane properties or the method of producing the cell compartment is characterized by being well-equipped with the following. 1) Obtain biological cells from natural tissue or natural biological species. 2) The cells described in Procedure 1) are cultured in a large volume in an appropriate environment. 3) After obtaining a lysed solution of the cells described in the procedure 2), various biological membranes or mixtures belonging to the present invention are obtained through separation and purification of different methods.

The method of obtaining the biomembrane of the present invention in vitro includes the fractionation centrifugation method, the density gradient centrifugation method, and the two-phase extraction method, and various methods may be used singly or in pairs, or three together, And extracts closure structures or cell compartments having biomembrane properties.

Preferably, the electrofractionation centrifugation method is performed by the following procedure.

The supernatant is obtained by centrifuging the cell liquor under the first high rotation speed condition and centrifuged under a second rotation speed condition lower than the first high rotation speed to obtain a precipitate to obtain the required biomembrane.

Preferably, the difference between the first high rotation speed and the second rotation speed may be 1.5 times, 1 time, 2 times, or 3 times.

Preferably, the first high rotation speed is generally 15,000-30,000 x g, and may be 20,000 x g, 25,000 x g, 30,000 x g, 35,000 x g, or even larger.

In addition to limiting the speed of rotation, centrifugation is required at low temperatures.

Some preferred conditions include high-speed centrifugation at 15,000-30,000 x g for 10-30 min at 1-6 DEG C, throwing up the precipitate, collecting the supernatant, adding the supernatant to 100,000-200,000 x g, 30-90 min, 1- Ultracentrifugal centrifugation is carried out under the condition of 6 캜, the supernatant is thrown, and the precipitate is collected to obtain a closure structure or cell compartment having the required biological membrane and biological membrane properties. Finally, the precipitate is resuspended in 15-30% glycerin in PBS / physiological saline and stored.

For some concrete implementations, the extraction method by the electrodensity gradient extraction method is performed by the following procedure.

The resulting cell lysis solution was resuspended and the re-suspended solution was put into a sucrose solution of different concentration and subjected to ultra-high-speed centrifugation under the conditions of 150,000-300,000 x g for 60-90 min at 1-6 DEG C to collect the supernatant, Is centrifuged at 100 to 200,000 x g for 30 to 90 minutes at 1-6 DEG C, the supernatant is thrown, and the precipitate is collected to extract the closure structure or cell compartment having the required biological membrane and biological membrane properties. Finally, the precipitate is resuspended in 15-30% glycerin in PBS / physiological saline and stored.

Alternatively, the resulting cell lysate solution suspension is suspended again, the sucrose solution of the first concentration and the sucrose solution of the second concentration are successively added to the re-suspensions, and then the solution of the third sucrose is added, wherein the first concentration is lower than the second concentration , And the third concentration is lower than the first concentration.

More preferably, the mass percent concentration range of the solution per eukaryotic cell is 10-70%, preferably, the unmixed mass percent concentration of the solution per three electrochemical procedures is 10%, 20%, 30%, 40% , 50%, 55%, 60%, 65%, 70%. The molar ratio of the first concentration of the concentration solution is 0.1-0.5 mol / L, the concentration of the second concentration solution is 1-3 mol / L, and the concentration of the third concentration solution is 0.01-0.3 mol / L.

In some specific embodiments, preferably, the extraction method by the electrodensity gradient centrifugation method comprises the following procedure.

The resulting cell lysate suspension is again suspended and the first low-speed centrifugation is repeated several times to collect the precipitate 1 and prepare a suspension 1. The second high-speed centrifugation of the suspension 1 is repeated several times to obtain a precipitate 2, Suspension 2 is obtained by suspending the precipitate 2 with a non-ionic surface active solution, suspension 2 is diluted with sucrose solution, diluted suspension 2 is placed in the bottom of the centrifugal tube, and then sucrose and low- Centrifugation is carried out at a high speed (for example, under the conditions of 28000 × g -45,000 × g, 10-24 h, and 4 ° C.), and collected at the interface at a low concentration and a high concentration to obtain a required biological membrane. The mass percentage of high sucrose is 20-35% and the mass percentage of low glucose is 2-10%.

In some specific embodiments, preferably, the electric two-phase extraction method has the following characteristics.

The aqueous two-phase mixture of the glucan / polyethylene glycol is homogeneously mixed in a separating funnel, and the mixture is left to stand at 4 ° C under a resting condition, and the upper and lower layers are carefully separated to obtain fresh upper and lower phases.

The cell lysate solution suspension is suspended again and placed in an aqueous two-phase mixture, which is then slowly transferred 30 to 40 times over and mixed homogeneously.

Centrifugation was carried out at 2,000-4,000 × g for 5 to 10 minutes at 4 ° C. The upper and lower phases were successively placed in a two-phase system, separated 3 times, merged with the upper phase, diluted 5 times, g, 30-90 min, 4 ° C, and collecting the precipitates, extracting closure structures or cell compartments having biological membranes and biological membranes, and reproducing the precipitates with 15-30% glycerin in PBS / physiological saline I keep it in the tack.

More preferably, the electrical two phase is a two phase mixture of glucan / polyethylene glycol.

More preferably, the two phases of the electric energy include an aqueous two-phase, an organic two-phase, an aqueous solution and an organic phase solution, and the electric solvent includes any one of water, acetonitrile, acetone, tetrahydrofuran, methanol, ethanols, Component.

The cell lysis solution to which it belongs is obtained by cell disruption, polishing, or chemical disruption.

In some preferred embodiments, density gradient centrifugation and two-phase extraction separation may be used as methods for separating and purifying cell compartments based on biological membranes obtained by the above method.

In some preferred embodiments, a biomembrane precipitate is prepared as a suspension after the preparation of a two-phase system, and cell division is proceeded. Of course, the cell compartment may be analyzed directly with naturally occurring tissues and organisms.

Secondary processing method of biomembrane

The membrane (cell membrane, cell compartment membrane or cell compartment membrane) extracted by the method of the present invention is generally used directly in an active material package, but in order to further enhance the properties of the membrane and stability after packaging, In the present invention, a secondary processing is carried out for a single substance or a mixture of various components such as a material of a membrane, a membrane fragment, a membrane component or a minimum unit extracted from an extracorporeal membrane. Such processing includes dissolution of the mixed components, A solvent, or the like is dissolved in an organic solvent such as chloroform, ethyl ether, etc. to mix together components such as a complete film, a film piece, or a component of a film, and then adhere to a solid surface to remove the organic solvent. .

The self-closing property, which is self-closing in a certain solvent, has various uses. For example, the active ingredient is automatically packaged in a membrane, or the pre-formed self-assembled membrane first forms a membrane, and then the active ingredient is packaged, inserted, adsorbed onto the membrane surface And so on.

Preferably, the resulting mixture of membrane components is dissolved in an organic solvent, placed in a container, and evaporated under reduced pressure to allow the biological membrane to adhere to the surface of the container and evaporated to normal weight. By completely volatilizing the organic solvent, it is very beneficial for the production of the factory.

Preferably, the buffer solution is evaporated to a normal weight and dissolved in a buffer solution. The so-called buffer solution can maintain the closure property of the secondary sclera or the membrane property, and a PBS buffer solution can be used as the buffer solution. After dissolution, centrifugation is carried out under a certain condition, and the precipitate is collected to obtain a closed structure and cell compartment having biomembrane properties with the secondary processed biological membrane of the present invention.

On the other hand, the present invention provides a method for producing a cell compartment also in a closure structure having biomembrane and biomembrane properties by self-assembly. In this method, a material having a closure structure and a cell compartment having a film-forming property obtained by the production method described in the various technical means is adhered to a container wall in the form of a dry film, and then water or a buffer is slowly poured into the container wall to lightly or strongly vibrate To obtain a cell compartment also in a closure structure having biomembrane and biological membrane properties required by the self-assembly.

Preferably, the material obtained by the electric procedure 3) is dissolved in a similar organic solvent (other organic solvent, ethyl ether, etc.) such as chloroform, placed in a container, adhered to the surface of the container by vacuum evaporation, After evaporation, PBS buffer solution was added, and the mixture was slowly shaken for 0.5-3 h, centrifuged at 100,000-200,000 × g for 30-90 min at 1-6 ° C., the supernatant was thrown and the precipitate was collected, The closure structure also has a cell compartment.

The container referred to here refers to the surface of a container such as glass, ceramics, or stainless steel.

Applications:

The closure structure or cell compartment (hereinafter abbreviated as "biological membrane") having biomembrane and biomembrane properties of the present invention is widely used in various aspects as a kind of base material. For example, the closure structure or cell compartment having biomembrane and biomembrane properties of the present invention is used in a package for various active materials, and the active material is an arbitrary substance. In general, when the active substance alone exists, Elements affect the absorption and storage of organisms, but once these are packaged in biological membranes, these adverse factors are improved. For example, these "active substances" are used in biomedical carrier technology, particularly in the research and product development of genetically modified, drug carrier and cosmetic active ingredient carriers, in the research and product development of vaccines and immunomodulators, It is also used for research and product development of polymer materials. The present invention is to provide a method of using a biodegradable polymer membrane obtained by naturally occurring and / or magnetic aggregation for the first time as a carrier and / or an immunomodulator in chemical, pharmaceutical and cosmetic fields, and to provide various polymer materials, medicines, Cosmetics raw materials and intermediates.

Citation of Drug Carriers : 1. Improved stability: The packing action between the lipid bilayer membrane and the membrane of the biomembrane alleviates the opportunity for the drug to contact the extraterrestrial instability elements, thereby improving stability. Penicillin G, for example, is unstable against acids. When penicillin G is orally administered, it is destroyed by gastric acid. However, when packaged in a biological membrane, its stability and oral absorption effect can be enhanced. 2. Improvement of solubility: Most of drugs are poorly soluble chemical molecules. In use, solvents are added to improve solubility by adding toxic and adverse effects. However, after packaging the drug with the carrier membrane, the poorly soluble drug component can be dissolved in the water-soluble drug or fat-soluble drug by the double affinity of the lipid bilayer. Taxol, for example, is widely used as a broad-spectrum antitumor drug, but its solubility in water is very low. In domestic and overseas markets, a large amount of kremopers are added to taxol recipes to increase solubility. Klammor has allergenicity, , Difficulty in breathing, tiredness, hypotension and the like. When packs are packaged as biological membranes, solubility is increased, and such allergic reactions hardly occur. 3. Enhancement of therapeutic effect: Biomembrane allows for the drug to penetrate sufficiently into the target tissue, due to the characteristics of the arresting affinity and tissue compatibility. For example, rifampicin can be packaged in a biological membrane to enclose the drug in human cells, killing the Mycobacterium tuberculosis and significantly improving the drug effect compared to conventional rifampicin agents. 4. Decreased toxicity or irritation: Some chemotherapy drugs have very strong vascular toxicity, which is a cause of common vascular complications such as venous inflammation due to chemotherapy. However, after the drug is packaged into a biological membrane, intravenous injection significantly reduces the vascular toxicity of the drug. In addition, some drugs, especially antibiotics and antineoplastic drugs, have relatively high nephrotoxicity. Since the drugs are packaged in biological membranes, the mononuclear macrophages such as liver and spleen and bone marrow are concentrated in the organs richly, The drug is less accumulated in the free drug than in the free drug, and the drug has toxicity to the heart and kidney, or the drug has toxicity to the normal cell, and then significantly reduces the toxicity of the drug. Amphotericin B, for example, is a traditional drug used for systemic fungal infections, but its toxicity is relatively large, especially for nephrotoxicity, so long-term use damages the kidneys and circulatory system, Is limited. However, packaging by biofilm can change the distribution in the body and significantly reduce the toxicity. 5. Persistence: Most of the drugs are metabolized or excreted in the body quickly, so the action time is short. Packaging the drug into the biomembrane reduces the excretion and metabolism and prolongs the residence time of the drug in the blood, allowing the drug to slowly release in the body, thereby extending the duration of action of the drug. For example, calcitonin is a multi-peptide produced by secretion from thyroid C cells, which is commonly used for the treatment of diseases such as osteoporosis. Since the half-life of a multi-peptide is short, it must be administered repeatedly. However, packaging calcitonin as a biomembrane can extend the half-life in the body more than twice. 6. Targetability: Two targets are included: passive target and main target. The passive target means a drug packaged in a biological membrane is swallowed by the macrophage in the human body to be regarded as an extraneous foreign matter and swallowed by the macrophages of the tiger mononuclear-macrophage system to produce the passive target for the reticuloendothelial system . For example, meglumine is a drug used in the treatment of hepatic resumanniosis, packaged in biological membranes, and then the drug concentration is increased 500 times. The main target refers to linking a target molecule such as a ligand to a biological membrane packaged with a drug and specifically binding with a receptor of the target cell to change the distribution of the microparticles to reach a specified target site. For example, doxorubicin is used as a broad-spectrum antiepileptic drug, but has a very strong cardiac toxicity and severely even causes heart failure. However, after packaging doesxorubicin into a biological membrane containing the tumor target RGD, it not only significantly reduces cardiac toxicity but also doubles the therapeutic effect on lung cancer.

Genetic modification Taran SPECIFICATION REAGENT: The term " genetic modification technology" refers to the introduction of a DNA fragment into a specific organism, binding it back to its own genome, and obtaining a genetic entity having stable expression characteristics through multiple artificial rearing of the recombinant. On one side, this technology can raise new traits that people want in living modified organisms and breed new varieties. In addition, the one - sided transgenic metamorphosis technique is not only an important tool for studying genetic modification and gene expression, but also a crucial step in current gene therapy. The ideal gene transfer reagent has the following characteristics. That is, high-efficiency tar culture, safety, low cytotoxicity, and simple, time-saving and economical methods. However, at present, the commonly used taransulfone reagent has too high cytotoxicity if the taranization efficiency is not too low. Therefore, it has become a challenge to find a new taranosulfation reagent which is capable of highly efficient tranfection and low cytotoxicity.

Biomembrane lipid bilayer has excellent superiority to cell membrane and can be used as a carrier to enter the cell in the form of foreign material such as DNA by adsorption to the cell and through membrane fusion or cell phagocytosis, have. DNA is introduced into the cell to form inclusion bodies or lysosomes, which are released from the DNA inclusion bodies in the small parts of them, enter the cytoplasm, enter the nucleus of the cells again, and express and express them. At the same time, the biomembrane is naturally derived and therefore has little cytotoxicity.

Cosmetics: The stratum corneum is the outer layer of the skin, the lipid in the stratum corneum is tightly wrapped and arranged alternately with the stratum corneum cell, and the two are mutually bonded by covalent bonds to form the main part of the stratum corneum, It forms the major material foundation of the function. Studies have shown that removal of lipids from the horny layer of the epidermis results in loss of barrier function, loss of moisture in the skin, and dryness of the skin. When the removal of lipids in the skin is stopped, the moisture content in the stratum corneum is immediately restored and the dryness of the skin is improved. This is because the lipid exists in the stratum corneum of the skin, it functions as an adhesive by filling the spaces between the cells, it prevents the moisture in the skin from spreading to the outside, keeps moisture and softens the skin, And barrier and moisturizing function.

The biomembrane has a double molecular layer structure and has the following functions in application to the field of cosmetics. 1. Moisturizing action: Lipid components such as cholesterol, ceramide, and palmitic acid in the biological membrane restore the barrier function of the skin, remarkably enhance the electrical conductivity of the skin, have a strong binding ability with water molecules, It forms a reticular structure in the stratum corneum, maintains moisture in the skin, and improves skin elasticity. 2. Whitening Action: Lipid components such as ceramides in biomembranes are often signal molecules that regulate the peroxides in cells and have whitening action. And the biomembrane contains a large amount of unsaturated fatty acids, melanin deposits on the skin relieves the skin is clear. 3. Anti-aging action: Phospholipids in the biological membrane penetrate into the deep layers of the skin and bind with the phospholipid origin of the deep-seated membrane to fluidize the membrane. For example, the unsaturated phospholipids of linoleic acid and? -Linolenic acid cause fluidity and permeability Thereby enhancing the metabolic function of the cells and activating the cells. It achieves the goal of retarding cell senescence by promoting the repair and growth of the epidermal growth factor in the event of tissue damage, disease or aging.

The biomembrane is added as an active ingredient in cosmetics and plays a unique role. At the same time, the biomembrane can be used as a carrier of a functional ingredient and has various effects for enhancing the following functional ingredients. 1. Improving Stability: The packing action between the membrane and the membrane of the biomembrane lipid bilayer membrane reduces the chances of contact between functional components and extrinsic instability, thereby enhancing stability. For example, the alcohol structure of vitamin A contains many unsaturated double bonds, and is easily oxidized when it is in contact with air because of its poor light and thermal stability. However, its stability is greatly enhanced after it is packaged in biological membranes. 2. Solubility enhancement: Most of the ingredients with cosmetic functions are poorly soluble substances, and it is difficult to penetrate the skin barrier during use. However, due to the double affinity of the lipid bilayer, the biomembrane is a poorly soluble functional ingredient Soluble material and liposoluble material. 3. Long-term efficacy: Biomembrane has a root effect as a carrier. Experimental membranes and packaged drugs, which I would like to prove, have a much longer retention time in the blood circulation than retention times of free drug. 4. Promoting absorption: Functional ingredients in cosmetics need to penetrate the stratum corneum and reach the corresponding site of action to improve skin care and skin condition. The stratum corneum of the human skin acts as a very strong barrier, and the functional ingredient of the large molecule is difficult to permeate. The functional ingredient packaged in a biomembrane has excellent affinity because the structure of the biomembrane and the stratum corneum are similar, and the functional ingredient is carried in the biomembrane, thereby increasing the percutaneous permeation amount.

Vaccine carriers and immunomodulators: With advances in biology, molecular immunology, and gene engineering, vaccines are becoming more and more important, and increasing numbers of people have to be vaccinated with safe and reassuring supplements and carriers. . Many of these have been used extensively, including complete prointe supplements, incomplete Freund's adjuvant, bacterial endotoxins, polyvalent anions and polyvalent anions, and mineral sorbents, both producing local and systemic toxicity, forming granulomas that are difficult to accept, Is short and is not being used by people gradually because of its low effectiveness. Aluminum hydroxyapatite adjuvant is safe and effective, but it has only drawbacks that humoral immunity is possible, cell immunity is poor, and the lot-to-lot difference of antigen binding is comparatively large. Therefore, vaccine carriers and adjuvant new technologies that can induce cell immunity and humoral immunity in a safe, effective, and efficient manner have already created new challenges in vaccine applications.

Since the lipid bilayer structure of the biomembrane is highly compatible with the cell membrane, it is adsorbed to the cell membrane, and through the fusion and intracellular uptake of the membrane, it can enter the cell carrier in the form of the foreign material by direct penetration . The biological membrane can be used as a carrier of a substance such as protein, nucleic acid, synthetic peptide, cell factor, bacterium, virus and the like in a package or adsorption manner, and the biological membrane is excellent in safety as a non-pathogenic carrier. And biomembrane is effectively presented by the organism 's antigen, ingested into the cells, and because it produces a strong immune response, itself has a natural immune supplement.

Biomembranes have the following advantages as vaccine carriers and immunosuppressants. 1. High safety: Biomembrane is a non-immunogenic and non-pathogenic carrier for antigen expression and presentation carrier of vaccine. It is not only excellent in safety, but also has no bad effect on repeated inoculation without granuloma at the inoculation site. At the same time, after the animal is immunized with the biological membrane, the occurrence of the antibody against the biological membrane carrier can not be detected, and the possibility of causing an organic immunodeficiency by the biological membrane itself is lowered. 2. Multifunctional: Biomembrane carriers are able to insert proteins, nucleic acids, synthetic peptides, cell factors, bacteria, viruses and other substances in a directionally oriented manner and are applied not only to the construction of traditional vaccines as carriers and adjuvants, but also to the new gene vaccines . 3. Complex formation: It is possible to insert different antigens, nucleic acids and other substances oriented on the biofilm, and to study multivalent complex vaccines and multiple vaccines. 4. Highly efficient hemolytic immunity: DCs are the most specialized antigen presenting cells (APCs) that have the strongest function among the cells so far discovered, and biomembranes are formed by their lipid bilayer structure, Facilitates the uptake of APCs to the antigen, thereby effectively activating antibodies produced by humoral immunity to the corresponding antigen, playing an important role in the prevention of numerous infectious diseases. 5. Strong cell immunity: While traditional vaccines mainly focus on humoral immunity, HIV, HCV, and tumors require a new therapeutic vaccine capable of inducing specific cell immunity, which can lead to higher levels of vaccine carriers and adjuvants Demand. The biomembrane carrier has natural advantages and can effectively activate DC cells and other antigen presenting cells. By presenting the exogenous antigen to MHC-1, it activates antigen CD8 + and CD4 + T lymphocytes, . 6. Delayed: Biomembrane carriers are linked to antigens, delaying the action of antigens, and produce a more thorough immune response, which enhances the immune response.

Applications of biosimilar vaccines: 1. Preventable infectious vaccine: In the prevention of bacterial, viral and protozoal infections, humoral immunity has a significant effect. It is possible to prevent and control the occurrence and epidemics of infectious diseases by modifying a small amount of biomolecule carriers and connecting antigen such as a pathogen to mobilize an immune response of an organism. 2. Therapeutic vaccine for infectious diseases: In the case of an organism already infected with a virus, the preventive vaccine is meaningless since the antibody produced by humoral immunity can not enter the cell and remove the pathogen. Although viral diseases have no effective therapeutic drug so far, their onset characteristics are mainly caused by intracellular infections. Therefore, it is possible to induce a cellular immune response having a strong specificity through a biological membrane carrier vaccine, Lt; / RTI > 3. Tumor Prevention Vaccine: Persistent High-risk HPV infection is the cause of almost all invasive cervical cancer. Helicobacter pylori infection is an important cause of disease such as gastric cancer, gastric mucosal lymphoma (MALT), and the World Health Organization already recognizes them as a primary carcinogen. There is a relatively clear pathologic relationship between these carcinogens and the tumor spleen, and a vaccine is prepared by linking the relevant antigen or gene to the biomembrane carrier, inoculating the susceptible healthy person or the hyalurus muscle group, The research and development of a tumor preventive vaccine can inhibit tumors from " clone ". 4. Tumor therapeutic vaccine: Tumor treatment vaccine has been concentrated on two aspects, immunotherapy and gene therapy. In addition to enhancing the immune response of the organism at different levels, The regeneration regulating ability is promoted to achieve the purpose of tumor treatment. The point of the study is how to effectively increase tumor immunity and break immunity tolerance. The volume of the tumor can be reduced by producing a peptide vaccine, a genetically modified carrier vaccine, a dendritic cell (DC) vaccine or the like through a biological membrane carrier and inducing a T cell response with strong specificity. 5. Own Immune Disease Vaccine: Immune impairment of the immune system itself causes pathological changes in the corresponding tissue organs. Therapeutic vaccines of their own immune diseases use a design that removes the memory T cells of the immune response mainly caused by their tissue antigens, while at the same time supporting the immune control method. For example, some immunomodulatory cellular factors regulate the entire immune system. DNA vaccines, mixed vaccines, fusion multi-peptide vaccines, etc. as biomembrane carriers have a good applied conduction in their immunity vaccines.

Polymeric materials: From ancient times, people have thought that if one or more of their organs becomes diseased, they can not change their organs as if they were to change parts of a machine. In 1954, Boston medical scientist Leland Hartwell and Joseph Head completed the first successful human organ transplantation - kidney transplant surgery, and the new era of transplantation of human organs. However, the obstacles to the development of organ transplantation are causing the worries of doctors around the world. One of the major obstacles is that when the organ is transplanted to the patient, his or her own rejection can not be avoided. Currently, patients who have undergone organ transplantation have to take lifelong immunosuppressive drugs to prevent rejection in the body. These drugs affect the entire immune system and reduce the patient's resistance to disease. To solve this problem, scientists are studying new methods. That is, the local tissue or cells of the patient are combined with a polymer material serving as a stand to cultivate the most suitable " magnetic " organs in the relevant part of the patient's body. This polymeric material stand is compatible with excellent cells and tissues and should provide a place for their growth. At the same time, it should have decomposition properties, and should not be harmful to human body after decomposition, and there should be no after effects in tissues and organs. However, the ideal material for artificial long-standing stands has not yet been found.

When cells or tissues are cultured on a biomembrane base, it is a stand for restoration and restoration of artificial organs or tissues. When used for organ transplantation, it is not only safe, it does not cause rejection in the body, , Which provides abundant technical support for the research and application of polymeric materials because of its high utilization.

Specific operating method

The biological film produced by the present invention can package various active substances. The packaging method includes reverse phase evaporation, ultrasonic emulsification, electrostatic adsorption, crosslinking, high pressure homogenization, and pH gradient.

The biomembrane as used herein refers to a biomembrane or cell compartment obtained by an electric method or a biomembrane obtained by secondary processing.

The reverse phase evaporation method includes the following procedure. Mixing a biomembrane with an amine compound, dissolving the compound in a volatile organic solvent, evaporating the volatile organic compound by a vacuum rotary evaporation method, and then mixing the mixture with an active substance.

Procedure for packaging the active material into the biomembrane by electrophoretic emulsification method: The active material is prepared, ultrasonication is performed on the organism, the solution of the active material is slowly poured in the process of disintegration, and centrifuged to obtain the precipitate. To obtain the packaged material. Preferably, the biological membrane is a biological membrane of a cell membrane. What is obtained by the electrostatic adsorption method is a biomembrane by self-assembly or a biomembrane through secondary processing.

BRIEF DESCRIPTION OF THE DRAWINGS In the following, the present invention will be described in more detail with reference to specific embodiments and accompanying drawings. These embodiments are intended to illustrate how the present invention may be realized and are not intended to limit the scope of the present invention. In the following examples, the test method in which the specific conditions are not known depends on the general conditions or the conditions proposed by the manufacturer.

Example 1 : Extraction and purification of biological membranes

And extracted and purified by a density gradient centrifugation method to obtain the required biological membrane. The concrete procedure is as follows.

(1) 18-24h After killing fasted New Zealand white rabbits, take the liver, remove the large blood vessels, cut the liver to about 2 mm ³ , and wash it with normal saline until the tissue lye is whitened.

(2) Preparation of homogenate: 1 mmol / L CaCl ₂ , 50 mmol / L HEPES (pH 7.4), 1 mmol / L PMSF, 2 μg / mL aprotinin, 2 μg / mL Antipain.

(3) Homogenize the liver tissue and the homogenate in a mass: volume ratio of 8 by volume, put in an ice bath, and homogenize under a condition of 15,000 rpm with a motorized homogenizer until the tissue is liquidated.

(4) The homogenate is filtered with four layers of gauze, and the filtrate is centrifuged at 1,000 x g, 10 min, and 4 ° C to collect the precipitate.

(5) The precipitate was sufficiently suspended and precipitated with sucrose solution A (0.3 mol / L sucrose, 50 mmol / L Tris, 3 mmol / L MgCl ₂ ), and then suspended in a suspension solution of 9 times volume of sucrose solution B (2 mol / , 50 mmol / L Tris and 3 mmol / L MgCl ₂ ), and the upper layer was filled with a sucrose solution C (0.25 mol / L glucose, 10 mmol / L HEPES, 1 mmol / L EDTA) Lt; 0 > C to collect the upper film layer.

(6) The membrane layer was centrifuged at 90,000 × g, 60 min, 4 ° C. in a washing liquid (50 mmol / L HEPES, 1 mmol / L PMSF, 2 μg / mL aprotinin and 2 μg / mL Antipain) .

Example 2 : Extraction and purification of biological membranes

By density gradient centrifugation Extracted and purified to obtain the required biological film. The concrete procedure is as follows.

(1) Remove 30 mL of fresh blood from the surface of blood plasma and erythrocyte sediment at 100 x g, 10 min, 4 ℃ under wash.

(2) Add 5 volumes of pH 8.0 PBS buffer, centrifuge under conditions of 2,000 × g, 15 min, 4 ° C, discard supernatant, and repeat 3 times.

(3) A pH 8.0 PBS buffer solution having a volume of 40 times the volume of the precipitate is added, and the mixture is allowed to stand for 2 hours at 4 ° C to cause hemolysis.

(4) Next, centrifuge under conditions of 22,000 × g, 20 min, 4 ° C, and repeat 4 times (resulting in a biological membrane in the conventional sense).

100 완충액(0.25 mmol/L자당 함유，150 mmol/LNaCl，1mmol/LEDTA，20 mmol/L Tris―HCl와1 % Triton X

100)으로 다시 현탁시킨 후, 같은 체적의 80%(W/V)자당용액으로 희석한다(콜레스테롤과 프핑고미엘린은 비교적 긴 지방산사슬을 가지고 있어, 분자간의 작용력이 강하기에, 이로 조성되는 구조는 긴밀하여, 그 밀도는 무질서 액체와 액정지간에 속하며, 저온하에서 표면활성제의 추출에 효과적으로 견딘다.).(Triton X

100밖에도 다른 비이온형 표면활성제도 가능함) .(5) The precipitate was pre-cooled in Triton X

100 buffer (containing 0.25 mmol / L sucrose, 150 mmol / L NaCl, 1 mmol / LEDTA, 20 mmol / L Tris-HCl and 1% Triton X

100), and then diluted with 80% (w / v) sucrose solution of the same volume (cholesterol and phingomyelin have a relatively long fatty acid chain, Tightly, the density belongs to disordered liquids and liquid crystals, and is effectively resistant to the extraction of surfactants at low temperatures.) (Triton X

100 other non-ionic surfactants are also possible).

(6) Take 4 mL of the membrane suspension, place it in the bottom of the centrifuge tube, add 4 mL 30% and 3 mL 5% sucrose solution sequentially, and centrifuge at 38,000 × g, 18 h, , 5% and 30% of the sucrose solution interface to obtain the required biological membrane.

Example 3 : Extraction and purification of biological membranes

And extracted and purified by a two-phase distribution method to obtain a required biological film. The concrete procedure is as follows.

(1) Seed corn seeds are selected, seeded in 1% NaClO for 10 min, and washed gently, germinated for 72 h under constant temperature and dark condition at 25 ° C.

(5 mmol / L EDTA, 25 mmol / L Tris, 0.25 mmol / L sucrose, 1 mmol / L MgSO ₄ , 0.2% (W / V)) in a volume ratio of 2: BSA, 0.5% (W / V) PVP-10, 10% (W / V) glycerin, 15 mmol / L β- mercaptoethanol, 1 mmol / L PMSF and 1 mmol / L DTT) Polish.

(3) The polishing solution was filtered with four layers of gauze, and the filtrate was centrifuged at 12,000 xg for 15 minutes at 4 DEG C to obtain a supernatant.

(4) The supernatant is centrifuged at 80,000 xg for 30 minutes at 4 ° C, and the precipitate is collected.

(5) The precipitate is sufficiently suspended and precipitated in a suspension (25 mmol / L Tris, 0.25 mmol / L sucrose, 0.2% (W / V) BSA, 10% (W / V) mannitol, 1 mmol / L DTT).

(6) The suspension was added to a two-phase system (1.7 g sucrose, 0.003 g DTT, 2.25 mL water, 50 mmol / L KCl 0.5 mL, 1.63 PEG, 3.25 g Dextran T-500, 0.5 mL of 200 mmol / L PBS in a two- ), Centrifuged at 4,000 × g, 5 min, and 4 ° C., and the upper and lower phases were successively taken in a two-phase system. After the three phases were separated, the upper phase was merged, And then the precipitate is collected under the conditions of 80,000 x g, 60 min, and 4 캜 to obtain the required biological film.

Example 4 : Extraction and purification of biological membranes

And extracted and purified by fractionation centrifugation to obtain the required biological membrane. The concrete procedure is as follows.

(1) Antarctic ice and snow birds ( Chlamydomonas Subcaudata is isolated from the Antarctic sea ice (the winter temperature of Antarctica falls from -2 ° C at the ice-water interface to -50 ° C at the ice-air interface.) When the ice is frozen, At the same time, as the formation of sea ice and the covering of the icy and snow cover are covered, the illuminance is significantly reduced. At low temperature, low light intensity, high saltiness, etc., Because all affect all major processes of photosynthesis, ice and snow algae have resulted in complex adaptive changes in physiology, metabolism and genetics to survive and breed in these harsh environments, and their membrane structure and membrane composition have also undergone major changes. )

(2) Antarctic ice and snow algae were cultured in a ratio of 1: 100 (21.2 g NaCl, 3.6 g NaSO ₄ , 0.6 g KCl, 0.3 g NaHCO ₃ , 0.1 g KBr, 0.1 g H ₃ BO ₃ , 0.1 g NaF, 9.6 g of MgCl ₂ .6H ₂ O, 1.0 g of CaCl ₂ , and 0.1 g of SrCl ₂ .6H ₂ O), and cultured in an incubator capable of adjusting the illuminance. -4 ° C, illuminance 1300-1900 lx, irradiation period 12 persons / 12 arms, 4-5 times a day for 14 days.

(3) The ice and snow algae culture was centrifuged at 4,000 rpm, 20 min, and 4 ° C to collect ice and snow algae sediments, and washed twice with preliminary cooling distilled water to remove impurities in the extracellular sticky part, .

(4) The collected frozen-thawed algae precipitate was suspended in a homogenous buffer (0.5 mol / L KOH, 0.5 mol / L sucrose, 3 mmol / L EDTA, 0.6% PVP, 1 mmol / L PMSF, 1 mmol / L DTT, 5 mmol / L ascorbic acid, 0.6% BSA), and the cells were disrupted using a compressed cell disruptor.

(5) The obtained cell homogenate is centrifuged at 8,000 rpm for 20 minutes at 4 ° C, and the supernatant is collected.

(6) The supernatant is centrifuged at 145,000 xg for 60 minutes at 4 DEG C, and the precipitate is collected to obtain the required biological film.

Example 5 : Extraction and purification of biological membranes

And extracted and purified by a fractionation centrifugation method and a density gradient centrifugation method (Example 8) to obtain a required biological membrane. The concrete procedure is as follows.

(1) Highly thermophilic bacteria ( Thermus Thermophillus ) has been isolated from the Yellowstone National Park in the United States. These microorganisms live in a high-temperature environment such as a volcanic area, a surrounding area, a hot spring, etc. In order for the microorganisms to survive under high temperature and undergo normal metabolism and grow, The chemical composition of the biomembrane inside the thermophilic bacteria increases with the change of the environmental temperature and not only increases the total content of the preservative but also increases the fatty acid with high melting point and increases the stability of the membrane. Since the double layer lipids in the flock consist of covalent bonds, this structure greatly increases heat resistance.)

(2) No. 1 a highly thermophilic bacteria: Among medium (10L medium in the proportion of 100 _{26 g (NH4) 2 SO 4} , 2.47g MgSO 4 ˙ 7H 2 O, 2.8g KH 2 PO 4, 0.74g CaCl 2 ˙ 2H _{2 O, 0.19g FeCl 3 ˙ 6H} 2 O, 0.018g MnCl 2˙ 4H 2 O, 0.044g Na 2 B 4 O 7 ˙ 10H 2 O, 0.002g ZnSO 4 ˙ inoculated in 7H containing ₂ O), incubator , And cultured at 60 DEG C and 150 rpm for 24 hours.

(3) The cells are collected by centrifugation at 4,000 rpm, 30 min, and 4 ° C.

(4) The cells were resuspended in a homogenous buffer (20 mmol / L Tris-Cl pH 8.0, 100 mmol / L NaCl, ₂ mmol / L MgCl ₂ , 1 mmol / L DTT) and centrifuged at 6,000 rpm, 10 min, To remove the supernatant.

(5) Resuspend the precipitate (containing 10 ml buffer per about 1 g) by adding a homogeneous buffer solution, add PMSF to a final concentration of 1 mmol / L, and shred for 30 min (amplitude 55%, ultrasonic 5 s, stop 8 s) in an ice bath.

(6) The crushed cells are centrifuged at 25,000 xg for 30 minutes at 4 DEG C to remove precipitates, and supernatant is taken.

(7) The supernatant is subjected to ultrahigh-speed centrifugation under the conditions of 145,000 x g, 1 h, and 4 캜, and the precipitate is collected to obtain a biological membrane.

Example 6 : Isolation and purification of cell compartments

The cells are separated and purified by density gradient separation to obtain the required cell compartment. The concrete procedure is as follows.

(1) Spinach leaves grown in 10 g of individual healthy, continuous weather for several consecutive days were washed thoroughly, and then the large stems were removed. A 6-fold volume of a homogenous buffer (50 mmol / L potassium phosphate buffer , 1 mmol / L MgCl ₂ , 1 mmol / L MnCl ₂ , 1% BSA, 1 mmol / L DTT) was added to each well and the mixture was polished in an ice bath.

(2 mmol / L HEPES-KOH, 0.3 mmol / L sorbitol, 2 mmol / L EDTA, 1 mmol / L MgCl ₂ , 1 mmol / L MnCl ₂ , 1% BSA, 3% PEG 6000, 1% Ficoll) And centrifuged beforehand under the conditions of 30,000 x g, 20 min, and 4 캜.

(3) The abrasive liquid was filtered with four layers of gauze and centrifuged at 3,000 xg for 15 minutes at 4 캜 to collect the precipitate. The precipitate was suspended in 2 ml of the homogeneous buffer, The cells are placed in a separate Percoll fraction, centrifuged at 15,000 × g for 20 minutes at 4 ° C., and the lower layer is collected to obtain the required cell compartment. It contains a large amount of chloroplasts.

Example 7 : Isolation and purification of cell compartments

And separated and purified by a two-phase extraction method to obtain the cell compartment required. The concrete procedure is as follows.

(1) Preparation of aqueous two-phase system: mixture (90 g 20% (W / W) Dextran T-500 in 300 g 45 g 40% (W / W) PEG 3350, 33.9 g sucrose, 7.5 g 0.2 mmol / L PBS, 0.45 g 2 mmol / L KCl) were homogeneously mixed in a centrifuge tube to prepare an aqueous two-phase mixture of the same concentration of glucan / polyethylene glycol (Dextran T-500 / PEG 3350), uniformly mixed in a separating funnel, Leave at 4 째 C overnight, carefully separate the upper and lower layers, and prepare fresh upper and lower layers. Each layer is stored at 4 째 C, and used for the post-elution.

(2) The biological membrane precipitate obtained in Example 4 was resuspended in a resuspension buffer (5 mmol / L PBS, 0.33 mol / L sucrose, 3 mmol / L KCl, 1 mmol / L DTT, 1 mmol / L PMSF, 0.1 mmol / L EDTA) .

(3) Put the ash suspension in a Dextran T-500 / PEG 3350 aqueous two-phase mixture prepared in procedure (1) in a proportion of 1: 3 by mass and conduct lightly 30-40 times.

(4) The mixture was centrifuged at 1,500 rpm, 10 min, and 4 ° C, and the supernatant and the supernatant were taken in succession. The supernatant was separated into 3 phases, , 100,000 × g, 60 min, and 4 ° C., and collecting the precipitate to obtain the required cell compartment and, at the same time, a lot of thylakoids. (The growth and propagation of ice and snow algae are promoted by photosynthesis The thylakoids of ice and snow algae are distributed not only in a simple "cup shape" but also in the whole cell other than the nucleus apical region in a flattened form, The photosynthetic action is effective regardless of which direction the cells are pointing, because it is a flat layer structure, and the thylakoids of the Antarctic ice and snow algae The content of unsaturated fatty acids in membranes is significantly higher than that of normal thylakoid membranes because the Antarctic ice and snow algae overcomes the adverse effects of low light by eliminating the lipid saturation of the thylakoid membranes.

Example 8 : Isolation and purification of cell compartments

The cells are separated and purified by the density gradient method to obtain the required cell compartment. The concrete procedure is as follows.

(1) Density gradient In a centrifuge, add 1.5 ml of 60% sucrose, 6 ml of 40% sucrose and 1.5 ml of 20% sucrose, respectively, from bottom to top.

(2) 1 ml of the biological membrane precipitate obtained in Example 5 is placed in a density gradient centrifuge tube and centrifuged at 38,000 rpm, 1.5 h, and 4 ° C to collect the middle layer film layer.

(3) The collected membrane layer is washed with a homogeneous buffer, and centrifuged at 145,000 × g, 90 min, and 4 ° C. to collect precipitates. In addition, the required cell compartments are obtained and many chromosomes are contained therein. The chromosome, which does not have a photosynthesis function, plays a role of collecting mainly the branching and lipids, and it contains many lipids and plastoquinone Plastoquinone is a natural fat-soluble quinone compound, which is similar in structure to vitamin K, vitamin E, and coenzyme Q10. It participates in energy production and activation in human cells, activates human cells and cell energy give. )

Example 9 : Production of biomembrane

It rewrites the membrane that is required for self-assembly technology. The concrete procedure is as follows.

(1) The biomembrane obtained in Example 2 is dissolved in a sufficient amount of ethyl ether at a ratio of 1:10.

(2) Put the biomembrane ethyl ether solution into a round bottom flask, attach the biomembrane to the surface of the flask by the reduced pressure evaporation method, evaporate to the normal weight, add 2 times the volume of the PBS buffer solution and shake it slowly for 2 h.

(3) The solution was centrifuged at 6,000 rpm, 20 min, and 4 ° C to collect the precipitate, and the re-suspension (20 mmol / L Tris, 0.1 mol / L NaCl, ₂ mmol / L MgCl ₂ , 1 mmol / ) To precipitate.

(4) The suspension is subjected to ultra-high speed centrifugation under the conditions of 150,000 × g, 60 min, and 4 ° C., the supernatant is thrown, and the precipitate is collected to obtain a biological membrane having the closure characteristics by the self-collection technique.

Of course, not only a biological film having a self-focusing technique can be obtained by using the organisms in Embodiment 1, but the biological film can be obtained by a similar method of another embodiment or the same method.

Example 10 : Inside membrane Package

DNA packaging is performed inside the biological membrane by reverse phase evaporation. Specifically, it is as follows.

(1) The biological film obtained in Example 2 and octadecylamine (or (2,3-dioleoyloxy-propyl) -trimethylammonium) were mixed in proportions of 10: 1 (volume ratio) Or ethyl ether, methanol, etc.) and then evaporated in a rotary evaporator in a rotary evaporator (37 ° C, 200 rpm) to remove the chloroform.

(2) Mix GFP eukaryotic expression plasmid having a concentration of 1 mL of 280 ug / mL and PBS having a concentration of 1 mL of 100 mmol / L in the same volume.

(3) The PBS containing the plasmids is slowly dropped into the biological membrane of the above procedure (1) and cultured in a water bath at 38 캜 for 20 minutes to obtain the gene transformed carrier in which the required GFP plaque is packaged.

Example 11 : Inside membrane  package

 The drug package is advanced inside the biological membrane by the ultrasonic emulsification method. Specifically, it is as follows.

(1) Quercumin and PEG-400 are sufficiently mixed at a ratio of 1: 4 (weight ratio) at room temperature.

(2) The cell compartment obtained in Example 5 was disrupted with an ultrasonic disrupter in an ice bath, and the solution of the quercumin solution obtained in the step (1) was slowly dropped. Ultimately, ultrasonic emulsification was carried out until the volume ratio of the two reached 1: .

(3) The amplitude of the ultrasonic probe is 50%, 30s of operation, 30s of arc, 45 times of cycle;

(4) The liquid is centrifuged under the conditions of 100,000 x g, 20 min, and 4 캜, the supernatant is thrown, and the precipitate is collected to obtain the carrier containing the required amount of the curcumin.

Example 12 : Surface adsorption

 The vaccine is prepared by adsorbing the virus on the surface of the biological membrane by the electrostatic adsorption method. Specifically, it is as follows.

(1) In Example 9, the biological film obtained by the self-assembly technique is dissolved again in a pH 5.8 PBS buffer solution at a volume ratio of 1: 2, and sufficient vibration is applied.

(2) The inactivated circovirus (PCV2) and the biomembrane dissolved again in PBS were thoroughly mixed at a ratio of 8: 1, frozen at -30 ° C, and then incubated overnight at -60 ° C Vaccine.

Example 13 : Surface bridge

The cells are crosslinked on the biological membrane surface by a crosslinking method to obtain artificial organs. Specifically, it is as follows.

(1) In Example 9, the biological film obtained by the self-assembly technique is crosslinked with a glutaraldehyde solution to prepare a biomembrane model plate.

(2) The cell suspension is prepared with F-12 culture medium (10% calf serum) by culturing the vincristine fibroblast chondrocytes in an incubator (5% CO ₂ , 37 ° C).

3, the membranes dried board sterilized in light of the ultraviolet light into a 6-hole culture plate, locked to the cell suspension, were added to 2h vibration put into a constant temperature incubator under 37 ℃, the culture plate a CO ₂ incubator (5% CO _2, 37 ° C) for 2 days to obtain the required artificial vandal plate.

Example 14 : Between membranes  Insert (cell organ)

The drug is inserted between the double molecular layers of the biological membrane by high pressure homogenization. Specifically, it is as follows.

(1) The cell compartment obtained in Example 7 and retinoic acid are melted at 65 占 폚 and sheared with a high shear dispersion emulsifier to obtain colostrum.

(2) The colostrum was mixed with distilled water having a twin -80 dissolved therein at 65 ° C, the homogenized liquid was circulated 15 times, the homogenized liquid pressure reached 80 MPa, and naturally cooled to room temperature, A drug carrier is obtained.

Example 15 : Inside membrane  Package + target

The pH gradient method packs the drug inside the biological membrane and connects the target molecule to the protein on the biological membrane. Specifically, it is as follows.

(1) The biological film obtained in Example 5 was dissolved in an aqueous solution of 0.3 mol / L citric acid (pH = 4), and then a thin film was formed on the vessel wall by vacuum rotation.

(2) The biomembrane obtained above was adjusted with a 1 mol / L sodium carbonate solution, and the pH of the suspension was adjusted to 7.8 to form a proton gradient in and out of the biomembrane, and the drug was actively contained as a carrier.

(3) Adriamycin is dissolved in 1 mol / L HEPES buffer (pH = 7.8) at 60 캜 to form a saturated solution of adriamycin.

(4) The biological membrane suspension and the adriamycin saturated solution were mixed and cultured in a water bath for 20 minutes under the condition of 60 DEG C, and the mixture was centrifuged at 80,000 x g for 60 minutes at 4 DEG C to collect the precipitate, To obtain the packaged drug carrier.

(5) expressing the target factor iRGD with avidin, decorating the biomembrane carrier containing the adriamycin, and ligating iRGD with the adiamycin-sealed biomembrane carrier using the biotin-avidin system, Adriamycin obtains a packaged target carrier.

Example 16 : drug Carrier  Beneficial effect (enhancing drug solubility)

(1) Carrier packed with the coumarin compound obtained in Example 11 was shaken gently at 37 DEG C with 5% Triton X-100 and de-emulsified while being diluted with methanol having a volume ratio of 100 times.

(2) Measure the content of curcumin by high performance liquid chromatography. As a result, the content of quercumin in the carrier was 7.2%.

(3) A carrier containing a simple mixture of a coumarin and a carrier and a carrier packed with a coumarin was stored in a sealed colorless glass bottle at a temperature of 37 ° C for one week and two weeks, respectively, based on the content of the curcumin of 7.2% .

(4) According to the results shown in Fig. 1, the solubility of the coumuccine packaged in the biological membrane is significantly higher than that of the mixture.

Example 17: Carrier  Beneficial effect (to improve drug stability)

Same as in Example 16.

Thread Tools : drug Carrier  Beneficial effect (reduce drug toxicity)

(1) Based on the above-mentioned 7.2% of the content of the quercumin, the carrier containing the quercumin and carrier is simply mixed and the carrier packed with the quercumin is diluted to different concentrations.

(2) Culturally different concentrations of the curcumin and the curcumin-biomembrane carrier are incubated with the epidermal cells.

(3) According to the results in Fig. 2, the cytotoxicity of the coumu- mine packaged in biological membranes is significantly lower than that of the mixture.

Example 19 : drug Carrier  Beneficial effect (enhancing therapeutic effect of drug)

The drug package is conducted inside the biofilm by reverse phase evaporation. Specifically, it is as follows.

(1) The biological membrane obtained in Example 5 is dissolved in chloroform having a volume of four times, and then evaporated in vacuo (37 DEG C, 200 rpm) using a rotary evaporator to remove chloroform.

(2) Rifampicin is dissolved in PBS, and ethyl ether is added to dissolve the biological membrane in procedure (1), and the mixture is mixed with the same volume as rifampicin and cultured in a water bath at 50 ° C for 20 minutes to obtain the required drug carrier packed with rifampicin .

(3) The drug carrier packed with rifampicin was shaken gently at 37 ° C with 5% Triton X-100, de-emulsified overnight, diluted with 100 times volume of methanol, and subjected to high performance liquid chromatography to remove rifampicin The content is measured. As a result, the content of rifampicin in the carrier was 10.2%.

4 ml of strong white mice, 20, without limitation, a female a male, body weight 25 ~ 35g, by injection of 1.75 × 0 ⁸ colony into the tail vein, to form a unit (CFU) / mL of the Mycobacterium tuberculosis H ₃₇ R _V suspension 0.1mL The mice are infected with rats and the rats are randomly divided into two groups A and B on the 14th day after injection, and the mice are divided into 10 rats.

(5) In group A, the drug carrier packed with rifampicin obtained above is intravenously injected once every 3 days with 10ul, and group B injects the same amount of rifampicin as a control.

(6) After 3 weeks of injection, the mice are sacrificed and the spleen is taken. The homogenate is cultured on an inclined plane of an appropriate concentration of L-J medium, and after 6 weeks, the number of colonies is calculated to calculate CFU.

(7) According to the results, rifampicin packaged in biomembranes was twice as effective as rifampicin.

Example 20 : Of the drug carrier  Beneficial effect Drug effect  Improvement, different formulations)

(1) 20 healthy white New Zealand white rabbits are divided into two groups of 1.5-2.0 kg, randomly divided into A and B groups, with no restriction on female males.

(2) After intravenous injection of 3% pentobarbital sodium, the vitreous cavity gas injection was performed at the temporal quadrant, and the vitreous cavity was injected. the C ₃ F ₈ gas is injected 0.1mL, and the gas is expanded after the prerequisite pressured 3d, then anesthetized in the same way, the mattress suture place of the original non-needle chamber 5-0 with the surrounding glass body, Gas permeation through the sclera with a cut of a brown glass viscometer and injection of a L929 cell suspension to construct a PVR experimental model.

(3) After the cells were injected, 0.2 mL of the drug carrier packed with retinoic acid packaged in Example 14 was injected into the group A, 0.2 mL of the commercially available retinoic acid silicone oil solution was injected to the group B, and the pre-installed suture line was caught.

(4) After injecting L929 cells into the cavity of a rabbit eye vitreous body, a cloudy ariary was formed in the posterior vitreous body immediately, and after about 1 week, it turned into a grayish white stripe and connected to the nipple and bone marrow line After procedure 28d, the patient is shaved with tropic amide, and the vitreous state is examined with an ophthalmoscope. Divide the turbidity of the vitreous into four classes of 0 to III. Level 0: No turbidity in the vitreous. Class Ⅰ: The vitreous body is slightly opaque, and there is no interference with fundus observation. Ⅱ: The vitreous body is moderately turbid, and the fundus is still visible through the opaque vitreous body. Class Ⅲ: The vitreous body is extremely cloudy, and fundus is not visible. I, II, and III. According to the experimental results, the turbidity of the sample is lower than that of the control, and it has statistical significance.

Rating Retinoic acid-biomembrane tank Retinoic acid-silicone oil tank 0 0 0 Ⅰ 8 5 Ⅱ 9 7 Ⅲ 3 8

Example 21 : Of the drug carrier  Beneficial effect (long term Drug effect  Improvement)

The drug is packaged inside the biofilm by reverse phase evaporation. Specifically, it is as follows.

(1) The biological membrane obtained in Example 6 was dissolved in chloroform having a volume of four times, and then evaporated by rotary evaporation under reduced pressure (37 ° C, 200 rpm) to remove chloroform.

(2) Calcitonin was dissolved in PBS, and ethyl ether was added to the biological membrane of procedure (1), mixed in the same volume as calcitonin, and incubated in a water bath at 50 ° C for 20 minutes to obtain the drug carrier packed with calcitonin .

(3) The drug carrier packed with calcitonin obtained above is dissolved in methanol having a volume ratio of 500 times, and then nitrogen is blown at 37 DEG C and dissolved again in PBS. The content of calcitonin is measured by immunoluminescence. As a result, the content of calcitonin in the carrier was 8.6%.

(4) Take 10 healthy white New Zealand white rabbits and divide them into two groups of 1.5-2.0 kg, randomly divided into A and B groups, with no restrictions on female males.

(5) Group A intravenously injects 10 ml of the drug carrier packaged with calcitonin obtained above, and Group B injects the same amount of calcitonin as a control.

(6) According to the results, the in vivo half-life of calcitonin in the body was 4 hours, but the half-life of calcitonin was usually only 2 hours and the long-term effect was doubled.

Example 22 : Of the drug carrier  Beneficial effects (passive Targetability / Toxicity reduction)

The drug is packaged inside the biological membrane by ultrasonic emulsification. Specifically, it is as follows.

(1) Amphotericin B is dissolved in ethanol at 4 캜 to form a saturator.

(2) The amphotericin B saturated solution and the cell compartment obtained in Example 6 were mixed and ultrasonically emulsified in an ice bath using an ultrasonic wave crusher. Ultrasonic probe amplitude 40%, operation 30s, interval 30s, cycle 20 times.

(3) The emulsion is centrifuged under the conditions of 100,000 xg, 20 min, and 4 캜, the supernatant is thrown, and the precipitate is collected to obtain the drug carrier packed with amphotericin B as required.

(4) The drug carrier packed with amphotericin B obtained above was dipped in 5% Triton X-100 at 37 占 폚 to be demineralized overnight, diluted with methanol at a volume ratio of 100 times, and then subjected to high performance liquid chromatography Measure the content of terrinsin B. As a result, the content of amphotericin B in the carrier was 8.3%.

(5) Thirty male healthy mice are divided into two groups of 18-22 g, randomly divided into A and B groups, with no restriction on female males.

(6) Group A mice are injected with 0.2 mg of the drug carrier packaged with amphotericin B in the tail vein, and group B mice are injected with the same dose of amphotericin B as a control. After 0.5, 1.0, 1.5, 2.0 and 5.0 h of each drug, mice were taken from each group and blood was drawn from the heart, and then the liver, kidney, spleen and other organs were taken out and the content of amphotericin B .

(7) According to the results, the drug distribution within the kidney of amphotericin B packaged in biomembranes is significantly lower than that of amphotericin B, effectively alleviating renal toxicity.

Table of rats in different time periods and in the long term Amphotericin B concentration ( ug / g ) serum Soy sauce kidney spleen Scanning time (h) Group A Group B Group A Group B Group A Group B Group A Group B 0.5 0.14 0.08 0.09 0.02 0.03 0.13 0.38 0.13 1.0 0.11 0.08 0.08 0.01 0.04 0.11 0.24 0.11 1.5 0.09 0.07 0.06 0.01 0.02 0.09 0.16 0.09 2.0 0.07 0.06 0.05 0.01 0.01 0.10 0.15 0.09 5.0 0.07 0.05 0.05 0.01 0.01 0.08 0.10 0.07

Example 23 : Of the drug carrier  Beneficial effects (active Targetability )

(1) Human liver cancer cell line BEL-7402 was inoculated subcutaneously into BALB / C nude mice and when the diameter of the tumor reached about 1 cm, fresh fish tissue was cut into 1 mm * 2 mm pellets, It is inserted into the tunnels under the outer cavernosum of the intervertebral disc of the centro nude mouse, lightly pressed with a cotton pad, and then the abdomen is sutured.

(2) After modeling, the abdomen is cut open at 12d, and 20 model mice with basically identical tumor size are randomly divided into two groups of A and B, and 10 animals are prepared per group. Group A administers 0.2 mg of doxorubicin drug carrier having the target property obtained in Example 15 with intraperitoneal administration per day and Group B administers 0.2 ml of commercially available doxorubicin per day intraperitoneally as a control.

(3) After 10d, the model mice are opened to measure the size of the tumor, and at the same time, the tumor tissue is subjected to the pathological section and the macroscopic observation. As a result, the tumor suppression rate of the sample group is higher than that of the control group.

Weight (g) Tumor (mm3) Before Treatment After treatment Before Treatment After treatment Doxorubicin-biomembrane 20.53 21.46 43.62 30.12 Doxorubicin 20.03 20.1 42.93 50.17

Example 24 : Genetically modified  Beneficial effect Torrance SPECIAL  Effectiveness of the reagent)

(1) U2OS cells are inoculated on 6-well plates and DMEM complete medium (containing serum) is cultured for 24 h at 37 ° C and 5% CO ₂ .

(2) Transferring the transplantation carrier packaged with the GFP plastid obtained in Example 10 into a serum-free DMEM medium at a volume ratio of 1: 9, and mix lightly and uniformly.

(3) A serum-free DMEM culture medium containing the GFP gene carrier was uniformly coated on CHO cells and cultured in a cell culture incubator (37 ° C, 5% CO ₂ ) for 6 h, and then cultured in DMEM containing 20% calf serum Add the culture medium and continue to incubate for 48 h.

(4) According to the results shown in Fig. 3, green fluorescence was observed under a capillary microscope after 48 h of cytoprotection. This signifies the success of the tarosulfation.

Example 25 The beneficial result of gene modification (enhancing the efficiency of tarosation)

(1) A commercially available lipid saturation reagent was purchased, and the proportion of Example 24 was used as a control.

(2) According to the results shown in Fig. 4, when the biomembrane is used as a taranspring reagent, the efficiency of the tarosulfation is effectively enhanced.

Example 26 Genetically modified  Beneficial results (alleviating cytotoxicity)

(1) The cells in Example 2 of Example 25 were subjected to 72 h of taranation and then digested with pancreatic juice, After staining with PI, flow cytometric analysis will reveal the different apoptosis status of the tarans.

(2) According to the results, the cell apoptosis rate of the taranspecific reagent genetically modified by the biomembrane effectively alleviates the cytotoxicity of the lipid body compared to the taranspersion reagent.

Example 27: Beneficial effect of cosmetic additives (moisturizing)

(1) randomly selected 10 persons, a position of 10 cm x 10 cm was selected in the middle of the left and right hands of each individual, and a biological film dissolved in the 500-volume PBS obtained in Example 4 was applied to any one hand, Contrast the same volume of PBS.

(2) After two weeks of continuous application to the same position of both hands, the moisture content of both hands is measured with a skin moisture analyzer.

(3) According to the results, the average skin moisture content of the hands with a bio film was 48%, and the average skin moisture content of the hands with PBS was 43%. When the bio film was used as an additive for cosmetics, the skin had an effective moisturizing effect .

Example 28 : Beneficial effects of cosmetic additives (whitening)

(1) In the activity analysis system of 1 mL 0.05 mol / L phosphoric acid buffer with 0.5 mmol / L L-DOPA as a substrate, 20 uL of the cell compartment obtained in Example 8 (dissolved in DMSO solution) , And the substrate solution kept in a constant-temperature water bath at 30 ° C was placed in advance of 400 μL. The volume of the substrate solution was adjusted to 970 uL with a buffer solution, and 30 μL of tyrosinase aqueous solution was added thereto. The absorbance at a wavelength of 475 nm under 1 min at constant temperature is measured, and the activity of the enzyme is calculated by the slope of the straight line.

(2) According to the results, the inhibitory activity against the tyrosinase activity of the cell compartment was 37%, and there was almost no inhibitory activity against the tyrosinase in the control group. This has a whitening effect when the biomembrane is used as a cosmetic additive.

Example 29 : Beneficial effects of cosmetic additives (anti-aging)

(1) randomly selecting 10 persons aged 45 to 55 years old, selecting a position of 10 cm x 10 cm in the middle of each of the right and left hands of each individual, and transferring the biomembrane dissolved in the 500-volume PBS obtained in Example 5 Apply the same volume of PBS to the other hand and contrast.

(2) Apply to both hands at the same position continuously for 4 weeks, and then examine the anti-aging condition with a skin elasticity analyzer.

(3) According to the results, the average skin elasticity of the hands with a biomembrane is 30%, and the average skin elasticity of the hands applied with PBS is 27%, which is effective when the biomembrane is used as a cosmetic additive.

Example 30: Cosmetic Carrier  Beneficial effect (to improve stability of functional ingredient)

Same as in Example 16.

Example 31: Cosmetic Carrier  Beneficial effect (enhancing solubility of functional ingredient)

Same as in Example 16.

Example 32: Cosmetic Carrier  Beneficial effect (enhances long-term efficacy of functional ingredient)

The drug is packaged inside the biological membrane by reverse phase evaporation. Specifically, it is as follows.

(1) The biomembrane obtained in Example 6 was dissolved in chloroform having a volume of four times, and then evaporated under reduced pressure (37 ° C, 200 rpm) with a rotary evaporator to remove chloroform.

(2) Phenyl-resorcinol was dissolved in ethanol, ethyl ether was added to the biological membrane in procedure (1), the phenyl was mixed with resorcinol at a volume ratio of 1:10, and the mixture was incubated in a water bath at 50 ° C for 20 minutes, A cosmetic carrier packaged with the requisite phenyl resorcinol is obtained.

(3) After de-emulsifying the cosmetic carrier packed with resorcinol on the phenyl obtained above, the content of phenyletereosinol is measured by high performance liquid chromatography and the concentration is diluted to 0.5% with PBS.

(4) Select a skin of 10 cm x 10 cm, and apply 1 mL of the cosmetic carrier packaged with the resorcinol to the phenyl obtained in the above procedure (3) one time each morning and evening.

(5) According to the results shown in Fig. 5, resorcinol, which is packaged in biological membranes, has a long-term whitening effect.

Example 33 : cosmetics Carrier  Beneficial effect (enhance absorption of functional ingredient)

(1) Alveolar growth medium B16 melanoma cells were collected, the concentration of the cell suspension was adjusted, and 500 μL was added per hole in the 24-well plate to adjust the density of unmeasured cells to 50000 cells / Incubate overnight at 37 ° C.

(2) The two sets of samples in Example 16 were each placed in a cell culture medium, the concentration was adjusted to 500 nmol / L, the state of cell inclusion after 5 hours was analyzed, and the fluorescence microscope was used to record and record.

(3) According to the results shown in Fig. 6, packaging of Kurubumin with a carrier of a biological membrane significantly improves the absorption rate of cells.

Example 34 : cosmetics Carrier  Beneficial effect Validity  Improvement)

(1) B16 melanoma cells are inoculated into 6-well plates and cultured in DMEM complete medium (containing serum) at 37 ° C under 5% CO ₂ for 24 h, and the supernatant is thrown.

(2) The carrier packed with the coumucin obtained in Example 11 is put into a serum-free DMEM medium, mixed lightly and evenly, and is usually in contrast with quercumin.

(3) A serum-free DMEM culture medium containing the above-mentioned quercumin carrier was uniformly spread on B16 melanoma cells, cultured in a cell culture machine (37 ° C, 5% CO ₂ ) in a 3-D culture, then the supernatant was thrown, The pancreatic digestive cells are put in and stored for 5 minutes, and the number of cells in each group is counted.

(4) The cell suspension was centrifuged at 20,000 × g for 15 minutes at 4 ° C., and the supernatant was thrown. The precipitate was immersed in a 1 mol / L NaOH solution and heated at 80 ° C. for 30 minutes to measure the absorbance at 475 nm using a spectrophotometer do.

(5) Calculation formula: Melanin synthesis inhibition rate is calculated by the inhibition rate of melanin synthesis = [1- (drug hole absorbance / number of drug hole cells) / control hole absorbance / number of control hole cells] × 100%. (Fig. 7)

Example 35 : Vaccine carrier  And Ajju Bunt's  Beneficial effect (safety)

When the biomembrane is used as a vaccine carrier or immunoadjuvant, it is determined whether or not it contains a substance causing an allergic reaction, and in order to secure the safety of the inoculation of the biomaterial, the present invention carries out an animal allergy experiment.

(1) Each of the cell compartments obtained in Example 7 and Example 8 was inoculated into a guinea pig, and subcutaneously inoculated at a rate of 2 mL per 1 mL of the medium, followed by a second subcutaneous injection at intervals of 1 week, and sensitized with guinea pigs to give 3 Later in the day, inject 500uL of the same sample into every guinea pig.

(2) Judgment of results: There was no irritability, shock, or death in the guinea pig after guinea pig. This explains that the injected material has no allergic reaction to guinea pigs.

Example 36 : Application of vaccine (anti-virus vaccine, Subcutaneous formulation )

(1) Choose healthy BALB / c mice at 6-8 weeks of age and multiply them by 8 in a teardrop.

(2) Immunization with the carrier packaged with the circovirus obtained in Example 12 into Group A, and comparison with the group of the commonly-appearing type of serovirus vaccine in the market. In group 2, 0.2 mL of each vaccine is injected into the abdominal cavity of the rats.

(3) Breeding under the same specifications for breeding management. After 21 days of immunization, 0.45 mL of PCV2 virus (10 ^7.0 TCID ₅₀ / mL) is injected intraperitoneally to attack the virus.

(4) After 21 days of the attack, kill the spleen, blind the spleen suspension with PK15, and proceed with IFA fluorescence analysis.

As shown in the table below, the greater the fluorescence yield, the stronger positive the separation of the varicose veins and the less immunogenic effect.

Group Fluorescence number of immunofluorescence sample (duplicate 2-hole statistical results) Group A (4,3) (2,1) (2,0) (1,1) (0, 1) (1,0) (0,0) (0,0) Group B (2,6) (2,5) (2,2) (2,2) (3, 0) (2,1) (1, 2) (2,0)

Example 37 : Application of vaccine (gene vaccine, intramuscular injection)

(1) Choose 10 beagle dogs, weighing 3 kg (2.5-3.5 kg), and randomly divide into two groups, A and B, without restriction of male and female.

(2) Biomass films (see method of Example 12) packaged with measles virus DNA plasmids were intramuscularly injected into group A, that is, a biosurfactant vaccine having a plasmid DNA concentration of 1 ug / Joe contrasts by injecting the dog measles DNA pla- matid with the same DNA concentration. The vaccine is injected once every two weeks.

(3) After the third immunization, dilute the brain tissue homogenate of dogs suffering from the measles outbreak, which has been treated with double-stranded organisms, and perform an attack experiment by administering 2 mL each to each experimental dog. Of these, 1 mL is dropped into the nose and 1 mL is administered intramuscularly.

(4) After the attack, measure the temperature of the rectum daily, record the incidence and mortality of the test dog in detail, and examine changes in the clinical symptoms of the animal.

(5) After the attack, two dogs were killed in group B, the other one had mild neurological symptoms, the maximum body temperature reached 41 ℃, and slowly recovered to normal. The body temperature of the test tube injected with the biomembrane was slightly higher, reaching a maximum of 39.4 ° C, but neurological symptoms and death did not occur.

(6) According to the results, it was found that carriers carrying the biological membrane as a gene vaccine and immunoadjuvant have a very strong immune effect.

Example 38  Application of vaccine (tumor vaccine, oral dosage form)

(1) Take 10 healthy mice and divide into 18-22 g, randomly divided into two groups, A and B, with no restriction on female males.

(2) Before immunization and inoculation, mice were fed with water and water for 12 hours. Polymerization with stomach acid was carried out by feeding 150 uL 0.01 mol / L NaHCO ₃ solution by oral administration 30 minutes before inoculation, and the biomembrane carrier packed with Helicobacter pylori- (See the method of Example 12). The vaccine is orally administered to group A, and a biosurfactant vaccine with a concentration of 100 uL Helicobacter pylori genetically modified protein of 10 uL is fed. Group B is fed with 100uL of PBS, and 10uL of Helicobacter pylori genetically modified protein is fed and contrasted.

(3) Experimental group and control group were immunized once on each of days 0, 7, 14, and 21, and fed and water was given 1 hour later. After 2 weeks of the last immunization (day 35), 10 ⁸ CFU / Fillroll attacks are conducted three times, once every two days, and the mice are killed four weeks after the last attack.

(4) Immediately after rats are killed, remove the stomach, remove the stomach and spleen, cut along the Taiwanese part, wash the stomach contents with sterile physiological saline, take the stomach tissue along the longitudinal axis, platinate, Proceed with the experiment.

(5) According to the urease experiment, the biosurfactant vaccine effectively protects mice from infection with Helicobacter pylori as compared to aseptic rats. Since Helicobacter pylori is an important cause of diseases such as gastric cancer and gastric mucosal tissue, it can effectively prevent diseases such as stomach cancer by using biocompatible Helicobacter pylori protein modified vaccine carrier and azuban.

Example 39 : vaccine carrier  And Ajju Bunt's  Beneficial effect (strong humoral immunity)

Same as Example 36.

Example 40 : vaccine carrier  And Ajju Bunt's  Beneficial effect (strong cell immunity)

와 IL-4 mRNA 발현을 비교한다. (1) For the rat spleen obtained in Example 38, splenic IMP cells INF-

And IL-4 mRNA expression.

)를 위주로 하는 증식이 생겼고, INF-

레벨은 대조조에 비해 현저한 차이가 있었다.(2) According to the results, Th1 cells (INF-1) were injected into rat splenic epithelial cells by using biomembranes as helicobacter pylori protein-modified vaccine carrier and adjuvant,

), And INF-

The level was significantly different from the control group.

Example 41 : Application of artificial organs

(1) 10 beagle dogs of 2 months age are selected and divided into two groups, randomly divided into two groups of A and B, without the restriction of male and female weighing 2.5kg (2-3kg).

(2) The right knee of the beagle dog was incised from the lateral side of the knee joint, and the lateral ligament was not cut, and the triangular scratch on the outer vane plate (the base was located at the edge and the tip direction occupied half of the width of the meniscus) Make a vertical scar on the poor supply base.

(3) Artificial meniscus obtained in Example 13 is implanted into scratches in Group A, and artificial meniscus with a commercially available collagen as a stand is implanted in Group B as a control. Both groups are fixed with an absorbable suture line, the incision is sutured, and postoperative restraint is maintained.

(4) After 12 weeks of operation, kill the animal and look at the specimen of the knee joint. The group A was well absorbed, and white new tissue was grown on the defect. The texture and color were similar to the surrounding normal meniscus and there was no clear boundary. Group B had similar texture and color to the normal meniscus tissue However, a white island scar tissue was seen in the defect, and the corresponding femur and tibial joint surface were slightly rough.

The invention described in this sentence can be realized without any device or limitation disclosed specifically by this sentence. It is to be understood that the terminology and expressions used herein are for the purpose of describing the invention and are in no way intended to limit the invention, the use of such terms and expressions, or the equivalents thereof, And it is to be understood that various modifications may be made without departing from the scope of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. .

The contents of sentences, patents, patent applications and all other documents and information obtained electronically contained in this sentence can be referred to in some extent as a comprehensive reference, and each publication that exists alone can be used as a reference . Applicants reserve the right to use and incorporate in this invention all data and information obtained from such statements, patents, patent applications or other documents.

Claims (25)

In the method for producing an in vitro membrane,
The method comprises the steps of 1) obtaining biological cells from natural or natural biological species,
2) The cells described in Procedure 1) are cultured in a large volume in a suitable environment,
3) After obtaining a lysed solution of the cells described in the procedure 2), various biological membranes or mixtures belonging to the present invention are obtained through separation and purification of different methods, and as a method for obtaining a biomembrane of the present invention from an in vitro system, Density gradient centrifugation, and two-phase extraction. A procedure for extracting closure structures or cell compartments having biological membranes and biomembrane properties by using various methods singly or in pairs or by using three together Wherein the method comprises the steps of:
The method according to claim 1,
The supernatant is obtained by centrifuging the cell liquor under the first high rotation speed condition through the electrofraction centrifugation extraction method and centrifuged under the second rotation speed condition lower than the first high rotation speed to obtain the precipitate, How to get there.
3. The method of claim 2,
Wherein the difference between the first high rotation speed and the second rotation speed is 1.5 times, 1 time, 2 times, or 3 times.
3. The method of claim 2,
The supernatant was collected, and the supernatant was centrifuged at 15,000-30,000 x g for 10 to 30 minutes under high-speed centrifugation at 1-6 ° C. The supernatant was centrifuged at 100,000-200,000 × g for 30-90 min at 1-6 ° C Centrifuging, centrifuging, throwing up the supernatant, and collecting the precipitate to obtain the required biological membrane.
The method according to claim 1,
The cell lysate thus obtained was resuspended in an electrodensity gradient centrifugation method. The resuspension was placed in a sucrose solution of different concentrations and subjected to ultra-high-speed centrifugation under the conditions of 150,000-300,000 x g, 60-90 min, 1-6 DEG C, Collecting the supernatant, ultracentrifugly centrifuging the collected liquid under the condition of 100,000-200,000 × g, 30-90 min, 1-6 ° C, throwing up the supernatant, collecting the precipitate, and extracting the required biological membrane .
The method according to claim 1,
The obtained cell lysate solution precipitate is resuspended, the sucrose solution of the first concentration and the sucrose solution of the second concentration are sequentially added to the re-suspension, and then the solution of the third sucrose is added. The first concentration is lower than the second concentration, 3 concentration is lower than the first concentration.
The method according to claim 5 or 6,
The mass percentage percent concentration of the solution in the sucrose solution is preferably 10-70%, and preferably the unmixed mass percent concentration of the solution per 3 electrical conduction steps is 10%, 20%, 30%, 40%, 45%, 50% , The molar ratio of the first concentration solution per concentration is 0.1-0.5 mol / L, the concentration of the second concentration solution is 1-3 mol / L, the concentration of the third concentration solution Wherein the molar ratio is 0.01-0.3 mol / L.
The method according to claim 1,
The resulting cell lysis solution precipitate was resuspended through the density gradient centrifugation method and the precipitate 1 was collected through several times of the first low speed centrifugation to prepare a suspension 1, followed by the second high speed centrifugation to the suspension 1 several times And the precipitate 2 is obtained by suspending the precipitate 2 in the nonionic surface active solution to obtain the suspension 2. At the same time, the suspension 2 is diluted with the sucrose solution, the diluted suspension 2 is taken in the lower part of the centrifugal tube, (For example, under the conditions of 28000 x g -45,000 x g, 10-24 h, and 4 ° C) by sequentially adding high-concentration sucrose and a low-concentration sucrose solution and collecting them at the interface of low concentration and high concentration, A method for obtaining a living biological membrane.
9. The method of claim 8,
Wherein the high sucrose (weight percent) is 20-35% and the low concentration sucrose solution is 2-10%.
The method according to claim 1,
The two-phase extraction method comprises the following steps: freshly disposing a two-phase system, then suspending the cell lysate precipitate again, putting it in a two-phase mixture, conducting 30 to 40 times ; Centrifugation was carried out at 2,000-4,000 × g for 5 to 10 minutes at 4 ° C. The upper and lower phases were successively placed in a two-phase system, separated 3 times, merged with the upper phase, diluted 5 times, g, 30-90 min, at 4 캜, and collecting the precipitate, thereby extracting the biological membrane.
11. The method of claim 10,
A method in which an aqueous two-phase, an organic two-phase, an aqueous solution and an organic phase solution are contained in an electric two phase, and an electric solvent is any one of water, acetonitrile, acetone, tetrahydrofuran, methanol, ethanols, .
11. The method of claim 10,
Wherein the electrical two phase consists of a two-phase mixture of glucan / polyethylene glycol.
A method of manufacturing a biological membrane having a magnetic assembly,
The closure structure and the cell compartment material having the obtained biological membrane and biological membrane properties are adhered to the container wall in the form of a dry film and then slowly water or buffer is poured slowly while applying a moderate or severe vibration, Wherein a closure structure and a cell compartment having a biomembrane and biomembrane properties are obtained.
14. The method of claim 13,
The material having the closure structure and the cell compartment having the biological membrane and biological membrane properties obtained was dissolved in an organic solvent and placed in a container. The biological membrane was attached to the surface of the container by the reduced pressure evaporation method and the mixture was evaporated to the normal weight. To 3 hours after centrifugation at 100 to 200,000 × g for 30 to 90 minutes under ultra-high-speed centrifugation at 1-6 ° C. to collect the precipitates and collect the supernatant to obtain the closure structure and cell compartment having the required biological membrane and biological membrane properties .
15. The method of claim 14,
Wherein the organic solvent is chloroform or ethyl ether.
15. The method of claim 14,
Wherein the biomembrane is obtained by any of the methods of claims 1 to 12.
17. The method according to any one of claims 1 to 16,
Wherein the biological membrane is derived from a natural plant, animal or microorganism, and the natural tissue is fresh blood or highly thermophilic.
The method according to claim 1 or 13,
The shape of the biological membrane includes a spherical shape, a cyst shape, a rod shape, a helical shape, a single layer, a plurality of layers, and various types of structures of a plurality of rooms. A cell membrane, a chloroplast membrane, a vacuole, and a peroxisome membrane are encompassed, or a cell compartment is encompassed in an electrophysiological membrane, and the cell compartment is distinguished from a cell organelle. Preferably, the cell compartment is a mitochondrial, chloroplast, Peroxisome, endoplasmic reticulum, nucleus, Golgi and cysts, and microtubules.
19. The method of claim 18,
A method comprising the above-mentioned method for producing a cell compartment and including the production of a two-phase system.
20. The method of claim 19,
The two-phase system is an aqueous two-phase system,
The production method is
(1) mixture (90 g 20% (W / W) Dextran T-500, 45 g 40% (W / W) PEG 3350, 33.9 g sucrose, 7.5 g 0.2 mmol / L PBS, 0.45 g 2 mmol / (Dextran T-500 / PEG 3350) in the same concentration was prepared, uniformly mixed in a separating funnel, allowed to stand overnight at 4 ° C, And the upper and lower layers were carefully separated and fresh upper and lower phases were prepared and each was stored at 4 캜 to be used for post-
(2) The biomembrane precipitate obtained in claim 1 was resuspended in resuspension buffer (5 mmol / L PBS, 0.33 mol / L sucrose, 3 mmol / L KCl, 1 mmol / L DTT, 1 mmol / L PMSF, 0.1 mmol / L EDTA) ,
(3) The resuspension was placed in a Dextran T-500 / PEG 3350 aqueous two-phase mixture prepared in Procedure (1) in a proportion of 1: 3 by mass,
(4) The mixture was centrifuged at 1,500 rpm, 10 min, and 4 ° C, and the supernatant and the supernatant were taken in succession. The supernatant was separated into 3 phases, , 100,000 x g, 60 min, 4 캜, and collecting the precipitate to obtain the required cell compartment.
19. The method of claim 18,
When the natural material is spinach,
The method of obtaining the cell compartment
(1) Spinach leaves which were grown in good weather for 10 consecutive days in which 10g of the growth was healthy were cleanly washed and then the large stems were removed, and a 6-fold volume of homogenous buffer (50 mmol / L potassium phosphate buffer , 0.3mmol / L sorbitol, 2mmol / L EDTA, 1mmol / L MgCl 2, 1mmol / L MnCl 2, 1% BSA, 1mmol / L DTT) to put, and polished on an ice bath,
(2 mmol / L HEPES-KOH, 0.3 mmol / L sorbitol, 2 mmol / L EDTA, 1 mmol / L MgCl ₂ , 1 mmol / L MnCl ₂ , 1% BSA, 3% PEG 6000, 1% Ficoll) And centrifuged beforehand under the conditions of 30,000 xg, 20 min and 4 캜,
(3) The abrasive liquid was filtered with four layers of gauze and centrifuged at 3,000 xg for 15 minutes at 4 캜 to collect the precipitate. The precipitate was suspended in 2 ml of the homogeneous buffer, In a separate Percoll fraction, centrifuging under the conditions of 15,000 xg, 20 min, 4 캜, and taking the lower layer to obtain the required cell compartment.
The method according to claim 1,
When the cells come from fresh blood, the method used is
(1) 30 ml of fresh blood was subjected to washing at 100 xg, 10 min, and 4 ° C to remove chorionic villus sediments from plasma and red blood cell surface,
(2) Add 5 volumes of pH 8.0 PBS buffer, centrifuge at 2,000 × g, 15 min, 4 ° C, throw the supernatant, repeat 3 times,
(3) A pH 8.0 PBS buffer solution having a volume of 40-fold was added to the precipitate, and the mixture was allowed to stand at 4 캜 for 2 hours,
(4) Next, centrifuge under conditions of 22,000 × g, 20 min, and 4 ° C. and repeat 4 times,
(5) The precipitate was transferred to a pre-cooled Triton X

100 buffer (containing 0.25 mmol / L sucrose, 150 mmol / L NaCl, 1 mmol / LEDTA, 20 mmol / L Tris-HCl and 1% Triton X

100) and then diluted with an 80% (w / v) sucrose solution of the same volume,
(6) Take 4 mL of the membrane suspension, place it in the bottom of the centrifuge tube, add 4 mL 30% and 3 mL 5% sucrose solution sequentially, and centrifuge at 38,000 × g, 18 h, , 5% and 30% of sucrose solution at the interface to obtain the required biomembrane.
The method according to claim 1,
When the bacterium is caused by highly thermophilic bacteria,
(1) Highly thermophilic bacteria ( Thermus Thermophillus ) was isolated from the Yellowstone National Park in the United States,
(2) No. 1 a highly thermophilic bacteria: Among medium (10L medium in the proportion of 100 _{26 g (NH4) 2 SO 4} , 2.47g MgSO 4 ˙ 7H 2 O, 2.8g KH 2 PO 4, 0.74g CaCl 2 ˙ 2H _{2 O, 0.19g FeCl 3 ˙ 6H} 2 O, 0.018g MnCl 2˙ 4H 2 O, 0.044g Na 2 B 4 O 7 ˙ 10H 2 O, 0.002g ZnSO 4 ˙ inoculated in 7H containing ₂ O), incubator And incubated for 24 hours at 60 DEG C and 150 rpm,
(3) The cells were collected by centrifugation under the conditions of 4,000 rpm, 30 min, and 4 캜,
(4) The cells were resuspended in a homogenous buffer (20 mmol / L Tris-Cl pH 8.0, 100 mmol / L NaCl, ₂ mmol / L MgCl ₂ , 1 mmol / L DTT) and centrifuged at 6,000 rpm, 10 min, To remove the supernatant,
(5) Immerse the precipitate (containing 10 ml buffer per about 1 g) in a homogenous buffer, add PMSF at a final concentration of 1 mmol / L, and disintegrate in an ice bath for 30 min (amplitude 55%, ultrasonic 5 s, stop 8 s)
(6) The crushed cells were centrifuged at 25,000 xg for 30 minutes at 4 DEG C, the precipitate was removed, the supernatant was taken,
(7) A method for producing a biomembrane by ultracentrifugal centrifugation under a condition of 145,000 x g, 1 h, and 4 캜, and collecting the precipitate.
The method according to claim 1 or 13,
A method of adding a target to an active ingredient of a prepared biological membrane, an inner membrane package, a surface adsorption, a surface crosslinking, a membrane intercalation, and a method of adding a target to a package inside the membrane.
25. The method of claim 24,
Wherein the active ingredient is a vaccine or an immunomodulating active ingredient, a cosmetic or cosmetic active ingredient, a drug active ingredient, a genetic material, and a cell or tissue.

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