KR20230026261A - Multi-layered vesicles for induction of immunity, pharmaceutical composition for inducing immunity comprising same, and method of manufacturing multi-layered vesicles for induction of immunity - Google Patents

Abstract

The present invention relates to a multi-layered endoplasmic reticulum for inducing immunity, a pharmaceutical composition for inducing immunity comprising same, and a method for manufacturing a multi-layered endoplasmic reticulum for inducing immunity. The multi-layered endoplasmic reticulum for inducing immunity according to the present invention can stably move into immune cells without chemical bonding, and a pharmaceutical composition for inducing immunity employing the same can effectively induce humoral immunity and cellular immunity at the same time, thereby exhibiting an improved immune-inducing effect.

Description

Multilayered vesicles for induction of immunity, pharmaceutical composition for induction of immunity comprising the same, and method for manufacturing multilayered vesicles for induction of immunity induction of immunity}

The present invention relates to multilamellar endoplasmic reticulum for induction of immunity, a pharmaceutical composition for induction of immunity comprising the same, and a method for preparing the multilamellar endoplasmic reticulum for induction of immunity.

BACKGROUND ART Vaccine antigens that inoculate all or part of pathogens in order to induce an immune response against pathogens are used for the prevention or treatment of various diseases. Protein antigens of vaccines used for induction of immunity are rapidly degraded in organs such as the liver and kidneys in the body, so it is difficult to effectively deliver them to immune cells. It is difficult to induce a high immune response.

In order to compensate for this, an adjuvant is used, and such an adjuvant can increase the efficacy of a vaccine by improving the reactivity of antigen-specific immune cells. However, protein antigens and adjuvants that are not in a single structural form are difficult to efficiently deliver to dendritic cells, and the ability to produce expression substances necessary for immunity is also reduced, resulting in a decrease in the efficiency of the immune response. Therefore, the protein antigen and the adjuvant must be made in a single structural form, must be protected from degradation in the body, and must be easily incorporated into dendritic cells.

For these challenges, polymeric micelles, hollow cages, and lipid-based nanoparticles are being studied as transporters of protein antigens and immune enhancers. Among them, lipid-based nanoparticles have low immunogenicity of lipids, high biocompatibility and protein It is in the spotlight for its excellent protective ability against antigens.

Since these lipid-based nanoparticles have poor immune-inducing performance due to the structural instability of lipids and low encapsulation efficiency, there have been studies to improve them by chemical bonding between protein antigens and lipid molecules. However, due to chemical bonding, chemically modified protein antigens may not cause a sufficient immune response or cause pharmacological side effects. Accordingly, the lipid-based nanoparticles for inducing immunity can induce a remarkable immune-inducing response by improving the structural stability of the lipid-based nanoparticles, achieving high encapsulation efficiency, and containing protein antigens that are not chemically bound at the same time. Particle development is required.

Korean Publication No. 10-2021-0087415 (2021.07.12.) Korean Publication No. 10-2021-0088594 (2021.07.14.)

The object of the present invention is to have excellent stability in serum without chemical binding, excellent ability to deliver into immune cells, and release of protein antigens in the endosome/lysosomal environment to enable environment-selective protein antigen release, resulting in excellent immunity. It is to provide a multilayer endoplasmic reticulum for inducing immunity having an inducing effect.

Another object of the present invention is to provide a pharmaceutical composition for inducing immunity, including the multilayer endoplasmic reticulum for inducing immunity, having an improved immune-inducing effect by increasing the expression of immune-related proteins in cell experiments and inoculation experiments.

Another object of the present invention is to provide a method for preparing multilayer endoplasmic reticulum for inducing immunity, which is economical and suitable for mass production as it can be produced by a simple process under mild conditions.

As a result of research to solve the problems described above, the inventors of the present invention have developed multilayer endoplasmic reticulum containing protein antigens produced by self-assembly of unilamellar endoplasmic reticulum meeting protein antigens. The multilamellar endoplasmic reticulum containing the protein antigen shows an excellent immune response-inducing effect due to the protective effect of the protein antigen and the enhanced incorporation effect into dendritic cells.

The multilamellar endoplasmic reticulum for induction of immunity according to the present invention includes two or more unit lamella layers, wherein the unit lamella layer includes an ionic lipid molecule charged with a first charge, and the unit lamella layer and the adjacent unit lamella layer are separated from each other. It is characterized in that a protein antigen charged with a second charge is located between them.

The first charge and the second charge of the multilayered endoplasmic reticulum for inducing immunity may have charges of different polarity, and the ionic lipid molecules and protein antigens of the multilayered endoplasmic reticulum for inducing immunity may form an ionic complex.

In addition, the weight ratio of the ionic lipid molecule to the protein antigen may be 1:0.01 to 1:1, and the ionic lipid molecule may be a cationic lipid molecule containing a C14-C20 acyl group.

The outermost lamellar layer of the multilamellar endoplasmic reticulum according to the present invention may further contain an immune enhancer, and specifically, the immune enhancer may be monophosphoryl lipid A.

The surface of the multilamellar vesicles according to an embodiment of the present invention may further include an anionic polysaccharide coating layer, and thus the surface charge of the multilamellar vesicles may be negatively charged.

In addition, the particle size of the multilayer vesicles according to the present invention may be 100 nm to 300 nm.

The present invention provides a pharmaceutical composition for inducing immunity comprising multilamellar vesicles for inducing immunity according to an embodiment of the present invention.

The method for producing multilayer endoplasmic reticulum for induction of immunity according to an embodiment of the present invention,

(a) preparing vesicles comprising ionic lipid molecules charged with a first charge;

(b) preparing unilamellar vesicles from the endoplasmic reticulum; and

(c) preparing a multilamellar endoplasmic reticulum by mixing a protein antigen charged with a second charge with the unilamellar endoplasmic reticulum.

In step (c) of the preparation method, the unilamellar endoplasmic reticulum and the protein antigen charged with the second charge are mixed at 4°C to 40°C, and may be converted into multilamellar endoplasmic reticulum through self-assembly.

After step (c) of the manufacturing method,

(d) injecting an immunostimulant into the solution containing the multilamellar vesicles.

may contain more

After step (d) of the manufacturing method,

(e) adding an anionic polysaccharide to a solution containing the multilamellar vesicles may be further included.

Multilayer endoplasmic reticulum for induction of immunity according to an embodiment of the present invention can maintain stability in serum only by electrostatic attraction without chemical bonding, has excellent ability to deliver into immune cells, and protein in an endosome/lysosome environment. By releasing the antigen, it is possible to release the environment-selective protein antigen, thereby exhibiting an excellent immune induction effect.

The pharmaceutical composition for induction of immunity comprising the multilayered endoplasmic reticulum for induction of immunity according to the present invention has high release of tumor necrosis factor, a major immune cytokine, in cell experiments, and excellent T cell-related protein expression in one short-term inoculation and three long-term inoculations. Thus, an improved immune induction effect can be exhibited.

In addition, the method for producing multilayer endoplasmic reticulum for induction of immunity of the present invention can be performed with a simple process under mild conditions, and thus has excellent economic feasibility and industrial feasibility in mass production.

1 shows the results of analyzing the sample of Example 1 using a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) instrument.
Figure 2 shows the result of measuring the transmittance of the sample of Example 1 using a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) device.
Figure 3 shows the results of analyzing the sample of Example 1 using a small-angle X-ray scattering method (4C SAXS II beamline, Pohang Accelerator Laboratory) instrument.
4 shows the results of analyzing the sample of Example 1 using a cryogenic transmission electron microscope (JEM-1400, JEOL Ltd., Tokyo, Japan).
5 shows the results of cryo-TEM analysis of the sample of Example 1 using a cryogenic transmission electron microscope (JEM-1400, JEOL Ltd., Tokyo, Japan).
6 shows the results of analyzing samples of Examples 1 and 2 using a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) instrument.
7 shows the results of analyzing the samples of Examples 1 and 2 over time in a serum environment using a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) device.
Figure 8 shows the results of the amount of OVA released from the sample of Example 2 according to the environment.
Figure 9 shows the morphological changes of bone marrow-derived dendritic cells treated with Example 2.
10 shows the result of analyzing the protein antigen of the cells treated with the sample of Example 2 using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA).
11 shows the result of analyzing the tumor necrosis factor of the cells treated with the sample of Example 2 using a tumor necrosis factor alpha quantification kit (Mouse TNF alpha ELISA Kit, abcam, Cambridge, UK).
12 shows the results of analyzing costimulatory molecules of cells treated with the samples of Example 2 using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA).
13 shows the results of analyzing the surface protein of the spleen of the rat inoculated with the sample of Example 2 using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA).
14 shows the results of analyzing OVA-specific immunoglobulin antibodies in the serum of mice inoculated with the sample of Example 2 using a Mouse Anti-OVA IgG Antibody Assay kit (Chondrex, Inc., Redmond, WA, USA). .
15 shows the result of analyzing immunoglobulin antibodies in the serum of mice inoculated with the samples of Example 2 using a Mouse total IgG Antibody Assay kit (Chondrex, Inc., Redmond, WA, USA).
16 shows the results of analyzing the IFN-γ in the spleen of mice inoculated with the sample of Example 2 using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA).
Figure 17 shows a schematic diagram of the preparation and immune response of multilamellar vesicles according to the present invention.

Hereinafter, the multilamellar vesicles for induction of immunity of the present invention, the pharmaceutical composition for induction of immunity comprising the same, and the method for preparing the multilamellar vesicles for induction of immunity will be described in detail. At this time, if there is no other definition in the technical terms and scientific terms used, they have meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and will unnecessarily obscure the gist of the present invention in the following description. Descriptions of possible known functions and configurations are omitted.

The singular form used in the present invention may be intended to include the plural form as well, unless the context specifically dictates otherwise.

Further, as used herein, numerical ranges include lower and upper limits and all values within that range, increments logically derived from the shape and breadth of the defined range, all values defined therebetween, and the upper limit of the numerical range defined in a different form. and all possible combinations of lower bounds. Unless otherwise specifically defined in the specification of the present invention, values outside the numerical range that may occur due to experimental errors or rounding of values are also included in the defined numerical range.

As described in the present invention, "comprising" is an open-ended description having the same meaning as expressions such as "comprises", "includes", "has" or "characterized by", elements not additionally listed, No materials or processes are excluded.

The present invention is a multilayered endoplasmic reticulum for immunity induction, which maintains stability only by electrostatic attraction without chemical bonding, has an excellent ability to deliver into immune cells, and is capable of releasing environmentally selective protein antigens, thereby exhibiting an excellent immune induction effect. In order to provide a pharmaceutical composition for induction and a method for preparing multilamellar vesicles for induction of immunity, multilamellar vesicles for induction of immunity having the following characteristics are provided.

The multilamellar endoplasmic reticulum for induction of immunity according to the present invention includes two or more unit lamella layers, wherein the unit lamella layer includes an ionic lipid molecule charged with a first charge, and the unit lamella layer and the adjacent unit lamella layer are separated from each other. It is characterized in that a protein antigen charged with a second charge is located between them.

The unit lamella layer refers to a lipid molecule bilayer unit structure formed by self-assembly of lipid molecules, and the surface of the unit lamella layer has hydrophilicity due to the head group of lipid molecules, and the inside has a tail group of lipid molecules (tail group). group) to have hydrophobicity. As the surface of the unit lamella layer is charged with a first charge, it has an electrostatic repulsive force between the unit lamella layers, but as a protein antigen charged with a second charge is located between the two unit lamella layers, the electrostatic repulsive force is offset. Thus, multilamellar vesicles can be stably formed. As the multilamellar endoplasmic reticulum for induction of immunity according to the present invention has the above-described structure, it can exhibit excellent stability without chemical binding, and can release protein antigens in the endosome/lysosomal environment, thereby enabling the release of environmentally selective protein antigens. It shows excellent immune induction effect.

The unit lamella layer may include 30 to 100 mol%, specifically 50 to 100 mol%, and more specifically 70 to 100 mol% of ionic lipid molecules charged with the first charge. The unit lamellar layer may further include an amphoteric lipid molecule, and the amphoteric lipid molecule may be a phosphocholine-based amphoteric lipid molecule containing a C14-C20 acyl group, preferably containing a C16-C20 acyl group. It may be a phosphocholine-based amphoteric lipid molecule, more preferably a phosphocholine-based amphoteric lipid molecule containing an unsaturated C16-C20 acyl group. Specifically, the amphoteric lipid molecule may be DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) containing a C18 acyl group, but is not limited thereto.

Since the unit lamella layer includes ionic lipid molecules charged with a first charge, the surface charge of the unit lamella layer has a first charge, which is the charge of the ionic lipid molecule.

The first charge and the second charge of the multilayered endoplasmic reticulum for inducing immunity may have charges of different polarity in the pH 6 to pH 9 region, preferably the first charge is positive charge, and the second charge may be negative charge. The ionic lipid molecules and protein antigens of the multilayered endoplasmic reticulum for inducing immunity may form an ionic complex, preferably an ionic complex consisting of cationic lipid molecules and anionic protein antigens.

The multilayer endoplasmic reticulum composed of ionic complexes according to the present invention solves the disadvantages of immunocompromised and pharmacological side effects of protein antigen-lipid complexes having chemical bonds, while enabling stable delivery to immune cells to induce more improved immunity have an effect

In addition, the weight ratio of the ionic lipid molecule and the protein antigen may be 1:0.01 to 1:1, preferably the weight ratio may be 1:0.1 to 1:0.5, more preferably the weight ratio is 1:0.2 to 1 : may be 0.3.

The ionic lipid molecule may be a cationic lipid molecule containing a C14-C20 acyl group, preferably a cationic lipid molecule containing a C16-C20 acyl group, more preferably a cationic lipid molecule containing an unsaturated C16-C20 acyl group. It can be a sexual lipid molecule. Specifically, the ionic lipid molecule may be 1,2-dioleoyl-3-trimethylammonium propane containing a C18 acyl group, but is not limited thereto.

The protein antigen charged with the second charge may be a protein antigen charged with a specific charge alone or a complex including a protein antigen charged with a specific charge. Specific examples of the charged protein antigens above, cationic proteins include lysozyme, avidin, antimicrobial proteins, RNA or DNA binding proteins, proteases, methylated collagen, cytochrome C, proteins involved in the aging process , platelet factor 4 and protamine sulfate. In addition, anionic proteins include wheat acidic esterase, alkaline phosphatase, beta-galactosidase, lactase, lipase, amylase, glycosidase, glucose oxidase, nitrate reductase, catalase, lactoglobulin, from the group consisting of carbonic anhydrase, casein protein in milk, trypsin inhibitor, proteins found in egg white, including ovalbumin, gamma-globulin and ovomucin, cathepsin and albumin. It may be a protein of choice. Preferably, the protein antigen may be ovalbumin, but is not limited thereto.

In the case of a complex including a protein antigen charged with a specific charge, it may be a complex of a polymer charged with a second charge and a protein antigen charged with a first charge, and the net charge of the complex is the second charge. can For example, it may be an anionic IgG complex prepared by combining IgG with an anionic polypeptide polymer such as polyglutamic acid or polyaspartic acid, but is not limited thereto.

The outermost lamellar layer of the multilamellar endoplasmic reticulum according to the present invention may further contain an immune enhancer, preferably, such as Alhydrogel ^® , Rehydragel ^TM and Adju-Phos ^® . aluminum salts, monophosphoryl lipid A (MPLA), CpG oligodeoxynucleotides, saponin-based adjuvants such as ISCOM, ISCOMATRIX and Matrix-M ^TM , or water-in-oil emulsions such as MF59 and AS03, but are not limited thereto . Specifically, the adjuvant may be monophosphoryl lipid A, and since monophosphoryl lipid A contains a plurality of long-chain aliphatic acyl groups, it is very effectively inserted into the lamellar layer containing ionic lipid molecules even by simple mixing, stably forming the lamellar layer. layer can be included.

The surface of the multilayer endoplasmic reticulum according to an embodiment of the present invention may further include an anionic polysaccharide coating layer, and the anionic polysaccharide used in the anionic polysaccharide coating layer may be heparin, alginic acid, hyaluronic acid, or chondroitin sulfate. Not limited.

The surface charge of the multilamellar vesicles for induction of immunity coated with the anionic polysaccharide according to an embodiment of the present invention may be negatively charged.

In addition, the particle size of the multilayer vesicles according to the present invention may be 50 nm to 1,000 nm, specifically, the particle size may be 100 nm to 500 nm, and more specifically, 100 nm to 300 nm.

The present invention provides a pharmaceutical composition for inducing immunity and an immune-inducing agent comprising multilamellar endoplasmic reticulum for inducing immunity according to one embodiment. The multilamellar vesicles for inducing immunity can easily deliver protein antigens and anionic drugs into cells, and, for example, when tumor-specific antigens are used, they can exhibit strong anticancer effects, but are not limited thereto. Therefore, the multilayer endoplasmic reticulum for inducing immunity can be used as a nano-vaccine for inducing immunity to prevent or treat various infectious diseases and cancers.

The present invention provides a method for inducing immunity in humans and animals by using the multilamellar endoplasmic reticulum for induction of immunity including the multilamellar endoplasmic reticulum for induction of immunity according to one embodiment. Immunity can be induced by administering the multilayer endoplasmic reticulum for inducing immunity to humans or animals. For example, when the multilamellar endoplasmic reticulum employs a tumor-specific antigen, it can exhibit strong anticancer effects, and when employing an antigen of an infectious disease, it can provide preventive and therapeutic effects on infectious diseases.

The present invention provides a method for preparing a multilamellar endoplasmic reticulum for inducing immunity, the method comprising: (a) preparing an endoplasmic reticulum comprising an ionic lipid molecule charged with a first charge; (b) preparing unilamellar vesicles from the endoplasmic reticulum; and (c) preparing a multilamellar endoplasmic reticulum by mixing a protein antigen charged with a second charge with the unilamellar endoplasmic reticulum.

Step (a) of the preparation method may be used without limitation as long as it is a method for preparing vesicles containing ionic lipid molecules, for example, giant vesicles. For example, in the step (a), preparing a solution by dissolving ionic lipid molecules in a solvent, preparing a film by drying the solution, and adding water to the film and then hydrating the film to prepare an vesicle. steps may be included. The vesicles prepared in step (a) may have a plurality of lamellar layers or form irregular lamellar layers, and may be dispersed in the aqueous phase in the form of a suspension or emulsion as large granular vesicles.

Step (b) of the preparation method may be used without limitation as long as it is a known method for micronizing vesicles, and may be specifically performed by ultrasonic treatment or an injection method. Sonication is typically performed repeatedly in an ice bath with a tip sonicator. Injection molding can be performed by a membrane extruder. A defined pore size in the injection molded filter allows the creation of unilamellar vesicles of a specific size.

In step (c) of the preparation method, the unilamellar endoplasmic reticulum and the protein antigen charged with the second charge may be mixed at 4 ° C to 40 ° C, preferably at 10 ° C to 35 ° C, more preferably at 15 ° C to 30 °C. The pH of the mixed solution may be between pH 6 and pH 9.

In addition, in step (c) of the preparation method, the unilamellar endoplasmic reticulum and the protein antigen charged with the second charge may be converted into the multilamellar endoplasmic reticulum through self-assembly, and the self-assembly causes the unilamellar endoplasmic reticulum to attach to the protein antigen charged with the second charge. It can be converted into a multilamellar endoplasmic reticulum having several layers through a morphological change process, such as folding or wrapping other unilamellar endoplasmic reticulum.

After step (c) of the manufacturing method, (d) injecting an immune enhancer into the solution containing the multilamellar vesicles; may further include, the injecting step may be carried out at 4 ° C to 40 ° C Preferably it may be carried out at 10 ℃ to 35 ℃, it may be carried out more preferably at 15 ℃ to 30 ℃.

After step (d) of the preparation method, (e) adding the anionic polysaccharide to the solution containing the multilamellar vesicles; , Preferably it may be carried out at 10 ℃ to 35 ℃, it may be carried out more preferably at 15 ℃ to 30 ℃.

The method for manufacturing multilayer endoplasmic reticulum for induction of immunity according to the present invention can be produced in a simple process under mild conditions, and is economical and easy to mass-produce because it is inexpensive and does not require complicated equipment.

Hereinafter, the multilamellar endoplasmic reticulum for induction of immunity according to the present invention, the pharmaceutical composition for induction of immunity including the same, and the method for preparing the multilamellar endoplasmic reticulum for induction of immunity according to the present invention will be described in more detail below through specific examples. However, the following examples are only one reference for explaining the present invention in detail, but the present invention is not limited thereto, and may be implemented in various forms. Also, the terms used in the description in the present invention are only for effectively describing specific embodiments, and are not intended to limit the present invention.

[Example 1] Preparation of multilamellar endoplasmic reticulum for induction of immunity

Step 1: Preparation of DOTAP unilamellar vesicles

20 mg of 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP), a cationic lipid molecule, was put into a 10 mL vial and mixed with 1 mL of chloroform. Chloroform was removed while flowing nitrogen gas at a slow rate. A DOTAP lipid film was formed by removing residual chloroform for 16 hours in a vacuum oven at 40 °C or higher. 1 mL of non-ionized water was added to the DOTAP lipid membrane and vigorously stirred every 15 minutes for 2 hours at 37° C. to obtain DOTAP vesicles.

The obtained DOTAP unilamellar vesicles were passed through a polycarbonate membrane (Avanti Polar Lipid, Inc., Alabaster, AL, USA) having a 100 nm pore using a mini-extruder to have a uniform size.

Step 2: Preparation of OVA-DOTAP multilamellar vesicles

OVA-DOTAP multilayer vesicles were prepared by mixing 0.5 mL of DOTAP unilamellar vesicles prepared in step 1 and 0.5 mL of OVA (Ovalbumin, Worthington Biochemical Corp., Lakewood, NJ, USA) solution, which is a protein antigen containing sodium phosphate.

[Example 2] Preparation of multilamellar vesicles coated with immunostimulant and anionic polysaccharide

0.5 mL of the OVA-DOTAP multilayer vesicles prepared in Example 1 was placed in a 10 mL vial and stirred at 750 rpm. Here, 0.5 mL of 50 μg mL ^-1 monophosphoryl lipid A (MPLA), an immunostimulant, was added for 10 minutes using an automatic injection device. It was left at 37 °C for 30 minutes. In this way, 0.5 mL of an aqueous solution of 200 μg mL ^-1 hyaluronic acid, an anionic polysaccharide, was injected into 0.5 mL of OVA-DOTAP multilayer vesicles (m-OVA-DOTAP multilayer endoplasmic reticulum) into which monophosphoryl lipid A (MPLA), an immune enhancer, was introduced. It was added for 10 minutes using an injection device to prepare multilamellar vesicles (hm-OVA-DOTAP multilamellar vesicles) coated with monophosphoryl lipid A (MPLA) as an immunostimulant and hyaluronic acid as an anionic polysaccharide.

[Example 3] Preparation of multilamellar vesicles for induction of immunity containing a mixture of lipid molecules

In Example 2, instead of 20 mg of DOTAP (1,2-dioleoyl-3-trimethyl ammonium propane), which is a cationic lipid molecule, 15 mg of DOTAP and 5 mg of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) The same process was performed except that the endoplasmic reticulum was obtained using a mixture of the final multilayer endoplasmic reticulum (hm-OVA-DOTAP) coated with monophosphoryl lipid A (MPLA) as an immunostimulant and hyaluronic acid as an anionic polysaccharide. /DOPC multilamellar vesicles) were prepared.

[Example 4] Preparation of multilamellar endoplasmic reticulum for induction of immunity containing CpG oligodeoxynucleotide as an immune enhancer

In Example 2, except for the fact that CpG oligodeoxynucleotide was used as an immune enhancer, the same process was performed to finally multilayer vesicles (hm -OVA (CpG oligodeoxynucleotide)-DOTAP multilamellar vesicles) were prepared.

[Experimental Example 1] Analysis of particle size and surface charge of unilamellar vesicles

Table 1 below shows the particle size and particle size of the DOTAP unilamellar vesicles prepared in step 1 of Example 1 with a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) instrument. is the surface charge value. As a result of the analysis, the particle size was measured to be 185.4 nm and the surface charge value was 46.0 mV. Accordingly, it was found that the unilamellar vesicles were unilamellar vesicles having a cationic surface charge.

Dynamic light scattering analysis of unilamellar endoplasmic reticulum Particle size (nm) surface charge (mV) 185.4 ± 7.0 46.0 ± 0.7

[Experimental Example 2] Analysis of particle size and surface charge value of multilayer vesicles

The OVA-DOTAP multilayer vesicles according to Example 1 were analyzed with a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) instrument, and the results of particle size and surface charge values are shown in FIG. .

As shown in Figure 1, the weight ratio of the ionic lipid molecule DOTAP and the protein antigen OVA was 1:0.05 to 1:0.95, and the particle size was 300 nm or less. When the weight ratio of DOTAP to OVA was 0.05 to 0.25, the particle size decreased as the weight of OVA increased, but the surface charge continued to maintain a positive charge. As a result, it can be seen that the anionic protein antigen OVA does not surround the DOTAP unilamellar endoplasmic reticulum, but causes the morphological change of the endoplasmic reticulum itself as OVA-DOTAP multilamellar endoplasmic reticulum is formed.

As shown in Figure 2, when the weight ratio of the protein antigen OVA based on the ionic lipid molecule DOTAP is 0.5 or more, the permeability to the multilayered endoplasmic reticulum was reduced and it was found to be aggregated. This is a phenomenon caused by the collection of a relatively large amount of DOTAP liposomes by a large amount of OVA, and it can be seen that stable multilayer vesicles can be made in an appropriate ratio range of OVA and DOTAP.

[Experimental Example 3] Analysis of encapsulation efficiency of OVA in multilayer vesicles

OVA-DOTAP multilayer vesicles according to Example 1 prepared by fixing the weight ratio of DOTAP, an ionic lipid molecule, and OVA, a protein antigen, to 1:0.25, and changing the concentration of sodium phosphate from 0 mM to 0.5 mM, were prepared in an ultracentrifuge. (Optima TLX Ultracentrifuge 120,000, Beckman, Pasadena, CA, USA) was centrifuged at 84,000 rpm at 4 °C for 2 hours. After centrifugation, the unencapsulated OVA was separated and quantified using a micro bicichoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). The results are shown in Table 2 below.

As shown in Table 2 below, the highest OVA encapsulation efficiency was 93.6% at 0.1 mM sodium phosphate concentration. From this, it can be seen that the electrostatic interaction between OVA and DOTAP unilamellar vesicles is regulated in an appropriate salt concentration environment, and it can be seen that it exhibits a very high encapsulation efficiency of 93.6%.

OVA encapsulation efficiency of OVA-DOTAP multilamellar endoplasmic reticulum Sodium Phosphate Concentration (mM) Encapsulation efficiency (%) 0 91.9 ± 1.5 0.05 86.3 ± 0.3 0.10 93.6 ± 0.1 0.25 91.3 ± 1.2 0.50 92.8 ± 3.1

[Experimental Example 4] Small-angle X-ray scattering analysis of multilamellar vesicles

The results of the small-angle X-ray scattering method (4C SAXS II beamline, Pohang Accelerator Laboratory) of OVA-DOTAP multilayer vesicles according to Example 1 are shown in FIG. 3 . As the weight ratio of DOTAP to OVA increases from 1:0 to 1:0.5, the scattering intensity increases and the scattering vector increases by an integer multiple, indicating that the OVA-DOTAP multilamellar vesicles have a multilayered structure. The repetitive layer distance calculated through the scattering vector was about 11.2 nm, which can be seen as the sum of the thickness of the lipid membrane of the DOTAP unilamellar vesicle and the thickness of the OVA solution.

[Experimental Example 5] Ultra-cold transmission electron microscopy analysis of multilamellar vesicles

The ultra-cold transmission electron microscope (JEM-1400, JEOL Ltd., Tokyo, Japan) analysis results of the OVA-DOTAP multilayer vesicles according to Example 1 are shown in FIG. 4 . As shown in Figure 4, it can be seen that the DOTAP unilamellar endoplasmic reticulum is folded by OVA or the morphological change process such as wrapping other unilamellar endoplasmic reticulum and the like, and it can be seen that multi-layered endoplasmic reticulum is produced. As shown in FIG. 5 in detail, it can be seen that OVA induces self-assembly of DOTAP unilamellar endoplasmic reticulum into multilamellar endoplasmic reticulum, and some hemispheres or bilayer endoplasmic reticulum were also observed.

The theoretical molecular size of OVA is about 7.0 nm in the major axis and about 3.6 nm in the minor axis, and as a result of ultra-low temperature transmission electron microscopy, the OVA-DOTAP multilayer vesicles have a thickness of about 6.8 nm. The interlayer distance of 11.2 nm analyzed by the small-angle X-ray scattering method (4C SAXS II beamline, Pohang Accelerator Laboratory) device according to Example 4 is the sum of the film thickness of OVA-DOTAP multilayer vesicles and the short axis length of OVA. It can be predicted.

[Experimental Example 6] Analysis of particle size and surface charge of vesicles

DOTAP unilamellar endoplasmic reticulum according to step 1 of Example 1, OVA-DOTAP multilamellar vesicles according to Example 1 and OVA-DOTAP multilamellar vesicles coated with an immunostimulant and anionic polysaccharide according to Example 2 (hm-OVA-DOTAP multilamellar vesicles) ) was analyzed with a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) instrument, and the results of the particle size and surface charge value are shown in FIG.

The particle size of DOTAP unilamellar vesicles was 185.4 ± 7.0 nm, the particle size of OVA-DOTAP multilamellar vesicles was 167.4 ± 4.7 nm, and the particle size of hm-OVA-DOTAP multilamellar vesicles was 200.1 ± 2.1 nm. It can be seen that the particle size decreased as multilamellar vesicles were formed together with OVA in unilamellar vesicles, and then increased as the immunostimulant and anionic polysaccharide were coated.

The surface charge value of DOTAP unilamellar vesicles was 46.0 ± 0.7 mV, the surface charge value of OVA-DOTAP multilamellar vesicles was 27.5 ± 0.6 mV, and the surface charge value of hm-OVA-DOTAP multilamellar vesicles was -32.8 ± 3.1 mV. As a result, it can be seen that the surface charge value decreased as multilamellar vesicles were formed together with OVA in unilamellar vesicles, and the surface charge became negative as the immunostimulant and anionic polysaccharide were coated.

[Experimental Example 7] Confirmation of stability of hm-OVA-DOTAP multilamellar endoplasmic reticulum in serum environment

DOTAP unilamellar vesicles according to step 1 of Example 1, OVA-DOTAP multilamellar vesicles according to Example 1 and hm-OVA-DOTAP multilamellar vesicles according to Example 2 were mixed with 0%, 10%, 30% and 50% fetal bovine serum. (FBS) The change in particle size was measured for 3 days while maintaining the temperature of 37 ℃ in the environment (pH 7.4). Figure 7 shows the results of analyzing the particle size change with a dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics, Osaka, Japan) instrument, DOTAP unilamellar vesicles are shown in Figure 7A, OVA-DOTAP multilayer vesicles are Figure 7 7B and hm-OVA-DOTAP multilamellar vesicles are shown in Figure 7C. In all the results, the particle size was constant and no aggregation occurred. In particular, the particle size of hm-OVA-DOTAP multilamellar vesicles did not change the most, and as a result, it can be seen that hm-OVA-DOTAP multilamellar vesicles are maintained very stably in the body environment only with electrostatic attraction without chemical bonding. there is.

[Experimental Example 8] Environment-selective antigen release effect of hm-OVA-DOTAP multilamellar endoplasmic reticulum

By creating an environment similar to endosomes/lysosomes in which protein antigens should be released, it was confirmed whether OVA, a protein antigen, was easily released in this environment. Fetal bovine serum (FBS) environment (pH 7.4), 50 mM sodium citrate (pH 5.0), ester hydrolase in HBSS (pH 7.4), ester hydrolase mixture with 50 mM sodium citrate (pH 5.0) and endo The amount of OVA released from hm-OVA-DOTAP according to Example 2 was analyzed in a som/lysosome-like environment (pH 5.0 containing 50 mM citric acid, esterase, and 140 mM sodium chloride).

As shown in FIG. 8, the amount of OVA released in the endosome/lysosome-like environment was the highest (*** p < 0.01). The reason for this result is the reason why OVA, a protein antigen, loses its anionic property and is released from cationic DOTAP under weakly acidic conditions, and the reason why ester hydrolase breaks down the ester bond of DOTAP to break DOTAP and release OVA. It is also believed that there is a synergistic effect of Therefore, the hm-OVA-DOTAP multilayer endoplasmic reticulum is thought to be highly effective in inducing immunity by selectively releasing a large amount of OVA, a protein antigen, in an endosome/lysosomal environment.

[Experimental Example 9] Induction of maturation of dendritic cells of hm-OVA-DOTAP multilamellar endoplasmic reticulum

To obtain bone marrow-derived dendritic cells, bone marrow was extracted from 6-week-old C57BL/6 mice and centrifuged. The washed cells were dispersed in a culture medium (RPMI-1640 + 10% fetal bovine serum + 20 mM penicillin/streptomycin) at a concentration of 10 ⁷ cells mL ^-1 . 9.6 mL of the culture medium, 0.2 mL of 10 ⁷ cell mL ^-1 cells, and 0.2 mL of 1 mg mL ^-1 rmGM-CSF were placed in a culture dish and cultured in a CO ₂ incubator for a total of 6 days.

The cultured bone marrow-derived dendritic cells were treated with fetal bovine serum (FBS), a single OVA, an OVA mixture containing monophosphoryl lipid A (MPLA), and the hm-OVA-DOTAP multilamellar vesicles according to Example 2 for 24 hours, Changes in their morphology were confirmed and shown in FIG. 9 .

Immature dendritic cells are matured by OVA and MPLA, especially as a result of treatment with an OVA mixture containing monophosphoryl lipid A (MPLA) and hm-OVA-DOTAP multilamellar endoplasmic reticulum coated with monophosphoryl lipid A (MPLA). As a result of the treatment, a number of branches extending from dendritic cells were observed. These results indicate that monophosphoryl lipid A (MPLA), an immunostimulant, is essential for induction of immunity.

[Experimental Example 10] Intracellular delivery effect of hm-OVA-DOTAP multilamellar vesicles

Bone marrow-derived dendritic cells cultured as in Experimental Example 9 were recovered and separated on the 6th day of culture and redispersed in the culture medium. A single OVA, monophosphoryl lipid A (MPLA) was added to a 24-well plate in 2 X 10 ⁵ cell well ^-1 and using AF647-OVA labeled with AF647 fluorescence based on 2 mg mL ^-1 OVA concentration. The included OVA mixture and the hm-OVA-DOTAP multilamellar vesicles according to Example 2 were treated for 24 hours. The bone marrow-derived dendritic cells thus treated were subjected to fluorescence analysis using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA), and are shown in FIG. 10 (*** p < 0.01).

It was found that about 18 times more OVA was incorporated into the cells in the result of hm-OVA-DOTAP multilamellar vesicles treatment according to Example 2 than in the result of treatment with the OVA mixture containing single OVA and monophosphoryl lipid A (MPLA). . Therefore, it can be seen that the hm-OVA-DOTAP multilamellar endoplasmic reticulum is more advantageous in delivering protein antigens into immune cells than a mixture of non-multilamellar endoplasmic reticulum and an adjuvant.

[Experimental Example 11] Cellular immunoassay of hm-OVA-DOTAP multilamellar endoplasmic reticulum (1)

The cells re-dispersed as in Experimental Example 10 were added to a 24-well plate in 2 X 10 ⁵ cell well ^-1 and added with fetal bovine serum (FBS) and 2 mg mL ^-1 OVA at a concentration of single OVA, monophosphoryl lipid A (MPLA) and hm-OVA-DOTAP multilamellar vesicles according to Example 2 were treated for 24 hours. The supernatant was separated and tumor necrosis factor, a cytokine, was quantified with a tumor necrosis factor alpha quantification kit (Mouse TNF alpha ELISA Kit, abcam, Cambridge, UK), and the results are shown in FIG. 11 (*** p < 0.01).

As shown in Figure 11, the result of hm-OVA-DOTAP multilamellar vesicles released 5.8 times more tumor necrosis factor than single OVA and 1.9 times more tumor necrosis factor than OVA mixture containing monophosphoryl lipid A (MPLA). appeared to be These results indicate that hm-OVA-DOTAP multilamellar vesicles are delivered to immune cells and induce a higher level of immune response than the mixture of protein antigens and immunostimulants that do not form multilamellar endoplasmic reticulum.

[Experimental Example 12] Cellular immunoassay of hm-OVA-DOTAP multilamellar endoplasmic reticulum (2)

The cells re-dispersed as in Experimental Example 10 were added to a 24-well plate in 2 X 10 ⁵ cell well ^-1 and added with fetal bovine serum (FBS) and 2 mg mL ^-1 OVA at a concentration of single OVA, monophosphoryl lipid A (MPLA) and hm-OVA-DOTAP multilamellar vesicles according to Example 2 were treated for 24 hours. After treatment, APC-CD40, APC-CD80, and PE-Cy7-CD86 were treated, respectively, and fluorescence was analyzed using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA), and the results are shown in FIG. 12 (*** p < 0.01).

As shown in Figure 12, the monophosphoryl lipid A (MPLA)-containing OVA mixture and the hm-OVA-DOTAP multilamellar vesicles were obtained from monophosphoryl lipid A (MPLA)-free fetal bovine serum (FBS) and a single OVA. It was confirmed that the expression of CD40, CD80, and CD86, which are co-stimulatory molecules, increased by 2.0 to 3.6 times compared to . Differences in the expression of co-stimulatory molecules due to the formation of multilayered endoplasmic reticulum were not confirmed in cell analysis, and this needs to be confirmed by in vivo complex analysis.

[Experimental Example 13] Short-term inoculation of hm-OVA-DOTAP multilamellar vesicles

Based on the amount of fetal bovine serum (FBS) and 10 μg OVA, a single OVA, an OVA mixture containing monophosphoryl lipid A (MPLA), and hm-OVA-DOTAP multilayer vesicles according to Example 2 were administered by tail vein injection. Mice were inoculated. Two days after inoculation, the spleen was removed from the mouse to obtain cells, and after removing red blood cells, APC-CD40, PE-Cy7-CD86, PE-Cy7-MHC class II, APC-OVA _257-264 (SIINFEKL) peptide binding H- 2Kb monoclonal antibody was treated for 30 minutes. Each surface protein of the treated sample was fluorescently labeled, and fluorescence was analyzed using a flow cytometer (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA), as shown in FIG. 13 (*** p < 0.01).

As shown in FIG. 13, the surface protein expression of the hm-OVA-DOTAP multilayer endoplasmic reticulum was the most excellent even after single inoculation. These results suggest that hm-OVA-DOTAP multilayer endoplasmic reticulum increased the possibility of interacting with CD4 T cells and CD8 T cells involved in humoral and cellular immunity, respectively.

[Experimental Example 14] Long-term inoculation of hm-OVA-DOTAP multilamellar vesicles (1)

Based on the amount of 10 μg OVA, a single OVA, an OVA mixture containing monophosphoryl lipid A (MPLA), and hm-OVA-DOTAP multilayer vesicles according to Example 2 were injected into mice by tail vein injection on Day 0, 21 A total of three doses were inoculated on the first day and the 35th day.

Blood for immunoglobulin antibody quantification was obtained on the 21st, 35th, and 56th day of breeding, and 1,500 g of blood solidified at room temperature for 1 hour was centrifuged at 4 ^° C. for 10 minutes to obtain serum. The obtained serum was quantified for immunoglobulin antibodies using Mouse Anti-OVA IgG Antibody Assay kit (Chondrex, Inc., Redmond, WA, USA), and the results are shown in FIG. 14 (* p < 0.1, *** p < 0.01).

As shown in FIG. 14, as the number of inoculations increased, OVA-specific immunoglobulin antibodies increased, and in particular, OVA-specific immunoglobulin antibodies increased rapidly in the hm-OVA-DOTAP multilayer endoplasmic reticulum. After a total of three inoculations, the formation of OVA-specific immunoglobulin antibodies did not occur at all in single OVA, and the hm-OVA-DOTAP multilayer vesicles were 3.8 times higher than the OVA mixture containing monophosphoryl lipid A (MPLA). Therefore, it can be seen that the result of inoculation with hm-OVA-DOTAP multilamellar vesicles significantly improves the formation of OVA-specific immunoglobulin antibodies compared to the result of inoculation with a mixture of protein antigen and adjuvant without multilamellar endoplasmic reticulum. As shown in FIG. 15, the concentration of total immunoglobulin antibody production was also found to increase the most in the case of hm-OVA-DOTAP multilamellar vesicles.

[Experimental Example 15] Long-term inoculation of hm-OVA-DOTAP multilamellar endoplasmic reticulum (2)

On the 56th day of breeding, 3 weeks after the 3rd inoculation according to Experimental Example 14, the spleen was extracted from the mouse to obtain cells, and red blood cells were removed, and then added to a 24-well plate as 10 ⁶ cell well ^-1 . Protein transporter inhibitors (10.6 mM brefeldin A and 2 mM monensin) and 10 mg mL ^-1 SIINFEKL peptide were added to the cells and treated in a CO ₂ incubator for 24 hours. Treated spleen cells were treated with FITC-CD8 and APC-IFN-γ for 30 minutes to fluorescently label IFN-γ in CD8 T cells and analyzed by flow cytometry (BD FACS Aria II, BD Bioscience, Franklin Lakes, NJ, USA). It is shown in Figure 16 by fluorescence analysis using (** p < 0.05).

As shown in Figure 16, the result of inoculation with hm-OVA-DOTAP multilamellar endoplasmic reticulum was 13.2 times higher than the result of inoculation with single OVA, compared to the result of inoculation with OVA mixture containing monophosphoryl lipid A (MPLA). and showed 5.2 times higher fluorescence intensity. Therefore, when inoculated with hm-OVA-DOTAP multilamellar endoplasmic reticulum, IFN-γ formation in CD8 T cells of spleen-derived dendritic cells compared to the case of inoculation with single OVA or OVA mixture containing monophosphoryl lipid A (MPLA). can be seen to improve.

In conclusion, it can be seen that the long-term inoculation of hm-OVA-DOTAP multilamellar vesicles according to Example 2 has an excellent immune response inducing effect. It was found that hm-OVA-DOTAP multilamellar vesicles granulated into multilamellar vesicles could induce humoral immunity and cellular immunity simultaneously and more effectively compared to single OVA or OVA mixture containing monophosphoryl lipid A (MPLA). can

Claims (15)

In multilamellar vesicles,
The multilayer endoplasmic reticulum includes two or more unit lamellar layers,
The unit lamellar layer contains ionic lipid molecules charged with a first charge,
A multilamellar endoplasmic reticulum for inducing immunity, characterized in that a protein antigen charged with a second charge is located between the unit lamella layer and another adjacent unit lamella layer. According to claim 1,
The first charge and the second charge have charges of different polarities, multilayer endoplasmic reticulum for inducing immunity. According to claim 1,
The ionic lipid molecule and the protein antigen form an ionic complex, multilayer endoplasmic reticulum for inducing immunity. According to claim 1,
The weight ratio of the ionic lipid molecule and the protein antigen is 1: 0.01 to 1: 1, multilayer endoplasmic reticulum for inducing immunity. According to claim 1,
The ionic lipid molecule is a cationic lipid molecule containing a C14-C20 acyl group, a multilayer endoplasmic reticulum for inducing immunity. According to claim 1,
The outermost lamellar layer of the multilamellar endoplasmic reticulum further comprises an immune enhancer, multilamellar endoplasmic reticulum for inducing immunity. According to claim 6,
The immune enhancer is monophosphoryl lipid A, a multilayer endoplasmic reticulum for inducing immunity. According to claim 1,
The surface of the multilayer endoplasmic reticulum further comprises an anionic polysaccharide coating layer, multilayer endoplasmic reticulum for inducing immunity. According to claim 8,
The surface charge of the multilayer endoplasmic reticulum is negatively charged multilayer endoplasmic reticulum for induction of immunity. According to claim 1,
The multilayer endoplasmic reticulum has a particle size of 100 nm to 300 nm for inducing immunity. A pharmaceutical composition for inducing immunity comprising the multilayer endoplasmic reticulum for inducing immunity of any one of claims 1 to 10. (a) preparing vesicles comprising ionic lipid molecules charged with a first charge;
(b) preparing unilamellar vesicles from the endoplasmic reticulum; and
(c) preparing a multilamellar endoplasmic reticulum by mixing a protein antigen charged with a second charge with the unilamellar endoplasmic reticulum;
Including, a method for producing a multilayer endoplasmic reticulum for inducing immunity. According to claim 12,
In the step (c), the unilamellar endoplasmic reticulum and the protein antigen charged with the second charge are mixed at 4 ° C to 40 ° C, and are converted into multilayer endoplasmic reticulum through self-assembly. According to claim 12,
After step (c),
(d) injecting an immunostimulant into the solution containing the multilamellar vesicles;
Further comprising, a method for producing a multilayer endoplasmic reticulum for inducing immunity. According to claim 14,
After step (d),
(e) injecting an anionic polysaccharide into a solution containing the multilamellar endoplasmic reticulum; further comprising a method for producing a multilamellar endoplasmic reticulum for inducing immunity.

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