WO2023113550A1 - Dendritic cell-mimicking functional nanostructure, and method for producing same - Google Patents

Abstract

The present invention relates to a dendritic cell-mimicking functional structure comprising: a non-spherical core particle; and a shell including a cell membrane of lipid molecules derived from a dendritic cell, and to a method for producing same, wherein the dendritic cell-mimicking functional structure significantly achieves the effect of stable T cell activation in the body, and can thus provide an enhanced immuno-anticancer treatment effect.

Description

Dendritic cell-mimicking functional nanostructure and its preparation method

The present invention relates to a dendritic cell-mimicking functional nanostructure and a method for preparing the same.

Cancer is the number one cause of death in Korea. Representative cancer treatment methods may include surgical operation to remove cancer, radiation treatment, and chemical drug treatment, but these treatment methods have a problem in that they have a poor prognosis and serious side effects.

The main cause of cancer is formation of tumors by cancer cells that have not been removed according to the immune response due to reduced immunity or immune evasion of cancer cells. Therefore, immuno-anticancer therapy that can help cancer treatment by increasing the patient's own immune response can be a fundamental cancer treatment. Most of the currently developed immuno-anticancer treatments directly inject drugs that enhance immune function, but drug injection still has limitations in that the delivery efficiency is very low and additional side effects may exist. Accordingly, research is focused on the development of therapeutic agents capable of dramatically increasing cancer treatment efficiency, reducing recurrence rates and side effects, and enhancing immune function.

On the other hand, research on nano-bio convergence technology that grafts nanotechnology to the biomedical field has been continued. However, most nanomaterials are obtained through synthesis, and surface modification using nanomaterials involves adding a compound, which can cause side effects such as toxicity in the body, induction of unwanted immune responses, and induction of cancer. Therefore, it is possible to overcome the limitations of existing nanotechnology by minimizing side effects by using a surface modification method that directly simulates a living body and implementing various functions according to the characteristics of a living body.

In particular, the nanoparticle-cellularization technology uses the entire cell membrane of a specific cell as a coating material to physically cloak the surface of the nanoparticle. It can maintain the complex properties of the cell membrane along with proteins, lipids, and carbohydrates, and thus the surface of the nanoparticle. The characteristics of a specific cell can be implemented as it is.

In 2015, the research team of Liangfang Zhang at the University of California first introduced platelets to the surface of PLGA (poly(lactic-co-glycolic) acid) particles, and it has been applied to various cells to date.

After supporting doxorubicin on the PLGA core, coat it with a red blood cell membrane to prepare an immunocompatible nanocarrier to remove solid tumors, or coat the red blood cell membrane and cancer cell membrane on melanin nanoparticles at the same time to nanoparticles with hybrid cell membranes. was manufactured to increase circulation time in the body and increase tumor targeting ability.

However, most of the conventional cell membrane coating technologies have been conducted mainly on cancer cells and blood cells, and their applications have also mainly focused on studies aimed at stable delivery in the blood. In addition, when cancer cell membranes are directly introduced as antigens and delivered into the body, there are disadvantages such as a decrease in immune response due to antigen resistance and a feeling of rejection against cancer cell-derived substances. Therefore, it is very necessary to develop an immunotherapeutic agent capable of directly functioning as an antigen-presenting immune cell and induce differentiation and proliferation of T cells without an intermediate process.

[Prior art literature]

[Non-Patent Literature]

(Non-Patent Document 0001) Hu, Che-Ming J., et al. Nature, 2015, 526(7571) 118-12

(Non-Patent Document 0002) Brian T. Luk et al., Theranostics. 2016, 6(7), 1004-1101

(Non-Patent Document 0003) Q. Jiang et al., Biomaterials 2019, 192, 292-308

The present disclosure seeks to more effectively implement the cancer treatment effect according to the increase in the immune response by using cells having an antigen presenting function beyond the material limitations of the prior art. Specifically, through a structure that mimics dendritic cells, cytotoxic T cells are directly activated according to the antigen presenting function of dendritic cells in the body, inducing selective death of cancer cells, and providing excellent immunotherapy effects by increasing the immune response. is to do

In addition, an object of the present disclosure is to impart functionality through the design of nanostructures to realize more marked activation of cytotoxic T cells and to provide more enhanced immunotherapeutic effects.

Dendritic cell-mimicking structure according to the present disclosure in order to achieve the above object is non-spherical core particles; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; the shell may be characterized in that it comprises a bilayer of the lipid molecules.

In one embodiment of the present invention, the non-spherical core particles may be prepared by template the shape of dendritic cells.

In one embodiment of the present invention, the non-spherical core particle may have an elliptical, rod-shaped, plate-shaped or irregular shape.

In one embodiment of the present invention, the non-spherical core particle may be a one-dimensional or two-dimensional nanostructure.

In one embodiment of the present invention, the surface of the non-spherical core particle may be negatively charged.

In one embodiment of the present invention, the surface of the non-spherical core particle may form a bond with at least one component included in the shell.

In one embodiment of the present invention, the non-spherical core particles are organic polymer nanoparticles, metal organic framework nanoparticles, metal nanoparticles, metal oxide nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, It may be one or two or more selected from the group consisting of photothermal conversion nanoparticles, nucleic acid-containing nanoparticles, and protein-containing nanoparticles.

In one embodiment of the present invention, the non-spherical core particles may have an average particle diameter of 50 nm to 50 μm.

In one embodiment of the present invention, the non-spherical core particle may have an aspect ratio of 1.1 to 6.0.

In one embodiment of the present invention, the shell may further include a component derived from a cell membrane of a cell different from dendritic cells.

In one embodiment of the present invention, the surface of the shell may be labeled with a physiologically active polymer, physiologically active component or protein.

In addition, the method for manufacturing a dendritic cell-mimicking structure according to the present disclosure includes (S10) purifying a cell membrane from dendritic cells; (S20) forming a cell membrane suspension by treating the cell membrane with ultrasonic waves; (S30) filtering the cell membrane suspension through a membrane filter to obtain liposomes; and (S40) mixing the non-spherical core particles and the liposomes, and then performing filter compression to obtain non-spherical particles having a cell membrane introduced therein.

In one embodiment of the present invention, before the step (S40), a step of negatively charging the surface of the non-spherical core particle may be further included.

The dendritic cell-mimicking structure according to the present disclosure introduces the antigen-presenting ability to the surface of the nanoparticle as it is, and the antigen-presenting function of the dendritic cell is preserved and at the same time, it is not killed, and the targeting function and the photothermal effect function of the nanoparticle are additionally imparted and strengthened. It can provide a functional immuno-anticancer treatment effect.

The dendritic cell-mimicking structure according to the present disclosure can provide an effect of increasing an immune response by staying in the body for a long period of time and continuously inducing proliferation and differentiation of T cells.

The dendritic cell-mimicking structure according to the present disclosure has the advantage of minimizing side effects through a method of stimulating the patient's own immune system and selectively removing cancer cells of a microscopic size or metastasis that are difficult to diagnose.

The dendritic cell-mimicking structure according to the present disclosure has anticancer activity by itself and can be used as an immunotherapeutic agent, and stronger anticancer activity can be expected through combination with other anticancer agents, so it is useful for the development of new anticancer agents that exhibit strong anticancer effects. can be utilized

The dendritic cell-mimicking structure according to the present disclosure is designed as a structure to minimize uptake due to being recognized as an external antigen by immune cells in the body, so that the activation effect of cytotoxic T cells is more excellently realized and the immunotherapeutic effect is further strengthened. can provide.

1 relates to gold nanorods having various colors prepared according to Preparation Example 1 of the present invention. Rods 1 to 5 have different colors, and accordingly, UV-Vis absorption wavelengths are all different.

2 shows a TEM image of gold nanorods prepared according to Preparation Example 1 of the present invention.

3 is a graph showing the results of ICP-MS analysis of gold nanorod particles prepared according to Preparation Example 1 of the present invention. It can be confirmed that a sufficient amount of particles was produced at a concentration of 50 μg/ml or more for all of Rods 1 to 5.

4 is a graph showing the surface charge of gold nanorods (GNRs) prepared according to Preparation Example 1, and finally negatively charged particles (Citrate GNRs) were obtained by surface modification on positively charged CTAB GNRs.

5 is a graph showing the results of analyzing cell viabilit of Rods 1 to 3 after CTAB treatment and citration.

6 shows a TEM image of gold nanorods coated with a dendritic cell membrane.

7 is a graph showing surface zeta potential values before and after coating dendritic cells (DC) and dendritic cell membranes.

8 shows the result of measuring protein quantification using BCA assay after coating the dendritic cell membrane on the gold nanorod, and it was verified that the cell membrane was successfully coated.

9 shows the results of FACS performance of bone marrow dendritic cells (BMDC) and dendritic cell membrane-coated gold nanorods (DC rod3) according to the present invention, through which the T cell proliferation effect was evaluated.

Hereinafter, with reference to the accompanying drawings and embodiments, a dendritic cell-mimicking functional structure and a manufacturing method thereof according to the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. However, the present invention may be implemented in many different forms, and is not limited to the embodiments described herein. Nor is it intended to limit the scope of protection defined by the claims.

Unless otherwise defined, the technical and scientific terms used in the description of the present invention have meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the gist of the present invention in the following description Descriptions of well-known functions and configurations that may be unnecessarily obscure are omitted.

Numerical ranges, as used herein, include lower and upper limits and all values within that range, increments logically derived from the form and breadth of the range being defined, all values defined therein, and the upper and lower limits of the numerical range defined in different forms. includes all possible combinations of 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.

The singular form used herein may be intended to include the plural form as well, unless the context dictates otherwise.

The term "comprises" in the present specification is an open 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.

As used herein, the term “aspect ratio (AR)” is defined as the ratio of the length divided by the width, where the length is the length of the longest dimension of the particle, and the width can be defined as the shortest length of the particle.

As used herein, the term “nanoparticle” or “nanostructure” may refer to a length, width or diameter ranging from 1 nm to 50 μm.

Hereinafter, the present invention will be specifically described.

The dendritic cell-mimicking structure according to the present disclosure preserves the antigen-presenting function of dendritic cells by introducing the antigen-presenting ability of dendritic cells to the surface of nanoparticles, and at the same time significantly increases immune anti-cancer treatment through the targeting function and photothermal effect of nanoparticles. to provide an effect.

Dendritic cells (DC) are powerful antigen presenting cells (APCs) that play an important role in inducing and regulating immune responses in the body. By activating naive T cells (naive ell) that have never encountered an antigen, they can induce a primary immune response and function as immune cells capable of inducing antigen-specific acquired memory immunity. In addition, major histocompatibility complex I/II (MHC I/II), as well as co-stimulatory molecules such as CD80 and CD86, and cell adhesion molecules such as ICAM-1 are present on the cell membrane surface. molecules) are highly expressed, and by secreting large amounts of various cytokines such as interferon, IL-12, and IL-18 related to T cell activation, it can exhibit a strong antigen presenting function.

A dendritic cell-mimicking structure according to the present disclosure for implementing the antigen presenting function of dendritic cells includes non-spherical core particles; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; the shell may be characterized in that it includes a bilayer of the lipid molecules.

Dendritic cells are present in peripheral tissues, receive activation signals by external stimulation signals, and mature. At the same time, external protein antigens are presented to MHC class I and II in the form of peptides. Thereafter, dendritic cells migrate to the draining lymphnode, bind to T cells having a T cell receptor (TCR) that can bind to the MHC-peptide complex on the surface of dendritic cells, and express CD80, CD86, etc. in dendritic cells. Costimulatory ligands complete activation of T cells by co-binding with CD28, a costimulatory receptor expressed on T cells. Activated T cells proliferate and differentiate, migrate from lymph nodes to peripheral tissues, and can eliminate cells expressing foreign antigens (eg, cancer cells).

The dendritic cell-mimicking structure according to the present disclosure functions as a strong T cell activator for activating the cellular immune function, and can further act as an immune cell therapeutic agent.

Non-spherical core particles according to the present disclosure may be one-dimensional or two-dimensional nanostructures. Specific examples of the one-dimensional or two-dimensional nanostructures include carbon nanoribbons, carbon nanotubes, graphene, graphene oxide, reduced graphene oxide, MXene, metal nanorods, metal nanowires, or metal nanoplatelets ( platelet), but is not limited thereto.

In addition, the non-spherical core particles include biocompatible organic polymer nanoparticles, metal organic framework nanoparticles, metal nanoparticles, metal oxide nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, and light-to-heat conversion nanoparticles. And it may be one or two or more selected from the group consisting of nucleic acid-containing nanoparticles and protein-containing nanoparticles. Specifically, it is more preferable if the nanomaterial itself has no toxicity and has photosensitivity, so that it can be applied to photothermal therapy.

Specific examples of organic polymer nanoparticles include polyacetylene, polyaniline, polypyrrole, polythiophene, poly(1,4-phenylenevinylene), poly(1,4-phenylene sulfide), and poly(fluorenylene). tinylene) and at least one selected from the group consisting of derivatives thereof.

Specific examples of metal nanoparticles include copper (Cu), nickel (Ni), cobalt (Co), iron (Fe), zinc (Zn), titanium (Ti), chromium (Cr), silver (Ag), gold It may be at least one selected from the group consisting of (Au), platinum (Pt), aluminum (Al), and composites thereof.

The metal oxide nanoparticles may be single metal oxide nanoparticles or multi-metal oxide nanoparticles, and the metal may be at least one selected from cerium (Ce), manganese (Mn), and iron (Fe). At this time, a ligand compound such as albumin or dextran may be additionally coupled for dispersibility in vivo.

In one example, the non-spherical core particle may be elliptical, rod-shaped, plate-shaped or irregular. When recognized as an external antigen, the uptake by macrophages increases as the particle size is larger and the particle is closer to a spherical shape. In this respect, the dendritic cell-mimicking structure including a shell including non-spherical core particles and a dendritic cell-derived cell membrane can be designed as a structure to minimize the uptake rate by immune cells according to recognition as a foreign antigen when introduced into the body. .

Non-spherical core particles according to the present disclosure may have an average particle diameter of 50 nm to 50 μm, 60 to 1000 nm, or 80 to 500 nm.

In a specific example, the non-spherical core particle may be a one-dimensional nanostructure, and the aspect ratio (length/width) of the non-spherical core particle may have a value greater than 1. Specifically, the aspect ratio is 1.1 to 10.0, 1.1 to 8,0, 1,1 to 6.0, 1.2 to 10.0, 1.2 to 8.0, 1.2 to 6.0, 1.5 to 10.0, 1.5 to 8.0, 1.5 to 6.0, 2.0 to 6.0 or 2.0 to 2.0. May be 5.0.

In one non-limiting example, the non-spherical core particle may be a gold nanorod, and may have a maximum absorption wavelength of 700 to 800 nm. As the non-spherical core particle has a non-spherical shape and has the maximum absorption wavelength as described above, it is possible to minimize uptake by macrophages and maximize T cell activation.

As a non-limiting embodiment according to the present disclosure, the non-spherical core particles may be gold nanoparticles that can maintain a safe state in the body and are easily surface-modified, but are not limited thereto.

The shell formed on the outside of the non-spherical core particle may include a cell membrane of lipid molecules derived from dendritic cells, and may specifically include a bilayer of lipid molecules. At this time, the first lipid molecule layer facing the outside of the shell may be oriented to exhibit a stronger negative charge compared to the second lipid molecule layer facing the surface of the non-spherical core particle.

The shell containing the cell membrane of the lipid molecule may be thinly and uniformly coated on the surface of the non-spherical core particle in the range of 2 to 50 nm, narrowly 5 to 20 nm.

The surface of the non-spherical core particle may be negatively charged for improved colloidal stability when the lipid molecule is coated with the cell membrane. Methods known in the art can be used to obtain negatively charged non-spherical core particles. As an example, a particle with a negatively charged surface can be obtained through citric acid treatment.

In addition, for stable bonding with the shell, the surface of the non-spherical core particle may form a bond with at least one component included in the shell. The bond may be a variety of chemical bonds such as a covalent bond, an ionic bond, or a complex.

The shell may further include components derived from cell membranes of cells different from dendritic cells.

The surface of the shell may be labeled with a bioactive polymer, a bioactive component, or a protein. Specifically, the physiologically active component may mean a cytokine for cell signaling. Non-limiting examples may include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monokines and colony stimulating factors. More specifically, cytokines include BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (C-X-C motif ligand ligand) family, GDF (Growth/differentiation factor) family, growth hormone, IFN (Interferon) family, IL (Interleukin) family, TNF (Tumor necrosis factors) family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6/uPAR-Related Protein 1), SLUPR-2 ( Secreted Ly-6/uPAR-Related Protein 2) and combinations thereof. The cytokine induces or inhibits transcription factors or growth factors essential for the differentiation of T cells, and mediates the growth and differentiation of other immune cells, thereby further enhancing the effect of immunotherapeutic treatment of the dendritic cell-mimicking structure according to the present disclosure. there is. The dendritic cell-mimicking structure according to the present disclosure may be used for various purposes such as cancer treatment, treatment or prevention of immune diseases, and may be specifically provided in the form of a pharmaceutical composition for cancer treatment.

According to one example, cancer is a general term for various malignant solid tumors that are expandable through local and metastasis by invasion, and specific examples include B-cell lymphoma, non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, and squamous cell carcinoma of the skin. , colorectal cancer, melanoma, head and neck squamous cancer, hepatocellular carcinoma, gastric cancer, sarcoma, gastroesophageal cancer, renal cell carcinoma, glioblastoma, pancreatic cancer, bladder cancer, prostate cancer, breast cancer, cutaneous T-cell lymphoma, Merkel cell carcinoma, or multiple myeloma may, but is not limited thereto.

According to one example, the pharmaceutical composition for treating cancer is used to modify, maintain or preserve pH, osmoticity, viscosity, sterility, transparency, color, isotonicity, odor, stability, dissolution rate or release rate, adsorption or penetration. Formulation materials may be included.

The pharmaceutical composition may be administered orally or parenterally. Examples of parenteral administration include intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intravascular, and intradermal administration to a patient, and may be administered to a patient through a skin patch or transdermally. It can also be administered through the skin using iontophoresis, respectively.

In addition, the present disclosure may provide a method for preparing a dendritic cell mimic structure.

The dendritic cell-mimicking nanostructure according to the present disclosure is prepared by extracting cell membranes of activated dendritic cells and filter extruding them together with the nanoparticles to induce the cell membranes to be introduced onto the surface of the nanoparticles.

Specifically, (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by treating the cell membrane with ultrasonic waves; (S30) filtering the cell membrane suspension through a membrane filter to obtain liposomes; and (S40) mixing the non-spherical core particles and the liposomes, and then performing filter compression to obtain non-spherical particles having a cell membrane introduced therein.

Purifying cell membranes from dendritic cells may be performed through rapid freezing-thawing and centrifugation. Thereafter, the purified cell membrane may be treated with ultrasonic waves to form a cell membrane suspension of hundreds of nanometers or several micrometers. When the cell membrane suspension is filtered through a membrane filter, cell membrane liposomes having a desired size can be obtained.

The prepared nonspherical core particles and cell membrane liposomes are mixed and filter compressed to obtain nonspherical core-shell particles into which cell membranes have been introduced. That is, a form in which the cell membrane of dendritic cells is coated on the surface of the non-spherical core particle is obtained.

At this time, (S50) pulsing the antigen to the non-spherical particles into which the cell membrane has been introduced; further including, a dendritic cell-mimicking functional structure that functions as a dendritic cell can be completed.

The step of pulsing the antigen into the nanoparticle into which the cell membrane is introduced is a step in which the antigen is loaded on the cell membrane by exposing the dendritic non-spherical particle to the antigen, which can induce activation of strong antigen-specific T cells. there is.

The antigen may be a tumor antigen-derived peptide or protein, and the tumor antigen may be a tumor-associated antigen or a tumor-specific antigen. Specifically, for example, in a mouse cancer model, it may be a protein or peptide derived from ovalbumin (OVA), lymphocytic choriomeningitis mammarenavirus (LCMV) glycoprotein, or retrovirus protein. More specifically, it may be a cancer antigen peptide or protein of OVA257-264, GP33-41, or p15E models.

In addition, in human cancer antigens, (1) HER2/Neu, tyrosinase, gp100, MART, HPV E6/E7, EBV EBNA-1, carcinoembryonic antigen, a-fetoprotein, GM2, GD2, testis antigen, prostate antigen, and CD20 and (2) tumor-specific antigens including neoantigens that can be produced by various mutations. Preferably, the antigen may be one or two or more different peptides of the aforementioned cancer antigen peptide.

The pulsing may be performed by various pulsing protocols known in the art, but more preferably, pulsing may be performed by mixing the tumor antigen-derived peptide or protein with a tumor antigen-derived peptide or protein for 0.5 to 6 hours under 5% CO ₂ and 37° C. humidified conditions.

Specifically, as a non-limiting example of the present invention, when treating OVA _257-264 and GP _33-41 antigens, 0.1 to 0.3 μg / ml, and when treating p15E antigen, treatment at a concentration of 2 to 7 μg / ml , the antigen can be pulsed by storing in a 37° C. incubator for 30 minutes after treatment.

The antigen-pulsed dendritic cell-mimicking functional construct has excellent cancer targeting function due to its nano-sized size, is stable, and has a large surface area to volume ratio, so it can easily contact T cells. Therefore, an effective anti-cancer immune response can be induced through the activation of the dendritic cell-mimicking functional structure and the activation of the specific T cell response accordingly. In addition, since there is no risk of death in the body, there is an advantage that the circulation time in the body is very long.

The dendritic cell-mimicking functional structure according to the present disclosure is prepared by fusing a liposome prepared by sonicating a dendritic cell-derived cell membrane and nanoparticles, and the fusion may mean mixing and co-extruding the liposome and the nanoparticle. .

Specifically, after ultrasonic treatment of the dendritic cell-derived cell membrane, it is filtered through a nano-sized membrane filter to obtain liposomes having a particle size of 200 nm or less. The particle diameter of the liposome may be specifically 50 to 150 nm.

The surface zeta potential of the dendritic cell-mimicking nanostructure according to the present disclosure may be -40 to -20 ㎷, specifically -35 to -25 ㎷.

In addition, the present disclosure may provide a drug delivery system or device including the above-described dendritic cell-mimicking nanostructure.

In another aspect, the present disclosure may provide a pharmaceutical composition comprising the above-described dendritic cell-mimicking nanostructure and a pharmaceutically acceptable carrier or excipient.

The present invention will be described in more detail through the following examples. However, the following examples are only references for explaining the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

Preparation Example 1. Preparation of non-spherical core particles

10 ml of 0.1 M CTAB and 2.5 ml of 0.001 M HauCl ₄ were added to the vial, stirred, and 600 μl of 0.01 M NaBH ₄ was added as a reducing agent. After mixing for 2 minutes by increasing the stirring speed, 3-5 nm gold nanoparticles were added. A gold seed was obtained. After aging the gold seeds for 30 minutes, CTAB 0.075 M, 0.075 M, 0.1 M, 0.1 M, and 0.1 M were added to five 250 ml Erlenmeyer flasks, respectively, and the temperature was adjusted to 30 °C. After adding 4.8 ml, 3.6 ml, 2.4 ml, 2.4 ml, and 4.8 ml of 0.004 M AgNO ₃ , respectively, and leaving it for 15 minutes, 250 μl of 0.064 M ascorbic acid was added to the prepared growth solution. 80 μl, 80 μl, 160 μl, 2 μl, and 40 μl were added, respectively, and stored at room temperature without stirring in the dark for 12 hours. When the transparent growth solution turns wine-colored, it means that gold nanorods grow. After the color change occurred, the crystal growth reaction of the gold nanorods was completed. After centrifugation and washing with distilled water twice, gold nanorods (CTAB-GNR) stabilized with 5 types of CTAB were obtained.

A suspension obtained by diluting 40 ml of CTAB-GNR with water was concentrated in an ultrafiltration cell using a membrane filter, the residue was diluted in water, and the suspension was centrifuged again. The residue was redispersed in 0.15% by weight of Na-PSS, the suspension was centrifuged again, and the residue was redispersed in 0.7% by weight of Na-PSS to obtain PSS-stabilized gold nanorods (PSS-GNR). was synthesized. This was redispersed by treatment with 30 ml of 5 mM sodium citrate and stored at room temperature for 12 hours. This method was repeated twice to obtain negatively charged citrate-GNR.

*GNR: Gold Nano Rod

*CTAB: cetyltrimethylammonium bromide

*PSS: polystyrenesulfonate

Experimental Example 1. Evaluation of stability of non-spherical core particles prepared according to Preparation Example 1

<1-1. surface zeta potential>

In the case of the prepared CTAB-GNR, colloidal stability was poor, and particle aggregation was observed with the naked eye even before a day had elapsed. The surface zeta potential of the gold nanorod particles before and after citric acid treatment for colloidal stability was measured using a dynamic light scattering nanoparticle size analyzer (DLS, SZ100, Horiba) and is shown in FIG. 4 .

Gold nanorods whose surface is negatively charged after citric acid treatment have a surface potential of -30 to -40 ㎷ in distilled water, so that coating can be achieved through interaction with cell membranes without problems due to electrostatic repulsion, and without aggregation. It can stably retain its shape.

<1-2. cell viability>

5 shows the results of evaluating the cell viability of human dermal fibroblasts with respect to CTAB-GNR and citric acid-treated gold nanorods. Cell viability, which was as low as 40% or less, reached 100% after treatment with citric acid, confirming that most of the cell toxicity was removed.

Example 1. Preparation of dendritic cell mimic structure

Dendritic cells differentiated from bone marrow cells isolated from femurs of 6- to 8-week-old Naive B6 mice (Orient Bio) were extracted and centrifuged at 2,000 rpm for 5 minutes to obtain pure dendritic cells. Thereafter, the Protease Inhibitor Tablet-PBS buffer was treated and dispersed at a concentration of 1-2×10 ⁶ cells/ml. Then, only cell membranes were purified through rapid freezing at -70 ° C, thawing at room temperature, and centrifugation. Thereafter, ultrasonic waves (VC505, Sonics & materials) performed with 20% amplitude, 3 sec on/off and 3 sec 1 rotation with 2 sec cooling between cycles for a total of 60 times were processed to obtain cell membrane suspension in micro units. , This was filtered with a polycarbonate membrane (pore size of 1 μm, 400 nm, and 100 nm) having a nano-sized filter to produce nano-liposomes having a diameter of each pore size.

After mixing 5 μl of the non-spherical core particle prepared according to Preparation Example 1 and 1 ml of the nano-liposome, they were subjected to filter extrusion together to prepare a dendritic cell-mimicking structure. By treating the prepared dendritic cell-mimicking structure with 0.2 μg/ml of OVA _257-264 antigen at 37° C. for about 30 minutes, the antigen is induced to be presented on the surface of MHC class I and II, mimicking dendritic cells pulsed with the antigen. A nanostructure was prepared.

Experimental Example 2. Characteristic analysis of dendritic cell mimic structure

In order to confirm whether the dendritic cell-mimicking structure according to Example 1 was properly prepared, physicochemical and biological analyzes were performed.

<2-1. TEM image analysis>

6 shows a TEM image after fabrication of the dendritic cell mimic structure. The dendritic cell mimic structure has a length-width of [1] 110 nm to 20 nm (Aspect Ratio 5.5), [2] 120 nm to 65 nm (Aspect Ratio 1.85), [3] 60 nm to 20 nm (Aspect Ratio 3.5) , [4] 180 nm ~ 100 nm (Aspect Ratio 1.8), and [5] 135 nm ~ 75 nm (Aspect Ratio 1.8) that a cell membrane coating having a thickness of about 10-20 nm is formed on the surface of the gold nanorod You can check.

<2-2. Surface zeta potential measurement>

The surface charge was confirmed by measuring the zeta potential of the dendritic cell membrane (DC), the dendritic cell mimicking structure (Rod1 to Rod5) and the spherical dendritic cell mimicking structure (S60), and the results are shown in FIG. 7 .

It can be confirmed that the strong negative zeta potential of the gold nanorods dispersed with citric acid before coating the dendritic cell membrane shows a relatively low value after coating the cell membrane. In addition, in the case of the dendritic cell mimicking structures according to the present invention, potential values similar to those of the dendritic cell membrane were shown, confirming that the cell membrane coating was successfully performed.

Experimental Example 3. T cell activation of dendritic cell mimic structure

Some of the dendritic cell-mimicking constructs (rod 3) prepared in Example 1 using 5 × 10 ⁴ CD8 T cells and 1 × 10 ⁴ DCs isolated from P14 mice were placed in a 96-well U bottom plate in the same well. Into the culture medium through 200 μl of 10% RPMI, and co-cultured at 37 ° C. for 3 days. After 3 days, the plate was recovered from the incubator, stained with a fluorescent antibody, and the degree of activation of CD8 T cells was analyzed by flow cytometry. The results are shown in FIG. 9 .

When the dendritic cell-mimicking nanostructure was not treated with OVA _257-264 and co-cultured with CD8 T cells, it was confirmed that CTV, a measure of T cell proliferation, did not increase. Similarly, CD44, an activation measure, and functional It can be seen that cytokines such as IFNγ, IL-2, TNFα, etc. are also rarely released from CD8 T cells. On the other hand, as a result of treating the dendritic cell mimicking nanostructure with OVA _257-264 so that it can bind to the T cell receptor of CD8 T cells isolated at P14, it can be confirmed that CD8 T cells proliferated as a result of CTV progression. As a control group, it can be seen that the expression rates of CD44, IFNγ, TNFα, and IL-2 were all high to the same extent as those of bone marrow-derived dendritic cells (BMDC), and the T cell proliferation and activation effects were excellent.

As described above, the present invention has been described with specific details and limited examples, but this is only provided to help a more general understanding of the present invention, the present invention is not limited to the above examples, and the field to which the present invention belongs Those skilled in the art can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention should not be limited to the described embodiments and should not be determined, and all things equivalent or equivalent to the claims as well as the following claims belong to the scope of the present invention.

Claims (13)

1. non-spherical core particles; and a shell comprising cell membranes of lipid molecules derived from dendritic cells, wherein the shell comprises a bilayer of the lipid molecules.
2. According to claim 1,

   The non-spherical core particles have an elliptical, rod-shaped, plate-shaped or atypical shape, dendritic cell-mimicking structure.
3. According to claim 1,

   The non-spherical core particles are one-dimensional or two-dimensional nanostructures, dendritic cell mimic structure.
4. According to claim 1,

   The surface of the non-spherical core particle is negatively charged, dendritic cell-mimicking structure.
5. According to claim 1,

   The surface of the non-spherical core particle forms a bond with at least one component included in the shell, dendritic cell-mimicking structure.
6. According to claim 1,

   The non-spherical core particles include organic polymer nanoparticles, metal organic framework nanoparticles, metal nanoparticles, metal oxide nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, photothermal conversion nanoparticles, and nucleic acid-containing nanoparticles. , And one or two or more selected from the group consisting of protein-containing nanoparticles, dendritic cell-mimicking structure.
7. According to claim 1,

   The non-spherical core particles have an average particle diameter of 50 nm to 50 μm, dendritic cell-mimicking structure.
8. According to claim 1,

   The non-spherical core particles have an aspect ratio of 1.1 to 6.0, dendritic cell-mimicking structure.
9. According to claim 1,

   The shell further comprises a component derived from the cell membrane of a cell different from dendritic cells, dendritic cell-mimicking structure.
10. According to claim 1,

    The surface of the shell is a physiologically active polymer, physiologically active component or protein is labeled, dendritic cell-mimicking structure.
11. (S10) purifying cell membranes from dendritic cells;

    (S20) forming a cell membrane suspension by treating the cell membrane with ultrasonic waves;

    (S30) filtering the cell membrane suspension through a membrane filter to obtain liposomes; and

    (S40) mixing the non-spherical core particles and the liposomes and filter-compressing them to obtain non-spherical particles having cell membranes;
12. According to claim 11,

    Prior to the step (S40), a method for manufacturing a dendritic cell-mimicking structure, further comprising the step of negatively charging the surface of the non-spherical core particle.
13. According to claim 11,

    The aspect ratio of the non-spherical core particles is 1.1 to 6.0, dendritic cell mimic structure manufacturing method.

Images