Metabolic anti-cancer gene delivery nanoparticles coated with mixed cell membrane dually targeting caner and cancer-associated adipocytes

Abstract

In order to overcome the limitation that the effectiveness of anticancer treatment targeting only cancer cells is reduced due to interaction with the tumor microenvironment, the present invention develops a system that simultaneously delivers therapeutic genes to cancer cells and tumor-associated fat cells, thereby inhibiting the proliferation of cancer cells by simultaneously suppressing the glucose metabolism of cancer cells and the lipid metabolism of tumor-associated fat cells, and is significant in that it amplifies the effect of conventional metabolic anticancer treatment that was previously performed by targeting only cancer cells.

Description

Technology Field

[0001] The present invention relates to mixed cell membrane-coated nanoparticles capable of simultaneously delivering anticancer genes into cancer cells and tumor-associated fat cells, a method for manufacturing the same, and a pharmaceutical composition comprising the same. Background Technology

[0003] Research results have shown that free fatty acids secreted by fat cells surrounding tumors intensify cancer cell metastasis. With obesity identified as a factor that exacerbates cancer, this finding is expected to provide new clues regarding the correlation between cancer cells and fat cells.

[0004] HIF-1α (Hypoxia-inducible factor-1α) is a transcription factor that induces the expression of various genes involved in Epithelial-Mesenchymal Transition (EMT), the phenomenon in which epithelial cancer cells transform into motile and invasive mesenchymal cells; it has been reported in various cancers that increased HIF-1α expression is associated with a poor prognosis.

[0005] Among the five FDA-approved drugs identified as candidate drugs exhibiting HIF-1α inhibitory efficacy, KSMC-T1 and KSMC-A4 were confirmed to have superior effects in in vitro cell experiments. Among these, KSMC-T1 was confirmed to exhibit cancer growth inhibitory effects and HIF-1α expression inhibitory effects in in vivo xenograft animal models. When the inhibitory mechanism was investigated in in vitro cell experiments, it was found that it inhibits the expression level of HIF-1α by suppressing protein synthesis through the regulation of mTOR and elF-2. However, due to the limitations of small molecule compounds, there was a problem in that they penetrated into tissues and cells other than cancer cells to inhibit HIF-1α, leading to metabolic issues.

[0006] To address the aforementioned problems, targeted anticancer therapy has been introduced, a treatment method that attacks specific cancer cells or their specific functions. Targeted anticancer therapy aims to maximize therapeutic efficacy and minimize side effects by utilizing the differences between cancer cells and surrounding normal cells to specifically destroy cancer cells or inhibit their growth, thereby suppressing the development and metastasis of cancer.

[0007] As such, existing research on the development of cancer treatments has been focused on the cancer cells themselves; however, anticancer treatments targeting only cancer cells have a limitation in that the effectiveness of the treatment is diminished due to interactions with the tumor microenvironment.

[0008] As research results continue to be published indicating that the development, growth, invasion, and anticancer drug resistance of cancer cells occur through interactions with the tumor microenvironment, attempts are being made to target the tumor microenvironment to overcome the limitations of existing anticancer drugs.

[0009] The aforementioned cancer microenvironment, also known as the tumor microenvironment (TME), is the totality of the environment in which cancer cells proliferate and evolve. It is a concept that encompasses not only cancer cells but also fibroblasts, immune cells, the extracellular matrix, and adipocytes present within the cancer tissue. Since the interactions between cancer cells and the cancer microenvironment contribute to tumor growth, metastasis, drug response, and resistance, the importance of the cancer microenvironment is being highlighted as it is known to be studied as a target for the development of novel anticancer drugs and plays a major role in research on resistance to existing anticancer drugs.

[0011] To overcome the aforementioned limitations, the present invention has developed a system for simultaneously delivering therapeutic genes to cancer cells and surrounding fat cells.

[0012] Specifically, after the mixed cell membrane coated nanoparticles (H-CMNP) target and intrude into cancer cells and peritumoral adipocytes, the internal siHIF1α-peptide complex separates the peptide into monomers through oxidation and releases siRNA into the cytoplasm.

[0013] siHIF-1α delivered to cancer cells inhibits the proliferation of cancer cells by inhibiting glycolysis within the cells and thereby inhibiting the use of glucose. However, there was a limitation in that the anticancer effect was halved as fatty acid beta-oxidation within the cancer cells was complementarily activated, using fatty acids as alternative energy.

[0014] To complement this, siHIF-1α delivered to adipocytes surrounding the tumor inhibits lipolysis within the adipocytes, thereby inhibiting the exocytosis of fatty acids and inhibiting the supply of fat energy sources to the cancer cells.

[0015] In other words, the siHIF-1α of the present invention is significant in that it amplifies the effects of conventional metabolic anticancer drugs, which were previously performed by targeting only cancer cells, by simultaneously inhibiting the glucose metabolism of cancer cells and the lipid metabolism of adipocytes surrounding the cancer to inhibit the proliferation of cancer cells. Prior art literature

[0017] Published Patent 10-2022-0165207 The problem to be solved

[0018] The present invention aims to provide mixed cell membrane coated nanoparticles coated with a mixed cell membrane containing a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle.

[0019] In addition, the present invention provides a method for manufacturing the above-mentioned mixed cell membrane coated nanoparticles, wherein

[0020] A step of preparing a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle;

[0021] A step of preparing a mixed cell membrane (H-CMNPs) comprising a cancer cell membrane and a tumor-peripheral fat cell membrane; and

[0022] A step of incorporating the above complex into a mixed cell membrane;

[0023] The purpose is to provide a method for manufacturing mixed cell membrane-coated nanoparticles including

[0024] In addition, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of solid tumors comprising the above-mentioned mixed cell membrane-coated nanoparticles. means of solving the problem

[0026] To achieve the above objective, the present invention provides a mixed cell membrane coated nanoparticle in which a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle is coated onto a mixed cell membrane.

[0027] In one embodiment of the present invention, the siRNA (siHIF-1α) for HIF-1α may have the nucleotide sequence of SEQ ID NO. 1.

[0028] In another embodiment of the present invention, the non-viral gene delivery vehicle may be a disulfide-linked poly(oligo-arginine) peptide (PAs-s).

[0029] In another embodiment of the present invention, the peptide may be a monomer composed of the amino acid sequence of SEQ ID NO. 2 polymerized.

[0030] In another embodiment of the present invention, the mixed cell membrane may be a cell membrane that is a mixture of a cancer cell membrane and a tumor-peripheral fat cell membrane.

[0031] In another embodiment of the present invention, the nanoparticles may simultaneously target cancer cells and tumor-associated fat cells to enhance the anticancer effect.

[0032] In another embodiment of the present invention, the nanoparticles may inhibit glycolysis in cancer cells.

[0033] In another embodiment of the present invention, the nanoparticles may inhibit periarticular adipogenesis.

[0034] In another embodiment of the present invention, the nanoparticles may inhibit cytokine exchange between cancer cells and tumor-peripheral fat cells.

[0035] In another embodiment of the present invention, the complex may be composed of siRNA (siHIF-1α) targeting HIF-1α and a non-viral gene delivery vehicle in a weight ratio of 1:2.

[0036] In another embodiment of the present invention, the nanoparticles may be composed of the complex and the mixed cell membrane in a weight ratio of 1:2.

[0038] In addition, the present invention provides a method for manufacturing the above-mentioned mixed cell membrane coated nanoparticles, wherein

[0039] A step of preparing a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle;

[0040] A step of preparing a mixed cell membrane (H-CMNPs) comprising a cancer cell membrane and a tumor-peripheral fat cell membrane; and

[0041] A step of incorporating the above complex into a mixed cell membrane;

[0042] A method for manufacturing mixed cell membrane-coated nanoparticles comprising

[0043] In one embodiment of the present invention, the complex may be composed of siRNA (siHIF-1α) targeting HIF-1α and a non-viral gene delivery vehicle in a weight ratio of 1:2.

[0044] In another embodiment of the present invention, the nanoparticles may be composed of the complex and the mixed cell membrane in a weight ratio of 1:2.

[0046] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of solid tumors comprising the above-mentioned mixed cell membrane-coated nanoparticles.

[0047] In addition, the present invention provides a method for preventing or treating solid tumors comprising the above-mentioned mixed cell membrane-coated nanoparticles.

[0048] In addition, the present invention provides a use for the prevention or treatment of solid tumors comprising the above-mentioned mixed cell membrane-coated nanoparticles.

[0049] In one embodiment of the present invention, the solid tumor may be one or more selected from the group consisting of brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendiceal cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.

[0050] In another embodiment of the present invention, the composition may be administered parenterally by one or more routes selected from the group consisting of oral administration or external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intra-intestinal injection, local injection, sublingual injection, or intrathoracic injection.

[0051] In another embodiment of the present invention, the composition may be administered in combination with an immunotherapeutic agent. Effects of the invention

[0053] To address the problems of traditional anticancer treatments that cause metabolic issues in tissues and cells other than cancer, and to overcome the limitations of conventional targeted anticancer treatments that inhibit glycolysis by targeting only cancer cells to solve these issues, the present invention has the effect of amplifying anticancer effects by developing a system that simultaneously delivers therapeutic genes to cancer cells and surrounding fat cells.

[0054] In other words, siHIF-1α has the effect of inhibiting cell proliferation by simultaneously inhibiting the glucose metabolism of cancer cells and the lipid metabolism of adipocytes surrounding the tumor.

[0055] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention. Brief explanation of the drawing

[0057] Figure 1 shows the results of measuring the surface charge and size according to the manufacturing process of a gene delivery system. Figure 2 shows the results of gene transfer rates within cancer cells and peritumoral fat cells according to the type of coated cell membrane. Figure 3 shows the results of the inhibitory effect on glycolysis in cancer cells according to the gene delivery system. Figure 4 shows the results of the inhibitory effect on adipocyte differentiation around cancer cells according to the gene delivery system. Figure 5 shows inhibition of interaction between cancer cells and peritumoral fat cells (reduction in cytokine secretion). This is the result showing the effects by gene delivery system. Figure 6 shows the results of measuring the size and weight of the tumor through animal experiments in Example 5-1, categorized by gene delivery system. Figure 7 is the result of analyzing the TUNEL expression level of cancer in Example 5-2 by immunohistochemistry. Figure 8 shows the results of measuring the cytokine expression levels of cancer cells in Example 5-3. Figure 9 shows the results of measuring the mRNA expression levels of adipocytes surrounding the tumor in Example 5-4. Figure 10 shows the results of measuring the cytokine expression levels of adipocytes surrounding the tumor in Example 5-4. Figure 11 shows the results of measuring the amount of Pyruvic Acid and Free Fatty Acid inside cancer cells in Example 5-5. Specific details for implementing the invention

[0058] The present invention provides mixed cell membrane coated nanoparticles coated with a mixed cell membrane containing a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle.

[0059] The term "siRNA" above refers to short double-stranded RNA capable of inducing the degradation of target mRNA or inhibiting the expression of target genes through the cleavage of target mRNA.

[0060] The term "non-viral gene carrier" above refers to a system that passes through the cell membrane and delivers genes into the cell through means other than viruses.

[0061] The term "mixed cell membrane" above refers to a cell membrane formed by mixing a cancer cell membrane and a fat cell membrane surrounding the tumor.

[0062] The term "nanoparticle" above refers to a very small particle of nanoscale size that forms a complex of siRNA (siHIF-1α) and a non-viral gene carrier, passes through the cell membrane, and delivers the siHIF-1α gene into the cell.

[0063] In one embodiment of the present invention, the siRNA for HIF-1α may have the nucleotide sequence of SEQ ID NO. 1.

[0064] The siRNA of the present invention may include a variant having one or more substitutions, insertions, deletions, and combinations thereof, which is a functional equivalent having a change that does not reduce its activity. For example, the siRNA of the present invention may exhibit 80% or more homology with the siRNA of each corresponding sequence number, preferably 90%, and more preferably 95% or more homology. Such homology can be easily determined by comparing the sequence of nucleotides with the corresponding portion of a target gene using computer algorithms widely known in the art, such as Align or BLAST algorithms.

[0065] In another embodiment of the present invention, the non-viral gene delivery vehicle may be a disulfide-linked poly(oligo-arginine) peptide (PAs-s).

[0066] The above peptide may be in the form of polymerized 9 arginine monomers having Cys bonded to one or both ends.

[0067] In another embodiment of the present invention, the peptide may be in the form of a monomer composed of the amino acid sequence of SEQ ID NO. 2 polymerized, wherein the degree of polymerization may be 2 to 100 monomers, preferably 50 to 60 monomers polymerized, but is not limited thereto.

[0068] The peptides used in this specification may include not only peptides but also derivatives thereof. For example, the peptides of the present invention may exhibit homology of 80% or more with the peptide of each corresponding sequence number, preferably 90%, and more preferably 95% or more. The derivatives may include peptides in which the N-terminus, C-terminus, etc. of the peptide are chemically modified, or in which amino acids are added, substituted, or deleted, and are not particularly limited.

[0069] In another embodiment of the present invention, the siRNA may simultaneously target cancer cells and tumor-associated fat cells to enhance the anticancer effect.

[0070] In Example 2 of this specification, siRNA composed of siHIF-1α conjugated with Cy5.5 was treated at a rate of 2 µg based on the gene in each chamber seeded with cancer cells and peritumoral adipocytes, and the degree of cell introduction was analyzed by Fluorescence-Activated Cell Sorting (FACS) to measure the gene delivery rate within cancer cells and peritumoral adipocytes according to the type of cell membrane.

[0071] As a result, as shown in Figure 2, it can be seen that only in the case of the mixed cell membrane gene delivery system, excellent results were shown in all parent cells that serve as components.

[0072] In another embodiment of the present invention, the nanoparticles may inhibit glycolysis in cancer cells.

[0073] In Example 3-1 of this specification, the inhibitory effect on glycolysis in cancer cells was confirmed by measuring the mRNA expression level of HIF-1α and the expression level of genes associated with the glycolysis pathway in cancer cells (EO771) co-cultured with tumor-associated adipocytes.

[0074] As a result, as shown in Figure 3, it can be seen that the inhibitory effect on glycolysis is most excellent in cancer cell membrane and mixed cell membrane gene delivery systems.

[0075] In another embodiment of the present invention, the nanoparticles may inhibit periarticular adipogenesis.

[0076] In Example 3-2 of this specification, the mRNA expression levels of HIF-1α and the expression levels of genes associated with cancer cell-friendly adipocyte differentiation were measured in cancer cells (EO771) co-cultured with cancer cells (3T3L1).

[0077] As a result, as shown in Figure 4, it can be seen that the inhibitory effect on peritumoral adipogenesis is most superior in the adipocyte membrane and mixed cell membrane gene delivery systems.

[0078] In another embodiment of the present invention, the nanoparticles may inhibit cytokine exchange between cancer cells and tumor-peripheral fat cells.

[0079] In Example 3-3 of this specification, 2 µg of siRNA composed of siHIF-1α conjugated with Cy5.5 was treated in each chamber seeded with cancer cells and peritumoral adipocytes, respectively, and then the entire amount of media between the two chambers was recovered and the cytokine release amount was measured by ELISA.

[0080] As a result, as shown in Figure 5, it can be confirmed that the mixed cell membrane gene delivery system causes simultaneous interference effects in both cell lines and significantly reduces cytokine release compared to the single cell membrane gene delivery system.

[0081] In another embodiment of the present invention, the complex may be composed of siRNA (siHIF-1α) targeting HIF-1α and a non-viral gene delivery vehicle in a weight ratio of 1:2.

[0082] The weight ratio of the siRNA and the non-viral gene delivery vehicle is not limited to the weight ratio described in the claims of this specification, and any ratio recognized as similar or equivalent is also included in the scope of the present invention.

[0083] In another embodiment of the present invention, the nanoparticles may be composed of the complex and the mixed cell membrane in a weight ratio of 1:2.

[0084] The weight ratio of the above complex and the mixed cell membrane is not limited to the weight ratio described in the claims of this specification, and those recognized as similar or equivalent are also included in the scope of the present invention.

[0086] In addition, the present invention provides a method for manufacturing the above-mentioned mixed cell membrane coated nanoparticles, wherein

[0087] A step of preparing a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle;

[0088] A step of preparing a mixed cell membrane (H-CMNPs) comprising a cancer cell membrane and a tumor-peripheral fat cell membrane; and

[0089] A step of incorporating the above complex into a mixed cell membrane;

[0090] A method for manufacturing mixed cell membrane-coated nanoparticles comprising

[0091] The terms used in this manufacturing method are consistent with the terms described in the above-mentioned mixed cell membrane coated nanoparticles.

[0092] In one embodiment of the present invention, the complex may be composed of siRNA (siHIF-1α) targeting HIF-1α and a non-viral gene delivery vehicle in a weight ratio of 1:2.

[0093] The weight ratio of the siRNA and the non-viral gene delivery vehicle is not limited to the weight ratio described in the claims of this specification, and any ratio recognized as similar or equivalent is also included in the scope of the present invention.

[0094] In another embodiment of the present invention, the nanoparticles may be composed of the complex and the mixed cell membrane in a weight ratio of 1:2.

[0095] The weight ratio of the above complex and the mixed cell membrane is not limited to the weight ratio described in the claims of this specification, and those recognized as similar or equivalent are also included in the scope of the present invention.

[0097] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of solid tumors comprising the above-mentioned mixed cell membrane-coated nanoparticles.

[0098] The term "cancer" above refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to invade and destroy normal body tissues.

[0099] The term “prevention” above may refer to any act of suppressing cancer in an individual or delaying its onset by administering a pharmaceutical composition according to one aspect.

[0100] The term “treatment” above may refer to any act in which the symptoms of cancer in an individual are improved or beneficially altered by the administration of a pharmaceutical composition according to one aspect.

[0101] The above pharmaceutical composition may be provided as a pharmaceutical composition comprising an active ingredient alone or comprising one or more pharmaceutically acceptable excipients or diluents.

[0102] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. For non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. For suppository bases, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0103] In one embodiment of the present invention, the solid tumor may be one or more selected from the group consisting of brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendiceal cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.

[0104] In another embodiment of the present invention, the composition may be administered parenterally by one or more routes selected from the group consisting of oral administration or external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intra-intestinal injection, local injection, sublingual injection, or intrathoracic injection.

[0105] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0106] The dosage varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the time, but can be appropriately selected by a person skilled in the art.

[0107] In another embodiment of the present invention, the composition may be administered in combination with an immunotherapeutic agent.

[0108] The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into consideration, and this can be easily determined by a person skilled in the art.

[0110] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0112] [Preparation Example]

[0113] Preparation Example 1: Preparation of therapeutic gene siHIF-1α

[0114] The therapeutic gene (siHIF-1α) of a gene delivery system targeting RNA molecules was constructed based on the NM_010431.2 sequence.

[0115] It was constructed based on the 21-base sequence 5'-CAGCUGACCAGUUACGAUUtt-3' (Sense sequence) (Sequence No. 1) and efficiently inhibits gene expression by adding the overhang sequence TT to the 3' end. This is achieved through the chemical synthesis of siTACE oligonucleotides.

[0116] Using a solid silica support, the basic base materials that make up genes—adenosine, guanosine, cytidine, and uracil—are synthesized by sequentially attaching them. As each base is sequentially attached, an oligonucleotide is synthesized through four steps: de-blocking, coupling, capping, and oxidation.

[0117] Subsequently, the oligonucleotides are recovered by reacting with ammonia water, and the synthesized oligonucleotides are purified using reverse-phase silica resin and the principle of chromatography.

[0118] The quantification process is obtained by measuring the absorbance of purified oligonucleotides using UV equipment (UV spectrophotometer) and calculating the number of moles of synthesized oligonucleotides from this.

[0120] Preparation Example 2: Preparation of peptide for internal gene loading (PAs-s)

[0121] Cys-(9Arg)-Cys monomer peptide (Sequence No. 2) can be synthesized using the solid-phase Fmoc peptide synthesis method, which is a synthesis method in which each amino acid is extended one by one according to a predetermined sequence order, using amino acids in which the α-amino group is protected by an Fmoc (Fluoreonylmethoxycarbonyl, 9-fluorenylmethoxycarbonyl) group.

[0122] After the elongation of the peptide chain is finished, the peptide is treated with TFA (Trifluoroacetic acid) to obtain a free form, and then the monomeric peptide in the form of a TFA salt is substituted with an acetate salt. Subsequently, the salt form is substituted with an acetate salt through an ion-exchange chromatography method mediated by AG1-X8 resin.

[0123] After freeze-drying the completed peptide, it is stirred at 150 rpm at a concentration of 57 mM in phosphate-buffered saline (PBS) containing 30% DMSO at room temperature for 6 days. Subsequently, the low molecular weight peptide is removed using a dialysis membrane, and the purified peptide is collected. This peptide can condense genes through positively charged arginine to form a complex, and when the complex is delivered into the cytoplasm, the peptide is degraded into monomers by oxidation, and the genes can be released into the cytoplasm.

[0125] Preparation Example 3: Preparation of a mixed cell membrane containing a cancer cell membrane and a peritumoral adipocyte membrane (H-CMNPs)

[0126] Breast cancer cells (EO771) and adipocytes (3T3L1) are co-cultured to extract the cancer cell membrane and the adipocyte membrane surrounding the cancer.

[0127] First, 2 × 10⁶ inside the flask to form peritumoral fat cells 7 3T3L1 is cultured in a medium composed of Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Bovine Serum and 1% Penicillin.

[0128] After 3 days, the cells are differentiated into adipocytes by replacing the DMEM medium with one containing 0.5 mM IBMX (3-isobutyl-1-methyl-xanthine, Sigma) + 1 µM Dexamethasone and 10 µg / ml Insulin.

[0130] After 3 days of administering differentiation media, replace it with DMEM containing 10 µg / ml insulin and culture for an additional 3 days.

[0131] Afterwards, 2 × 10 7 EO771 cells are administered a medium composed of 10% Fetal Bovine Serum and 1% Penicillin in which Dulbecco's modified Eagle's medium (DMEM) is added, and cultured for an additional 3 days.

[0132] In this way, cancer cells and fat cells are co-cultured to create an environment for the cancer cells and surrounding fat cells, and the cell membranes are separated.

[0133] For cell membrane separation, whole cells are recovered via Trypsin-EDTA and cultured in hypotonic lysis buffer containing 25 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, and EDTA-free SIGMAFAST Protease Inhibitor Cocktail.

[0134] Afterwards, the cells are destroyed using an ultrasonic homogenizer, and the lysate is separated by centrifugation at 20,000 g and 4 ℃.

[0135] Afterward, the supernatant is ultracentrifuged at 100,000 g and 4 ℃ to obtain the cell membrane. The obtained cell membrane is suspended in 0.2 mM EDTA buffer.

[0137] Preparation Example 4: Preparation of a gene delivery system coated with a mixed cell membrane

[0138] siHIF-1α and peptide (PAs-s) are reacted in phosphate-buffered saline (PBS) in a weight ratio of 1:2 for 20 minutes, and the completed complex is mixed with a mixed cell membrane in a weight ratio of 1:2.

[0139] Through a process of extruding the mixed liquid 8 to 10 times in sequence using filters of 0.8 μm, 0.4 μm, and 0.2 μm, the siHIF-1α and peptide (PAs-s) complex is stably encapsulated inside the cell membrane.

[0141] [Example]

[0142] Example 1: Measurement of Surface Charge and Size of a Gene Delivery System

[0143] The size and zeta potential values ​​of the nanoparticles were analyzed using a Zeta-Sizer (Malvern) after dilution in distilled water.

[0144] As a result, as shown in Figure 1, it can be confirmed that the siRNA condenses due to PAs-s, shrinks in size, and takes on a positive charge, and after coating the composite cell membrane, it takes on a negative charge due to the cell membrane coating.

[0146] Example 2: Verification of the cell targeting effect of a mixed cell membrane-based gene delivery system

[0147] Cancer cells (EO771) and differentiated adipocytes (3T3L1) were seeded into the upper and lower chambers of a Transwell plate, respectively, and co-cultured for 3 days to induce peritumoral adipocyte formation in the 3T3L1 cells. Subsequently, the upper chamber was transferred to another plate to separate the cultures of the two cell types. Afterward, 2 µg of siRNA composed of Cy5.5-conjugated siHIF-1α was administered to each chamber based on the gene, and the degree of cell induction was analyzed using Fluorescence-Activated Cell Sorting (FACS). The study was conducted by dividing the cells into four groups: a group administered siRNA alone (Naked siRNA), a group coated with peritumoral adipocyte membranes (A-CMNPs), a group coated with cancer cell membranes (C-CMNPs), and a group coated with mixed cell membranes (H-CMNPs).

[0148] As a result, as shown in Figure 2, the gene delivery system coated with a single cell membrane showed excellent results for each individual single cell, but the degree was negligible for other cells. However, it can be confirmed that the mixed cell membrane gene delivery system showed excellent results for all constituent parent cells.

[0150] Example 3: Measurement of mRNA in cancer cells and peritumoral adipocytes

[0151] Cancer cells (EO771) and differentiated adipocytes (3T3L1) were seeded into the upper and lower chambers of a Transwell plate, respectively, and co-cultured for 3 days to convert 3T3L1 cells into peritumoral adipocytes. Subsequently, 2 µg of siHIF-1α (based on gene) was administered simultaneously to both the upper and lower chambers. The study was conducted by dividing the groups into a siRNA-only administration group (Naked siRNA), a peritumoral adipocyte membrane-coated nanoparticle administration group (A-CMNPs), a cancer cell membrane-coated nanoparticle administration group (C-CMNPs), and a mixed cell membrane-coated nanoparticle administration group (H-CMNPs).

[0152] After 24 hours of gene treatment, cells were uniformly ruptured using the RNeasy Mini kit (Qiagen), and only RNA was isolated. The isolated RNA was reacted with reverse transcriptase using the iScript cDNA synthesis kit (Bio-Rad) to synthesize cDNA complementary to 1 μg of RNA from each group. Subsequently, the relative amount of TACE mRNA to the endogenous control group GAPDH was measured via Real-time PCR using the Cyber ​​premix Ex taq RT-PCR kit.

[0154] Example 3-1: Confirmation of inhibitory effect on glycolysis in cancer cells

[0155] The mRNA expression levels of HIF-1α and the expression levels of genes associated with the glycolysis pathway were measured according to gene delivery systems in cancer cells (EO771) co-cultured with tumor-associated adipocytes. As shown in Figure 3, the inhibitory effect on glycolysis was found to be most pronounced in cancer cell membrane and mixed cell membrane gene delivery systems.

[0156] This is the result confirming that the mixed cell membrane normally delivers genes to cancer cells and functions.

[0158] Example 3-2: Confirmation of inhibitory effect on peritumoral adipogenesis

[0159] The mRNA expression levels of HIF-1α and the expression levels of genes associated with cancer cell-friendly adipocyte differentiation were measured according to the gene delivery system in cancer cells (EO771) and pericancer adipocytes (3T3L1) co-cultured. As shown in Figure 4, the adipocyte membrane and mixed cell membrane gene delivery systems showed the most superior inhibitory effect on pericancer adipocyte differentiation.

[0160] This is the result confirming that the mixed cell membrane normally delivers genes to fat cells surrounding the tumor and exerts its effects.

[0162] Example 4: Confirmation of Inhibitory Effects on Gene Delivery System Interactions via Cytokine ELISA Measurement in Cancer Cells and Peritoneal Adipocytes

[0163] Cancer cells (EO771) and differentiated adipocytes (3T3L1) were seeded into the upper and lower chambers of a Transwell plate, respectively, and co-cultured for 3 days to induce peritumoral adipocyte formation in the 3T3L1 cells. Subsequently, 2 µg of siHIF-1α (based on gene) was simultaneously administered to both the upper and lower chambers. The treatment was divided into four groups: a siRNA-only group (Naked siRNA), a group administered nanoparticles coated with peritumoral adipocyte membranes (A-CMNPs), a group administered nanoparticles coated with cancer cell membranes (C-CMNPs), and a group administered nanoparticles coated with mixed cell membranes (H-CMNPs). 48 hours after gene treatment, the entire amount of media located between the upper and lower chambers was collected, and the supernatant was used as a sample after centrifugation at 4°C, 13,000 rpm, and 5 minutes.

[0164] Peritumoral adipocytes (3T3L1) co-cultured with cancer cells (EO771) exchange cytokines with each other, promoting cancer cell proliferation and peritumoral adipogenesis; the effect of inhibiting this interaction between cancer cells and peritumoral adipocytes was confirmed for each gene delivery system.

[0165] As a result, as shown in Figure 5, it can be confirmed that the mixed cell membrane gene delivery system induces simultaneous interference effects in both cell lines and significantly reduces cytokine release compared to the single cell membrane gene delivery system (the leftmost PBS is the result of analyzing the culture medium after 1 hour of co-culture).

[0167] Example 5: Orthopedic breast cancer animal model experiment using EO771 (in vivo experiment)

[0168] 2.5 × 10⁶ in C57BL / 6 experimental animals 5Canine EO771 cells were diluted in 20 μl of Hank's balanced salt solution (HBSS) and injected into the fourth inguinal mammary gland. After observing tumor formation for one week, 20 μg of the therapeutic gene was administered via tail vein at 3-day intervals twice to the gene-based groups: a group of nanoparticles coated with the gene-containing peritumoral adipocyte membrane (A-CMNPs), a group of nanoparticles coated with the cancer cell membrane (C-CMNPs), and a group of nanoparticles coated with a mixed cell membrane (H-CMNPs). Three weeks after the administration of the cancer cells, the animal model was dissected to obtain organs, including blood. For organs requiring PCR analysis, the RNA was obtained after homogenizing with RLT buffer (Qiagen), and cDNA was synthesized using an iScript cDNA synthesis kit. The relative amount of TNF-α mRNA to the endogenous control, GAPDH, was then measured via Real-time PCR. When protein and metabolite analysis was required, organ tissue samples were physically pulverized and treated with Reporter lysis 5X buffer (Promega) and 0.1 mM PMSF (Protease inhibitor), and a protein-containing supernatant was obtained by centrifugation at 16,800 rpm at 4 ℃ for 30 minutes.

[0170] Example 5-1: Measurement of Size and Weight of Cancer Tissue

[0171] When looking at the results of measuring the size and weight of the cancer tissue, as shown in Figure 6, it can be seen that the size and weight of the cancer tissue were smallest in the group of nanoparticles coated with mixed cell membranes (H-CMNPs) compared to the control group (PBS treatment group), the group of nanoparticles administered with nanoparticles coated with adipocyte membranes around the cancer (A-CMNPs), and the group of nanoparticles administered with nanoparticles coated with cancer cell membranes (C-CMNPs).

[0173] Example 5-2: Measurement of Apoptosis Rate in Cancer Tissue

[0174] Cancer tissue was prepared as a slide with a thickness of 6 μm in a paraffin block, and the cancer cell death rate was analyzed by immunohistochemistry using TUNEL staining. As a result, as shown in Figure 7, the TUNEL expression level was strongest in the group administered nanoparticles coated with a mixed cell membrane (H-CMNPs).

[0175] This result indicates that the apoptosis rate of cancer tissue was highest in the group administered nanoparticles coated with a mixed cell membrane (H-CMNPs).

[0177] Example 5-3: Measurement of cytokine expression levels in cancer tissue

[0178] When looking at the results of measuring the cytokine expression levels in the cancer tissue, as shown in Figure 8, it can be confirmed that the amounts of cancer proliferation-derived factors TGF-β, IL-10, and VEGF were significantly reduced in the group of nanoparticles coated with a mixed cell membrane (H-CMNPs) compared to the group of nanoparticles coated with a tumor-peripheral adipocyte membrane (A-CMNPs) and the group of nanoparticles coated with a cancer cell membrane (C-CMNPs).

[0180] Example 5-4: Measurement of mRNA and cytokine expression levels in peritumoral adipocytes

[0181] When looking at the results of measuring mRNA expression levels in peritumoral adipocyte tissue, as shown in Figure 9, it can be confirmed that gene markers related to peritumoral adipogenesis (C / EBP ~ CCL5) and markers related to lipid metabolism (Lipolysis marker, ATGL ~ FATP) were significantly reduced in the group of nanoparticles coated with mixed cell membranes (H-CMNPs) compared to the control group (PBS treatment group), the group of nanoparticles coated with peritumoral adipocyte membranes (A-CMNPs), and the group of nanoparticles coated with cancer cell membranes (C-CMNPs).

[0182] This result confirms that the anticancer metabolic effect can be amplified by reducing the amount of cytokines and fatty acids provided to cancer cells.

[0183] When looking at the results of measuring the cytokine expression levels in the adipocyte tissue surrounding the tumor, as shown in Figure 10, it can be confirmed that the amounts of cancer proliferation-derived factors IL-6, IL-1β, and CCL2 were significantly reduced in the group of nanoparticles coated with a mixed cell membrane (H-CMNPs) compared to the control group (PBS-treated group), the group of nanoparticles administered with nanoparticles coated with an adipocyte membrane surrounding the tumor (A-CMNPs), and the group of nanoparticles administered with nanoparticles coated with a cancer cell membrane (C-CMNPs).

[0185] Example 5-5: Measurement of Pyruvic Acid and Free Fatty Acid Amounts in Cancer Cells

[0186] When looking at the results of measuring the amounts of Pyruvic Acid and Free Fatty Acid in cancer cells, as shown in Figure 11, it can be confirmed that compared to the control group (PBS treatment group), the group administered nanoparticles coated with a tumor-peripheral adipocyte membrane (A-CMNPs), and the group administered nanoparticles coated with a cancer cell membrane (C-CMNPs), the group administered nanoparticles coated with a mixed cell membrane (H-CMNPs) showed the greatest reduction in the amount of products resulting from the conversion of glucose into Pyruvic Acid due to the inhibition of glycolysis, and at the same time, the accumulation rate of fatty acids released from adipocytes showed the greatest reduction.

[0187] This result indicates that the group administered with nanoparticles coated with a mixed cell membrane (H-CMNPs) exhibits the most superior inhibitory effects on glycolysis in cancer tissue and lipolysis in peritumoral adipocytes.

[0189] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A mixed cell membrane coated nanoparticle, wherein a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle is coated onto a mixed cell membrane, wherein the siHIF-1α is siRNA having the nucleotide sequence of SEQ ID NO. 1, the non-viral gene delivery vehicle is a disulfide-linked poly(oligo-arginine) peptide (PAs-s) polymerized by disulfide bonds, the peptide is polymerized from a monomer having the amino acid sequence of SEQ ID NO. 2, and the mixed cell membrane is a cell membrane formed by mixing a cancer cell membrane and a peritumoral adipocyte membrane. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 A mixed cell membrane coated nanoparticle according to claim 1, characterized in that the nanoparticle simultaneously targets cancer cells and tumor-associated fat cells to enhance the anticancer effect. Claim 7 A mixed cell membrane coated nanoparticle according to claim 1, wherein the nanoparticle inhibits glycolysis in cancer cells. Claim 8 A mixed cell membrane coated nanoparticle according to claim 1, wherein the nanoparticle inhibits peri-cancer adipogenesis. Claim 9 A mixed cell membrane coated nanoparticle according to claim 1, characterized in that the nanoparticle inhibits cytokine exchange between cancer cells and tumor-peripheral fat cells. Claim 10 A mixed cell membrane coated nanoparticle according to claim 1, characterized in that the complex comprises siRNA (siHIF-1α) targeting HIF-1α and a non-viral gene delivery agent in a weight ratio of 1:

2. Claim 11 A mixed cell membrane coated nanoparticle according to claim 1, characterized in that the complex and the mixed cell membrane are composed of a weight ratio of 1:

2. Claim 12 A method for manufacturing mixed cell membrane coated nanoparticles according to claim 1, comprising the steps of: manufacturing a complex of siRNA (siHIF-1α) targeting hypoxia-inducible factor-1alpha (HIF-1α) and a non-viral gene delivery vehicle; and manufacturing a mixed cell membrane (H-CMNPs) comprising a cancer cell membrane and a peritumoral adipocyte membrane. A method for preparing mixed cell membrane coated nanoparticles comprising the step of incorporating the above complex into a mixed cell membrane, wherein the siHIF-1α is siRNA having the nucleotide sequence of SEQ ID NO. 1, the non-viral gene delivery vehicle is a disulfide-linked poly(oligo-arginine) peptide (PAs-s) polymerized by disulfide bonds, the peptide is polymerized from a monomer having the amino acid sequence of SEQ ID NO. 2, and the mixed cell membrane is a cell membrane formed by mixing a cancer cell membrane and a peritumoral adipocyte membrane. Claim 13 A method for preparing mixed cell membrane coated nanoparticles according to claim 12, wherein the complex is composed of siRNA (siHIF-1α) targeting HIF-1α and a non-viral gene delivery vehicle in a weight ratio of 1:

2. Claim 14 A method for manufacturing mixed cell membrane coated nanoparticles according to claim 12, characterized in that the complex and the mixed cell membrane are composed of a weight ratio of 1:

2. Claim 15 A pharmaceutical composition for the prevention or treatment of breast cancer comprising mixed cell membrane-coated nanoparticles according to any one of claims 1, 6 to 11. Claim 16 delete Claim 17 A pharmaceutical composition for the prevention or treatment of breast cancer according to claim 15, wherein the composition is administered parenterally by one or more routes selected from the group consisting of oral administration, external application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intra-intestinal injection, local injection, sublingual injection, or intrathoracic injection. Claim 18 A pharmaceutical composition for the prevention or treatment of breast cancer according to claim 15, characterized in that the composition is administered in combination with an immunotherapeutic agent.

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