Ectosome comprising corona-virus spike protein and use thereof

Abstract

The present invention provides: an ectosome comprising a corona-virus spike protein, wherein the ectosome exhibits improved drug delivery efficiency to lung cancer cells without side effects, and thus exhibits excellent anticancer efficacy; and a use thereof.

Description

Ectosomes containing coronavirus spike protein and uses thereof

[0001] The present invention relates to ectosomes targeting lung cancer and respiratory diseases, and more particularly to ectosomes comprising a coronavirus spike protein and uses thereof.

[0002] Nanomedicine has emerged as a groundbreaking approach in medical treatment, utilizing nanocarriers as sophisticated drug and gene delivery systems. The primary goal of these nanocarriers is to enhance therapeutic efficacy while minimizing side effects. This strategic paradigm is inspired by the natural processes observed in viral infections, where efficient targeting, cell penetration, and precise payload release into the cytoplasm are essential for optimal therapeutic outcomes. Inspired by the mechanisms of viral infection, the present invention focuses on the use of nanocarriers for targeted therapy, particularly targeting lung cancer cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the global COVID-19 pandemic, provides a notable example of precise tissue tropism mediated by its spike protein. This viral protein interacts with angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which are highly expressed in lung tissue. The fusogenic activity of the spike protein further promotes endosomal escape, enhancing cargo delivery to the cytoplasm, similar to the early stages of viral infection. In this regard, Korean Patent No. 2748069 discloses an ectosome-biodegradable polymer nanoparticle complex with enhanced targeting ability to a lesion and its use.

[0003] However, in the case of the above prior art, there is a problem that the drug delivery efficiency is not high in lung cancer target treatment.

[0004] The present invention aims to address various issues, including those described above, by providing an ectosome containing the coronavirus spike protein, which exhibits excellent anticancer efficacy by demonstrating enhanced drug delivery efficiency to lung cancer cells without side effects, and its use. However, these tasks are exemplary and are not intended to limit the scope of the present invention.

[0005] According to one aspect of the present invention, there is provided an ectosome membrane-coated biodegradable polymer nanoparticle comprising a biodegradable polymer nanoparticle coated with a host cell-derived ectosome membrane, wherein a coronavirus spike protein is presented on the surface of the ectosome membrane.

[0006] According to another aspect of the present invention, a drug delivery vehicle comprising the ectosome membrane-coated biodegradable polymer nanoparticles as an active ingredient is provided.

[0007] According to another aspect of the present invention, a pharmaceutical composition for treating lung cancer is provided, comprising the ectosome membrane-coated biodegradable polymer nanoparticles as an active ingredient.

[0008] According to another aspect of the present invention, there is provided a method for isolating an ectosome membrane from a host cell overexpressing a spike protein of a coronavirus: and

[0009] A method for producing biodegradable polymer nanoparticles with enhanced targeting ability to lung tumor tissue is provided, comprising a step of coating the above ectosome membrane on a biodegradable polymer surface.

[0010] According to another aspect of the present invention, use of the biodegradable polymer nanoparticles for the manufacture of a medicine for preventing and treating lung cancer is provided.

[0011] According to another aspect of the present invention, a method for treating lung cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of the biodegradable polymer nanoparticles.

[0012] As described above, the ectosome containing the coronavirus spike protein of the present invention was isolated from cells overexpressing the spike protein (HEK-S) and used as a nanocarrier for targeted treatment of lung cancer cells. The ectosome exhibited high anticancer efficacy without any side effects. Therefore, it can be utilized as a new and promising strategy for targeted cancer treatment. Of course, the scope of the present invention is not limited by these effects.

[0013] Figure 1a is a schematic diagram schematically illustrating a lung cancer targeting strategy using the spike protein of SARS-CoV-2.

[0014] Figure 1b is a schematic diagram schematically illustrating the molecular mechanism of entry in a lung cancer targeting strategy using the spike protein of SARS-CoV-2.

[0015] Figure 2a is a schematic diagram schematically showing a method for preparing HEK-S-PLGA-DOX using ectosomes derived from HEK293T cells overexpressing the coronavirus spike protein.

[0016] Figure 2b is a graph (top) and gel image (bottom) showing the results of analyzing the expression of HEK293T-S cells induced to overexpress the coronavirus spike protein.

[0017] Figure 2c is a gel photograph analyzing the protein profile of HEK293T-S cells induced to overexpress the coronavirus spike protein.

[0018] Figure 2d is a gel photograph showing the expression of spike protein in ectosomes isolated from HEK293T-S cells induced to overexpress coronavirus spike protein, analyzed by Western blot.

[0019] Figure 2e is a graph showing the results of analyzing the physicochemical properties of ectosome nanoparticles containing the coronavirus spike protein, such as size distribution (left) and surface potential (right).

[0020] Figure 2f is an image showing the results of observing the morphology of PLGA nanoparticles (HEK-PLGA) in which PLGA nanoparticles (PLGA NPs) and HEK293T cell-derived materials are combined using a transmission electron microscope (TEM).

[0021] Figure 2g is an image showing the results of observing the morphology of nanoparticles HEK-S-PLGA using a transmission electron microscope (TEM).

[0022] Figure 3a is a fluorescence image photograph observing the selective uptake ability of ectosome nanoparticles containing coronavirus spike protein using HUVEC cells with high ACE2 expression.

[0023] Figure 3b is a fluorescence image photograph showing the selective absorption ability of ectosome nanoparticles containing the coronavirus spike protein using the A549 cell line, which has low expression levels of both ACE2 and TMPRSS2, and the Calu-3 cell line, which has high expression levels of ACE2 and TMPRSS2 among lung cancer cell lines.

[0024] Figure 3c is a fluorescence microscopy image showing the intracellular pathway of ectosomal nanoparticles containing the coronavirus spike protein by treating lung cancer cells with an endocytosis inhibitor.

[0025] Figure 4a is a graph showing the results of analyzing the cell killing efficacy of ectosome nanoparticles containing coronavirus spike protein using CCK-8 analysis.

[0026] Figure 4b is a graph showing the results of analyzing the cell killing efficacy of ectosome nanoparticles containing the coronavirus spike protein using Annexin V analysis.

[0027] Figure 5a is a photograph showing the results of analyzing the tumor tissue targeting ability by intravenously injecting an ectosome containing the coronavirus spike protein of the present invention into a lung cancer xenograft model.

[0028] Figure 5b is a photograph showing the fluorescence signal of ectosomes containing the coronavirus spike protein at the tumor site. The enhanced cancer-targeting ability of HEK-S-PLGA-DiO administered intravenously in vivo was confirmed.

[0029] Figure 5c is a photograph showing the fluorescent signal of ectosomes containing coronavirus spike proteins in the tumor area.

[0030] Figure 6a is a photograph showing the results of analyzing the tumor tissue targeting ability by injecting an ectosome containing the coronavirus spike protein of the present invention into the peritoneal cavity of a lung cancer xenograft model.

[0031] Figure 6b is a photograph showing the fluorescent signal of ectosomes containing the coronavirus spike protein at the tumor site. Precision targeting of xenograft tumors by intraperitoneally injected HEK-S-PLGA-DiO was observed.

[0032] Figure 6c is a photograph showing the fluorescent signal of ectosomes containing coronavirus spike proteins in the tumor area.

[0033] Definition of terms:

[0034] The term "ectosome" used in this document refers to a nano-vesicle with a phospholipid membrane structure having a diameter of 100 to 500 nm, which is produced by budding of the plasma membrane, unlike 'exosomes' among extracellular vesicles.

[0035] The term "biodebgradable polymer" as used in this document refers to a polymer that is physiologically harmless and can be broken down by enzymatic action within the body. Biodegradable polymers include natural polymers such as starch, chitin, cellulose, polyalginate, and collagen, as well as artificial polymers such as PLGA (poly(lactic-co-glycolic) acid), PGA (poly(glycolic acid), PLA (poly(lactic acid), PCL (poly(caprolactone), and PHA (polyhydroxyalkanoate).

[0036] The term "drug delivery carrier" used in this document refers to a material used to deliver a drug to the required lesion and maintain it for an appropriate period of time, and a method of effectively delivering a drug to the lesion using a drug delivery carrier is called a drug delivery system.

[0037] Detailed description of the invention:

[0038] According to one aspect of the present invention, there is provided an ectosome membrane-coated biodegradable polymer nanoparticle comprising a biodegradable polymer nanoparticle coated with a host cell-derived ectosome membrane, wherein a coronavirus spike protein is presented on the surface of the ectosome membrane.

[0039] In the above polymer nanoparticles, an anticancer drug may be loaded, and the anticancer drug may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin.

[0040] In the above polymer nanoparticles, it may be a natural biodegradable polymer or an artificial biodegradable polymer, and the artificial biodegradable polymer may be PLGA {poly(lactic-co-glycolic) acid)}, PGA {poly(glycolic acid)}, PLA {poly(lactic acid)}, PCL {poly(caprolactone)}, or PHA (polyhydroxyalkanoate), and the natural biodegradable polymer may be starch, chitin, cellulose, polyalginate, or collagen.

[0041] In the above polymer nanoparticles, the host cell may be COS-7, CHO, HKB11, BHK21, HeLa, HEK293, HEK293T, HT-1080, PER.C6, or F2N78, and the anticancer agent may be bound to the surface or interior of the biodegradable polymer nanoparticles by covalent bonds or non-covalent bonds or may be encapsulated inside nanoparticles having a core-shell structure, and may have a size of 100 to 350 nm in diameter.

[0042] According to another aspect of the present invention, a drug delivery vehicle comprising the ectosome membrane-coated biodegradable polymer nanoparticles as an active ingredient is provided.

[0043] According to another aspect of the present invention, a pharmaceutical composition for treating lung cancer is provided, comprising the ectosome membrane-coated biodegradable polymer nanoparticles as an active ingredient.

[0044] In the above pharmaceutical composition, the lung cancer may be ACE2 or TMPRSS2 positive lung cancer.

[0045] According to another aspect of the present invention, there is provided a method for isolating an ectosome membrane from a host cell overexpressing a spike protein of a coronavirus: and

[0046] A method for producing biodegradable polymer nanoparticles with enhanced targeting ability to lung tumor tissue is provided, comprising a step of coating the above ectosome membrane on a biodegradable polymer surface.

[0047] In the above manufacturing method, the host cell may be COS-7, CHO, HKB11, BHK21, HeLa, HEK293, HEK293T, HT-1080, PER.C6, or F2N78, and the biodegradable polymer nanoparticle may contain an anticancer agent, and the anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin.

[0048] According to another aspect of the present invention, use of the biodegradable polymer nanoparticles for the manufacture of a medicine for preventing and treating lung cancer is provided.

[0049] According to another aspect of the present invention, a method for treating lung cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of the biodegradable polymer nanoparticles.

[0050] The pharmaceutical composition of the present invention may vary depending on the type of the patient's affected area, application site, number of treatments, treatment time, formulation, patient's condition, type of adjuvant, etc. The dosage is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The above daily dose may be administered once a day, or divided into 2 to 3 times a day at appropriate intervals, or intermittently at intervals of several days.

[0051] In the pharmaceutical composition of the present invention, the compound can be administered orally or parenterally, and preferably parenterally, by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intramuscular injection, or intraperitoneal injection.

[0052] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, solid or liquid formulation additives may be used in the manufacture of the pharmaceutical composition. The formulation additives may be either organic or inorganic. Examples of excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is generally approved for addition to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar, gelatin, or other agents as needed. In addition, preservatives, antioxidants, and the like can be added as needed. In addition, when the pharmaceutical composition is a drug, it can additionally contain one or more selected from fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, or preservatives. Meanwhile, the formulation of the pharmaceutical composition of the present invention may be in a desirable form depending on the method of use, and in particular, it is preferable to formulate it by adopting a method known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.Examples of specific dosage forms include: PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, POWDERS, SYRUPS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, DECOCTIONS, INFUSIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, CAPSULES, TROCHES, TINCTURES, PASTES, PILLS, SOFT or any one of hard gelatin capsules.

[0053] The pharmaceutical composition of the present invention may further include additional ingredients commonly used in the composition, such as conventional auxiliary agents such as stabilizers, solubilizers, and flavoring agents, and carriers.

[0054] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences (19 th Ed., 1995) is described in detail.

[0055] The present invention aims to utilize the specific tissue tropism exhibited by the SARS-CoV-2 spike protein for targeted therapy against lung cancer cells. Ectosomes isolated from human embryonic kidney 293T cells overexpressing the spike protein (HEK-S) were used as nanocarriers. Specifically, doxorubicin-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA-DOX) were coated with HEK-S to produce HEK-S-coated PLGA-DOX nanoparticles (HEK-S-PLGA-DOX). The active delivery of the coated nanoparticles to lung cancer cells was investigated in vitro and in vivo, with a particular focus on cells overexpressing ACE2 and TMPRSS2. Figures 1A and 1B of the present invention illustrate the mechanism by which the SARS-CoV-2 virus binds to the ACE2 receptor on human cells using the spike protein, followed by activation by the TMPRSS2 protein, leading to intracellular penetration. Based on this experimental theory, the present inventors fabricated nanoparticles (HEK-S-PLGA-DOX) coated with ectosomes containing the viral spike protein, which possess tumor-targeting capabilities. The active delivery of the coated nanoparticles to lung cancer cells, upon treatment with ACE2 and TMPRSS2, demonstrates the efficacy of HEK-S-coated nanocarriers in actively targeting lung cancer cells, suggesting a novel and promising strategy for cancer-targeted therapy. Incorporating the SARS-CoV-2 spike protein into nanocarrier design opens new avenues for precision medicine and offers a potential breakthrough in the development of targeted therapies for lung cancer and other malignancies.

[0056] In summary, nanomedicine often uses nanocarriers as drug or gene delivery systems to maximize therapeutic efficacy while minimizing side effects. From drug administration to therapeutic activation, nanocarriers must appropriately target cells, penetrate them, and release their payload into the cytoplasm, reflecting the initial stages of viral infection. Specifically, SARS-CoV-2 leverages its specific tissue tropism through its spike protein, which interacts with ACE2 and TMPRSS2, which are highly expressed in lung tissue. Furthermore, the fusion activity of the spike protein induces endosomal escape, enhancing cargo delivery to the cytoplasm. The present invention focuses on targeting lung cancer cells overexpressing ACE2 and TMPRSS2 using ectosomes isolated from HEK293T cells overexpressing the spike protein (HEK-S). Doxorubicin-loaded PLGA nanoparticles (PLGA-DOX) coated with HEK-S (HEK-S-PLGA-DOX) exhibited active delivery to lung cancer cells both in vitro and in vivo, and the coating enhanced the anticancer efficacy of the nanoparticles. These results suggest that utilizing spike protein-mediated lung cancer targeting represents a novel and promising strategy for cancer-targeted therapy.

[0057] In conclusion, the present invention presents the investigation of the utilization of ectosomes derived from human embryonic kidney 293T cells overexpressing the Spike protein (HEK-S) as nanocarriers for targeted lung cancer therapy. The synthesis of HEK-S-coated poly(lactic-co-glycolic acid) nanoparticles loaded with doxorubicin (HEK-S-PLGA-DOX) demonstrates a novel approach to enhance drug delivery efficiency. Physicochemical characterization of HEK-S-PLGA-DOX demonstrates the successful integration of Spike protein-derived ectosomes into the nanocarrier system, demonstrating their potential as a tailored drug delivery platform. In vitro studies demonstrated selective and enhanced uptake of HEK-S-PLGA-DiO by lung cancer cells and provided important insights into the targeting mechanism by analyzing the expression levels of ACE2 and TMPRSS2. Notably, coating PLGA-DOX with HEK-S-derived ectosomes significantly enhanced the anticancer efficacy of the nanoparticles, demonstrating their potential to enhance cancer therapeutic efficacy. Furthermore, in vivo studies demonstrated enhanced tumor-targeting ability of HEK-S-PLGA-DiO administered intravenously (iv), and precise targeting was observed in xenograft tumors following intraperitoneal (ip) injection. These results suggest that ectosomes containing the coronavirus spike protein of the present invention represent a promising and innovative strategy for the treatment of lung cancer.

[0058] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0059] Example 1: PLGA and doxorubicin content and encapsulation of PLGA-DOX

[0060] The present inventors analyzed the PLGA and doxorubicin content and the encapsulation efficiency of PLGA-DOX. Specifically, PLGA nanoparticles were prepared using 0.67 dL / g acid-terminated 50:50 poly(dl-lactide-coglycolide) via the emulsion solvent evaporation method. The PLGA polymer was dissolved in acetone at a concentration of 5 mg / ml, and the polymer solution was sequentially added dropwise to 5 ml of 1% polyvinyl alcohol (PVA) under tip sonication conditions. The acetone in the emulsified solution was evaporated using a rotary evaporator. The resulting PLGA nanoparticle solution was then centrifuged at 17,000 g at 4°C and used. Next, PLGA nanoparticles containing doxorubicin (PLGA-DOX) were synthesized by adding doxorubicin to a 1% PVA solution in advance and dropping the PLGA solution into the PVA solution containing doxorubicin one drop at a time under tipsonation conditions. Afterwards, the synthesized PLGA and PLGA-DOX were quantified using UV-vis to confirm the encapsulation efficiency. As a result, the doxorubicin encapsulation efficiency was 21.8%. The results of the encapsulation efficiency analysis are summarized in Table 1 below.

[0061] PLGA and doxorubicin content and encapsulation efficiency of PLGA-DOX Mass of PLGA after encapsulation (mg) 0.972 Normalized percentage loss of PLGA (%) -93.2 Mass of bound doxorubicin (mg) 0.1176 mg Encapsulation efficiency (%) (bound doxorubicin / input dox) 21.8 Drug loading rate (%) (bound doxorubicin / PLGA (mg)) 12.1

[0062] Example 2: Preparation of ectosomes containing coronavirus spike protein

[0063] The present inventors isolated ectosomes from HEK293T cells overexpressing the coronavirus spike protein and used them to target lung cancer cells overexpressing ACE2 and TMPRSS2. Stable overexpression of the spike protein was confirmed in HEK-S cells overexpressing the spike protein in HEK293T cells and in the isolated ectosomes.

[0064] 2-1: Preparation and isolation of spike protein-overexpressing ectosomes

[0065] A plasmid containing the spike protein of SARS-Cov-2 was transfected into HEK293T cells using the Lipofectamine™ 3000 Transfection kit to induce gene overexpression. 24–72 hours after overexpression, 5 μl of 10 μg / μl cytochalasin B was added to the plate, and the cells were detached and vortexed for 3 minutes. The cells were then centrifuged at 1,000 rpm for 10 minutes to collect the supernatant. The supernatant was removed by centrifugation at 4,500 rpm for 15 minutes to obtain an ectosome pellet, which was resuspended in 10 μl of PBS and quantified using a nanodrop (Fig. 2a).

[0066] 2-2: Verification of Spike Protein Overexpression via Western Blot

[0067] HEK293T cells and HEK293T-S cells overexpressing Spike protein were washed with PBS, lysed in RIPA buffer at 4°C for 30 minutes, and centrifuged at 12,000 rpm for 15 minutes to extract proteins. Proteins were then quantified using a BCA assay, and equal amounts of protein were separated by SDS-PAGE electrophoresis. The proteins separated by size were transferred to PVDF membranes, blocked with 5% skim milk for 1 hour, and incubated with anti-Spike protein primary antibody at 4°C for 24 hours. The membranes were then washed three times for 10 minutes each with TBST buffer and incubated with secondary antibody for 1 hour at room temperature. Afterwards, the membranes were washed three times for 10 minutes each with TBST buffer, and protein expression signals were detected using ECL solution. GAPDH was used as a control to compare the amount of Spike protein in the same amount of protein. As a result, it was confirmed that spike protein was stably overexpressed in HEK293T-S cells that induced spike protein overexpression compared to HEK293T cells (Fig. 2b).

[0068] 2-3: Verification of membrane protein maintenance through electrophoresis

[0069] Electrophoresis was performed using HEK293T cells and HEK293T-S cells in which spike protein overexpression was induced, and ectosomes from each of the above cells, and it was confirmed that all samples had the same protein profile (Fig. 2c).

[0070] 2-4: Verification of Spike protein overexpression in ectosomes by Western blotting.

[0071] Ectosomes were extracted from HEK293T cells and HEK293T-S cells overexpressing Spike protein, washed with PBS, lysed in RIPA buffer at 4°C for 30 minutes, and centrifuged at 12,000 rpm for 15 minutes to extract proteins. Proteins were then quantified using a BCA assay, and equal amounts of protein were separated by SDS-PAGE electrophoresis. Proteins separated by size were transferred to PVDF membranes and blocked with 5% skim milk for 1 hour. The membranes were then incubated with anti-Spike protein primary antibody at 4°C for 24 hours, washed three times for 10 minutes each with TBST buffer, and reacted with secondary antibody for 1 hour at room temperature. Subsequently, the membranes were washed three times for 10 minutes each with TBST buffer, and protein expression signals were detected using ECL solution. As controls, NA / K ATPase and b-actin were used to compare the amount of spike protein in the same amount of protein. As a result, it was confirmed that spike protein was stably overexpressed in HEK293T-S cells and ectosomes compared to HEK293T cells where spike protein overexpression was induced (Fig. 2d).

[0072] 2-5: Preparation of ectosome nanoparticles containing coronavirus spike protein

[0073] The present inventors prepared HEK-S-coated PLGA-DOX (HEK-S-PLGA-DOX) by coating PLGA-DOX nanoparticles with HEK-S ectosomes prepared in Example 2-1. Specifically, to proceed with PLGA synthesis, a tip sonicator was operated at 30 Hz, and 1 ml of a PLGA solution prepared by dissolving 5 mg / ml of PLGA copolymer ([CAS#: 26780-50-7], 50:50 Carboxylated End Group (nominal), Lactel, Part#B6013-2P) in acetone as a solvent was added dropwise to 3 ml of 1% PVA. The solution was concentrated in a vacuum for 10 minutes using a rotary evaporator to remove the acetone as a solvent. Next, centrifugation was performed at 4°C, 17,000 xg for 10 minutes using a centrifuge to remove the supernatant, thereby removing the remaining PLGA that did not form nanoparticles. Afterwards, the ectosome PLGA nanoparticles extracted in Example 2-1 were mixed at a mass ratio of 1:10 and sonicated in a bath sonicator for 5 minutes.

[0074] 2-6: Physicochemical Characterization of Ectosome Nanoparticles Containing Coronavirus Spike Protein

[0075] The physicochemical properties of the ectosome nanoparticles containing the coronavirus spike protein of the present invention, such as size distribution and surface potential, were analyzed using a nanoparticle size analyzer (Anton Paar LiteSizer 500). As a result, as shown in Fig. 2e, the size distributions were 152 nm for PLGA, 158 nm for PLGA-DOX, 174 nm for HEK-PLGA-DOX, and 185 nm for HEK-S-PLGA-DOX, and the surface potentials were measured as -20 mV for PLGA-DOX, -10 to -15 mV on average for ectosomes, and -13 to -15 mV for ectosome-coated nanoanticancer agents (Fig. 2e).

[0076] 2-7: Morphological comparison using transmission electron microscopy (TEM)

[0077] The present inventors compared the morphology of PLGA nanoparticles (PLGA NPs) and HEK293T cell-derived PLGA nanoparticles (HEK-PLGA) using transmission electron microscopy (TEM). As a result, PLGA NPs maintained a relatively uniform size and round shape, had a clear particle surface, and appeared to have a hollow interior, whereas HEK-PLGA had a somewhat irregular or thickened membrane structure on the particle surface, and in the area indicated by the white arrow, an additional layer (membrane structure) appeared to be attached to the surface of some nanoparticles, indicating that ectosomes were coated on the PLGA nanoparticles (Fig. 2f). In addition, a thicker outer layer was observed for HEK-S-PLGA than for HEK-PLGA, confirming the possibility that the spike protein exists on the PLGA surface. In addition, in the area indicated by the white arrow, a specific structure was confirmed to be covered or attached to the surface (Fig. 2g).

[0078] Example 3: Analysis of the selective uptake capacity of ACE2 and TMPRSS2 of ectosome nanoparticles containing coronavirus spike protein.

[0079] The present inventors investigated the cell uptake capacity of ectosomal nanoparticles containing the coronavirus spike protein of the present invention. Specifically, the uptake capacity was compared between BM-MSC cells, which are known to have low ACE2 expression, and HUVEC cells, which have high ACE2 expression. First, poly-D-lysine coating was performed by placing cover glasses in 150 mm dishes, treating them with 70% ethanol, and exposing them to ultraviolet (UV) light for 1 day. The cover glasses were then washed four times with distilled water (DW) for 5 minutes each. After washing, the cover glasses were transferred to a 24-well plate and exposed to UV light until the PBS in the plate evaporated. The cover glasses were then stored at 4°C. The poly-D-lysine-coated cover glasses were placed in a 24-well plate and preincubated for 30 minutes in DMEM + 10% FBS + 1% penicillin medium. Next, 1×10 6Cells were plated and treated with PLGA-DiO, HEK-PLGA-DiO, and HEK-S-PLGA-DiO at a concentration of 250 ng / ml each and incubated for 2 hours. Afterwards, the medium was removed, and the cells were washed three times with 1X PBS, fixed with 500 μl of 4% paraformaldehyde (PFA) at 4°C for 24 hours, and a mounting solution containing DAPI was added. The cover glass with the cells was then turned over and mounted. After incubation for 30 minutes at room temperature, the GFP (DiO) and DAPI fluorescence intensities were confirmed using Evos M7000 (high-magnification field, HPF). As a result, the GFP signal was stronger in the ectosome nanoparticles containing the spike protein, confirming better uptake (Fig. 3a). In addition, the uptake capacity of lung cancer cell lines, A549, which has low expression levels of both ACE2 and TMPRSS2, and Calu-3, which has high expression levels of ACE2 and TMPRSS2, was compared for 6, 12, and 24 hours. As a result, the GFP signal was the strongest when Calu-3 cells were exposed to ectosomal nanoparticles containing spike proteins for 6 hours (Fig. 3b). In addition, to investigate the intracellular pathway of nanodrugs, lung cancer cells were treated with endocytosis inhibitors such as chlorpromazine (CPZ, clathrin pathway), ethylisopropylamylolide (EIPA, macropinocytosis pathway), and genistein (caveolin pathway). As a result, the main intracellular uptake pathway of nanodrugs was confirmed to be the endocytosis pathway via caveolin (Fig. 3c).

[0080] Example 4: Evaluation of tumor cell killing efficacy of ectosome nanoparticles containing coronavirus spike protein.

[0081] The present inventors performed MTT assay and Annexin V staining to evaluate the anticancer ability of ectosomal nanoparticles containing coronavirus spike protein against ACE2 and TMPRSS2-expressing tumor cells.

[0082] 4-1: Evaluation of apoptosis efficacy using the CCK-8 assay

[0083] The present inventors performed a CCK-8 assay to evaluate the apoptotic efficacy of ectosomal nanoparticles containing the coronavirus spike protein. Specifically, benign lung cancer cells, Calu-3, were seeded in a 96-well culture plate at a density of 5 × 10 3 After distributing individually, they were cultured at 37°C for 24 hours. Then, the ectosome nanoparticles containing the synthesized coronavirus spike protein were pretreated at concentrations of 0.125 μg / ml, 0.25 μg / ml, 0.5 μg / ml, and 1 μg / ml, respectively. As a negative control, the same volume of ectosomes containing the coronavirus spike protein or the coronavirus spike protein treated with nanomaterials was treated and cultured at 37°C for 24 hours. 10 μl of CCK-8 solution was added to each well and incubated for an additional 2 hours at 37°C. The solution was removed and the absorbance was measured at 460 nm using a microplate reader (UVM 340, Biochrom). As a result, compared to the control group, the ectosome containing the coronavirus spike protein of the present invention showed the best efficacy at a concentration of 0.25 to 1 μg / ml (Fig. 4a).

[0084] 4-2: Evaluation of apoptosis efficacy using Annexin V

[0085] To evaluate the apoptosis efficacy through Annexin V, 3×10 benign lung cancer cells, Calu-3 5Cells were divided into groups. Ectosome nanoparticles containing the coronavirus spike protein were added at a concentration of 0.5 μg / ml and cultured for 24 h at 37°C and 5% CO₂. After washing the cells once with PBS, 200 μl of 1X TE was added and cultured for 5 minutes to detach them. The detached cells were transferred to a 2 ml e-tube with the medium and centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, washed once with PBS, and centrifuged again at 500 g for 5 minutes at 4°C. After that, 100 μl of FACS buffer (95 μl of 1X Annexin V binding buffer + 5 μl of Annexin V) was added to the pellet for flow cytometry (FACS), suspended, and incubated at room temperature for 10 minutes. After 10 minutes, the reaction was stopped by adding Annexin V binding buffer (1 ml). After centrifugation at 500 g for 5 minutes at 4°C, the supernatant was removed, the cells were resuspended in 1X staining buffer, and finally, the cells were transferred to a FACS tube and analyzed by flow cytometry (FACS). As a result, it was confirmed that the anticancer ability of the ectosome nanoparticles containing the coronavirus spike protein of the present invention was enhanced by more than 25% (Fig. 4b).

[0086] Example 5: Analysis of tumor targeting ability after iv administration in a lung cancer xenograft model

[0087] The present inventors analyzed the tumor tissue targeting ability of ectosomes containing the coronavirus spike protein of the present invention using a lung cancer xenograft model. Specifically, the lung cancer xenograft model was performed by inoculating lung tumor cells genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) (Calu-3-luciferase-GFP, 1 × 10) into BALB / c nude mice. 6) was prepared by subcutaneously injecting it into the dorsal area of ​​the mouse. Thereafter, the size of the lung tumor was observed using an in vivo fluorescence imaging device (IVIS imaging system), and after confirming that the lung tumor had formed, the ectosome containing the coronavirus spike protein was intravenously injected through the tail vein of the lung tumor model mouse (Fig. 5a). After the experimental animal was sacrificed, the tumor was extracted and the targeting ability to the tumor tissue was analyzed through ex vivo imaging. As a result of examining the signal of the ectosome containing the coronavirus spike protein at the tumor site, a strong signal was observed from the nanoparticle containing the spike protein (Figs. 5b and 5c). The above results suggest that the nanoparticle of the present invention containing the spike protein has excellent targeting ability to the tumor.

[0088] Example 6: Analysis of tumor targeting ability after ip administration in a lung cancer xenograft model

[0089] The present inventors analyzed the tumor tissue targeting ability of the ectosome containing the coronavirus spike protein of the present invention using a lung cancer xenograft model. Specifically, the lung cancer xenograft model was formed by inoculating lung tumor cells genetically engineered to express fluorescent protein and bioluminescent enzyme (luciferase) (Calu-3-luciferase-GFP, 1 × 10) into BALB / c nude mice. 6) was prepared by subcutaneously injecting it into the dorsal region of the mouse. Thereafter, the size of the lung tumor was observed using an in vivo fluorescence imaging device (IVIS imaging system), and after confirming that the lung tumor had formed, the ectosome containing the coronavirus spike protein was injected intraperitoneally into the lung tumor model mouse (Fig. 6a). After sacrificing the experimental animal, the tumor was extracted and the targeting ability to the tumor tissue was analyzed through ex vivo imaging. As a result, the nanoparticle containing the spike protein of the present invention showed a strong signal even when administered intraperitoneally (Figs. 6b and 6c). The above results suggest that the nanoparticle of the present invention containing the spike protein has excellent targeting ability to the tumor.

[0090] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. An ectosome membrane-coated biodegradable polymer nanoparticle comprising a biodegradable polymer nanoparticle coated with a host cell-derived ectosome membrane, wherein a coronavirus spike protein is presented on the surface of the ectosome membrane.

2. In paragraph 1, The above biodegradable polymer nanoparticles are ectosome membrane-coated biodegradable polymer nanoparticles loaded with an anticancer agent.

3. In paragraph 1, The above biodegradable polymer nanoparticles are ectosome membrane-coated biodegradable polymer nanoparticles, which are natural biodegradable polymers or artificial biodegradable polymers.

4. In paragraph 3, The above artificial biodegradable polymer is PLGA{poly(lactic-co-glycolic) acid)}, PGA{poly(glycolic acid)}, PLA{poly(lactic acid)}, PCL{poly(caprolactone)}, or PHA(polyhydroxyalkanoate), an ectosome membrane-coated biodegradable polymer nanoparticle.

5. In paragraph 3, The above natural biodegradable polymer is starch, chitin, cellulose, polyalginate, or collagen, and the ectosome membrane-coated biodegradable polymer nanoparticles.

6. In paragraph 1, Ectosome membrane-coated biodegradable polymer nanoparticles, wherein the host cell is COS-7, CHO, HKB11, BHK21, HeLa, HEK293, HEK293T, HT-1080, PER.C6, or F2N78.

7. In paragraph 2, Ectosome membrane-coated biodegradable polymer nanoparticles, wherein the anticancer agent is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin.

8. In paragraph 2, An ectosome membrane-coated biodegradable polymer nanoparticle, wherein the anticancer agent is bound to the surface or interior of the biodegradable polymer nanoparticle by covalent or non-covalent bonds or is encapsulated inside a nanoparticle having a core-shell structure.

9. In paragraph 1, Ectosome membrane-coated biodegradable polymer nanoparticles having a diameter of 100 to 350 nm.

10. A drug delivery system comprising an ectosome membrane-coated biodegradable polymer nanoparticle according to any one of claims 1, 3 to 6, and 9 as an active ingredient.

11. A pharmaceutical composition for treating lung cancer, comprising the ectosome membrane-coated biodegradable polymer nanoparticle of any one of claims 2, 7 and 8 as an active ingredient.

12. In paragraph 11, A pharmaceutical composition wherein the above lung cancer is ACE2 or TMPRSS2 positive lung cancer.

13. Step of isolating ectosome membranes from host cells overexpressing the spike protein of coronavirus: and A method for producing biodegradable polymer nanoparticles with enhanced targeting ability to lung tumor tissue, comprising a step of coating the above ectosome membrane on a biodegradable polymer surface.

14. In paragraph 13, A manufacturing method wherein the host cell is COS-7, CHO, HKB11, BHK21, HeLa, HEK293, HEK293T, HT-1080, PER.C6, or F2N78.

15. In paragraph 13, A manufacturing method wherein the biodegradable polymer nanoparticles contain an anticancer agent.

16. In paragraph 15, A method for manufacturing the anticancer agent, wherein the anticancer agent is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin.

17. Use of the biodegradable polymer nanoparticles of any one of paragraphs 2, 7 and 8 for the manufacture of a medicine for preventing and treating lung cancer.

18. A method for treating lung cancer in a subject, comprising administering to the subject a therapeutically effective amount of the biodegradable polymer nanoparticle of any one of claims 2, 7 and 8.

Images