Hybrid nanocomposite of polymer nanoparticles and cell membrane and medical use thereof

Abstract

The present invention relates to: a hybrid nanocomposite of polymer nanoparticles and cell membrane; and medical use thereof. More specifically, the present invention provides a hybrid nanocomposite formed by fusion of biocompatible polymer nanoparticles and cell membrane, and a composition for drug delivery using same. The hybrid nanocomposite according to the present invention can deliver a drug, a vaccine, or a gene into cells with improved efficiency by synergistically exhibiting the properties of polymer nanoparticles and the properties of cell membrane.

Description

Nanocomposites hybridized with polymer nanoparticles and cell membranes and their medical uses

[0001] The present invention relates to a nanocomposite hybridized by fusing polymer nanoparticles and cell membranes, and its medical use.

[0002] The cell membrane has both protective and adaptive functions, can transport cargo and antigens, and its lipid content influences the formation of membrane vesicles and the stability of membrane release, making it a target for modern vaccination methods. Phospholipid bilayers and membrane proteins interact with and modulate immune responses, and thus can be used to improve vaccine delivery. Cell membrane markers and ligands can enhance vaccine efficacy and prevent off-target effects, which can lead to unexpected side effects, making them excellent candidates for multipurpose vaccine delivery systems.

[0003] Nanoparticles have also been studied for use in vaccine delivery, with some success. Nanoparticle-delivered vaccines can help reduce the scope of nonspecific immune responses through controlled release, and surface modifications can help target specific antigens to enhance immune responses. Nanoparticles may be more effective than other vaccine delivery methods due to their unique interactions with immune cells across cell membranes and their relative stability in vivo, where responses to environmental signals influence vaccine delivery. However, nanovaccine carriers must be biocompatible, exhibit extended in vivo circulation, and deliver vaccines to specific immune cells for effective uptake.

[0004] Top-down cell membrane processing utilizes existing cell membranes and incorporates lipid membranes into them to enable vaccine delivery. Membrane-based nanovaccines, which incorporate cell membranes into nanoparticles, can enable programmable immune responses. While concerns remain regarding the biocompatibility and suitability of this technology, membrane-based nanoparticle vaccine delivery is a promising technology. First, developing a cell membrane-based platform requires large-scale manufacturing and purification capabilities. This necessitates the development of reproducible processes. Second, maintaining cell membrane properties requires expanded methodologies for cell membrane extraction, along with rigorous quality control and validation procedures.

[0005] Conventional vaccines have been distributed by coating core nanoparticles with cell membranes. However, due to limitations in cell membrane fluidity, core nanoparticles often result in uneven coating. More than 60% of nanoparticles coated using this method had less than 20% cell membrane coverage. The inability to control cell membrane coating can limit the efficacy of this method for vaccine delivery. Uneven coating reduces intracellular delivery efficiency, and nanoparticles partially coated with the cell membrane can absorb unwanted biomolecules and rapidly degrade, reducing particle stability.

[0006] The purpose of the present invention is to provide a drug delivery platform having high biocompatibility and excellent intracellular drug or gene delivery efficiency.

[0007] To achieve the above purpose, the present invention provides a hybrid nanocomposite formed by fusing biocompatible polymer nanoparticles and a cell membrane.

[0008] The present invention provides a composition for drug delivery comprising the above hybrid nanocomposite; and a therapeutic or prophylactic agent.

[0009] In addition, the present invention provides a method for producing a hybrid nanocomposite, comprising the steps of: preparing a polymersome by dissolving an amphiphilic polymer in an organic solvent and then hydrating the polymersome into a thin film; suspending cells in a storage dissolution buffer to disrupt them, centrifuging them, and extruding the collected supernatant to obtain cell membrane vesicles; and mixing the prepared polymersomes with the obtained cell membrane vesicles and then extruding them to induce self-assembly.

[0010] The hybrid nanocomposite according to the present invention is the first technology to present a biomimetic platform by fusing biocompatible polymer nanoparticles and cell membranes, and can synergistically demonstrate the properties of polymer nanoparticles and cell membranes.

[0011] The hybrid nanocomposite according to the present invention can have the biocompatibility, structural stability, encapsulation efficiency, etc. of polymer nanoparticles, and at the same time, exhibit the surface properties and antigen expression properties inherent to cell membranes, thereby improving the problems of conventional cell membrane-coated nanoparticles and being utilized as a drug, vaccine, or gene delivery platform with improved efficiency, thereby exhibiting excellent efficacy.

[0012] Figure 1 schematically illustrates the formation process of a cell membrane hybrid polymersome (HyPSome) for mRNA delivery according to one embodiment of the present invention and the mRNA transfection process by endocytosis of the mRNA-loaded HyPSome.

[0013] FIG. 2 shows the characteristics of various cell membrane vesicles (CMVs) and methoxy-polymersomes (PSome) manufactured according to one embodiment of the present invention, wherein (a) is a transmission electron microscope (TEM) image (scale bar is 200 nm), (b) is a size distribution curve measured by dynamic light scattering (DLS), and (c) is a zeta potential measurement result, showing (i) Raji cell membrane vesicles (Raji CMV), (ii) MDCK cell membrane vesicles (MDCK CMV), (iii) MCF-7 cell membrane vesicles (MCF-7 CMV), and (iv) PSome.

[0014] Figure 3 shows TEM images and size distribution curves of HyPSomes according to the mixing ratio to determine the optimal hybridization conditions for CMVs and PSomes.

[0015] Figure 4 shows the size stability of HyPSomes with different ratios of PSomes to cell membranes under various conditions, from left to right: Raji cell-based HyPSome (Raji HyPSome), MCF-7 cell-based HyPSome (MCF-7 HyPSome), and MDCK cell-based HyPSome (MDCK HyPSome) (mean ± sd, n = 3).

[0016] Figure 5 shows the analysis of fluorescence characteristics according to the mixing ratio of CMVs and PSomes, and confirms the membrane compatibility and fusion efficiency of Raji HyPSome, MCF-7 HyPSome, and MDCK HyPSome from the left in order (mean ± sd, n = 5).

[0017] Figure 6 shows the characteristics of HyPSomes. (a) Confocal microscopy images of Raji HyPSome, MCF-7 HyPSome, and MDCK HyPSome (scale bar is 10 μm), (b) Western blotting analysis of major protein receptors in Raji, MCF-7, and MDCK membranes incorporated into HyPSomes, (c) Zeta potential distributions of Raji HyPSome, MCF-7 HyPSome, and MDCK HyPSome (mean ± sd, n = 5), (d) Flow cytometry dot plots of DiI-labeled PSomes, DiO-labeled CMVs, and DiO and DiI-labeled HyPSomes.

[0018] Figure 7 shows the results of in vitro assays for cell viability, cellular uptake, and endosomal escape of HyPSomes. (a) shows the in vitro cell viability of Raw 264.7 cells at 6 h, 12 h, and 24 h, i: Raji HyPSome, ii: MDCK HyPSome, iii: MCF-7 HyPSome iv: PSome, v: Raji CMV, vi: MDCK CMV, and vii: MCF-7 CMV (mean ± sd, n = 5). 50 μg / mL HyPSome loaded with 0.001 mM OVA-FITC was used. (b) shows the in vitro cellular uptake efficiency of Raw 264.7 cells (mean ± sd, n = 5, p<.001,***), (c) is the fluorescence visualization of Raw 264.7 cells incubated with OVA-FITC, Raji HyPSome, MCF-7 HyPSome and MDCK HyPSome for 6 h, 50 μg / mL HyPSome loaded with 0.001 mM OVA-FITC was used, the respective colors represent nuclei (blue), endosomes (red) and nanoparticles (green), and the scale bar is 10 μm.

[0019] Figure 8 shows the analysis of EGFP expression efficiency of EGFP mRNA-loaded HyPSome, (a) is a CLSM image of Raw 264.7 cells transfected with EGFP treated in various groups, (b) is a histogram analysis of in vitro transfection efficiency using FlowJo software (n = 5, p<.001,***), (c) shows the in vitro transfection efficiency measured as the percentage of EGFP positive cells by flow cytometry (mean ± sd, n = 5, p<.001,***), and the scale bar is 20 μm.

[0020] Hereinafter, the present invention will be described in detail.

[0021]

[0022] In order to improve the problems of conventional cell membrane coating technology, the inventors of the present invention fused synthetic polymer nanoparticles with cell membranes to produce cell membrane hybrid polymersomes (HyPSomes) for improved vaccine delivery, and confirmed that the produced cell membrane hybrid polymersomes exhibited excellent ability to deliver external mRNA to antigen-presenting cells with improved cellular uptake and translation efficiency, thereby serving as an excellent vaccine delivery system, thereby completing the present invention.

[0023]

[0024] The present invention provides a hybrid nanocomposite formed by fusing biocompatible polymer nanoparticles and a cell membrane.

[0025] In this specification, the "hybrid nanocomposite" may be expressed as a "cell membrane hybrid polymersome (HyPSomes)".

[0026]

[0027] The above biocompatible polymer nanoparticle may be a polymersome made of an amphiphilic polymer.

[0028] The above amphiphilic polymer may be formed by synthesizing a hydrophilic polymer and a hydrophobic polymer.

[0029] The hydrophilic polymer of the above amphiphilic polymer may be at least one selected from the group consisting of polyalkylene glycol (PAG), polyacrylic acid (PAA), polyacrylonitrile (PAN), polyethylene oxide (PEO), polyvinylacetate (PVAc), polyvinyl alcohol (PVA), polyvinylpyrrolidone, polyacrylamide, hydrophilic polyamino acids and derivatives thereof, but is not limited thereto. For example, the hydrophilic polymer may be selected from (mono)methoxypolyethylene glycol, (mono)acetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, polyvinylpyrrolidone, polyglutamine, polyglutamic acid, polythreonine, polyasparagine, polyarginine or polyserine. In addition, the hydrophilic polymer may include derivatives thereof. For example, the derivative of the polyethylene glycol may be methoxypolyethylene glycol (mPEG).

[0030] The hydrophobic polymer of the above amphiphilic polymer can be used without limitation as long as it is a substance that can form amphiphilic particles together with the hydrophilic polymer. For example, the hydrophobic polymer may be at least one selected from the group consisting of polyester, polyanhydride, hydrophobic polyamino acid, polyorthoester, and polyphosphazine. The polyamino acid may include at least one selected from the group consisting of polyleucine, polyisoleucine, polyvaline, polyphenylalanine, polyproline, polyglycine, polytryptophan, polyalanine, polylactide, polyglycolide, polycaprolactone, and polymethionine. In addition, the hydrophobic polymer may include derivatives thereof.

[0031] The above amphiphilic polymer may be selected from a group of materials formed by synthesizing the above hydrophilic and hydrophobic polymers, and may preferably be a methoxy-poly(ethylene glycol)-block-polylactic acid (mPEG-b-PLA) copolymer, but is not limited thereto.

[0032] The above cell membrane may be a vesicle derived from the cell membrane of various cells such as cancer cells or normal cells, and the cancer cells may be selected from B lymphocyte Raji cells, breast cancer cell line MCF-7 cells, or MDA-MB-231 cells, and in addition, may be selected from non-cancerous cells such as canine kidney epithelial-derived cells (Madin-Darby canine kidney, MDCK), monkey kidney cells (Vero cells), etc., but are not limited thereto.

[0033] In particular, when the cell membrane is a vesicle derived from the cell membrane of a cancer cell, the cancer cell membrane contains specific molecules and antigens that can stimulate the immune system to generate a strong cytotoxic T lymphocyte (CTL) response. Therefore, the vaccine's efficacy can be enhanced by utilizing its immune-stimulating properties. When the immune cell membrane is used as a material for a hybrid nanocomposite, the presence of costimulatory molecules or receptors on the immune cell membrane can induce the activation of T lymphocytes or dendritic cells, maximizing the efficacy of the mRNA vaccine.

[0034] The above hybrid nanocomposite may be a fusion of the polymer nanoparticles and the cell membrane at a volume ratio of 1: (0.1 to 10), and this fusion ratio may be appropriately controlled depending on the type of the polymer and the type of cell from which the cell membrane is derived.

[0035] The above hybrid nanocomposite may have an average diameter of 50 to 200 nm, and the higher the fusion ratio of the polymer nanoparticles, the lower the average diameter within the above range.

[0036] The hybrid nanocomposite according to the present invention can enhance structural stability and increase encapsulation efficiency by applying mPEG-b-PLA polymersomes, and can exhibit the unique properties of each cell membrane across various cell lines. Therefore, the hybrid nanocomposite according to the present invention can be utilized as a platform for drug or gene delivery.

[0037]

[0038] The present invention provides a composition for drug delivery comprising the above hybrid nanocomposite; and a therapeutic or prophylactic agent.

[0039] The therapeutic or prophylactic agent may be a vaccine or a compound capable of inducing an immune response, and the compound may include a drug commonly used for various diseases.

[0040] Alternatively, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0041] The drug delivery composition according to the present invention can more effectively deliver a vaccine, drug, or gene into cells by loading the hybrid nanocomposite described above.

[0042]

[0043] In addition, the present invention provides a method for producing the above hybrid nanocomposite.

[0044] The method for manufacturing a hybrid nanocomposite according to the present invention may include the steps of dissolving an amphiphilic polymer in an organic solvent and then hydrating the polymer to form a thin film to prepare a polymersome; the steps of suspending cells in a storage dissolution buffer to disrupt them, centrifuging them, and extruding the collected supernatant to obtain cell membrane vesicles; and the steps of mixing the manufactured polymersomes and the obtained cell membrane vesicles and then extruding them to induce self-assembly.

[0045] The step of manufacturing the above polymersome can be performed by synthesizing an amphiphilic polymer, dissolving the synthesized polymer in an organic solvent, removing the organic solvent to obtain a polymer in the form of a thin film, and hydrating the same.

[0046] The above amphiphilic polymer may be selected from a group of materials formed by synthesizing the above hydrophilic and hydrophobic polymers, and may preferably be a methoxy-poly(ethylene glycol)-block-polylactic acid (mPEG-b-PLA) copolymer, but is not limited thereto.

[0047] The organic solvent may be selected from, but is not limited to, chloroform, toluene, or tetrahydrofuran.

[0048] The step of obtaining the above cell membrane vesicles can be performed by suspending the cells in a storage lysis buffer, disrupting them with an ultrasonic grinder, performing gradient centrifugation at least once, collecting the supernatant, and then extruding the cells.

[0049] The extrusion may be performed through nanopores having a diameter of 200 to 400 nm.

[0050] The step of inducing self-assembly of the manufactured polymersome and the obtained cell membrane vesicle can be performed by mixing the manufactured polymersome and the obtained cell membrane vesicle in a volume ratio of 1: (0.1 to 10), and at this time, the manufactured polymersome and the obtained cell membrane vesicle can self-assemble to form a hybrid nanocomposite.

[0051] The extrusion may be performed through nanopores having a diameter of 200 to 400 nm.

[0052] In addition, the step of inducing the self-assembly can form a hybrid nanocomposite loaded with a therapeutic or preventive agent by further including the polymersome and a therapeutic or preventive agent outside the cell membrane vesicle.

[0053] The corresponding features can be replaced by those described above.

[0054] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0055]

[0056] <Experiment preparation>

[0057] 1. Materials

[0058] 3,6-dimethyl-1,4-dioxane-2,5-dione [3,6-dimethyl-1,4-dioxane-2,5-dione; D,L-lactide] (Sigma-Aldrich, USA), 2000 Da molecular weight methoxy-poly(ethylene glycol); mPEG] (Sigma-Aldrich, USA), Tin(II) 2-ethylhexanoate [Tin(II) 2-ethylhexanoate; Sn(Oct)2] (Sigma-Aldrich, USA), diethyl ether (Sigma-Aldrich, USA), DNase (Roche, Switzerland), Amicon ® Ultra-15 centrifugal filter 50 K (Millipore Corporation, USA), phosphate-buffered saline 1X (PBS) (WELGENE, Republic of Korea), ReadyShield® protease inhibitor cocktail (Sigma-Aldrich, USA), Bradford reagent (Sigma-Aldrich, USA), ovalbumin-fluorescein conjugate (OVA-FITC) (Thermo Fisher Scientific, USA), CleanCap® enhanced green fluorescent protein mRNA (5-methoxyuridine) (EGFP mRNA (5moU)) (TriLink BioTechnologies, USA, #L-7201), DiO (Biotium, USA), DiI (Biotium, USA), SnakeSkin™ dialysis membrane 10 K (Thermo Fisher Scientific, USA), Trans-Blot Turbo RTA Mini 0.2 μm nitrocellulose transfer kit (Bio-Rad, USA) CD19 mouse monoclonal antibody, clone OTI3B10, Catalog NO.TA506236 (Origene, USA, #F003), CD47 mouse monoclonal antibody, clone B6H12.2, Catalog NO. AM33292PU-S , purified (Origene, USA, #3P220803), recombinant HRP anti-sodium potassium ATPase antibody, clone EP1845Y (Abcam, United Kingdom, #ab185065), Goat anti-rabbit IgG H&L (horseradish peroxidase-conjugated) (Abcam, United Kingdom, #ab6721), Rabbit anti-mouse IgG H&L (horseradish peroxidase-conjugated, #ab6709) (Abcam, United Kingdom), EZ-cytox (DoGenBio, Republic of Korea), Hoechst 33342 (Thermo Fisher Scientific, USA), and LysoTracker Red DND-99 (Thermo Fisher Scientific, USA) were used.

[0059]

[0060] 2. cells

[0061] The Raw 264.7 cell line was purchased from the Korean Cell Line Bank (KLCB No. 40071). MCF-7, MDCK, and Raji cell lines were purchased from the American Type Culture Collection (#HTB-22TM, #CCL-34, and #CCL-86). MCF-7, MDCK, and Raji cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic [amphotericin B, penicillin, streptomycin]. Raji cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic [amphotericin B, penicillin, streptomycin]. Cell culture media and supplements were purchased from Thermo Fisher Scientific (San Jose, CA, USA).

[0062]

[0063] <Example 1> Synthesis of amphiphilic mPEG-b-PLA copolymer and preparation and characterization of polymersomes (PSomes)

[0064] 1-1. Synthesis and characterization of mPEG-b-PLA copolymer

[0065] mPEG-b-PLA copolymers were synthesized through ring-opening polymerization of D,L-lactide. Methoxy-poly(ethylene glycol) (mPEG) terminated with a hydroxyl group was used as an initiator, and Tin(II) 2-ethylhexanoate (Sn(Oct)2) was used as a catalyst (water-soluble fraction = 0.275). Subsequently, various concentrations of D,L-lactide and Sn(Oct)2 (0.50 wt% of mPEG and D,L-lactide) were injected into a three-necked flask and heated under nitrogen at 120°C for 24 h under reflux. After completion of the reaction, the resulting solution was removed using a rotary evaporator and precipitated in an excess of cold diethyl ether to produce mPEG-b-PLA copolymers. The copolymer product was filtered under vacuum using a Buchner funnel, precipitated three times, and then dried at 25°C for 24 h.

[0066] Subsequently, the molecular weight and mPEG content of the synthesized mPEG-b-PLA were determined using a 600 MHz nuclear magnetic resonance spectrometer software (Avance Neo 600; Bruker, Bremen, Germany). 1 It was characterized by H nuclear magnetic resonance spectrum. The chemical structure was determined using Fourier transform infrared spectroscopy (Excalibur series, Varian Inc., Palo Alto, CA, USA).

[0067]

[0068] 1-2. Preparation of polymersomes (PSomes)

[0069] 10 mg of mPEG-b-PLA copolymer was dissolved in 1 mL of chloroform. The chloroform was removed using a rotary evaporator, forming a thin film of mPEG-b-PLA copolymer on the wall of a round-bottom flask. The film was then placed under high vacuum for 6 hours to remove any residual chloroform. The dried film was hydrated in 2 mL of PBS at 50°C for 6 hours and then stirred magnetically for an additional 12 hours.

[0070] After hydration, the particles were diluted in 1X PBS to a concentration of 1 mg / mL. The size distribution and zeta potential of the PSomes were measured by dynamic laser scattering and zeta potential analysis (ELS-Z; Otsuka Electronics, Osaka, Japan), respectively, and the morphological characteristics were investigated by TEM using a JEM-1011 microscope (JEOL, Tokyo, Japan).

[0071]

[0072] <Example 2> Preparation and Characterization of Cell Membrane Vesicles (CMVs)

[0073] Raji, MCF-7, and MDCK cells were suspended in a storage lysis buffer and sonicated six times at 150 W for 5 s at 4°C using an ultrasonicator (VCX 500, sonic&materials, USA). DNase (1 mg / mL) was added to the solution and centrifuged at 5,300 rpm for 5 min to harvest the supernatant. The harvested supernatant was centrifuged at 20,000 × g for 30 min and then filtered using a 50 K membrane by centrifugation at 3,000 × g for 10 min. The collected supernatants were washed with 0.1X PBS supplemented with 1X protease inhibitor, sonicated for 3 min, and finally extruded through a polycarbonate membrane with 400 nm pores using a mini-extruder (Avanti Polar Lipids) to form cell membrane vesicles (e.g., Raji CMVs, MCF-7 CMVs, and MDCK CMVs). The protein concentration of CMVs was measured using Bradford reagent (Sigma-Aldrich).

[0074]

[0075] <Example 3> Preparation and characterization of cell membrane hybrid polymersomes (HyPSomes)

[0076] Raji CMVs, MCF-7 CMVs, and MDCK CMVs were mixed with PSomes at various ratios (PSome [1 mg / mL]:CMV = 1:9, 3:7, 5:5, 7:3, and 9:1). Then, the mixture was extruded through a 200 nm pore in a mini-extruder. OVA-FITC-loaded hybrid polymersomes (HyPSomes) and EGFP mRNA-loaded HyPSomes were prepared as previously described. PSomes, each CMV, and OVA-FITC (0.001 mM) or EGFP mRNA (15 μg / mL) were added, and then extruded through a 200 nm pore in a mini-extruder.

[0077]

[0078] The hydrodynamic diameter of HyPSomes was analyzed using dynamic light scattering (DLS; ELSZ-2000S, Otsuka Electronics Korea Co., Ltd., Seongnam-si, Republic of Korea), and the zeta potential was measured using a Zetasizer (ZSP, Malvern, Australia). Their morphological characteristics were investigated using field emission TEM with a JEM-2100F microscope (JEOL, Tokyo, Japan). Before TEM characterization, samples were prepared by contacting droplets containing HyPSomes with a copper grid, followed by negative staining with uranyl acetate for 3 min.

[0079] Referring to Fig. 2, transmission electron microscopy (TEM) images confirm that CMVs and PSomes present in the cell membrane have a spherical shape and are composed of two layers of two cell membrane lipids (Fig. 2a). Dynamic light scattering (DLS) analysis revealed that the size was less than 200 nm (Fig. 2b), and the surface charge was confirmed to exhibit a zeta potential of -10 mV while maintaining the original surface charge (Fig. 2c).

[0080]

[0081] In addition, the bilayer membrane was observed using TEM to determine the optimal hybridization conditions for CMVs and PSomes. As shown in Fig. 3 and Table 1 below, HyPSomes with various configurations were formed, and the size of HyPSomes was found to decrease as the amount of polymer increased.

[0082] Table 1 below shows the analysis of the diameters of PSomes and cell membranes according to the hybridization ratio.

[0083]

[0084]

[0085] <Experimental Example 1> Stability Analysis of Cell Membrane Hybrid Polymersomes

[0086] To analyze the stability of HyPSomes, changes in their hydrodynamic diameters were measured using dynamic light scattering (DLS). The hydrodynamic diameter changes of HyPSomes mixed at various ratios were observed over 4 weeks using DLS analysis in a 10% fetal bovine serum solution.

[0087] Referring to Figure 4, as the polymer ratio increased in the three variants of HyPSomes, the original nanoparticle size was more effectively maintained.

[0088]

[0089] <Experimental Example 2> Fluorescence Characterization of Cell Membrane Hybrid Polymersomes

[0090] To investigate the membrane compatibility and fusion efficiency of HyPSomes, PSomes and CMVs were labeled with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) (5 μM in DMSO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) (5 μM in DMSO), respectively, and FRET analysis was performed. Before hybridization, PSomes and CMVs labeled with the FRET dye pair (DiI and DiO) were dialyzed for 5 days using SnakeSkin™ dialysis membrane 10 K. Then, the labeled PSome and CMVs were extruded through a 200 nm pore polycarbonate membrane in a mini-dialysis machine at a specified ratio and dialyzed for 48 h using SnakeSkin™ dialysis membrane 10 K.

[0091] Fluorescence was measured at 470 nm using a microplate reader (Synergy H1; BioTek, USA). Immunofluorescence imaging was analyzed using the same procedure using a Super Sensitive High Resolution CLSM (LSM880 with Airyscan, Carl Zeiss, Germany). Fluorescence characterization of HyPSomes by flow cytometry confirmed that HyPSomes and CMVs were taken up into the source cells. After labeling PSomes and CMVs with DiI and DiO, respectively, HyPSomes were formed and cultured in 5 × 10 Raji, MCF-7, and MDCK cells. 5 After introduction into cells, each fluorescence distribution was analyzed using a flow cytometer and confirmed as a dot plot.

[0092] As a result, referring to Figure 5, hybridization due to physical self-assembly of the two particles reduced the distance between DiO and DiI, increasing the energy transferred to the acceptor and generating intense fluorescence at 565 nm. In particular, the fluorescence intensity reached a maximum at a ratio of 9:1 for HyPSomes derived from Raji and MCF-7 cells, and at a ratio of 7:3 for HyPSomes derived from MDCK cells. These results are related to the fact that as the polymer ratio increases, the particle size decreases and the distance between the FRET dye injections shortens, resulting in a higher intensity of fluorescence emitted at a wavelength of 565 nm. Particle hybridization was effectively achieved at all ratios, except for MDCK HyPSomes, which required ratios of 1:9 and 3:7.

[0093]

[0094] <Experimental Example 3> Confirmation of hybridization characteristics between cell membrane nanoparticles and PSomes

[0095] 3-1. Hybridization Confirmation through Confocal Microscopy Analysis

[0096] Hybridization was verified by labeling each nanoparticle membrane with a unique fluorescent dye.

[0097] Referring to Fig. 6(a), after hybridization, fusion can be confirmed by the overlap of fluorescence observed using confocal microscopy (CLSM). The green fluorescent dye, DiO, labeled the cell membrane, and the red fluorescent dye, DiI, labeled the polymersome membrane. These two particles were hybridized using a nano-extrusion method, and the residual dye was subsequently dialyzed before observation using CLSM. Due to the photostability and potential FRET effect between DiO and DiI, the unique colors of the dyes were confirmed to combine within the nanostructured membrane, resulting in an indistinguishable yellow color.

[0098]

[0099] 3-2. Western Blot Analysis

[0100] Western blotting was performed to confirm successful fusion. First, samples containing CMVs and HyPSomes were loaded onto a 10% polyacrylamide gel after denaturation. Proteins were transferred from the polyacrylamide gel to a nitrocellulose membrane, which was blocked with skim milk for 1 hour at 25°C and then incubated overnight at 4°C with primary antibodies CD19, CD47, and ATPase. Finally, the nitrocellulose membrane was probed with horseradish peroxidase-conjugated secondary antibodies and developed using Clarity Western ECL substrate.

[0101] As a result, as shown in Fig. 6(b), it was confirmed that each particle was successfully fused, which proved that the protein receptor was preserved on the surface of the hybrid nanoparticle.

[0102]

[0103] 3-3. Surface charge characteristic analysis

[0104] Zeta potential distributions of Raji cell-based HyPSome (Raji HyPSome), MCF-7 cell-based HyPSome (MCF-7 HyPSome), and MDCK cell-based HyPSome (MDCK HyPSome) were measured.

[0105] As a result, as shown in Fig. 6(c), HyPSomes were slightly negatively charged at approximately -10 mV, which is an expected result when the intrinsically negatively charged cell membrane binds to the surface-neutral PSomes.

[0106]

[0107] 3-4. Flow cytometry

[0108] Individual cell membranes were labeled with specific fluorescent markers and cellular uptake was assessed using flow cytometry.

[0109] As a result, as shown in Fig. 6(d), among the various hybridizations, MCF-7 HyPSomes recorded a low efficiency of 56.9%, unlike Raji and MDCK HyPSomes, which recorded 77.1% and 99.8%, respectively. In particular, this difference in efficiency of MCF-7 HyPSomes is due to the degree of bending rigidity resulting from the difference in the cholesterol component of the cell membrane of metastatic cancer cell lines.

[0110]

[0111] These results suggest that the fusion of membrane nanoparticles with PSomes leverages both the biological functionality of the cell membrane and the structural integrity of the synthetic polymer. Notably, the differences in hybridization efficiency across different cell types highlight the need for optimization tailored to the cell membrane being used. The inherent characteristics of large cells present challenges in achieving higher hybridization efficiencies. However, the successful integration of these entities into HyPSomes demonstrates their potential as a vaccine delivery platform.

[0112]

[0113] <Experimental Example 4> In vitro analysis of cell viability, cellular uptake, and endosomal escape of HyPSomes

[0114] 4-1. Cell viability analysis

[0115] To determine cell viability in HyPSomes, Raw 264.7 cells (10 4) were seeded in 96-well plates and cultured at 37°C in a 5% CO2 atmosphere for 12 hours. After 12 hours of culture, HyPSomes, CMVs, and PSomes were added, and the cells were further cultured for 6, 12, 24, and 48 hours. After treatment, water-soluble tetrazolium salt (WST) was added, and the cells were further cultured at 37°C in a 5% CO2 atmosphere for 1 hour. After culture, the absorbance was measured at 450 nm using a microplate reader (Synergy H1, BioTek, USA).

[0116] As a result, as shown in Fig. 7(a), HyPSomes did not show toxicity to immune cells at 6, 12, or 24 hours, confirming that they were not toxic as mRNA delivery vehicles.

[0117]

[0118] 4-2. Cell uptake analysis

[0119] Uptake of HyPSomes loaded with OVA-FITC by antigen-presenting cells is considered an essential step for enhancing immune responses. Therefore, the intracellular antigen uptake efficiency of HyPSomes was analyzed. The Raw 264.7 cell line can be used to display exogenous antigens to both helper T cells and B cells and to evaluate the uptake capacity of HyPSomes. In this experimental example, OVA was used as a model antigen, and FITC was used to detect intracellular antigens, thereby examining the intracellular antigen uptake efficiency of HyPSomes.

[0120] First, for flow cytometry analysis, Raw 264.7 cells (5 × 10 5) were seeded in 6-well plates and pre-cultured for 24 h at 37°C in a 5% CO2 atmosphere. After 24 h, HyPSomes and OVA-FITC solutions were added, and the cells were cultured for 6 h at 37°C in a 5% CO2 atmosphere. After treatment, the obtained cells were washed and collected. Flow cytometry was performed using an LSR II flow cytometer (FACSymphony, Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software version 10.9.0 (Tree Star, Inc., Ashland, OR, USA).

[0121] Raw 264.7 macrophages (4×10 5 ) were seeded on cover glass bottom dishes and pre-incubated for 24 h at 37°C in a 5% CO2 atmosphere. After 24 h, the cells were treated with OVA-FITC-loaded HyPSomes and OVA-FITC solution, and then incubated for 6 h at 37°C in a 5% CO2 atmosphere. Cells were stained with Hoechst 33342 to visualize nuclei and LysoTracker Red DND-99 to visualize lysosomes. After staining, the cells were washed with DMEM without phenol red, and the cellular uptake of the particles was visualized using an ultrasensitive high-resolution CLSM (LSM880 with Airyscan, Carl Zeiss, Germany).

[0122] As a result, as shown in Fig. 7(b), HyPSomes exhibited significantly higher cellular uptake efficiency than the experimental group treated with OVA-FITC alone. In particular, fluorescence intensity was higher in Raji cell-based HyPSomes and MCF-7 cell-based HyPSomes. These results suggest that the cellular uptake efficiency of cancer cell surface markers and B-cell lymphoma surface markers was increased.

[0123]

[0124] 4-3. Endosomal escape analysis

[0125] The cytosolic delivery ability of HyPSomes was evaluated in vitro. Fluorescence visualization was performed using a confocal microscope to observe Raw 264.7 cells cultured with OVA-FITC, Raji HyPSomes, MCF-7 HyPSomes, and MDCK HyPSomes for 6 hours. As shown in Fig. 7(c), when only the antigen (green) was delivered to macrophages, weak fluorescence was observed due to low cellular uptake efficiency, and the fluorescence uptake was found to be combined with the uptake of lysosomes (red). These results indicate that when only OVA-FITC was delivered, the antigen was not delivered to the cytosol of macrophages but was degraded by lysosomes.

[0126] In contrast, when antigen (green) was transported intracellularly by HyPSomes, the fluorescence was clearly distinct from that of lysosomes (red). This distinction indicates that HyPSomes, composed of the cell membrane and PSomes, successfully escaped the endosomal environment and released the antigen into the cytoplasm. While the cell membrane, as a cell-derived material, inherently promotes more efficient endosomal bypass, this is complemented by biocompatible, biodegradable, polymer-based nanoparticles, or PSomes, known for their robust structural integrity and efficient endosomal bypass ability. Therefore, hybridized HyPSomes not only encapsulate antigens with high efficiency during self-assembly, but also enhance their ability to deliver antigens into the cytoplasm. The combination of efficient cellular uptake and successful endosomal escape makes HyPSomes a potential platform for mRNA delivery.

[0127]

[0128] <Experimental Example 5> Confirmation of EGFP Expression Efficiency of EGFP-mRNA-Loaded HyPSomes

[0129] The role of mRNA is to help translate DNA into proteins within the cytoplasm, and for delivery systems such as HyPSomes, ensuring efficient mRNA loading and subsequent intracellular translation is crucial.

[0130] In this experimental example, EGFP mRNA was loaded into HyPSomes using nanoextrusion. The mRNA was loaded into the hydrophilic core of HyPSomes, which was formed by self-assembly of PSomes and membrane vesicles. The translational capacity of EGFP-mRNA-loaded HyPSomes was visually confirmed using CLSM. As previously discussed, HyPSomes exhibit efficient endocytosis and endosomal escape, enabling stable loading of EGFP-mRNA. This ensures successful release of mRNA loaded into HyPSomes into the cytoplasm. The mRNA was then transfected with fluorescent proteins by ribosomes, the mRNA translation proteins of RAW 264.7.

[0131] Specifically, Raw 264.7 cells (5 × 10 5 ) were seeded in 6-well plates and pre-cultured in complete medium for 24 h at 37°C in a 5% CO2 atmosphere. After 24 h, the cell culture medium was replaced with opti-MEM. EGFP mRNA-loaded HyPSome, PSome, and naked mRNA solutions were added and cultured for 48 h at 37°C in a 5% CO2 atmosphere. After culture, the obtained cells were washed and collected. Flow cytometry was performed using an LSR II flow cytometer (FACSymphony, Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software version 10.9.0 (Tree Star, Inc., Ashland, OR, USA). To maintain the validity and reliability of the flow cytometry results, voltage parameters were maintained consistently across all experimental groups.

[0132] Raw 264.7 macrophages (5×10 5 ) were seeded on cover glass bottom dishes and pre-cultured in complete medium for 24 h at 37°C in a 5% CO2 atmosphere. After 24 h, the cell culture medium was replaced with opti-MEM, and EGFP mRNA-loaded HyPSome, PSome, and naked mRNA solutions were added and cultured for 48 h at 37°C in a 5% CO2 atmosphere. After 24 h, opti-MEM was removed, and the medium was replaced with DMEM without phenol red. Cells were stained with Hoechst 33342 to visualize nuclei. After staining, cells were washed with DMEM without phenol red, and EGFP mRNA translation was visualized using an ultrasensitive high-resolution CLSM (LSM880 with Airyscan, Carl Zeiss, Germany).

[0133] As a result, referring to Fig. 8(a), a distinct green fluorescence signal can be observed in mRNA-loaded HyPSomes, which is higher than the corresponding signals in naked mRNA and EGFP-mRNA-loaded PSomes. This finding suggests that HyPSomes, which are essential for the cell membrane, have a higher translational efficiency compared to naked mRNA.

[0134] To support this and quantitatively analyze the cellular expression of EGFP mRNA, flow cytometry analysis was used. Figures 8(b) and 8(c) show a clear increase in EGFP expression in all HyPSomes variants, in contrast to naked mRNA and PSomes. Raji HyPSomes, MCF-7 HyPSomes, and MDCK HyPSomes showed a 6.74-, 7.44-, and 7.03-fold increase, respectively, compared to PSomes. These results demonstrate the efficiency of the HyPSomes mRNA expression system in inducing robust EGFP expression across a significant portion of the cells under investigation.

[0135] Raw 264.7 cells can recognize and respond to foreign genetic sequences, reducing the efficiency of foreign genome translation. However, because the cell membrane is adept at evading these immune defenses, HyPSomes can enhance translation efficiency. HyPSomes optimize entry into immune cells through their integrated cell membrane, thereby increasing EGFP expression. These results suggest that HyPSomes can deliver foreign mRNA to antigen-presenting cells and support optimal expression.

[0136]

[0137] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A hybrid nanocomposite formed by fusing biocompatible polymer nanoparticles and cell membranes.

2. In paragraph 1, The above polymer nanoparticles are, A nanocomposite characterized by being a polymersome made of an amphiphilic polymer, methoxy-poly(ethylene glycol)-block-polylactic acid (mPEG-b-PLA) copolymer.

3. In paragraph 1, The above cell membrane, A nanocomposite characterized by being a vesicle derived from the cell membrane of a cancer cell or a normal cell.

4. In paragraph 1, The above nanocomposite is, A nanocomposite characterized in that the polymer nanoparticles and the cell membrane are fused at a volume ratio of 1: (0.1 to 10).

5. In paragraph 1, The above nanocomposite is, A nanocomposite characterized by an average diameter of 50 to 200 nm.

6. A composition for drug delivery comprising a nanocomposite according to any one of claims 1 to 5; and a therapeutic or prophylactic agent.

7. In paragraph 6, The above therapeutic or preventive preparations, A composition for drug delivery, characterized in that it is a vaccine or compound capable of inducing an immune response.

8. In paragraph 6, The above therapeutic or preventive preparations, A drug delivery composition characterized in that it is selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

9. A step of preparing a polymersome by dissolving an amphiphilic polymer in an organic solvent and then hydrating the thin film; A step of suspending cells in a storage lysis buffer, disrupting them, centrifuging them, and extruding the collected supernatant to obtain cell membrane vesicles; and A method for producing a hybrid nanocomposite, comprising a step of mixing the manufactured polymersome and the obtained cell membrane vesicle and then extruding the mixture to induce self-assembly.

10. In paragraph 9, The above extrusion is, A manufacturing method characterized in that it is performed through nano-pores having a diameter of 200 to 400 nm.

11. In paragraph 9, The step of inducing the above self-assembly is: A manufacturing method characterized in that the above-mentioned manufactured polymersome and the obtained cell membrane vesicle are mixed in a volume ratio of 1: (0.1 to 10).

12. In paragraph 9, The step of inducing the above self-assembly is: A manufacturing method characterized in that it is performed by further including a therapeutic or preventive agent.

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