KR20230073035A - Janus cells asymmetrically coated with metal-organic framework nanoparticles encapsulated with active substances and uses thereof - Google Patents

Abstract

The present invention relates to Janus cells asymmetrically coated with metal-organic framework nanoparticles encapsulated with active substances and to uses thereof, and specifically, provides cells with a novel Janus structure, of which internalization of cytotoxic drugs is prevented while preserving the cell's unique ability to bind to the microenvironment by maintaining the biological and structural characteristics of normal living cells, and a manufacturing method thereof. In addition, a pharmaceutical composition comprising the cells of the novel Janus structure is provided.

Description

Janus cells asymmetrically coated with metal-organic framework nanoparticles encapsulated with active substances and uses thereof

The present invention relates to a Janus cell asymmetrically coated with metal-organic framework nanoparticles encapsulated with an active material and its use, which maintains the biological and structural characteristics of a typical living cell, thereby maintaining the cell's unique binding to the microenvironment. Provided are cells having a novel Janus structure in which internalization of a cytotoxic drug is prevented while preserving the ability, and a method for producing the same. In addition, a pharmaceutical composition comprising the cell having the novel Janus structure is provided.

Nanotechnology offers a promising strategy to improve the efficacy of various drugs to enhance cancer treatment. Among them, nanoscale metal-organic frameworks (MOFs), a type of hybrid material developed through self-assembly of metal ions and organic ligands, have emerged as a platform for the effective delivery of therapeutic drugs to tumor tissues. did Compared with other traditional porous materials, MOFs have several clear advantages, such as high loading efficiency of drugs and biological macromolecules (DNA and proteins), tunable and designable functions, stimuli-responsive degradability and biocompatibility. Due to these unique properties, the MOF-based drug delivery system (DDS) has presented an unprecedented opportunity for cancer treatment [Liang, K, et al. "Biomimetic Mineralization of Metal-Organic Frameworks as Protective Coatings for Biomacromolecules." Nat. Commun. 2015, 6, no. 7240.; Liang, Kang, et al. "Enzyme Encapsulation in Zeolitic Imidazolate Frameworks: A Comparison Between Controlled Co-precipitation and Biomimetic Mineralization." Chem. Commun. 2016, 52, 473-476.; Poddar, Arpita, et al. "Encapsulation, Visualization and Expression of Genes with Biomimetically Mineralized Zeolitic Imidazolate Framework-8 (ZIF-8)." Small 2019, 15, No. 1902268.]. However, the application of MOF-based DDS is limited due to its short in vivo cycle time and rapid phagocytic clearance [Rosenblum, Daniel, et al. "Progress and Challenges Towards Targeted Delivery of Cancer Therapeutics." Nat. Commun. 2018, 9, no. 1410.]. In recent years, significant efforts have been devoted to the development of novel drug delivery systems based on living circulating cells that deliver drug-laden nanoparticles linked to the cell's outer surface. Due to the many attractive and unique features that arise from circulating cells, such as enhanced mobility and long cycle life, novel cell-mediated drug delivery systems (c-DDS) demonstrate improved drug delivery and controlled drug release. [Rosenblum, Daniel, et al. "Progress and Challenges Towards Targeted Delivery of Cancer Therapeutics." Nat. Commun. 2018, 9, no. 1410.; Agrahari, V., Agrahari, V., Mitra, AK “Next Generation Drug Delivery: Circulatory Cells-Mediated Nanotherapeutic Approaches.” Expert Opin. Drug Delivery 2017, 14, 285-289.]. For example, Mitragotri's group demonstrated the use of red blood cells (RBCs) as a carrier to prolong the resistance time of nanoparticles in the blood [Chambers, E., Mitragotri, S. "Prolonged Circulation of Large Polymeric Nanoparticles by Non- covalent Adsorption on Erythrocytes." J. Controlled Release 2004, 100, 111-119.; Chambers, E., Mitragotri, S. "Long Circulating Nanoparticles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation." Exp. Biol. Med. 2007, 232, 958-966.]. See Stephan, Matthias T., et al., "Therapeutic Cell Engineering with Surface-conjugated Synthetic Nanoparticles." Nat. Med. 2010, 16, 1035-1041.] by covalently attaching drug-containing nanoparticles to therapeutic T cells and stem cells to activate T cells and increase the stem cell population in vivo .

Although these systems take advantage of both the unique properties of cells and the ability of nanoparticles for enhanced drug loading and controlled drug release, these existing methods still have two major limitations. First, interactions between nanoparticles and cell membranes lead to nanoparticle accumulation and lead to internalization by phagocytosis or micropinocytosis [Fleischer, C. C., Payne, C. K. “Nanoparticle-Cell Interactions: Molecular Structure of the Protein Corona and Cellular Outcomes." Acc. Chem. Res. 2014, 47, 2651-2659.18]. This natural uptake mechanism of mammalian cells limits the types of therapeutic agents that can be delivered by circulating cells because highly cytotoxic drugs can induce cell death before reaching the target tissue [Verma, A., Stellacci , F. "Effect of Surface Properties on Nanoparticle-Cell Interactions." Small 2010, 6, 12-21.; Nel, Andre E., et al., "Understanding Biophysicochemical Interactions at the Nano-bio Interface." Nat. Mater. 2009, 8, 543-557.]. To overcome the internalization problem, cellular backpacks, i.e., microscale patches hundreds of nanometers thick, have been proposed [Shields, C. Wyatt, et al., "Cellular Backpacks for Macrophage Immunotherapy." Sci. Adv. 2020, 6, No. eaaz6579.; Klyachko, Natalia L., et al., "Macrophages with Cellular backpacks for Targeted Drug Delivery to the Brain." Biomaterials 2017, 140, 79-87.; Xia, Junfei, et al., "Asymmetric Biodegradable Microdevices for Cell-Borne Drug Delivery." ACS Appl. Mater. Interfaces 2015, 7, 6293-6299.]. Due to their microscopic size, the cell backpack is not swallowed by the carrier cells. However, manufacturing involves a costly and time-consuming layer-by-layer (LbL) process, which limits the amount of drug-laden "backpacks" that can adhere to cells. Second, most of the reported cell-mediated drug delivery systems are designed to maximize the drug loading capacity by randomly coating the entire surface of cells with drug-containing nanoparticles [Chambers, E., Mitragotri, S. "Prolonged Circulation of Large Polymeric Nanoparticles by Non-covalent Adsorption on Erythrocytes." J. Controlled Release 2004, 100, 111-119.; Chambers, E., Mitragotri, S. "Long Circulating Nanoparticles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation." Exp. Biol. Med. 2007, 232, 958-966.]. Such complete and non-specific cell surface blockade using a layer of nanoparticles may prevent cells from physically interacting with the microenvironment and thus hinder cell binding to the target.

Under this background, the present inventors have developed a new c-DDS system called MOF-coated Janus carrier cells that solves the problem of nanoparticle internalization and cell-microenvironment binding.

Accordingly, an object of the present invention is to provide a Janus cell in which a part of the surface is asymmetrically coated with drug metal-organic framework nanoparticles encapsulated with an active material.

Another object of the present invention is to provide a pharmaceutical composition comprising the aforementioned Janus cells.

Another object of the present invention is to provide a method for preparing the above-described Janus cells.

In order to solve the above problems, the present invention is a Janus cell in which metal-organic framework nanoparticles encapsulated with an active material are asymmetrically coated on a part of the cell surface by tannic acid. to provide.

According to a preferred embodiment of the present invention, the metal-organic framework may be zeolitic imidazolate framework-8 (ZIF-8; 2-methylimidazole, zinc salt).

According to another preferred embodiment of the present invention, the zinc of the ZIF-8 forms a coordination bond with tannic acid to induce binding between the ZIF-8 nanoparticles.

According to another preferred embodiment of the present invention, the metal-organic framework nanoparticles may be coated on 30 to 80% of the entire cell surface.

According to another preferred embodiment of the present invention, the metal-organic framework nanoparticles may encapsulate one or two or more different active materials.

According to another preferred embodiment of the present invention, the encapsulated active material may be at least one selected from the group consisting of compounds, DNA, proteins, peptides, anticancer agents, fluorescent particles, lipids and cells.

According to another preferred embodiment of the present invention, the type of Janus cells may be at least one selected from the group consisting of circulating tumor cells, immune cells, red blood cells, and stem cells.

According to another preferred embodiment of the present invention, the Janus cell may have characteristics in which the internalization of the metal-organic framework encapsulating the active material is prevented and the ability to bind to the microenvironment is preserved.

According to another preferred embodiment of the present invention, the Janus cells can release the encapsulated active substance in an acidic environment.

The present invention also relates to a method in which zeolitic imidazolate framework-8 (ZIF-8; 2-methylimidazole, zinc salt) nanoparticles encapsulated with proteinase K are asymmetrically coated on a portion of the cell surface by tannic acid. Janus cells are provided.

According to another preferred embodiment of the present invention, the proteinase K may be encapsulated at a concentration of 0.05 μg/mL to 2 μg/mL.

Additionally, the present invention provides pharmaceutical compositions comprising the various types of Janus cells described above.

According to a preferred embodiment of the present invention, the pharmaceutical composition can be used for anticancer.

In addition, the present invention provides a composition for delivering an active substance comprising the above-described various types of Janus cells.

Furthermore, the present invention provides a method for producing Janus cells of various types described above, comprising the following steps:

a) preparing metal-organic framework nanoparticles in which an active material is encapsulated;

b) monodispersing metal-organic framework nanoparticles encapsulated with an active material in a solution;

c) adding a solution in which metal-organic framework nanoparticles encapsulated with an active material are monodispersed to the cells attached to the plate;

d) adding tannic acid to the cells attached to the plate of step c);

e) removing metal-organic framework nanoparticles that are not bound to cells; and

f) Separating cells adhered to the plate by treating with trypsin.

The MOF-coated Janus cell according to the present invention preserves the cell's unique binding ability to the microenvironment by maintaining the biological and structural characteristics of general living cells, and the use of tannic acid allows the internalization of MOF nanoparticles encapsulated with active substances. can be prevented to protect carrier cells from cytotoxic drugs. In addition, MOF nanoparticles encapsulated with one or two or more different active substances are asymmetrically coated on cells and are very useful as a general platform for drug delivery in various applications including combination therapy.

Figure 1 is a schematic diagram showing the development process of the MOF-Janus cell-based drug delivery system.
Figure 2 shows the results of verifying the degradation of tannic acid (TA)-induced ZIF-8 nanoparticles, (Ai) and (A-ii) are the original ZIF-8 (Ai) and TA-coated ZIF-8, respectively. (A-ii) is a high-resolution scanning electron microscopy (HRSEM) photograph (scale bar: 200 nm), and (B) is an X-ray diffraction (X-ray) of ZIF-8 and TA-coated ZIF-8. -ray diffraction, XRD) spectrum, (c) is a N ₂ adsorption graph of original ZIF-8 and TA-coated ZIF-8, (D) is a pore of original ZIF-8 and TA-coated ZIF-8 (pore) It shows the size distribution.
Figure 3 is a result of confirming the formation of ZIF-8 and tannic acid coating on MDA-MB-231, (A) is a SEM micrograph of MOF nanoparticles, and (Bi) and (B-ii) are original cells, respectively. (Bi) and SEM/EDS micrographs of cells coated with MOF and tannic acid (TA) (B-ii). MOF particle size and morphology are maintained before and after MOF coating on the cell surface. (C) is the FT-IR result of intact MDA-MB-231 cells, tannic acid, ZIF-8 NP and ZIF-8/TA-coated MDA-MB-231 cells, and (D) is the original MDA-MB-231 cells without TA. Z-stack confocal microscopy images of MDA-MB-231 cells and MOF-coated MDA-MB-231 cells.
Figure 4 shows the development results of MOF-coated Janus cells, (A) is a Z-stack confocal microscope image of ZIF-8-coated glass adherent cells (blue: cell nuclei, green: FITC-labeled ZIF-8, Scale bar: 5 μm), (B) is the result of cell viability assay of glass-adhered mammalian cells coated with ZIF-8 nanoparticles, and (I) and (II) of (C) are native Confocal micrographs of cells (I) and Janus cells (II) (scale bar: 5 μm), (D) is the result of flow cytometry analysis of Janus cells carrying FITC-labeled ZIF-8 nanoparticles, and (E) are SEM micrographs of MOF nanoparticle patches attached on intact cells and Janus cells (bars: 1 μm). The p values in (B) were calculated using the t-test (***p<0.001, **p<0.01, *p<0.05).
Figure 5 shows integrin-mediated cell-microenvironmental interactions, (A) to (C), in order, cells with free attachments (A), MOF-Janus cells (B), and cells fully coated with ZIF-8. Photomicrographs of (C), (D) and (E) are photomicrographs of MOF-Janus cells cultured with DMEM for 1 hour at neutral pH (D) and pH 6 (E), respectively. (F) shows the survival of MOF-Janus cells after removal of ZIF-8 nanoparticles at pH 6. (G) is the result of an in vitro assay of translocation of native MDA-MB-231 cells and MOF-Janus cells across endothelial cell monolayers. The p values in (G) were calculated using the t-test (***p < 0.001, **p < 0.01, *p < 0.05).
Figure 6 shows the effect of reduced pH on cell viability, (A) and (B) are micrographs of intact cells cultured with serum-free cell culture medium at pH 7.4 (A) and pH 6 (B), respectively. It is a picture.
Figure 7 shows the results of MOF encapsulation of proteinase K at different concentrations, (A) is a SEM / EDS micrograph of ZIF-8 nanoparticles with and without encapsulated proteinase K enzyme , and (B) shows the UV-Vis standard curve for proteinase K at different concentrations.
Figure 8 shows the in vitro drug delivery and cancer spheroid targeting of MOF-Janus cells, (A) is a graph showing the encapsulation efficiency of proteinase K at various concentrations in ZIF-8 nanoparticles, (Bi) and (B-ii) is a photomicrograph of cells treated with native proteinase K (Bi) and proteinase K@MOF nanoparticles (B-ii) for 1 hour. (C) is a graph showing cell viability (%) after treatment with native proteinase K or proteinase K@MOF nanoparticles for 1 hour, and (D) is a graph showing proteinaseK@MOF attached to cells. (Blue: cell nuclei, red: proteinase K), (E) are MOF-Janus cells after attaching proteinase K to the surface of Janus cells for 1 hour and 6 hours. This is a graph showing the survival rate. (F) is a graph showing the survival rate of target breast cancer tissues after culturing with Janus cells containing various concentrations of proteinase K for 1, 3 and 5 hours, and (G) is Matrigel-embedded ) Confocal micrographs showing cell viability (green: live cells, red: dead cells, blue: all cell nuclei) of MDA-MB-231, (H) is MOF-Janus cell target site attachment and Matrigel-embedded Confocal micrographs showing release of proteinase K killing MDA-MB-231 (green: MOF-Janus cells, red: dead cell nuclei, blue: all cell nuclei). The p values for (C) and (F) were calculated using t-test (*** p < 0.001, ** p < 0.01, * p < 0.05).
Figure 9 shows the cell viability of MDA-MB-231 cells after completely coating with Proteinase K@MOF nanoparticles for 6 hours, (A) completely coated with Proteinase K@MOF (1.5 mg/ml) for 6 hours. It is a graph showing the cell viability of MDA-MB-231 cells, and (B) is a graph showing the release profile of proteinase K from MOF nanoparticles at pH 6.
Figure 10 shows the attachment of MOF-Janus cells to Matrigel-trapped MDA-MB-231 tissue, (A) shows MOF-Janus cells seeded on original MDA-MB-231 trapped in Matrigel. (B) is a photomicrograph of MOF-Janus cells cultured for 6 hours and washed with serum-free DMEM (green: MOF-Janus cells).
Figure 11 shows the encapsulation results of BSA and urease in ZIF-8 nanoparticles, (A) and (B) are graphs showing the concentration-dependent encapsulation efficiency of BSA (A) and urease (B) in MOF nanoparticles, respectively. , (C) and (D) show the PXRD spectra of BSA@MOF and (D) Urease@MOF, respectively.
Figure 12 shows multiple proteins loaded into MOF-Janus carrier cells, (A) shows the results of double protein loading on carrier cells, (B) and (C) triplex protein loading on carrier cells at two different magnifications. It shows the protein loading result (red: proteinase K, green: BSA, yellow: urease, blue: cell nucleus).

As described above, existing drug delivery systems based on living circulating cells that deliver drug-containing nanoparticles linked to the outer surface of cells are limited in the types of therapeutic agents that can be delivered due to nanoparticle internalization, and cell -There was a limitation in that the therapeutic was not effectively delivered to the target cell due to the microenvironmental binding problem, which prevented the carrier cell from binding to the target.

Accordingly, the present inventors sought a solution to the above problems by developing a novel c-DDS system called MOF-coated Janus carrier cells, which solves the problems of nanoparticle internalization and cell-microenvironment coupling. MOF-coated Janus carrier cells are prepared by immobilizing nanoparticles of zinc-based MOF (ZIF-8) asymmetrically with cytotoxic enzymes encapsulated on the surface of carrier cells (Fig. 1). Tannic acid (TA) is used to prevent the classical nanoparticle internalization pathway and to establish highly stable interactions between MOF nanoparticles and MOF-cell membranes via multidentate tannic acid complexation. The MOF-coated Janus cells developed in the present invention preserve the cell's unique ability to bind to the microenvironment by maintaining the biological and structural characteristics of normal living cells. In addition, upon reaching the cancer cells, the MOF nanoparticles on the carrier cells are rapidly degraded in acidic conditions, as shown in FIG. 1, to release cytotoxic enzymes.

Accordingly, a first aspect of the present invention relates to a Janus cell in which a portion of the cell surface is asymmetrically coated with a metal-organic framework nanoparticle encapsulated with an active material by tannic acid. .

In the present invention, the "metal-organic framework (MOF)" is a porous material obtained by a self-organizing reaction of metal ions and organic ligands. By linking metal ions with organic ligands, a crystalline polymer structure having spaces (pores) inside can be obtained. MOFs have a very large specific surface area in the crystal. In addition, the pore diameter and topology can be controlled by appropriately selecting and combining metal ions and organic ligands.

Metal ions for forming the MOF may be selected according to a target structural design. In general, Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Al ³⁺ , Fe ²⁺ , Cr ³⁺ and the like. Preferably, it may be Zn ²⁺ , but is not limited thereto.

Organic ligands for forming the MOF may also be selected according to a target structural design. In general, terephthalic acid, 4,4'-bipyridyl, imidazole, 2-imidazole carbaldehyde, 1,4-benzene dicarboxylate, 1,4-diazabicyclo [2,2,2] octane, etc. there is Preferably, it may be 2-methylimidazolate, but is not limited thereto.

A preferred MOF in the present invention may be zeolitic imidazolate framework-8 (ZIF-8; 2-methylimidazole, zinc salt), but is not limited thereto. The ZIF-8 (zeolitic imidazolate framework-8; 2-methylimidazole, zinc salt) contains Zn ²⁺ cation as a metal node and 2-methylimidazolate (2-mIM) as a linker. can do.

In the Janus cell of the present invention, when the MOF is ZIF-8, zinc of ZIF-8 forms a coordinate bond with tannic acid to induce binding between ZIF-8 nanoparticles.

The tannic acid serves to establish a highly stable interaction between the MOF nanoparticles and between the MOF and the cell membrane through multidentate tannic acid complexation.

In the Janus cells of the present invention, the MOF nanoparticles are coated only on a portion of the entire surface of the cell, and it is preferable that the coating is coated at a level capable of preserving the cell's inherent binding ability to the microenvironment. For example, the MOF may be coated on 30 to 80%, preferably 40 to 70%, and more preferably 40 to 60% of the entire cell surface.

In the Janus cell of the present invention, internalization of the metal-organic framework in which the active substance is encapsulated is prevented by the use of tannic acid, and the MOF is asymmetrically coated on a part of the cell surface, thereby entering the microenvironment through the non-encoded cell surface. binding capacity is preserved.

In the Janus cell of the present invention, the MOF nanoparticles may encapsulate one or two or more different active substances. Through this, it is possible to deliver various active substances (enzymes) as well as various proteins (enzymes) to target cells, and this ability is a cornerstone of cancer treatment, which is receiving great attention, for combination therapy, which is a treatment combining two or more types of treatments. expected to be useful.

In the Janus cell of the present invention, the active substance encapsulated in the MOF may be at least one selected from the group consisting of compounds, DNA, proteins, peptides, anticancer agents, fluorescent particles, lipids, and cells. In particular, since the Janus cell of the present invention exhibits the property of preventing the internalization of the MOF coated on the surface, the cells can be protected from substances encapsulated in the MOF, and thus substances exhibiting cytotoxicity can also be encapsulated in the MOF. There is an advantage.

The type of Janus cell of the present invention can be used without limitation as long as it binds to the target cell and can effectively deliver the active substance encapsulated in the MOF. For example, it may be at least one selected from the group consisting of circulating tumor cells, immune cells, red blood cells, and stem cells, but is not limited thereto. The immune cells may be, for example, T cells, NK cells, dendritic cells, or macrophages.

When the Janus cells of the present invention reach the target cells in an acidic environment, they release the encapsulated active substances and deliver them effectively to the target cells.

In a specific embodiment of the present invention, a portion of the surface of MDA-MB-231 cells, a breast cancer cell line, was asymmetrically coated with MOF encapsulated with proteinase K through multi-site tannic acid complexation to prepare Janus cells. At this time, the optimal dose of proteinase K to be encapsulated may be 0.05 μg/mL to 2 μg/mL, preferably 0.1 μg/mL to 1 μg/mL, but is not limited thereto.

A second aspect of the present invention relates to a drug delivery composition and/or pharmaceutical composition comprising the above-described various types of Janus cells.

The composition for drug delivery and/or the pharmaceutical composition of the present invention may be used for various diseases or disorders depending on the type of drug encapsulated in the MOF. For example, when the drug encapsulated in the MOF is proteinase K or an anticancer drug, the drug delivery composition and/or pharmaceutical composition of the present invention can be used for anticancer purposes.

Pharmaceutically acceptable carriers included in the drug delivery composition and/or pharmaceutical composition of the present invention include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol, as well as combinations thereof. Such compositions may be formulated to provide rapid, sustained or delayed release of the active after administration.

A pharmaceutically acceptable carrier can be prepared according to a number of factors well known to those skilled in the art, such as the specific bioactive substance used, its concentration, stability and intended bioavailability; diseases and disorders or conditions to be treated with the Janus cells of the present invention; the individual to be treated, age, size and general condition; Factors such as, but not limited to, the route used to administer the composition, such as nasal, oral, ocular, topical, transdermal and intramuscular, should be considered. In general, pharmaceutically available carriers used for administration of bioactive substances other than oral administration routes include aqueous solutions containing D5W, dextrose and physiological salts in an amount of less than 5% by volume. In addition, pharmaceutically usable carriers may contain additional ingredients capable of enhancing the stability of active substances such as preservatives and antioxidants.

The drug delivery composition and/or pharmaceutical composition of the present invention may be suspended in a physiologically compatible carrier. In the present invention, the term "physiologically compatible carrier" refers to a carrier that is compatible with other ingredients of the formulation and is not harmful to its recipients. The person skilled in the art is familiar with physiologically compatible carriers. Examples of suitable carriers include cell culture medium (e.g., Eagle's Minimum Essential Medium), phosphate buffered saline, Hank's balanced salt solution +/−glucose (HBSS), and multielectrolyte solutions such as Plasma-Lyte ?? A (Baxter) included.

In addition, the dose of the composition for drug delivery and/or the pharmaceutical composition of the present invention is administered in an effective dose for the intended treatment. The therapeutically effective amount required to treat or inhibit the progression of a particular medical disease can be readily determined by one skilled in the art using preclinical and clinical studies known in the medical arts. In the present invention, the therapeutically effective amount refers to an amount of an active substance that induces a biological or medical response in a specific tissue, system, animal, or human, which is desired by a clinician or researcher. Any suitable route of administration of the composition for drug delivery of the present invention may be used.

The present invention also provides a method for treating a disease comprising administering the various types of Janus cells described above to a subject. Administration of the Janus cells of the present invention is applicable to any animal, and the animal may include not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, mice, rats, and cats.

In the present invention, the term "administration" means introducing the Janus cells of the present invention into a subject by any suitable method. Since the route of administration of the composition for drug delivery and/or pharmaceutical composition of the present invention is the same as described above, description thereof is omitted.

A third aspect of the present invention relates to a method for preparing the above-described various types of Janus cells.

Specifically, the method for preparing Janus cells of the present invention includes the following steps:

a) preparing a metal-organic framework nanoparticle encapsulated with an active material (Metal-organic framework, MOF);

b) monodispersing metal-organic framework nanoparticles encapsulated with an active material in a solution;

c) adding a solution in which metal-organic framework nanoparticles encapsulated with an active material are monodispersed to the cells attached to the plate;

d) adding tannic acid to the cells attached to the plate of step c);

e) removing metal-organic framework nanoparticles that are not bound to cells; and

f) Separating cells adhered to the plate by treating with trypsin.

In the production method of the present invention, step a) is a step of preparing an active material encapsulated MOF, which can be performed according to a known method for preparing MOF nanoparticles, and a person skilled in the art can use MOF nanoparticles can be prepared using known methods according to the types of metal ions and organic ligands.

In the production method of the present invention, step b) is a step of monodispersing the MOF prepared in step a) in a solution before adding it to cells, and the MOF prepared according to a known method such as fractional centrifugation, fractional precipitation, etc. can be uniformly monodispersed in the solution.

In the manufacturing method of the present invention, step c) is a step of contacting the MOF encapsulated with the active material and the cells to coat the surface of the cell with the MOF encapsulated with the active material. The material-encapsulated MOF comes into contact with the surface of cells that are not attached to the plate, and thus the active material-encapsulated MOF can be asymmetrically coated.

In the production method of the present invention, step d) is a step of adding tannic acid to achieve a highly stable interaction between the encapsulated MOF and the encapsulated MOF and the cell membrane, through which the active material Internalization is prevented.

In the manufacturing method of the present invention, step e) is a step of removing MOF nanoparticles that are not bound to cells, and is repeated washing using the same culture medium contained in the plate to which cells are attached in step c). Unbound MOF nanoparticles can be removed.

In the manufacturing method of the present invention, step f) is a step of separating cells attached to the plate by treating with trypsin. It takes the form of a Janus structure.

In summary, in the present invention, Janus carrier cells coated with MOF interwoven on only one side of the Janus structure were developed as cell-mediated DDS. MOFs scrambled onto carrier cells cannot be internalized, thus protecting carrier cells from cytotoxic proteins contained in MOFs. The interaction of the target cell with the carrier cell is ensured by the bare surface of the Janus cell. Cytotoxic proteins in MOF are released when MOF reaches target cells in an acidic environment. Effective removal of cancer cells was confirmed quantitatively by introducing MOF-Janus cells containing proteinase K into a similar cancer environment in vivo . The entire process of preparing Janus cells takes less than 15 minutes, including synthesis of drug-loaded MOF nanoparticles, asymmetric attachment of nanoparticles to the cell surface, and separation of MOF-coated cells from the substrate. The ability of MOF-Janus cells to deliver a variety of proteins and multiple enzymes makes the system suitable as a general platform for cell-mediated drug delivery. It also enables co-delivery applications including combination therapy.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Synthesis of enzyme-encapsulated or non-encapsulated ZIF-8 nanoparticles

2-Methylimidazole (Hmim, 0.1 g/mL) dissolved in 1X PBS was added to an equal volume of 0.07 g/mL zinc acetate (Zn(OAc) 2 (aq.)) dissolved in 1X PBS. After stirring at room temperature for 10 minutes, particles were collected by centrifugation at 10,000 rpm for 5 minutes. The collected particles were washed several times with ethanol and water. For the synthesis of enzyme encapsulated ZIF-8, the enzyme of interest was added to 0.1 g/mL Hmim and 0.07 g/mL Zn(OAc) 2 (aq.). After stirring at room temperature for 10 minutes, centrifugation and washing with ethanol and water were repeated to collect particles.

Verification of stability of MOF nanoparticles after application of tannic acid

From a DDS perspective, the stability of drug-loaded MOF nanoparticles is an important issue, since unwanted leakage of the encapsulated drug can seriously impair the cell viability of the carrier. Therefore, before proceeding with the study on the formation of MOF-coated Janus cells, the stability of MOF nanoparticles after TA application was investigated in terms of nanoparticle morphology, crystallinity and porosity.

TA-coated ZIF-8 nanoparticles (ZIF-8-TA) were prepared by incubating ZIF-8 nanoparticles in TA solution (0.4 mg/mL) for 10 minutes with gentle agitation. As shown in (A) of FIG. 2, the average size of the ZIF-8 nanoparticles was 160 nm. Figure 3 (A) shows the polydisperse particle size distribution. After incubating ZIF-8 nanoparticles in TA solution (ZIF-8-TA) for 10 min, no noticeable morphological change was observed. Also, Fig. 2 (B) shows that the crystallinity of ZIF-8 is hardly affected by culturing. Changes in porosity and pore size were investigated through nitrogen adsorption-desorption measurements. As confirmed in (C) of FIG. 2, in both ZIF-8 and ZIF-8-TA, stable N ₂ adsorption was observed at low relative pressure, and the BET of ZIF-8 and ZIF-8-TA (Brunauer- Emmett-Teller) surface areas were 1599 and 1574 m ² /g, respectively. Also, as shown in (D) of FIG. 2 , transition of the porous structure did not occur due to the TA engraving process. These results show that the stability of MOF nanoparticles is maintained even after MOF coating with TA on cells. These results are encouraging because the stability of the nanoparticle encapsulation is important for safe protection of the carrier cells against the cytotoxic effects of drug molecules.

Preparation of MOF-coated Janus cells

3-1. Development of MOF-coated Janus cells

A breast cancer cell line (MDA-MB-231 cells) was chosen as a model circulating cell because it has been shown to homing to the source tumor and has been suggested as an excellent cancer cell-targeted drug delivery vehicle. First, MDA-MB-231 cells attached to extracellular matrix (ECM)-coated plates interact with ZIF-8 nanoparticles in aqueous conditions, facilitating the formation of multiple non-covalent bonds between nanoparticles and cell membrane components. . TA, which is widely used for various pharmacological and biomedical applications, has been introduced into the system to achieve highly stable multiple MOF-cell membrane and MOF-MOF interactions through polyvalent tannic acid complexation.

3-2. Tannic Acid Binder Assisted ZIF-8 Coating on Living Cells (ZIF-8/TA Coated Cells) and Formation of MOF-Janus Cells

Coating of ZIF-8 nanoparticles on living cells is described by Verma, A., Stellacci, F. "Effect of Surface Properties on Nanoparticle-Cell Interactions." Small 2010, 6, 12-21.] was performed with minor modifications. About 1Х10 ⁵ MDA-MB-231 cells (KCLB30026) were plated in a 12-well plate and cultured in DMEM (Dulbecco Modified Eagle's medium) at 37°C in a humidified 5% CO ₂ incubator. After reaching 80% confluence, the cells were rinsed with 1x PBS and then 500 μL of 20 mg/mL ZIF-8 nanoparticles monodispersed in 1x PBS solution was added to the cells. After shaking gently for 15 seconds, 500 μL of 32 μg/mL tannic acid dissolved in 1x PBS solution was added to the system for 30 seconds. Then, unbound ZIF-8 nanoparticles were removed from the system by repeated washing with DMEM. Formation of MOF-Janus cells was achieved by introducing trypsin (0.25 mg/mL) into ZIF-8 coated cells for 3 min with gentle shaking. The separated cells were collected by centrifugation and fresh DMEM was introduced into the MOF-Janus cells. The formation of ZIF-8 coated MOF-Janus cells was confirmed using an LSM 700 microscope (Zeiss, Axio Observer) and a flow cytometer (BD CytoFLEX).

B(i) and B(ii) of FIG. 3 show nanoparticles well attached to the cell surface. EDS (Energy-dispersive spectrometry) mapping showed a uniform distribution of Zn ²⁺ metal and confirmed that the ZIF-8 nanoparticles were successfully attached to the cell surface.

Fourier-transform infrared (FT-IR) spectroscopy was further performed on ZIF-8 and TA-coated MDA-MB-231 cells to confirm the formation of coordination bonds between TA and ZIF-8 nanoparticles. As shown in Figure 3C, Zhu et al., "SupraCells: Living Mammalian Cells Protected within Functional Modular Nanoparticle-Based Exoskeletons." Adv. Mater. 2019, 31, No. 1900545.], the characteristic peaks at 1179 and 994 cm ^-1 assigned to vibrations of C≡N and CN in the imidazole ring of ZIF-8 and at 1080 cm assigned to the stretch vibration of CO in tannic acid. Characteristic peaks at ^-1 were identified, confirming the presence of TA and zinc coordination bonds.

Next, the asymmetric coating of the MOF nanoparticles was confirmed using confocal microscopy. To visualize the location of the MOF nanoparticles with a fluorescence microscope, FITC-labeled MOF nanoparticles were used. Figure 4(A) shows that the MOF nanoparticles are asymmetrically attached to the particles. As shown in (B) of FIG. 4 , cell viability of MDA-MB-231 cells after asymmetric surface modification was 85.2, 82.4, and 88.8% at 0, 24, and 48 hours after coating, respectively.

When these MOF-coated carrier cells are removed from the plate, the MOF-coated Janus cells contain the portion of the cell in contact with the plate that becomes the bare surface of the Janus structure. These collected Janus cells were observed under a fluorescence microscope and SEM.

To observe ZIF-8 coated cells by SEM, ZIF-8 coated MOF-Janus cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich). Cells were then sequentially dehydrated with 25, 50, 75, 85, 95 and 100% ethanol solutions in PBS. They were then completely lyophilized overnight. Samples were sputtered with Pt before imaging. SEM micrographs of the samples were taken using high-resolution FE-SEM at an accelerating voltage of 10.0 keV.

Asymmetric fluorescence signals originating from MOF were maintained in free-floating MOF-coated cells as shown in (C) of FIG. 4 . Flow cytometry analysis demonstrated that more than 80% of the collected Janus cells carried MOF nanoparticles labeled with FITC, as shown in Fig. 4(D). The SEM micrograph shown in (E) of FIG. 4 shows the presence of nanoparticles in the form of fine-sized patches on the cell surface. In this regard, the developed nanoparticle patch shows that the drug is more stably attached to the carrier cell with the help of TA by making conformal contact around the curved cell surface, unlike conventional micro-sized backpacks.

Microenvironment binding and movement ability through endothelial monolayer

4-1. Confirmation of microenvironment bonding ability

In circulating cells such as circulating tumor cells or T-cells, membrane proteins such as integrin proteins play a central role in extravasation and migration through the endothelium or lymph nodes. Despite the aforementioned importance of integrin proteins, existing designs of c-DDS have attempted to fully conjugate drug molecules to the cell surface [Chambers, E., Mitragotri, S. "Prolonged Circulation of Large Polymeric Nanoparticles by Non-covalent Adsorption on Erythrocytes." J. Controlled Release 2004, 100, 111-119.; Chambers, E., Mitragotri, S. "Long Circulating Nanoparticles via Adhesion on Red Blood Cells: Mechanism and Extended Circulation." Exp. Biol. Med. 2007, 232, 958-966.; Shields, C. Wyatt, et al., "Cellular Backpacks for Macrophage Immunotherapy." Sci. Adv. 2020, 6, No. eaaz6579.; Klyachko, Natalia L., et al., "Macrophages with Cellular backpacks for Targeted Drug Delivery to the Brain." Biomaterials 2017, 140, 79-87.; Xia, Junfei, et al., "Asymmetric Biodegradable Microdevices for Cell-Borne Drug Delivery." ACS Appl. Mater. Interfaces 2015, 7, 6293-6299.].

Unlike previous designs, the MOF-coated Janus cells developed in the present invention preserve the biological and structural features of MDA-MB-231 cells. Therefore, in this example, naked MDA-MB-231 cells, Janus cells, and MDA-MB-231 cells coated with ZIF-8 nanoparticles on the entire surface were compared for cell-microenvironment interaction. To this end, the cells were separately seeded in ECM-coated commercial 12-well plates and cultured for 6 hours in a CO ₂ incubator at 36° C. with cell culture medium and 5% fetal bovine serum. After washing the cells several times with PBS, cell adhesion on the matrix was observed under a microscope.

5 (A) to (C) show that intact MDA-MB-231 cells and Janus cells adhered well to the substrate, but cells completely coated with ZIF-8 nanoparticles hardly adhered to the substrate. Differences in morphology were observed between intact and Janus cells, with adherent Janus cells typically retaining a confined morphology, whereas intact cells display an elongated morphology. The restricted morphology of Janus cells may be due to the tight ZIF-8 nanoparticles coated on the cell membrane that hold the cells tightly.

To confirm that the degradation of MOF did not affect the morphology and viability of Janus cells, the MOF coated cells were lysed by placing the Janus cells in an acidic culture medium at pH 6 for 1 hour. The medium was then exchanged with fresh cell culture medium at neutral pH. As shown in (D) and (E) of FIG. 5 , when the ZIF-8 nanoparticles were removed, the Janus cells started to elongate like the original MDA-MB-231, whereas the untreated Janus cells were round. shape was maintained. The restored cell viability was also confirmed by Calcein AM, a cell-permeable fluorescent probe that interacts with esterases present in the cytoplasm of healthy cells. As shown in FIG. 5(F) and FIG. 6 , Janus cells showed good cell viability even after removing the ZIF-8 nanoparticles.

4-2. Check your mobility

The migration activity of Janus cells was investigated using an in vitro model of confluent monolayers of HUVEC cells grown on poly(ethylene terephthalate) (PET; 8 μm pore) inserts. FITC-labeled Janus cells (1.5 Х 10 ⁵ cells/mL) suspended in medium (50% DMEM, 50% EBM, 0.1% bovine serum albumin) were added to the HUVEC cells contained in the upper chamber. Native MDA-MB-231 cells were used as controls. EGF, a well-known cancer cell chemoattractant, was loaded into the lower chamber. After culturing for 12 hours at 37° C., 5% CO ₂ , the cell insert was removed. Cells in the bottom chamber were collected and counted. The rate of Janus cell transduction across the HUVEC monolayer was calculated by subtracting the number of Janus cells originally recovered. Each experiment was repeated three times.

As shown in Fig. 5(G), the number of Janus cells transferred through the monolayer was slightly less than the number of original MDA-MB-231 cells, but the original MDA-MB-231 cells as well as the Janus cells Able to pass across HUVEC cell monolayers in vitro .

Determination of Proteinase K Encapsulation Efficiency and Release Profile by UV-Vis Spectroscopy

Confirmation of the successful formation and performance evaluation of Janus cells, we tested their potential use in cytotoxic drug delivery and cancer targeting. To this end, Proteinase K, a non-specific serine protease known to rapidly digest cell surface proteins and lyse cells, was selected as a mock drug delivered to target cancer tissues. However, before proceeding with the test, a few things should be checked and clarified. For example, of interest is encapsulation efficiency, defined as the amount of proteinase K encapsulated by MOF or the percentage of proteinase K successfully entrapped in MOF nanoparticles. Proteinase K-containing MOF of ZIF-8 by adding 0.07 mg/mL Zn(OAc)2 (aq.) to 2-methylimidazole (Hmim) aqueous solution containing various concentrations of proteinase K. A one-pot synthesis method was used to prepare (proteinase K@MOF), and the morphology of the proteinase K-encapsulated MOF nanoparticles was investigated by SEM.

As confirmed in Fig. 7A, the particle size of Proteinase K@MOF was slightly larger than that of original ZIF-8, which may be due to the effect of the enzyme on the nucleation and growth process of ZIF-8 [Wu, Xiaoling , et al., Facile synthesis of multiple enzyme-containing metal-organic frameworks in a biomoleculefriendly environment. Chem. Commun. 2015, 51, 13408-13411.].

As shown in (B) of FIG. 7, the amount captured or encapsulated by MOF was determined by dissolving the MOF in an acidic solution and then UV-vis and UV-vis for quantification using a standard curve generated with a known concentration of proteinase K. It was determined using Bradford analysis.

Specifically, SDS-washed proteinase K-encapsulated ZIF-8 nanoparticles were suspended in 1 mL of citric acid (pH 6) for 6 hours to release the encapsulated protein through acidic degradation. The resulting solution was treated using an ultrafiltration device (Millipore, 100 kDa). After washing twice with ultrapure water, the resulting solution (100 μL) was mixed with 1 mL of Bradford solution (Sigma-Aldrich), and the solution was incubated at room temperature for 5 minutes. Spectra were collected on a UV-2550 spectrometer (Shimadzu, Japan). The efficiency of protein encapsulation was calculated using the ratio of the absorbance of the protein collected from the protein encapsulated ZIF-8 nanoparticles to the absorbance of the protein at the concentration initially provided in the reaction mixture for proteinase K@MOF nanoparticle synthesis. For determination of the release profile, washed samples were incubated in citric acid (pH 6). Every 5-10 minutes, 100 μL of the sample solution was withdrawn and mixed with 1 mL of Bradford's solution. The amount of protein released from the MOF nanoparticles was measured using a UV-2550 spectrometer (595 nm). All experiments were performed in triplicate. Then, the total amount of protein content of the biocomposite was compared with the amount of protein obtained by dissolution.

Figure 8 (A) shows that the average efficiency was 30.0, 46.6 and 75.2% for proteinase K at concentrations of 0.5, 1 and 1.5 mg/mL, respectively. Encapsulation efficiency of protein concentration dependent increase was described by Zhu, Guixian et al., "A metal-organic zeolitic framework with immobilized urease for use in a tapered optical fiber urea biosensor." Microchim. Acta 2020, 187, no. 72.].

In vitro ( in vitro ) Delivery of proteinase K to cancer models

6-1. Delivery of Proteinase K to 2D Breast Cancer Cell Tissue Using MOF-Janus Cells and Measurement of Cell Viability

In the presence of bare proteinase K and proteinase K contained in MOF nanoparticles, cell viability was investigated by introducing them into MDA-MB-231 cells attached to a glass substrate. To this end, proteinase K@MOF nanoparticles containing 0.15 mg of proteinase K were mixed with (i) 1 mL of serum-free cell culture medium and (ii) 0.15 mg of exposed proteinase K dispersed in the same medium. only dispersed.

As shown in (B-i) of FIG. 8, within 1 hour, most of the cells cultured with the exposed proteinase K were detached from the glass substrate, but the MDA-MB-incubated with proteinase K@MOF nanoparticles 231 cells established stable attachment to the substrate as shown in (B-ii) of FIG. 8 .

Additionally, an MTS assay was performed to quantify cell viability. Two-dimensional (2D) breast cancer tissue was prepared by seeding 1Х10 ⁴ MDA-MB-231 cells in a 96-well plate. When a confluent monolayer of tissue was formed, MOF-Janus cells containing proteinase K at concentrations of 1, 0.1 and 0.01 μg/mL were introduced into each well. To induce the release of proteinase K from the MOF nanoparticles, fresh DMEM at pH 6 was introduced into the system. Cancer tissue viability at various incubation times was measured using the MTS assay kit. As a control, 2D breast cancer tissue cultured with the same number of MOF-Janus cells without proteinase K was used. All experiments were performed in triplicate.

The survival rate determined by the MTS assay was 39.7% for the exposed proteinase K group and 93.9% for the proteinase K@MOF exposed group, as shown in FIG. 8(C). These results confirm the stable encapsulation of proteinase K inside the MOF nanoparticles and the successful blocking of the direct interaction between the proteinase K enzyme and MDA-M-231 cells.

Figure 8 (D) shows TA-assisted stable binding of proteinase K@MOF to cells identified through confocal micrographs. To better visualize proteinase K localization in cells, proteinase K was labeled with Cy5 fluorophore prior to MOF encapsulation and cell surface attachment.

In addition, in order to quantify the amount of proteinase K loaded on the carrier cells, MDA-MB-231 cells (1Х10 ⁶ ) were cultured in each well of a 12-well plate, and about 20 mg of proteinase K containing MOF was introduced into each well to asymmetrically coat cells with proteinase K@MOF. Unbound MOF nanoparticles were collected from the supernatant, and the nanoparticles were digested in a citric acid phosphate solution (pH 6) for 6 hours. The amount of recovered protein was measured using UV-vis and Bradford assay. The amount of proteinase K loaded into the carrier cells was calculated by subtracting the amount of proteinase K recovered from the original amount of proteinase K treated with 1Х10 ⁶ MDA-MB-231 cells. Each experiment was performed in triplicate. As a result, the total amount of proteinase K attached per 1Х10 ⁶ cells was calculated to be 0.167 mg, and 14.8% of the total amount of proteinase K@MOF treated was confirmed to be loaded per 1x10 ⁶ carrier cell.

Protection of MDA-MB-231 carrier cells against cytotoxic drugs provided by MOF was achieved by culturing carrier cells coated with MOF nanoparticles containing proteinase K in a CO ₂ incubator with serum-free cell culture medium. Confirmed. An MTS assay was performed to quantify cell viability, and exposed MOF-nanoparticle-connected cells were used as controls.

Figure 8(E) shows that the cell viability after 1 hour and 6 hours were 98.1% and 86.2%, respectively, confirming successful carrier cell protection against drug toxicity. Figure 9 (A) shows that the cell viability of all coated cells was 74.6% after 6 hours. The slight decrease in cell viability during longer incubation times was presumed to be due to the release of proteinase K loosely bound to the surface of MOF nanoparticles.

Finally, to test the MOF-Janus cell-based c-DDS for cancer treatment, the release behavior of proteinase K from MOF and the required dose were investigated. For release behavior, proteinase K@ZIF-8 containing 0.15 mg of proteinase K was dissolved in DMEM at pH 6, a pH known to be common in the extracellular and interstitial spaces of tumors, and the amount of protein in the solution was Measured over time using UV-vis and Bradford analysis.

As confirmed in (B) of FIG. 9, 100% protein release was observed within 60 minutes. Although the release behavior is pH dependent, the rapid dissolution tendency of ZIF-8 nanoparticles can be found in [(a) Carraro, Francesco, et al., "Phase dependent encapsulation and release profile of ZIF-based biocomposites." Chem. Sci. 2020, 11, 3397-3404.; (b) Badea, Madalina Andreea, et al., "Infuence of Matrigel on Single- and Multiple-Spheroid Cultures in Breast Cancer Research." SLAS Discovery 2019, 24, 563-578.].

To optimize the dose, three batches of Janus cells containing 1, 0.1, and 0.01 μg/mL proteinase K, respectively, were tested for confluentity of MDA-MB-231 cells (2D breast cancer tissue) cultured in 96-well plates. introduced into the monolayer. To mimic the cancer microenvironment, the cell culture medium was adjusted to pH 6 and cell viability was quantified by the MTS assay over time.

Figure 8 (F) shows that the viability of 2D breast cancer tissues exposed to Janus cells with 1 μg/mL proteinase K decreased from 92.0% after 1 hour to 32.5% and 27.2% after 3 and 5 hours, respectively. When the dose was reduced to 0.1 μg/mL, the corresponding survival rates were 89.2, 69.2, and 30.9% for 1, 3, and 5 hour incubations, respectively. When further decreased to 0.01 μg/mL, no significant change in cell viability was observed, and cell viability rates were 79.9, 78.5 and 80.9% for 1 hour, 3 hours and 5 hours of incubation, respectively.

6-2. Transfer of proteinase K to cancer spheroids using MOF-Janus cells and measurement of cell viability

For in vivo ( in vivo ) similar cancer treatment, three-dimensional tumor spheroids were generated by culturing MDA-MB-231 mixed with Matrigel through a hanging drop technique (hanging drop technique) [Hu, CMJ , Zhang, L. "Nanoparticle-based Combination Therapy Toward Overcoming Drug Resistance in Cancer." Biochem. Pharmacol. 2012, 83, 1104-1111.].

Specifically, formation of cancer spheroids was performed with 3D tissue models. Matrigel (Corning) was first diluted with DMEM containing 5% FBS, 1x Pen Strep to a final concentration of 0.1 mg/mL. 1 ml of diluted Matrigel was mixed with (8-10)Х10 ⁵ MDA-MB-231 cells. Using a 100 μL pipette, a drop of 30 μL was added to the bottom of a 24-well plate. The well plate was inverted and incubated overnight at 37° C./5% CO ₂ to form a gel. After culturing for 2 days in a CO ₂ incubator with a cell culture medium containing FBS, MOF-Janus cells having proteinase K at a concentration of 1 μg/mL were introduced into the system and co-cultured for 4 hours. Then, fresh DMEM at pH 6 was added to the system to induce the release of proteinase K from the MOF nanoparticles. Cancer tissue viability at various incubation times was investigated through a live/dead assay using Calcein AM and PI staining as shown in FIG. 8 (G). A confocal microscope was used to visualize live and dead cells. Z-stacking was performed to further investigate the location of dead cells in the 3D gel.

As shown in FIG. 10 , when Janus cells containing 1 μg/mL proteinase K were introduced into the cancer spheroid system, stable attachment of the Janus cells to the surface of the Matrigel-embedded cancer spheroids was observed. As shown in FIG. 8(H), when conditions such as the tumor extracellular environment were mimicked in the system, rapid death of tumor spheroid cells and Janus cells occurred within 3 hours. These results demonstrate the toxic therapeutic protein delivery effect and cancer targeting efficiency of the c-DDS developed in the present invention.

Development of polyprotein-loaded Janus cells

A variety of enzymes and the ability to transport and deliver multiple enzymes will help establish Janus cells as a general platform for cell-mediated drug delivery. To prove this, in this example, an MOF encapsulating proteinase K, urease, and bovine serum albumin (BSA) was prepared.

7-1. Preparation of fluorophore-labeled proteins

Proteinase K, BSA, and urease were separately prepared by labeling with Cy5, FITC, and Cy5.5 fluorophores, respectively. The enzyme of interest was first dissolved at 1 mg/mL in PBS (1x; pH 7.4), then 4 μL of sulfo-fluorophore-NHS ester (1 mM; Lumiprobe) was added and incubated for 30 minutes. Then, the modified protein was purified three times with PBS through a 100 kDa filter (Millipore) while centrifuging at 3600 rpm for 10 minutes.

7-2. Formation of MOF-Janus cells loaded with multiple proteins

The MOF encapsulated enzyme was prepared separately by mixing fluorescently labeled proteinase K, BSA and urease with the ZIF-8 precursor. The collected nanoparticles were resuspended in fresh DMEM and equal volumes of different ZIF-8 encapsulated MOFs were mixed together. Then, 500 μL of the mixed ZIF-8 nanoparticle encapsulated enzyme was added to the 12-well plate containing MDA-MB-231 cells. After gently agitating for 15 seconds, 500 μL of 32 μg/mL tannic acid dissolved in 1x PBS solution was added to the system for 30 seconds. Then, unbound ZIF-8 nanoparticles were removed from the system by repeated washing with DMEM. The formation of MOF-Janus cells coated with ZIF-8 was confirmed using an LSM 700 microscope (Zeiss, Axio Observer).

Simple mixing of MOF-containing proteins with mammalian cells and subsequent TA-assisted ligation resulted in cells loaded with several enzymes. Figure 12 shows the results of successful co-loading of two and three proteins of BSA@MOF, proteinase K@MOF and urease@MOF into cells. The ability to transport various enzymes in the MOF of the Janus cell DDS developed in the present invention is expected to be useful for combination therapy, which is a treatment combining two or more therapeutic agents as a cornerstone of cancer treatment, which is receiving great attention.

statistical analysis

The statistical significance of the results obtained was determined using Student's t-test. The significance level was chosen at a minimum p-value of 0.05.

Claims (15)

A Janus cell in which a part of the surface is asymmetrically coated with metal-organic framework nanoparticles encapsulated with an active material,
The metal-organic framework nanoparticles are bound to the cell surface by tannic acid, Janus cells. The Janus cell according to claim 1, wherein the metal-organic framework is zeolitic imidazolate framework-8 (ZIF-8; 2-methylimidazole, zinc salt). The Janus cell according to claim 2, wherein the ZIF-8 zinc forms a coordinate bond with tannic acid to induce binding between ZIF-8 nanoparticles. The Janus cell according to claim 1, wherein the metal-organic framework nanoparticles are coated on 30 to 80% of the entire cell surface. The Janus cell according to claim 1, wherein the metal-organic framework nanoparticles encapsulate one or two or more different active substances. The Janus cell according to claim 1, wherein the encapsulated active substance is at least one selected from the group consisting of compounds, DNA, proteins, peptides, anticancer agents, fluorescent particles, lipids, and cells. The Janus cell according to claim 1, wherein the type of Janus cell is at least one selected from the group consisting of circulating tumor cells, immune cells, red blood cells, and stem cells. The Janus cell according to claim 1, wherein the Janus cell has the characteristics of preventing internalization of the metal-organic framework in which the active material is encapsulated and preserving the ability to bind to the microenvironment. The Janus cell according to claim 1, wherein the Janus cell releases the encapsulated active substance in an acidic environment. A Janus cell whose surface is asymmetrically coated with zeolitic imidazolate framework-8 (ZIF-8; 2-methylimidazole, zinc salt) nanoparticles encapsulated with Proteinase K,
The Janus cell type is a blood circulating tumor cell, and the ZIF-8 nanoparticles are bound to the cell surface by tannic acid. 11. The Janus cell of claim 10, wherein the proteinase K is encapsulated at a concentration of 0.05 μg/mL to 2 μg/mL. Claims 1 to 11, wherein any one of the Janus cells, comprising a composition for active substance delivery. A pharmaceutical composition comprising the Janus cells of any one of claims 1 to 11. A pharmaceutical composition for anticancer, comprising the Janus cells of any one of claims 1 to 11. A method for preparing the Janus cells of claim 1, comprising the following steps:
a) preparing metal-organic framework nanoparticles in which an active material is encapsulated;
b) monodispersing metal-organic framework nanoparticles encapsulated with an active material in a solution;
c) adding a solution in which metal-organic framework nanoparticles encapsulated with an active material are monodispersed to the cells attached to the plate;
d) adding tannic acid to the cells attached to the plate of step c);
e) removing metal-organic framework nanoparticles that are not bound to cells; and
f) Separating cells adhered to the plate by treating with trypsin.

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