WO2023128566A1 - Microrobot including anti-cancer bacteria and magnetic nanoparticles and manufacturing method therefor - Google Patents

Abstract

The present invention relates to a porous microsphere having anticancer bacteria, etc. loaded thereto and a use thereof. A porous microsphere according to an aspect has excellent bacterial spore encapsulation efficiency and exhibits improved ability to targeting tumor sites due to the magnetic nanoparticle loaded therein with the aid of an external magnetic navigation system, and its movement in the human body can be monitored by bio-imaging.

Description

Microrobot including anti-cancer bacteria and magnetic nanoparticles and manufacturing method thereof

The present invention relates to porous microspheres capable of loading anti-cancer bacteria and the like, a manufacturing method thereof, and a use thereof.

Cancer is the leading cause of death worldwide, accounting for 7.6 million deaths in 2008 (about 13% of all deaths). Cancer is a general term for a large group of diseases that can affect specific areas of the body. Other terms used are malignancy and neoplasia. Cancer is the uncontrolled growth and spread of abnormal cells. Growths often invade surrounding tissue and can metastasize to distant sites. Metastasis is the leading cause of death from cancer. Cancer-related deaths worldwide continue to rise and are estimated to kill 13.1 million people by 2030.

Cancer treatment requires careful selection of one or more interventions such as surgery, radiotherapy and/or chemotherapy. The goal is to cure disease or significantly prolong life while improving the quality of life of patients. Various forms of radiation such as X-rays, gamma-rays, UV-rays, laser light, microwaves, electron beams and particle beams of eg neutrons, carbon ions and protons have been used to treat malignant diseases. Some of these radiations have been used in these applications, along with radiation sensitizers. Electromagnetic and ionizing radiation can actually protect cells from growth and differentiation by destroying their "DNA" molecules. This effect can be explained by the action of particles or waves that will move within a defined volume and create ionization that releases electrons and free radicals that create energetic deposits into that volume.

On the other hand, Nano Technology (NT) is a technology for manipulating nanometer- sized materials, which is one billionth of a meter. It refers to the technology of synthesizing, assembling, controlling, or measuring and identifying the properties of atomic , molecular and supramolecular materials. Nanotechnology is included in various scientific fields such as surface science, organic chemistry, molecular biology, semiconductor physics, and microfabrication, and has a very wide range of applications.

Accordingly, the present inventors developed porous microspheres loaded with anticancer bacteria and magnetic nanoparticles, and completed the present invention by confirming that the microspheres can target and control tumor sites for effective cancer treatment.

One aspect provides porous microspheres comprising a biocompatible polymer and a cationic polymer.

Another aspect is 1) preparing a first solution containing a biocompatible polymer and a pore-forming agent; 2) preparing a second solution containing a surfactant; 3) preparing microspheres using the first solution and the second solution; and 4) removing the pore-forming agent from the prepared microspheres.

Another aspect provides anticancer microspheres comprising the porous microspheres and oncolytic bacteria or spores thereof.

Another aspect provides a pharmaceutical composition for treating or preventing cancer, including the anticancer microspheres.

Another aspect provides a method for treating or preventing cancer, comprising administering the pharmaceutical composition for treating or preventing cancer to a subject.

One aspect is to provide porous microspheres comprising a biocompatible polymer and a cationic polymer.

The term "microspheres" used herein generally means a material in the form of a sphere having a diameter of 1 to 1000 μm. At this time, the sphere can be used in the sense of encompassing not only the sphere of mathematical definition, which is a three-dimensional shape consisting of all points at the same distance from one point, but also those of an outwardly round shape. That is, it may include an error range generated during the measurement or manufacturing process.

As used herein, the term "biocompatible polymer" refers to a polymer having in vivo safety that does not cause high cytotoxicity and inflammatory reactions when administered in vivo, and may refer to a biodegradable polymer. there is.

The biocompatible polymer is polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyethylene glycol (PEG), polyamino acid, polylactite, polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate, chondroitin sulfate, pullulan and Any one selected from chitosan; a polymer in which two or more of these are blended; And it may be selected from the group consisting of these two or more copolymers, but is not limited thereto.

In one embodiment, the biocompatible polymer may include polylactic acid (PLA).

The porous microspheres may further include magnetic nanoparticles.

The term "hydrophilic amine-coated magnetic nanoparticle" used herein refers to a nanometer-sized structure or material exhibiting magnetic properties. The term "magnetism" refers to the magnetic properties exhibited by a material. All materials interact with a magnetic field to generate an attractive or repulsive force. That is, when a magnetic field is applied to a material, it is magnetized, and the material is classified into a ferromagnetic material, a paramagnetic material, a diamagnetic material, a ferrimagnetic material, and the like, depending on how the material is magnetized. The term "nanoparticle" refers to a structure or material having a size of nanometers (nm). The size of a nanometer is a reduction of the size of a micron meter (10-6) to 1/1,000, and when the size of a material is reduced to the nanometer level, it exhibits various and unique physical, chemical, mechanical and electronic properties.

The magnetic nanoparticles may be one or two or more selected from the group consisting of iron, cobalt, nickel, oxides thereof, and alloys thereof, but are not limited thereto.

The magnetic nanoparticles are composed of iron, iron oxide selected from among Fe ₂ O ₃ , Fe ₃ O _{4 ,} Fe ₄ O _{5 ,} Fe ₅ O _{6 ,} Fe ₅ O _{7 ,} Fe ₁₃ O _{19 ,} and Fe ₂₅ O ₃₂ and CoFe ₂ It may be at least one selected from the group consisting of ferrite selected from O ₄ , and MnFe ₂ O ₄ .

The porous microspheres may include a cationic polymer, and specifically, a surface of the porous microspheres may be coated or modified with the cationic polymer.

The cationic polymer is polyethylenimine (PEI), polyamidoamine (PAA), poly-L-lysine (PLL), poly-D-lysine (PDL), protamine, poly [2-(N,N-dimethylamino)ethyl methacrylate] (Poly[2-(N,N-dimethylamino)ethyl methacrylate], (PDMAEMA)), Poly(amino-co-ester, PAE ), chitosan, dextran, cationic gelatin, cationic cyclodextrin, and polypropylenimine.

In one embodiment, the cationic polymer may be polyethyleneimine (polyethylenimine, PEI).

The microspheres are porous microspheres having a porous structure.

The size of the pores of the porous microspheres may be 1 to 10 μm, specifically, the size of the pores is 1 to 10 μm, 1 to 8 μm, 1 to 6 μm, 1 to 5 μm, 1 to 4 μm, 1 to 10 μm. It may be 3 μm, 1 to 2 μm, 2 to 10 μm, 2 to 8 μm, 2 to 6 μm, 2 to 5 μm, 2 to 4 μm, or 2 to 3 μm.

The number of open pores/area per 100 μm ² of the porous microsphere may be 0.1 to 6, specifically 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 0.5 to can be 1

The particle size (diameter) of the porous microspheres may be 50 to 500 μm, specifically, 50 to 500 μm, 50 to 400 μm, 50 to 300 μm, 50 to 250 μm, 50 to 200 μm, 100 to 500 μm. μm, 100 to 400 μm, 100 to 300 μm, 100 to 250 μm, 100 to 200 μm, 150 to 500 μm, 150 to 400 μm, 150 to 300 μm, 150 to 250 μm, or 150 to 200 μm.

In one embodiment, the size of the pores of the microspheres may be large enough to enclose bacteria or bacterial spores.

The microspheres may encapsulate bacteria, their dead cells, their dried products, and/or their spores, and may be encapsulated in the pores of the microspheres.

The bacterium is a bacterium having anticancer activity, and specifically, may be an oncolytic bacterium.

The oncolytic bacteria are Clostridium perfringens, Clostridium histolyticus, Clostridium butyricum , Clostridium oncolyticum, Clostridium oncolyticum , and Clostridium oncolyticum. Lithium sporogenes ( Clostridium sporogenes ), Clostridium novyi ( Clostridium novyi ) and Clostridium novyi -NT ( Clostridium novyi -NT) may include one or more selected from the group consisting of, specifically Clostridium It may be novyi-NT ( Clostridium novyi -NT).

The microspheres may have movement and/or position of particles controlled by an external magnetic field such as a magnet. In addition, the microspheres can be identified and located through magnetic resonance imaging (MRI), and specifically, they can be identified and/or located in vivo.

In one embodiment, since the microspheres contain magnetic nanoparticles, the movement of the particles can be controlled by an external magnetic field, and the position in the living body can be determined through magnetic resonance imaging (MRI). there is

The porous microspheres may be provided in a form included in a dispersion, and the dispersion may have a concentration capable of maintaining a properly dispersed state of the microspheres. Specifically, the dispersion may include one or more selected from carboxymethyl cellulose (CMC) and alginate (Alg). In addition, the concentration of the dispersion may be 3 wt% or less, specifically 0.1 to 3 wt%, 0.1 to 2.5 wt%, 0.1 to 2 wt%, 0.5 to 3 wt%, 0.5 to 2.5 wt%, 0.5 to 2 wt% %, 1 to 3 wt%, 1 to 2.5 wt% or 1 to 2 wt%.

The porous microspheres may be prepared by the method described below.

Another aspect is 1) preparing a first solution containing a biocompatible polymer and a pore-forming agent; 2) preparing a second solution containing a surfactant; 3) preparing microspheres using the first solution and the second solution; and 4) removing the pore forming agent from the prepared microspheres. The same parts as described above are also applied to the method.

In the method, step 1) may be a step of preparing an oil phase (first solution) by mixing and/or dissolving the biocompatible polymer and the pore-forming agent in an organic solvent.

The organic solvent may be used without limitation as long as it can mix and/or dissolve the biocompatible polymer and the pore-forming agent, and a highly hydrophobic and volatile organic solvent may be used as a preferred organic solvent. Specifically, the organic solvent may be one selected from the group consisting of chloroform, dichloromethane, dimethylformamide, ethyl acetate, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, and mixed solvents thereof. At this time, the content of the organic solvent may be 1 to 99% by weight based on the weight of the total oil phase, but is not limited thereto.

The pore-forming agent is added to impart an open pore structure to the finally prepared porous microspheres, and is not particularly limited as long as it can be removed after manufacturing the microspheres.

The pore formers are camphene, camphor, naphthalene, menthol, thymol, coumarin, vanillin, salicylamide, 2-aminopyridine, t-butanol, trichloro-t-butanol, imidazole, dimethylsulfone, urea, 2- It may contain one or more selected from the group consisting of amidopyridine and undecane (UD).

In one embodiment, the pore former may be undecane.

The first solution may include 0.1 to 5 wt% (mass percentage) of a biocompatible polymer, specifically, the biocompatible polymer is 0.1 to 5 wt%, 0.1 to 4 wt%, 0.1 to 3 wt%, 0.1 to 2 wt%, 0.1 to 1 wt%, 0.5 to 5 wt%, 0.5 to 4 wt%, 0.5 to 3 wt%, 0.5 to 2 wt%, 0.5 to 1 wt%, 0.6 to 2 wt% or 0.6 to 2 wt% It may be included in a concentration of 1 wt%.

The first solution may include 0.1 to 10 wt% of the pore former, and specifically, 0.1 to 10 wt%, 0.1 to 8 wt%, 0.1 to 6 wt%, or 0.1 to 4 wt% of the pore former. %, 1 to 10 wt%, 1 to 8 wt%, 1 to 6 wt%, 1 to 4 wt%, 2 to 10 wt%, 2 to 8 wt%, 2 to 6 wt% or 2 to 4 wt% It may be included in concentration.

In the above method, the pore structure of the porous microspheres can be controlled by adjusting the content of the pore-forming agent, specifically, the ratio of the pore-forming agent and the biocompatible polymer.

In the above method, the first solution may include the pore-forming agent and the biocompatible polymer in a ratio (w:w) of 1:1 to 6:1, specifically 1:1 to 6:1, 1 :1 to 5.83:1, 1:1 to 5.5:1, 1:1 to 5:1, 1:1 to 4.5:1, 1:1 to 4:1, 1:1 to 3.5:1, 1:1 to 3:1, 2:1 to 6:1, 2:1 to 5.83:1, 2:1 to 5.5:1, 2:1 to 5:1, 2:1 to 4.5:1, 2:1 to 4 :1, 2:1 to 3.5:1, 2:1 to 3:1, 2.5:1 to 6:1, 2.5:1 to 5.83:1, 2.5:1 to 5.5:1, 2.5:1 to 5:1 , 2.5:1 to 4.5:1, 2.5:1 to 4:1, 2.5:1 to 3.5:1, 2.5:1 to 3:1, 3:1 to 6:1, 3:1 to 5.83:1, 3 :1 to 5.5:1, 3:1 to 5:1, 3:1 to 4.5:1, 3:1 to 4:1, or 3:1 to 3.5:1.

The first solution may further include magnetic nanoparticles.

The first solution may contain 0.01 to 0.1 wt% of magnetic nanoparticles, and specifically, 0.01 to 0.1 wt%, 0.01 to 0.08 wt%, 0.01 to 0.06 wt%, or 0.01 to 0.04 wt% of the magnetic nanoparticles. %, it may be included in a concentration of 0.02 to 0.1 wt%, 0.02 to 0.08 wt%, 0.02 to 0.06 wt% or 0.02 to 0.04 wt%.

In one embodiment, the first solution may include a biocompatible polymer, a pore forming agent, an organic solvent, and magnetic nanoparticles.

In the method, step 2) may be a step of preparing an aqueous phase (second solution) by mixing and/or dissolving a surfactant in a hydrophilic solvent.

The hydrophilic solvent may be water, C1-4 alcohol or a mixed solvent thereof, but is not limited thereto.

The second solution is an aqueous phase and may contain a surfactant to adjust the interface between the oil phase polymer solution and the hydrophilic solvent. As the surfactant, any surfactant having a hydrophilic-lipophilic balance (HLB) value of 10 or more may be used.

The surfactant may include polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl butyral, polyvinyl methyl ether, polyvinyl ether, and mixtures thereof, and may specifically include polyvinyl alcohol. The amount of the surfactant may be preferably 0.1 to 10% by weight in the aqueous phase in consideration of the particle size of the porous microspheres to be produced.

The second solution may include 0.1 to 5 wt% of a surfactant, specifically, the surfactant is 0.1 to 5 wt%, 0.1 to 4 wt%, 0.1 to 3 wt%, 0.1 to 2 wt%, 0.1 to 1 wt%, 0.5 to 5 wt%, 0.5 to 4 wt%, 0.5 to 3 wt%, 0.5 to 2 wt%, 0.5 to 1 wt%, 1 to 5 wt%, 1 to 4 wt%, 1 to 2 wt% It may be included in a concentration of 3 wt% or 1 to 2 wt%.

In one embodiment, the second solution may include a hydrophilic solvent and a surfactant.

Steps 1) and 2) may be performed sequentially, in reverse order, or simultaneously.

In the method, step 3) is to prepare microspheres using the first solution and the second solution, and specifically, a) mixing the first solution and the second solution to form an emulsion, and b) ) obtaining microspheres from the emulsion.

The step a) may be to prepare oil-in-water emulsions by using the first solution as a discontinuous phase and flowing the second solution into a fluid device as a continuous phase.

In step a), the flow rate of the discontinuous phase may be set to 0.01 to 1 mL/min, specifically 0.01 to 1 mL/min, 0.01 to 0.8 mL/min, 0.01 to 0.5 mL/min, and 0.01 to 0.3 mL/min. mL/min, 0.01 to 0.2 mL/min, 0.01 to 0.1 mL/min, 0.05 to 1 mL/min, 0.05 to 0.8 mL/min, 0.05 to 0.5 mL/min, 0.05 to 0.3 mL/min, 0.05 to 0.2 mL /min, 0.05 to 0.1 mL/min, 0.1 to 1 mL/min, 0.1 to 0.8 mL/min, 0.1 to 0.5 mL/min, 0.1 to 0.3 mL/min, or 0.1 to 0.2 mL/min. can

In step a), the flow rate of the continuous phase may be set to 0.5 to 5 mL/min, specifically 0.5 to 5 mL/min, 0.5 to 4 mL/min, 0.5 to 3 mL/min, and 0.5 to 2 mL/min. mL/min, 0.5 to 1.5 mL/min, 1 to 5 mL/min, 1 to 4 mL/min, 1 to 3 mL/min, 1 to 2 mL/min, 1 to 1.5 mL/min, 1.3 to 5 mL /min, 1.3 to 4 mL/min, 1.3 to 3 mL/min, 1.3 to 2 mL/min, or 1.3 to 1.5 mL/min.

The step b) is to prepare microspheres including the biocompatible polymer and the pore-forming agent by removing the organic solvent from the emulsion, and the organic solvent may be removed by evaporation.

Step 4) may include forming pores on the surface and/or inside of the body of the microspheres by removing the pore-forming agent from the microspheres containing the biocompatible polymer and the pore-forming agent.

Removal and/or elution of the pore-forming agent from the microspheres may include lyophilization, sublimation under atmospheric conditions, and/or a solvent capable of dissolving the pore-forming agent.

In addition, the step of removing the pore forming agent may be performed for 10 minutes to 100 hours, but is not limited thereto.

The method may further include drying the obtained porous microspheres, and the drying may include lyophilization.

The method may further include modifying or coating the surface of the prepared microspheres with a cationic polymer.

Microspheres prepared by the above method can effectively enclose bacteria or bacterial spores. Accordingly, the method may further include encapsulating bacteria or bacterial spores.

Another aspect is 1) the porous microspheres; and 2) anticancer microspheres comprising oncolytic bacteria or their spores. The same parts as described above also apply to the microspheres.

The anticancer microspheres may have a form in which tumor lytic bacteria and/or their spores are encapsulated in pores of the porous microspheres.

According to one embodiment, it was confirmed that the porous microspheres have excellent encapsulation efficiency of oncolytic bacteria and/or their spores, and can also effectively release tumor lytic bacteria and/or their spores.

Another aspect is to provide a pharmaceutical composition for treating or preventing cancer comprising the anticancer microspheres. The same parts as described above also apply to the composition.

As used herein, the term "cancer" refers to a tumor that grows abnormally due to uncontrolled excessive growth of body tissue, or a disease that forms a tumor. The cancer is gastric cancer (intestinal gastric cancer, diffuse gastric cancer), liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, colon Cancer, perianal cancer, colon cancer, breast cancer, cervical cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system It may be a lymphoma, a spinal cord tumor, a brainstem glioma or a pituitary adenoma.

As used herein, the term "treatment" refers to all activities that improve or beneficially change the symptoms of cancer disease by administration of the composition of the present invention.

As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of a cancer disease or disease by administration of the composition of the present invention.

The composition may include a dispersion, and the dispersion may have a concentration capable of maintaining an appropriately dispersed state of the microspheres. Specifically, the dispersion may include one or more selected from carboxymethyl cellulose (CMC) and alginate (Alg). In addition, the concentration of the dispersion may be 3 wt% or less, specifically 0.1 to 3 wt%, 0.1 to 2.5 wt%, 0.1 to 2 wt%, 0.5 to 3 wt%, 0.5 to 2.5 wt%, 0.5 to 2 wt% %, 1 to 3 wt%, 1 to 2.5 wt% or 1 to 2 wt%.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms. Here, the meaning of "pharmaceutically acceptable" means that the application (prescription) does not have toxicity more than is adaptable without inhibiting the activity of the active ingredient. Any type of carrier that can be used in the pharmaceutical composition may be used as long as it is commonly used in the art and is pharmaceutically acceptable. Non-limiting examples of the carrier include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or Mineral oil etc. are mentioned. These may be used alone or in combination of two or more. The pharmaceutical composition may be prepared as an oral formulation or parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods.

When formulating the pharmaceutical composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but may not be limited thereto.

When the pharmaceutical composition is prepared as an oral dosage form, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc. It can be made into a formulation. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil etc. are mentioned. In the case of formulation, if necessary, diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included.

When the pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. In the case of formulation as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterilization for injection water, isotonic solutions such as 5% dextrose, and the like can be used. When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated into suppositories, the base is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like may be used.

The pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dose level is dependent on the severity of the disease, the activity of the drug, and the patient's Age, weight, health, sex, sensitivity to the drug of the patient, administration time of the composition of the present invention used, route of administration and excretion rate, treatment period, factors including drugs used in combination or simultaneous use with the composition of the present invention used, and others It can be determined according to factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with components known to exhibit therapeutic effects on known cancer diseases. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

The dose of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition herein is particularly Although not limited, it may be administered once a day or administered several times by dividing the dose. The dosage or frequency of administration is not intended to limit the scope of the present application in any way.

Another aspect is to provide a method for treating or preventing cancer, comprising administering the anti-cancer microspheres or pharmaceutical composition to a subject. The same parts as described above are also applied to the method.

As used herein, the term "subject" may include, without limitation, mammals, birds, reptiles, farmed fish, etc., including dogs, cats, mice, livestock, humans, etc. that have or are at risk of developing cancer diseases, and , the subject may be a human.

The pharmaceutical composition may be administered singly or in multiple doses in a pharmaceutically effective amount. At this time, the composition may be formulated and administered in the form of a liquid, powder, aerosol, injection, infusion (intravenous gel), capsule, pill, tablet, suppository or patch. The administration route of the anticancer microspheres or the pharmaceutical composition containing them may be administered through any general route as long as it can reach the target tissue.

The pharmaceutical composition is not particularly limited thereto, but as desired, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. It can be administered through the route of. However, oral administration can be administered in an unformulated form, and since the active ingredients of the pharmaceutical composition can be denatured or decomposed by gastric acid, the oral composition is formulated to coat the active agent or protect it from degradation in the stomach. It can also be administered orally in the form of a localized form or an oral patch. In addition, the composition may be administered by any device capable of transporting active substances to target cells.

Porous microspheres according to one aspect have excellent bacterial spore encapsulation efficiency, and due to the magnetic nanoparticles contained therein, they can improve targeting ability to tumor sites using an external magnetic field induction device and monitor movement in the human body through bio-imaging. there is.

1 is a diagram showing SEM images of microspheres according to the UD/PLA ratio.

2 is a diagram showing the size and pore size of the microspheres (n = 100, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001; ns, not significant ).

3 is a view showing SEM images of microsphere pores according to the UD/PLA ratio.

4A is a diagram showing the pore size of the microspheres, and B is a diagram showing the number of open pores per 100 μm ² area of the microspheres (n = 100, *p <0.05, **p <0.01, ***p < 0.001, ****p <0.0001; ns, not significant).

FIG. 5 shows brightfield and SEM images of C. novyi -NT spores separated by a Percoll density gradient.

6 is a view showing the reactivity of microspheres containing magnetic nanoparticles to a magnet.

7 is a diagram showing the mobility of microspheres including magnetic nanoparticles by a magnet.

8 is a diagram showing a T2-weighted magnetic resonance (MR) phantom image of microspheres including magnetic nanoparticles.

9 is a diagram showing a T2-weighted magnetic resonance (MR) phantom image of microspheres not containing magnetic nanoparticles.

10 shows T2-weighted MR images of CT26 tumor-bearing mice before (top) and after (bottom) intratumoral injection of microspheres containing magnetic nanoparticles. Red arrows indicate the MR signal of microspheres at the tumor site.

11 is a view showing the results of confirming the dispersion force of microspheres in CMC.

12 is a view showing the results of confirming the dispersion force of microspheres in alginate.

13 is a view showing the results of confirming the bacterial spore encapsulation efficiency according to cationic polymer modification (MS: non-porous microspheres, PMS: porous microspheres, PEI-PMS: porous microspheres modified with cationic polymers) (n = 100, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant).

14 is a diagram showing the encapsulation efficiency of FITC-labeled bacterial spores through fluorescence images (MS: non-porous microspheres, PMS: porous microspheres, PEI-PMS: porous microspheres modified with cationic polymers).

15 is a diagram confirming spore germination efficiency after 24 hours in microspheres encapsulated with bacterial spores.

16 is a view confirming the level of colonies of spores released from microspheres.

17A is a diagram showing the result of confirming the cytotoxicity of PEI-PMS to CT26 cells, B is a diagram showing the result of confirming the cytotoxicity of C. novyi -NT supernatant to CT26 cells, and C is a diagram showing the result of confirming the cytotoxicity of C. novyi -NT supernatant to CT26 cells. This is a diagram showing the results of confirming the cytotoxicity of the novyi -NT supernatant to RAW264.7 cells (n = 100, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001; ns, not significant).

18A is E. coli DH5α B is a diagram showing the result of confirming the cytotoxicity of the supernatant to CT26 cells, and B is a diagram showing the result of confirming the cytotoxicity of the heat-inactivated C. novyi -NT supernatant to CT26 cells (n = 100, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001; ns, not significant).

19 is a view showing images of live and dead cells according to C. novyi -NT supernatant treatment in CT26 cells.

20A and B are views showing the results of confirming cell viability of CT26 cells according to C. novyi -NT supernatant treatment by colony formation assay.

21 is a view showing the apoptotic/necrotic cell population (%) of CT26 cells treated with C. novyi -NT supernatant as a result of flow cytometry.

22 is a graph showing the results of apoptotic/necrotic cell population (%) of CT26 cells treated with C. novyi -NT supernatant.

23A is a diagram showing the result of analyzing the ATP released by treating CT26 cells with C. novyi -NT supernatant, and B is a diagram showing the result of analyzing the cells expressing calreticulin (CRT) (n = 100 , *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001; ns, not significant).

24A shows the result of analyzing CD80-expressing cells according to C. novyi -NT supernatant treatment in indirect co-culture of CT26 cells and RAW264.7 cells, and B shows the result of analyzing CD86-expressing cells. and C shows the results of analyzing cells expressing CD80 and CD86 (n = 100, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001; ns, not significant).

25 is a schematic diagram of the initiation of an immune response by C. novyi -NT spores.

26 is a schematic diagram of microspheres using electrostatic attraction between cationic polymers and bacterial spores.

27 is a diagram showing the tumor-targeting functionality of the microspheres of the present invention by an external magnetic field.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Fabrication of porous microspheres with multifunctionality

In order to fabricate the porous microspheres having multifunctionality according to the present invention, the following experiments were conducted.

Specifically, a fluidic device for fabricating microspheres is Tygon tube (inner diameter 1/32 inch, outer diameter 3/32 inch), glass capillary tube (5922-10, 5 IN PIPETS microcapillary, Ace Glass, USA) ), and a needle (30 G).

Injection of a dichloromethane (DCM) solution containing polylactic acid (PLA), undecane (UD) and iron oxide nanoparticles (0.03 wt%) as magnetic nanoparticles as a discontinuous phase did In addition, a 2 wt% polyvinyl alcohol (PVA) aqueous solution was used for the continuous phase and the collection phase. The ratios of PLA, UD, and magnetic nanoparticles (IONP) are specifically described in Table 1 below.

UD/PLA ratio (w:w)	UD (wt.%)	PLA (wt.%)	IONP (wt.%)
0	0	One	0.03
3	3	One	0.03
3.5	3.5	One	0.03
4.375	3.5	0.8	0.03
5	3.5	0.7	0.03
5.83	3.5	0.6	0.03

Next, in order to prepare oil-in-water emulsions, a syringe pump was used to set flow rates of the discontinuous phase and the continuous phase to 0.1 mL/min and 1.5 mL/min, respectively.

The emulsion collected in the collection phase was converted to UD/PLA microspheres by evaporating the DCM overnight, and the microspheres were washed three times with deionized water (DW) to remove residual UD and PVA. Next, the UD/PLA microspheres were gently agitated with ethanol to infiltrate the ethanol to dissolve and remove the UD. Thereafter, the microspheres were lyophilized to finally prepare porous PLA microspheres.

Example 2: Synthesis of Magnetic Nanoparticles

In order to fabricate the porous microspheres having multifunctionality according to the present invention, the following experiments were performed.

Specifically, as magnetic nanoparticles, 423.6 mg of iron(III) acetylacetonate and 120 mg of zinc chloride were mixed in a 50 mL round bottom flask to synthesize iron oxide nanoparticles (IONP). put in Then, 1.2 mL of oleic acid, 4.8 mL of oleylamine, and 4.8 mL of trioctylamine were added to the flask. The flask inlet was connected to a nitrogen gas inlet and stirred at 300 rpm for 3 minutes. A flask connected to a heating mantle was gradually heated at 200° C. for 25 minutes and maintained at a constant temperature of 200° C. for 1 hour. Then, it was slowly heated again at 330 °C for 1 hour and maintained at 330 °C for 1 hour. It was then cooled slowly at room temperature for 1 hour. After the reaction, 1.92 mL of toluene and 30 mL of ethanol were added to the solution. After transferring the solution to a 50 mL centrifuge tube and centrifuging at 1600 g for 5 min at room temperature, the supernatant was discarded and the pellet was redispersed by adding 8 mL of toluene and 30 uL of oleylamine. After the dispersed solution was centrifuged at 650 g for 3 minutes at room temperature and the supernatant was collected, 4 mL of ethanol was added to the supernatant and centrifuged at 1600 g for 5 minutes at room temperature. After this, the supernatant was discarded and redispersed in 4 mL of DCM to obtain final particles.

Example 3: Evaluation of porous microsphere properties

In order to evaluate the characteristics of the porous microspheres prepared in Example 1, the following experiments were performed.

First, FIG. 1 shows that uniform porous microspheres were prepared by the O/W emulsion method using a fluidic device. In addition, it can be seen that the microsphere size and pore size can be controlled through the ratio of UD/PLA, and specifically, as the ratio of UD in UD/PLA increases, the size of microspheres and pore size increase can (Fig. 2).

However, it was confirmed that when the UD/PLA ratio exceeds 5.83 (PLA 0.6%, UD 3.5%), too many pores adversely affect the mechanical properties of the microspheres, resulting in morphological instability. In addition, it can be seen that the number of open pores per area of 100 μm ² increased as the ratio of UD increased ( FIGS. 3 and 4A to 4B ).

On the other hand, as it was confirmed that the size of bacterial spores is generally about 1.5 um (Fig. 5), microspheres having a stable and uniform pore size and a UD/PLA ratio of 3 (UD/PLA) having a pore size capable of sufficient spore encapsulation It can be seen that PLA 3) is suitable.

Example 4: Magnetic Evaluation of Porous Microspheres

In order to evaluate the magnetism of the porous microspheres prepared in Example 1, the following experiment was performed.

Specifically, after dispersing the porous microspheres prepared in Example 1 in distilled water, it was confirmed that the porous microspheres had magnetism using a permanent magnet (FIG. 6), and a 3D printer (Ender-5, China, Creality) ), it was confirmed that the porous microspheres with magnetism could be moved as desired using a magnet (FIG. 7).

Next, the porous microspheres were dispersed at various concentrations in an agarose gel for magnetic resonance (MR) imaging and quantitative evaluation. As a result, a T2-weighted MR signal was not detected in microspheres without magnetic nanoparticles (FIG. 8), whereas in porous microspheres containing magnetic nanoparticles, as the particle concentration increased, It was confirmed that the T2-weighted MR signal increased (FIG. 9).

Next, the magnetic resonance imaging performance of the porous microspheres in vivo was evaluated. To this end, a mouse tumor model was prepared by subcutaneously inoculating CT26 colorectal cancer cells into mice. After 8 days, when the tumors grew, porous microspheres containing magnetic nanoparticles were intratumorally injected, followed by magnetic resonance imaging. As a result, it was confirmed that a T2-weighted signal was seen when porous microspheres containing magnetic nanoparticles were injected into the tumor (FIG. 10).

Example 5: Evaluation of dispersion force of microspheres having a porous structure

For uniform injection of the porous microspheres prepared in Example 1, an experiment was conducted to find an appropriate concentration of the dispersion while maintaining a dispersed state and passing through a small injection needle.

Specifically, a dispersion force confirmation experiment was performed using UD/PLA 3 microspheres. The microspheres were sterilized by soaking in 70% ethanol for 24 hours, ethanol was replaced with distilled water, and the process of washing the ethanol through ultrasonic waves was repeated three times. Finally, various concentrations of carboxymethyl cellulose (CMC), alginate , Alg) microspheres at a concentration of 10 mg/mL were dispersed in the solution.

As a result, in the 3 wt% and 4 wt% CMC and 3 wt% and 5 wt% Alg dispersions, the microspheres could not be dispersed through voltexing, and were dispersed using ultrasonic waves. When ultrasonic waves are used, it was confirmed that the concentration of CMC and alginate should be carried out at a maximum of 2% or less because there is a concern that bacteria may be eliminated during subsequent bacterial encapsulation. In addition, the time at which the microspheres in the dispersed state start to sink was shown (red arrow), and it was confirmed that the sinking time was delayed as the concentration of the dispersion increased (FIG. 11 and FIG. 12).

Example 6: Fabrication of porous microspheres coated with cationic polymers

In order to encode the porous microspheres prepared in Example 1 with a cationic polymer, the following experiment was performed.

Specifically, polyethyleneimine (PEI) was used as an example of a cationic polymer, and porous microspheres were dispersed in a 10 wt % PEI aqueous solution and coated for 1 hour at room temperature. The PEI coated porous microspheres were washed 5 times with distilled water. The surface morphology of the microspheres was observed with a scanning electron microscope (S-4800, HITACHI, JAPAN), and the average size of particles and pores was calculated by analyzing at least 100 microspheres for each sample using ImageJ software (USA). did.

Example 7: Evaluation of bacterial encapsulation efficiency and release performance of porous microspheres modified with cationic polymers

In order to evaluate the encapsulation ability of the microspheres modified with the cationic polymer and the release behavior of the encapsulated bacterial spores, the following experiment was performed. At this time, the UD/PLA 3 condition was determined as a carrier model in order to prepare uniformly porous microspheres having pores larger than bacterial spores. In addition, specifically, non-porous microspheres (MS) were used as a control, UD / PLA 3 porous microspheres (PMS) not coated with PEI and UD / PLA 3 porous microspheres (PEI-PMS) coated with PEI ) was selected as the experimental group.

7-1: Preparation of bacterial spores

As an example of bacterial spores, Clostridium novyi -NT ( Clostridium novyi -NT, C. novyi -NT) spores were prepared, C. novyi -NT spores were 5 g Na ₂ HPO ₄ per 1 L, 30 g peptone, They were cultured anaerobically and at 37 °C in a sporulation medium containing 0.5 g L-cysteine, 10 g maltose and 5% wt/vol cooked meat particles (Difco). Spores cultured for at least 2 weeks were isolated by percoll density gradients (55 and 70%).

In addition, to prepare FITC-labeled C. novyi -NT spores, 100 uL FITC DMSO solution (1 mg/mL) was added to 1 mL C. novyi -NT solution (1Х10 ⁹ spores) and stirred overnight at 25 °C. . Thereafter, the reaction solution was centrifuged at 4 °C and 8000 rpm for 10 minutes and washed three times with PBS.

7-2: Evaluation of bacterial spore encapsulation efficiency

In order to confirm the bacterial spore encapsulation efficiency of porous microspheres modified with cationic polymers, the following experiment was performed.

Specifically, after dispersing 10 mg of MS, PMS or PEI-PMS in PBS, 1 Х 10 ⁸ FITC-labeled C. novyi -NT spores were added, vacuum was applied, and the mixture was mixed for 4 hours. Thereafter, the microspheres were separated using magnetism, and a portion of the supernatant was taken to measure the fluorescence sensitivity of each. The fluorescence sensitivity of the supernatant was regarded as the amount of material not encapsulated into the microspheres, and the encapsulation rate was obtained by subtracting the value from the control group.

As a result, in the MS group, no spores were encapsulated at all because there was no electrostatic attraction and stomata. In the PMS group, about 20% were encapsulated. It was confirmed that the encapsulation was performed twice or more (FIG. 13). In addition, as a result of observing the bacterial spores encapsulated into each microsphere with a fluorescence microscope, it was confirmed that the encapsulation efficiency was improved according to the presence of pores and surface modification with cationic polymers (FIG. 14).

7-3: Evaluation of bacterial spore release ability

In order to confirm the ability of the porous microspheres modified with cationic polymers to release bacterial spores, the following experiment was performed. This was done to confirm that the spores encapsulated in the particles by internal pores and electrostatic attraction safely move to the target site in vivo and then are effectively released and germinate in a hypoxic environment.

Specifically, the spore-encapsulated PEI-PMS microsphere suspension was inoculated into the RCM broth. After removing the supernatant containing unencapsulated spores, the microspheres were re-dispersed in 1 mL of PBS, inoculated into RCM liquid medium, and absorbance was measured after 24 hours. In addition, in order to germinate spores of C. novyi -NT, an anaerobic bacterium, EC-oxyrase (Oxyrase Inc.) was added to RCM broth at 10% v/v and then cultured in BD GasPak. Then, after 4, 8, and 12 hours, 200 uL of the culture medium was spread on RCM solid medium, and colony images were obtained after 24 hours.

As a result, it was confirmed that the PEI-PMS group (control group) in which spores were not encapsulated did not germinate, while germination occurred while the spores were slowly released from the medium (FIG. 15). In addition, microspheres containing spores were inoculated into the RCM medium, and the culture medium was collected every 4, 8, and 12 hours and spread on an RCM oxyrase agar plate. As a result, it was confirmed that the spores were continuously released over time ( Figure 16).

Based on the above results, it can be seen that bacterial spores can be effectively encapsulated by the induction of electrostatic attraction with the pores of microspheres, and the encapsulated spores can be released and germinated in an appropriate environment without functional limitations or interference by the particles. .

Example 8: Cytotoxicity evaluation of porous microspheres encapsulated with bacterial spores

In order to confirm the cytotoxic and antitumor effects of the porous microspheres encapsulated with bacterial spores, the following experiments were performed.

8-1: Evaluation of cytotoxicity of porous microspheres

In order to confirm whether the porous microspheres themselves are cytotoxic, when CT26 cells were treated with PEI-PMS, it was found to be non-toxic compared to the control group (FIG. 17A).

8-2: Evaluation of cytotoxicity of bacterial spores

In order to evaluate the cytotoxicity of C. novyi -NT, the following experiment was performed.

Specifically, CT26 cells (3Х10 ⁵ cells/mL) or RAW264.7 cells (4Х10 ⁵ cells/mL) were seeded in a 24-well plate and cultured overnight. Thereafter, the C. novyi -NT supernatant and the E. coli DH5α supernatant were treated at the same concentration. After 4 hours, cells were washed with PBS and CCK-8 solution was added. After 1 hour, cytotoxicity was assessed using a microplate reader with the CCK-8 assay.

As a result, it was confirmed that the killing effect appeared in a concentration-dependent manner of the C. novyi -NT culture supernatant, and when treated with 5 mg/mL of C. novyi -NT spore culture supernatant, it was observed that most cells were killed ( 17B and C).

Next, in order to confirm that the toxicity is a specific characteristic of C. novyi -NT spores, the culture supernatant of the E. coli DH5α strain was treated at the same concentration, and it was confirmed that the E. coli DH5α strain did not exhibit toxicity (FIG. 18A). .

Next, in order to confirm whether the liposomase secreted from C. novyi -NT induces toxicity, the C. novyi -NT culture supernatant was inactivated by heat treatment, and then CT26 cells were treated with heat treatment. In one case, it was confirmed that there was no toxicity (FIG. 18B).

Next, in the live and dead assay and colony formation assay, it was confirmed that the proportion of dead cells increased as the concentration of the C. novyi -NT culture supernatant increased (FIG. 19, 20A and 20B).

8-3: Analysis of cell death mechanism by bacterial spores

In order to analyze cell death by C. novyi -NT culture, the following experiment was performed.

Specifically, CT26 cells (3Х10 ⁵ cells/mL) were seeded in a 12-well plate and cultured overnight. Thereafter, the cells were treated with C. novyi -NT culture medium at different concentrations. After 4 hours, cells were stained using the FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend) and then analyzed by flow cytometry (CytoFLEX S, Beckman Coulter, Miami, FL, USA).

As a result, it was confirmed that the ratio of late apoptotic/necrotic cells increased as the concentration of the C. novyi -NT culture supernatant increased (FIGS. 21 and 22).

Based on the above results, it can be seen that liposomes secreted by bacterial spores directly cause cancer cell death, and bacteria-based anti-tumor treatment can be performed using this.

Example 9: Assessment of immune activation by bacterial spores

In order to evaluate immune activation due to bacterial spores, the following experiment was performed.

9.1: DAMP expression analysis

In order to confirm immunogenic cell death (ICD)-associated molecular patterns (Damage-associated molecular patterns, DAMPs) caused by bacterial spores, the following experiment was performed.

Specifically, CT26 cells (3Х10 ⁵ cells/mL) were seeded in a 12-well plate and cultured overnight. Thereafter, the cells were treated with C. novyi -NT culture medium at different concentrations. After 4 hours, the cell pellet was washed with PBS and stained with Alexa Fluor 488-conjugated calreticulin antibody (dilution 1:500) at 4°C for 30 minutes to stain the calreticulin molecule expressed on the cell surface. processed. After washing the cells with PBS, the cells were redispersed in a PBS solution containing 2% FBS and analyzed by flow cytometry.

Next, CT26 cells (3Х10 ⁵ cells/mL) were seeded in a 12-well plate and cultured overnight. Thereafter, the cells were treated with C. novyi -NT culture medium at different concentrations. After 1 hour, the amount of ATP released from the cell medium was measured using an ATP bioluminescence kit.

As a result, as the concentration of C. novyi -NT supernatant treatment increased, the ATP release increased. In the case of 5 mg/mL treatment, the ATP release increased about 12 times compared to the control group, and the percentage of cells expressing calreticulin (CRT) ( %) increased about 37-fold (Fig. 23A and B).

9.2: Assessment of immune cell activation

In order to evaluate the activity of immune cells by cancer cells killed by bacterial spores (evaluating the degree of M1 polarization of M0 macrophages), the following experiment was performed. M1 macrophages are known to play a positive role in the elimination of pathogens and tumor cells and to activate the adaptive immune response by expressing high levels of antigen-presenting MHC complexes.

Specifically, CT26 colorectal cancer cells (3Х10 ⁵ cells/mL) and RAW264.7 cells (3.5Х10 ⁵ cells/mL) were seeded in a 12-well plate and indirectly co-cultured. First, CT26 colorectal cancer cells were treated with C. novyi -NT supernatant at each concentration and cultured for 4 hours. Thereafter, the culture medium of CT26 colorectal cancer cells was treated with RAW264.7 cells and cultured for 24 hours. Next, the cell pellet was washed with PBS, blocked with Fc receptors for 10 minutes at 4° C., and the cells were washed with PBS. Then, CD80 and CD86 molecules expressed on the cell surface were treated with APC-conjugated CD80 antibody, PE-conjugated CD86 antibody and zombie aqua dye (diluted 1:500) for 1 hour at 4°C for staining. did After washing the cells with PBS, the cells were redispersed in a PBS solution containing 2% FBS and analyzed by flow cytometry.

As a result, as the concentration of C. novyi -NT supernatant treatment increased, the ratio of CD80 and CD86 positive cells significantly increased (FIGS. 24A to C).

Based on the above results, it can be seen that cancer cells destroyed by bacterial spores activate immune cells by releasing tumor antigens or DAMPs (FIG. 25).

Bacterial-mediated tumor treatment, which applies oncolytic bacteria to cancer treatment, has a disadvantage in that the proportion of spores delivered to the tumor is small, and when the delivered spores escape into the bloodstream, they can cause harmful side effects such as systemic infection. Therefore, it is important to control the concentration of bacteria and target them to tumors for the therapeutic effect and reduction of side effects of the bacteria-mediated tumor treatment.

Since the surface of the porous microspheres of the present invention is modified with a cationic polymer, negatively charged bacterial spores can be effectively encapsulated through electrostatic attraction (FIG. 26). In addition, since the microsphere of the present invention contains magnetic nanoparticles, it has the advantage of being controllable with an external magnetic field and confirming its location in the living body through MRI (FIG. 27).

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (13)

1. Porous microspheres comprising biocompatible polymers and cationic polymers.
2. The porous microsphere of claim 1 , wherein the microsphere further comprises magnetic nanoparticles.
3. The porous microsphere of claim 1, wherein the surface of the microsphere is modified with a cationic polymer.
4. The porous microsphere of claim 1, wherein the microsphere is capable of encapsulating bacteria or bacterial spores.
5. The porous microsphere of claim 1, wherein the microsphere has a pore size of 1 to 10 μm.
6. The porous microsphere of claim 1, wherein the number of open pores/area per 100 μm ² of the microsphere is 0.1 to 6.
7. 1) preparing a first solution containing a biocompatible polymer and a pore forming agent;

   2) preparing a second solution containing a surfactant;

   3) preparing microspheres using the first solution and the second solution; and

   4) removing the pore forming agent from the prepared microspheres

   A method for producing porous microspheres comprising a.
8. The method according to claim 7, wherein the first solution includes the pore-forming agent and the biocompatible polymer in a ratio (w:w) of 1:1 to 6:1.
9. The method according to claim 7, wherein the first solution further comprises magnetic nanoparticles.
10. The method of claim 7 , wherein the method further comprises modifying the surface of the porous microspheres with a cationic polymer.
11. The method according to claim 7, wherein the method further comprises encapsulating bacteria or bacterial spores in the porous microspheres.
12. 1) the porous microspheres of any one of claims 1 to 6; and

    2) Oncolytic bacteria or their spores

    Including, anti-cancer microspheres.
13. A pharmaceutical composition for treating or preventing cancer, comprising the anticancer microspheres of claim 12.

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