Engineered cell composition as well as preparation method and application thereof

Abstract

The invention discloses an engineered cell composition as well as a preparation method and application thereof. The cell composition disclosed by the invention is macrophages or mononuclear cells. The preparation method of the composition comprises the following steps: constructing nanoparticles / microparticles, introducing cell specific recognition molecules to obtain specific nanoparticles / microparticles capable of being recognized by macrophages / monocytes, and co-incubating the specific nanoparticles / microparticles and the macrophages / monocytes to prepare the cell composition. The cell composition disclosed by the invention is simple and convenient in preparation method, is easy to carry drugs and probes, and can realize targeted delivery and diagnosis and treatment of infected or tumor parts.

Description

Technical Field

[0001] The present invention relates to an engineered cell composition and a preparation method and application thereof. The cell composition of the present invention is a macrophage or a monocyte. Background Art

[0002] Adoptive cell therapy involves the isolation, ex vivo activation and / or expansion of immune cells and their reinfusion into the patient, and its effect can be significantly increased by incorporating molecular-specific chimeric antigen receptors expressed on tumor cells. Adoptive cell therapy has revolutionized the clinical approach to treating cancer. The most prominent example to date is CAR-T cell therapy. Over the past decade, CAR-T cell therapy has transformed the field of oncology, showing exciting efficacy in previously incurable blood tumors. However, the treatment of solid tumors with CAR-T faces multiple obstacles, including an unfavorable tumor microenvironment, intra- / extra-tumor toxicity, and undesirable antigen specificity. There are relatively many types of adverse reactions to CAR-T therapy, among which cytokine storm (CRS) is the inevitable primary treatment risk in CAR-T therapy. In addition, off-target effects and neurotoxicity are also the most common and thorny safety issues. Therefore, exploring innate immune cells, including natural killer cells, macrophages, etc., to kill tumor cells makes it possible for them to succeed where T cell therapy is insufficient.

[0003] Monocytes and macrophages are able to target inflamed tissues and deliver therapeutic drugs. Macrophages have been reported to be used for targeted delivery to the brain, sites of inflammation, infection, or tumors. Unlike macrophages, which are terminally differentiated cells that are permanently present in various tissues, monocytes are circulating cells and can therefore navigate the vasculature and target difficult-to-reach pathological tissues. Macrophages are plastic and can assume a variety of roles, such as defense against foreign pathogens, promotion of wound healing and repair, and regulation of tissue homeostasis.

[0004] Traditionally, it is believed that drugs engulfed by macrophages and entering the phagosome will degrade the drugs and reduce the drug release rate. In addition, anti-tumor drugs can reduce the survival or migration ability of macrophages. In order to improve the above defects, efforts have been made to design to weaken the effects of macrophage engulfment and isolate the drugs outside the cells to protect the cells and drugs from degradation. Reports have shown that the size and shape of polymer particles become factors in regulating the above cell functions, the most important of which is phagocytosis. By changing the shape of the designed particles through physical parameters, particles that can avoid macrophage phagocytosis are developed, which bind to the cell surface and retain the basic cell functions of the cells.

[0005] Previously, we reported an engineered particle called a “backpack” that can firmly adhere to the surface of macrophages and modulate cell phenotype in vivo. The backpack evades phagocytosis for several days and releases cytokines, continuously directing macrophage polarization toward an anti-tumor phenotype. This represents a new approach to control and maintain the phenotype of adoptive cell immunotherapy.

[0006] In order to enhance the interaction with the cell surface, some specific molecules with cell surface recognition ability have attracted much attention. Among them, lectin has become one of them. It is a protein or glycoprotein purified from various plants, invertebrates and higher animals that contains at least one catalytically inactive domain. It can non-covalently reversibly bind to specific monosaccharides or oligosaccharides, has no enzyme activity or immunological activity characteristics, and is widely present in various organisms from viruses to mammals. Lectins are mainly divided into five categories: fucose, galactose / N-acetylgalactosamine, glucose / N-acetylglucosamine, mannose and N-acetylneuraminic acid. Plant lectins have a wide range of applications in biomedical research, diagnosis and treatment, and play a key role in immunology.

[0007] Liposomes were discovered by British scholars Bangham and Standish in 1965 through electron microscopy. Phospholipids can spontaneously form bilayer vesicles in water and were named liposomes, becoming one of the major discoveries in the pharmaceutical field in the 20th century. Liposomes are self-assembled bodies of lipid molecules or lipids, and are bilayer structures spontaneously formed by lipid molecules or lipids in water. Liposomes can encapsulate drugs of various properties and exhibit multiple functions. Liposomes are divided into single-chamber, multi-chamber and multivesicular liposomes. Preparation methods include thin film dispersion method, reverse evaporation method, double emulsion method, active drug loading method (ammonium sulfate gradient, pH gradient, ion gradient), freeze-thaw cycle method, solvent injection method, etc.

[0008] Lipid nanoparticles are usually composed of amphiphilic lipid materials, structural lipid materials, PEG lipid materials, and ionic lipid materials. These components form a nanoscale structure, the core of which is a lipid bilayer composed of amphiphilic lipid materials, which can encapsulate drugs, such as mRNA vaccines. This structure has excellent biocompatibility and stability, which helps protect the drug from degradation. In addition, the surface of the preparation can be modified with PEG (PEG lipid material) to increase blood circulation time and reduce the chance of being cleared by the immune system. The preparation can also improve in vivo biodistribution, increase its cellular uptake efficiency, and promote its delivery to target cells. In addition, the surface of the preparation can be targeted by changing the lipid composition and surface modification. Tumor-specific surface markers can increase its affinity for tumor tissue, enrich in tumor cells, and reduce its adverse effects on healthy tissues.

[0009] Polylactic acid-glycolic acid (abbreviated as PLGA) is a copolymer formed by lactic acid (LA) and glycolic acid (GA). When PLGA enters the body, it hydrolyzes to generate corresponding lactic acid and glycolic acid through ester bonds, and finally transforms into carbon dioxide and water after the tricarboxylic acid cycle. PLGA is non-irritating to the human body, non-toxic and has good biocompatibility and biodegradability. Drug delivery systems based on PLGA carriers provide great opportunities for biomedicine. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved several new drugs based on PLGA, which are widely used in clinical treatment or diagnosis of diseases. The preparation methods of PLGA nanoparticles or PLGA microparticles mainly include emulsification solvent evaporation method, spray drying method, nanoparticle precipitation method, phase separation method, dialysis method and microfluidics method.

[0010] Compared with CAR technology, which has a high starting point, large investment, and requires multidisciplinary collaboration, the use of liposomes, lipid nanoparticles, or PLGA nanoparticles / microparticles to modify macrophages / monocytes is more mature in technology, easier to achieve mass production, more feasible, and has a greater advantage in input-output ratio. Summary of the invention

[0011] The present invention modifies either macrophages or monocytes.

[0012] The macrophages and monocytes provided by the present invention are derived from human or mouse sources.

[0013] The present invention uses nanoparticle / microparticle preparations to modify cells and exert biological effects.

[0014] The nanoparticle / microparticle preparation provided by the present invention is located on the surface of the monocytes or macrophages.

[0015] The nanoparticle / microparticle preparation provided by the present invention is specifically a liposome preparation, a lipid nanoparticle, and a PLGA nanoparticle / microparticle. In addition to the above preparations, the technology of the present invention can also be extended to other micro-nano preparations, such as polymer micelles, lipid microspheres, microbubbles, nanosuspensions, etc.

[0016] The liposomes, lipid nanoparticles and PLGA nanoparticles / microparticles provided by the present invention all contain specific recognition molecules, drugs and carrier materials.

[0017] The specific recognition molecules used in the present invention come from lectins. The present invention selects animal lectins, plant lectins and microbial lectins. It is further selected from Leguminosae lectins, monocotyledon mannose binding lectins, chitin binding lectins, Lanaceae lectins, type II ribosome inactivating lectins, japanese lectins, and Cucurbitaceae phloem lectins. Specific types of plant lectins are such as: lentil lectin, wheat germ agglutinin (WGA), sessile elderberry lectin (SSL), peanut lectin, Sophora japonica lectin (MAL); Datura lectin (DSL), succinyl-concanavalin A (succ-ConA), snowdrop lectin (GNL). The present invention is particularly suitable for lectins from soybeans, wheat germ, lotus seeds, kidney beans, onions, lentils, tomatoes, sword beans, potatoes, and two or more combinations thereof. In addition, the present invention can also select lectins in natural compositions or purified (to any extent) therefrom, chemically synthesized lectins, modified lectins or derivatives thereof (natural or synthetic). Lectin derivatives include one or more subunits of multi-subunit lectins. Methods for producing lectins are known in the art, including extraction from natural sources, purification, or using biotechnology. Many lectins are known. Any lectin can be used in the present invention.

[0018] The specific recognition molecule provided by the present invention is characterized in that the specific molecule is a polymer containing lectin, such as a conjugate of wheat germ agglutinin and polyethylene glycol-phosphatidylethanolamine (WGA-PEG-DSPE) and a conjugate formed by wheat germ agglutinin and polyethylene glycol-polylactic acid copolymer (WGA-PEG-PLA). These lectin polymers are generated by the reaction of the primary amine groups carried by the lectin molecules themselves with DSPE-PEG-NHS or NHS-PEG-PLA.

[0019] The term "PEG" as used herein refers to polyethylene glycol or polyoxyethylene polymer. PEG2k, which can also be expressed as PEG2000, 2000 is the number average molecular weight of PEG. PEG5k is also used in the present invention.

[0020] The specific recognition molecule provided by the present invention is selected from wheat germ agglutinin (WGA) functional materials, specifically a conjugate of wheat germ agglutinin and polyethylene glycol 2k-phosphatidylethanolamine (WGA-DSPE-PEG2k) and a conjugate formed by wheat germ agglutinin and polyethylene glycol-polylactic acid copolymer (WGA-PEG5k-PLA5k).

[0021] The preparation of the present invention can encapsulate any anti-tumor active molecule for diagnosis or treatment of tumors. Specific non-limiting examples include: alkylating agents (such as cisplatin, carboplatin, oxaliplatin, carmustine, semustine, cyclophosphamide, chlorambucil, ifosfamide), purine and pyrimidine analogs and derivatives (such as methotrexate, pemetrexed, 5-fluorouracil, capecitabine, Segall, cytarabine, gemcitabine, hydroxyurea, floxuridine, cytarabine, mercaptopurine, thioguanine, thioazapurine, fludarabine, pentostatin, cladribine), topoisomerase inhibitors (such as etoposide, teniposide, amsacrine, irinotecan, topotecan), taxanes (such as paclitaxel, docetaxel, cabazitaxel), antifolates (such as methotrexate, oxadiazine ... , trimethoprim, pyrimethamine, pemetrexed), angiogenesis inhibitors (such as axitinib, everolimus, lenalidomide, pazopanib, sorafenib), anti-tumor monoclonal antibodies (such as bevacizumab, vandetanib, vatalanib, alemtuzumab, cetuximab, gemtuzumab, ibritumomab tiuxetan, panitumumab, rituximab, tositumomab, trastuzumab), cytotoxic antibiotics (such as actinomycin, bleomycin, plicamycin, mitomycin), anthracycline antibiotics (such as doxorubicin, epirubicin, daunorubicin, valrubicin, idarubicin), and physiologically acceptable salts, co-crystals, macromolecule (including antibody) conjugates and prodrugs thereof.

[0022] The preparation of the present invention can encapsulate any antimicrobial active molecule for the diagnosis or treatment of pathogenic bacteria infection. Specific non-limiting examples include: penicillins (such as penicillin, penicillin V potassium, oxacillin, cloxacillin, ampicillin, benzylpenicillin, furocillin, amoxicillin, mezlocillin, nafcillin), cephalosporins (such as cephalexin, cefotiam, cefadroxil, cefoxitin, cefazolin, cephradine, cefaclor, cefuroxime, cefpiramide, cefathiamidine, cefpirome, cefepime, cefazolin, ceftriaxone, cefixime, cephalosporin ... Minoxidil, ceftazidime, cefdinir, cefoperazone, ceftizole, cefmetazole, cefotaxime, cefoperazone, cefamandole), β-lactamase inhibitors (such as amoxicillin-clavulanate potassium, amoxicillin-sulbactam, cefoperazone-sulbactam, meropenem, imipenem, faropenem, penicillin-sulbactam, cefoperazone-tazobactam, cilastatin, panipenem-betamipron), aminoglycosides (such as amikacin, gentamicin, amikacin, streptomycin, netilmicin, tobutyric acid), azithromycin), tetracyclines (such as tetracycline, doxycycline, minocycline), sulfonamides (such as sulfamethoxazole, trimethoprim), quinolones (such as ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, fleroxacin, enoxacin, lomefloxacin, gatifloxacin, sparfloxacin, moxifloxacin, pazufloxacin) ), furans (such as nitrofurantoin, furazolidone), antifungal drugs (such as fluconazole, miconazole, nystatin, clotrimazole, bifonazole, terbinafenamide, ketoconazole, flucytosine, itraconazole, amphotericin B), nitroimidazoles (such as metronidazole, metronidazole benzoate, tinidazole, ornidazole), amides (such as chloramphenicol), glycopeptides (such as vancomycin, norvancomycin, teicoplanin), and physiologically acceptable salts, co-crystals, macromolecular (including antibody) conjugates and prodrugs thereof.

[0023] Suitable amphiphilic lipid materials for the liposome formulation of the present invention include but are not limited to: (1) Zwitterionic phospholipids, including phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine and lysophosphatidylethanolamine; (2) anionic amphiphilic phospholipids, such as phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin; (3) Cationic amphiphilic lipids, such as acyl trimethyl propyl ammonium, diacyl dimethyl propane ammonium, stearylamine, etc. (4) Natural phospholipids, such as natural soybean lecithin (SPC) and egg phosphatidylcholine (EPC). Non-limiting examples of phosphatidylcholines include dioleoylphosphatidylcholine (DOPC), 1,2-dierucoylphosphatidylcholine or 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dimethyloleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-myristoyl-2 -palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). Non-limiting examples of phosphatidylglycerols include dipalmitoylphosphatidylglycerol or 1,1-dipalmitoyl-sn-glycero-2-phospho-rac-(3-glycerol) (DPPG), 1,1-dierucoyl-sn-glycero-2-phospho-rac-(3-glycerol) (DEPG), 1,1-dilauroyl-sn-glycero-2-phospho-rac-(3-glycerol) (DLPG), 1,1-dimyristoyl-sn-glycero-2-phospho-rac-(3-glycerol) (DMPG), 1,1-dioleoyl-sn-glycero-2-phospho-rac-(3-glycerol) (DOPG), 1,1-distearoyl-sn-glycero-2-phospho-rac-(3-glycerol) (DSPG), 1-palmitoyl-15-oleoyl-sn-glycero-1-phospho-rac-(20-glycerol) (POPG) or a salt thereof, such as the corresponding sodium salt, ammonium salt or a combination of salts thereof.

[0024] The ionizable lipid of the present invention is selected from the group consisting of: 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid triheptacontane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 2,2-dioleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2- ({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)).

[0025] The "PEG lipid" provided herein refers to a lipid modified by polyethylene glycol (PEG). The lipid portion of the PEG lipid includes a lipid portion with a length of about C14 to about C22. In some embodiments, the lipid portion of the PEG lipid includes a lipid portion with a length of about C14 to about C16. In some embodiments, the PEG portion (e.g., mPEG-NH 2) is about 1000 Da, 2000 Da, 5000 Da, 10,000 Da, 15,000 Da or 20,000 Da in size. The PEG lipid is selected from, but not limited to, PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-dilauroylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)amide-3',6'-dioxoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-tetracosyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(

[00136] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG, poly(ethylene glycol)-2000-dimethylacrylate (PEG2k-DMA) and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA), PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amine. In some embodiments, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0026] "Structured lipids" provided by the present invention refer to sterols, or lipids containing sterol moieties. Incorporating structured lipids into lipid nanoparticles can help alleviate the aggregation of other lipids in the particles. Structural lipids can also be selected from groups including but not limited to cholesterol, fecal sterol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, α-tocopherol, hopane, phytosterol, steroids and mixtures thereof. In some embodiments, the structured lipid is a mixture of two or more components, each component being independently selected from cholesterol, fecal sterol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, α-tocopherol, hopane, phytosterol and steroids. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid is an analog of cholesterol.

[0027] The present invention uses neutral lipids to function to stabilize and improve lipid nanoparticles or microparticles. The phospholipid moiety can be selected from, for example, a non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid moiety can be selected from, for example, a non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.

[0028] In the unilamellar / multilamellar liposome, the mass ratio of the drug to the liposome lipid material is 1:8 to 1:400, or 1:10 to 1:300, or 1:10 to 1:200, or 1:10 to 1:150, or 1:15 to 1:150;

[0029] Phospholipid concentration can change in liposome provided by the invention.In some embodiments, the concentration of phospholipid can be the scope from about 5mg / ml to about 100mg / ml, preferably between 8-80mg / ml, more preferably between 10-50mg / ml, and most preferably between 10-30mg / ml.In separately preferred embodiment, the concentration of lipid can be about following scope: 5-8mg / ml, 8-15mg / ml, 15-20mg / ml, 20-30mg / ml, 30-40mg / ml, 40-50mg / ml, 50-60mg / ml, 60-70mg / ml, 70-80mg / ml, 80-90mg / ml, 90-100mg / ml.

[0030] The single-chamber / multi-chamber liposomes provided by the present invention are prepared by a thin film dispersion method and a reverse phase evaporation method. Phospholipids, cholesterol, WGA functional materials, and fat-soluble drugs are weighed and dissolved in an organic solvent to prepare a lipid solution. The lipid solution is placed in an eggplant-shaped bottle, and the lipid solution is subjected to a rotary vacuum treatment with a rotary evaporator to remove the organic solvent. After the film is successfully laid, an aqueous solution containing a buffer salt and / or an osmotic pressure regulator is added for hydration, which is a thin film dispersion method. Alternatively, the above-mentioned lipid solution and an aqueous solution of a water-soluble drug are prepared by using probe ultrasound or high-speed shearing under ice bath conditions to prepare an oil-in-water emulsion, and the organic solvent is removed by rotary evaporation, which is a reverse phase evaporation method. The above two methods can produce multi-chamber liposomes.

[0031] The multilamellar liposomes are treated by probe ultrasound, extrusion or high-pressure homogenization to prepare unilamellar liposomes. Generally, ultrasound is performed in an ice bath using a probe ultrasonic oscillator, with an ultrasound power of 50 to 300 W for 1 to 10 minutes. It can also be completed by a high-pressure homogenizer, with a homogenization pressure of 200 to 800 bar, 2 to 12 times of homogenization, and each homogenization time of 10 to 50 seconds.

[0032] The present invention can select active drug loading method to load drugs, utilize pH gradient, ammonium sulfate gradient, metal ion concentration gradient and other means to achieve drug encapsulation, and then remove unencapsulated drugs through appropriate techniques such as diafiltration, dialysis, centrifugation, molecular exclusion chromatography, and ion exchange.

[0033] The multivesicular liposome provided by the invention comprises phospholipid, cholesterol, WGA functional material, medicine, triglyceride, osmotic pressure regulator and buffer salt.

[0034] In the multivesicular liposome formulation: the triglyceride is selected from triolein, myristic acid triglyceride, tricaprylin, tripalmitin, trilaurin, tricaprin, tricaprylin, tricaproin or a combination thereof;

[0035] The inner water phase / outer water phase of the present invention may be selected from osmotic pressure regulators and / or buffer salts, and cholesterol for regulating the fluidity of the bilayer membrane may be selectively added or not added;

[0036] In the multivesicular liposome formulation provided by the present invention, the ratio of drug mass to total lipid material mass is 1:8-220, or 1:10-200, or 1:10-180, or 1:10-160, or 1:10-1:150, or 1:15 to 1:120, or 1:15-100, or 1:15-80.

[0037] In some embodiments, the mass ratio of phospholipids to triglycerides is 3 to 10:1;

[0038] In some embodiments, the mass ratio of phospholipids to cocoa ionized lipids is 5 to 10:1;

[0039] In some embodiments, the concentration of glucose or sucrose in the external aqueous phase is 0-5%, and the concentration of lysine is 3-5 mg / ml;

[0040] The molar ratio of phospholipid to cholesterol is 1:0.5~1.

[0041] The preparation method of multivesicular liposomes provided by the present invention is a double emulsion volatilization method. An organic solvent is used to dissolve a lipid material (including WGA functional material and a fat-soluble drug) as an oil phase, water or a solution containing a certain concentration of glucose (or sucrose) is used to dissolve a water-soluble drug as an aqueous phase, the oil phase and the aqueous phase are mixed, and a water-in-oil (W / O) emulsion is prepared by high-speed shearing or probe ultrasound, the emulsion is mixed with an external aqueous phase (containing a certain concentration of an emulsifier, a stabilizer, and an osmotic pressure regulator), and a W / O / W double emulsion is prepared by low-speed shearing; nitrogen is introduced to remove the organic solvent, and multivesicular liposomes are obtained.

[0042] The speed of high-speed shearing is 5000-22000rpm, the optional ultrasonic power is 100-300W, and the ultrasonic time is 30 seconds-20 minutes; the speed of low-speed shearing is 3000-4000rpm;

[0043] The volume ratio of the internal water phase to the oil phase is 0.1 to 1.1:1, preferably 0.5 to 2:2;

[0044] The volume ratio of the water-in-oil emulsion to the external aqueous phase is 0.2 to 1:1, preferably 0.25 to 1:1;

[0045] The organic solvent is selected from one of chloroform, dichloromethane, acetone, and ether, or a mixture of any two of them.

[0046] The lipid nanoparticles provided by the present invention include a main drug, a WGA functional material and a lipid material;

[0047] The lipid materials include ionizable lipid materials, neutral phospholipids, structural lipid materials and PEG lipid materials;

[0048] The lipid material contains about 30-60 mol% ionizable lipid material, about 10-30 mol% neutral phospholipid, 15-50 mol% structural lipid material, and about 1-10 mol% PEG lipid material;

[0049] The molar ratio of the drug to the lipid material is about 1:10 to 500, preferably 1:15 to 200;

[0050] The molar ratio of the WGA functional material to the lipid material is about 1:100-10000.

[0051] The lipid nanoparticle preparation method provided by the present invention includes: ethanol injection method, microfluidics method, T-tube mixing method, and membrane extrusion method, wherein the microfluidics method and the ethanol injection method are preferred.

[0052] The present invention provides a microfluidic method for preparing lipid nanoparticles, the steps of which are as follows:

[0053] Weigh an appropriate amount of ionizable lipid material, neutral phospholipid, structural lipid material, PEG lipid material, and WGA functional material and dissolve them in an organic solvent as the organic phase. Dissolve the drug in a buffer (such as 10mM pH 4 citrate buffer) as the aqueous phase. The two phases are mixed simultaneously through a microfluidic device at a flow rate of 0.02 to 6ml / min and a ratio of 1:1 to 10:1, and dialyzed with ultrapure water for 2 to 8 hours at room temperature to obtain a lipid nanoparticle solution, which is stored at 4°C for later use.

[0054] The organic solvent includes but is not limited to methanol, ethanol, isopropanol, propanol, butanol, isoamyl alcohol, tert-butanol, 2-methoxyethanol, dichloromethane, chloroform, ether, acetone, cyclohexane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide;

[0055] The mixing step is carried out at a temperature of less than about 30°C, less than about 28°C, less than about 26°C, less than about 24°C, less than about 22°C, less than about 20°C, less than about 18°C, less than about 16°C, less than about 14°C, less than about 12°C, less than about 10°C;

[0056] The present invention also provides an ethanol injection method for preparing lipid nanoparticles, the steps of which are as follows:

[0057] Weigh appropriate amounts of ionizable lipids, neutral phospholipids, WGA functional materials, structural lipids and PEG lipids, and dissolve them in anhydrous ethanol as the ethanol phase. Dissolve the drug in 10mM citrate buffer (pH4) as the aqueous phase. Rapidly inject the ethanol phase into the aqueous phase under vigorous stirring, and the volume ratio of the ethanol phase to the aqueous phase is 1:5 to 5:1. Then, dialyze against ultrapure water at room temperature for 2 to 8 hours to obtain lipid nanoparticles, which are stored at 4°C for later use.

[0058] The drug-loaded PLGA nanoparticles and PLGA microparticles provided by the invention comprise drugs, PLGA, WGA functional materials, emulsifiers, osmotic pressure regulators and buffer salts.

[0059] The present invention provides PLGA for preparing PLGA nanoparticles and PLGA microparticles. The molar ratio of lactic acid to glycolic acid of PLGA is in the range of 50:50 to 95:5, such as 50:50, 55:45, 65:35, 75:25, 80:20, 85:15, 90:10 or 95:5; the intrinsic viscosity of a 0.1% chloroform solution of the PLGA is in the range of 0.1 to 2.0 dL / g; the terminal group of the PLGA may be a carboxyl group or an ester group;

[0060] The emulsifier is polyvinyl alcohol (PVA), poloxamer or vitamin E polyethylene glycol succinate (TPGS), wherein the molecular weight of PVA (number average) is between 10000 and 50000 Da, preferably between 13000 and 23000 Da; the alcoholysis degree of PVA is 70 to 90%, more preferably 88%; the concentration of PVA in the aqueous phase is 0.1 to 1.5%, preferably 0.5 to 1.0%;

[0061] The (number average) molecular weight of the poloxamer is in the range of 7000 to 20000 Da, preferably 7600 to 15000 Da; the ethylene oxide content of the poloxamer is in the range of 78% to 85%; the concentration of the poloxamer in the aqueous phase is 0.1 to 1.5%, preferably 0.5 to 1.0%;

[0062] The drug-loaded PLGA nanoparticles provided by the present invention have a drug (PLGA) ratio of 1:10 to 500, preferably 1:12 to 300; the WGA material to PLGA mass ratio is 1:10 to 300;

[0063] The drug-loaded PLGA microparticles provided by the present invention have a drug material ratio of 1:10-500, preferably 1:50-300; and a WGA material to PLGA mass ratio of 1:30-150.

[0064] The invention provides a method for preparing PLGA nanoparticles. The method comprises the following steps: accurately weighing a drug and PLGA, dissolving them in an organic solvent as an oil phase, preparing an emulsifier aqueous solution of appropriate concentration as a water phase, slowly dripping the organic phase into the water phase under magnetic stirring conditions, and after the dripping is completed, continuously stirring at a constant temperature of 40 to 50° C. for 2 to 4 hours to volatilize and remove the organic solvent, centrifuging, washing the precipitate with distilled water, centrifuging to remove the supernatant, repeating the washing and centrifugation for 3 times, and freeze-drying to obtain drug-loaded PLGA nanoparticles. Before use, an isotonic solvent is added to resuspend the nanoparticles for standby use.

[0065] The organic solvent is selected from any one or a combination of acetonitrile, acetone, tetrahydrofuran, methanol, ethanol, dimethylformamide, and dimethyl sulfoxide;

[0066] The volume ratio of the oil phase to the water phase is 1:3 to 1:50, preferably 1:5 to 20;

[0067] The stirring condition may be mechanical stirring or magnetic stirring, with a stirring speed of 200 to 1000 rpm and a stirring time of 0.5 to 6 hours. Preferably, the stirring speed is 300 to 600 rpm and the stirring time is 0.5 to 4 hours.

[0068] The present invention provides a method for preparing drug-loaded PLGA microparticles: weigh PLGA and dissolve it in an appropriate amount of organic solvent as an oil phase, weigh an appropriate amount of water-soluble drug and an osmotic pressure regulator and dissolve them in an appropriate amount of water for injection, and shake well to form an internal aqueous phase (W 1 ), ultrasonically oscillate or homogenize at high speed under ice bath conditions at 4°C to make water-in-oil (W 1 / O) type primary emulsion. 1 / O) type primary emulsion is added to a certain volume of emulsifier aqueous solution (W 2 ) and then ultrasonicated again at 37°C to form water-in-oil-in-water (W 1 / O / W2 ) type emulsion. The organic solvent is evaporated and removed under stirring conditions at room temperature and normal pressure. Then, the solidified microparticles are collected by low-temperature high-speed centrifugation (4°C, 20000rpm, 20 minutes), washed with double distilled water to remove free drugs on the surface of the microparticles, repeated 3 times, and low-temperature high-speed centrifugation for 20 minutes again to remove the supernatant, refrigerated or freeze-dried at 4°C, and resuspended in an isotonic solvent before use.

[0069] The osmotic pressure regulator is any one or a combination of glucose, sucrose, mannitol, trehalose or sodium chloride;

[0070] The organic solvent is selected from any one or more of dichloromethane, chloroform, ethyl acetate, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, ether, tetrahydrofuran, cyclohexane, and n-hexane;

[0071] The volume ratio of the oil phase to the water phase is 6 to 20:1, preferably 8 to 12:1;

[0072] The volume ratio of colostrum to external aqueous phase is 1:2-20, preferably 1:5-10;

[0073] The concentration range of the PLGA solution in the oil phase is 10-200 mg / ml, more preferably 10-120 mg / ml, and preferably 20-120 mg / ml;

[0074] The stirring condition may be mechanical stirring or magnetic stirring, with a stirring speed of 200 to 1000 rpm and a stirring time of 0.5 to 6 hours. Preferably, the stirring speed is 300 to 600 rpm and the stirring time is 0.5 to 4 hours.

[0075] The ultrasonic oscillation has an ultrasonic power of 100 to 300 W, preferably 200 to 300 W; the ultrasonic time is 1 to 30 minutes, preferably 1 to 20 minutes, and more preferably 1 to 15 minutes;

[0076] The high-speed shearing is performed at a speed of 10,000 to 30,000 rpm for 10 to 60 seconds, preferably 20,000 to 25,000 rpm for 40 to 60 seconds.

[0077] Unless otherwise specified, the particle size of the nanoparticle preparations mentioned in the present invention is the average diameter measured by a dynamic light scattering instrument. The average particle size of the microparticle preparations is measured by a laser particle size analyzer.

[0078] In a specific embodiment, the average diameter of the nanoformulation is greater than 50 nm. In a preferred embodiment, the average particle size of the nanoformulation is between 50 and 450 nm; in a preferred embodiment, the average particle size of the nanoformulation is between 60 and 400 nm; in a preferred embodiment, the average particle size of the nanoformulation is between 70 and 350 nm; in a preferred embodiment, the average particle size of the nanoformulation is between 80 and 300 nm. In a particular embodiment, the nanoparticles have an average diameter in the range of 80-90 nm, 90-100 nm, 100-110 nm, 110-120 nm, 120-130 nm, 130-140 nm, 140-150 nm, 150-160 nm, 160-170 nm, 170-180 nm, 180-190 nm, 190-200 nm, 200-210 nm, 210-220 nm, 220-230 nm, 230-240 nm, 240-250 nm, 250-260 nm, 260-270 nm, 270-280 nm, 280-290 nm or 290-300 nm. The present invention also includes the use of nanoparticles having an average diameter less than 50 nm and preferably greater than 300 nm and less than 500 nm.

[0079] In a specific embodiment, the average particle size of the micronized preparation is 1-50 μm, or 1-40 μm, or 1-30 μm, or 1-28 μm, or 1-26 μm, or 1-24 μm, or 1-22 μm, or 1-20 μm, or 1-18 μm. In other embodiments, the average particle size is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 μm.

[0080] The polydispersity index (abbreviated as PDI) can be used to characterize the particle size distribution width of nanoformulations (such as unilamellar liposomes, lipid nanoparticles, PLGA nanoparticles), and a smaller PDI (e.g., less than 0.3) means a narrower particle size distribution. PDI can be measured using a dynamic light scattering instrument. The PDI of the above-mentioned nanoformulation can be about 0 to 0.28, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27 or 0.28. In some embodiments, the polydispersity index of the nanoformulation can be about 0.10 to 0.25.

[0081] The particle size distribution of the microparticle preparation is represented by the span (SPAN). The smaller the span, the narrower the distribution, that is, the more uniform the particle size. Span = (D90-D10) / D50, where D10, D50, and D90 represent the particle sizes (μm) corresponding to 10%, 50%, and 90% in the cumulative distribution diagram of the particle size, respectively. The span of the multivesicular liposome, multilamellar liposome, and PLGA microsphere preparation described in the present invention can be 0.3 to 3.0, for example 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0.

[0082] The encapsulation rate of the nanoformulation of the present invention is determined by gel column chromatography, and the encapsulation rate of the microparticle preparation is determined by high-speed centrifugation. In some embodiments, the drug encapsulation rate of the preparation is 50% to 100%; in some embodiments, the drug encapsulation rate of the preparation is 50% to 70%; in some embodiments, the drug encapsulation rate of the preparation is 70% to 90%; in some embodiments, the drug encapsulation rate of the preparation is 90% to 100%; in some embodiments, the drug encapsulation rate of the preparation is 75% to 95%. With respect to the preparation described in the present invention, the encapsulation rate of the drug can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 60%; in some embodiments, the encapsulation efficiency can be at least 65%; in some embodiments, the encapsulation efficiency can be at least 70%; in some embodiments, the encapsulation efficiency can be at least 75%; in some embodiments, the encapsulation efficiency can be at least 80%; in some embodiments, the encapsulation efficiency can be at least 85%; in some embodiments, the encapsulation efficiency can be at least 90%; in some embodiments, the encapsulation efficiency can be at least 95%.

[0083] In certain embodiments of the invention, the formulation may comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 or 10% lyoprotectant. In certain embodiments, the formulation comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 or 10% sucrose, glucose, trehalose or mannitol.

[0084] The preparations provided by the present invention may contain buffer salts, and buffer salts suitable for the present invention include but are not limited to sodium bicarbonate, sodium borate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, HEPES, Tris, and mixtures thereof, etc. In some embodiments, the concentration of the buffer salt is about 0.1-100mM, about 0.5-90mM, about 1.0-80mM, about 2-70mM, about 3-60mM, about 4-50mM, about 5-40mM, about 6-30mM, about 7-20mM, about 8-15mM, about 9-12mM buffer salt. In some embodiments, the concentration of the buffer salt is or is greater than about 0.1mM, 0.5mM, 1mM, 2mM, 4mM, 6mM, 8mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM of the salt buffer. In certain embodiments, the buffer comprises NaCl. The exemplary amount of NaCl can be in the range of from about 30mM to about 55mM. The amount of salt, buffer and lyophilization protectant can be changed so as to maintain the milliosmotic pressure molar concentration of the overall preparation. For example, the final milliosmotic pressure molar concentration can be maintained at less than 450mOsm / kg. In other embodiments, the milliosmotic pressure molar concentration is between 350 and 250mOsm / kg. Certain embodiments have a milliosmolarity of 300 ± 20 mOsm / kg.

[0085] In some embodiments, the pH of the buffer can range from about 4.5 to about 7.0, about 4.6 to about 7.0, about 4.8 to about 7.0, about 5.0 to about 7.0, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5. In some embodiments, the pH of the buffer can range from about 7.0 to about 8.0, about 7.1 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5.

[0086] The ratio of the number of cells to the number of particles in the preparation provided by the present invention is 1:100 to 50000. In some embodiments, the ratio of the number of cells to the number of particles in the preparation is 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000.

[0087] The incubation temperature of the cells and the nano / micro preparation provided by the present invention is 2 to 32° C. In some embodiments, the mixed incubation of the cells and the nano / micro preparation is performed at less than about 32° C., less than about 30° C., less than about 28° C., less than about 26° C., less than about 24° C., less than about 22° C., less than about 20° C., less than about 18° C., less than about 16° C., less than about 15° C., less than about 14° C., less than about 12° C., less than about 10° C., less than about 8° C., less than about 6° C., less than about 5° C., less than about 4° C., less than about 2° C.

[0088] The mixed incubation time of the cells and the nano / micro preparation provided by the present invention is 10 minutes to 180 minutes. In some embodiments, the mixed incubation time of the cells and the nano / micro preparation is 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes.

[0089] The therapeutic application of the composition of the present invention includes but is not limited to the following tumors: basal cell carcinoma, medulloblastoma, liver cancer, esophageal cancer, rhabdomyosarcoma, lung cancer, bone cancer, head and neck cancer, pancreatic cancer, skin cancer, skin or eye melanoma, endocrine system cancer (including gastric cancer, breast cancer, thyroid cancer, parathyroid cancer, etc.), rectal cancer, anal cancer, penile cancer, prostate cancer, colon cancer, endometrial cancer, vulvar cancer, uterine cancer, fallopian tube cancer, Hodgkin's lymphoma, cervical cancer, ovarian cancer, vaginal cancer, small intestine cancer, adrenal cancer, soft tissue sarcoma, chronic or acute leukemia, lymphoma, bladder cancer, renal cell carcinoma, urethral cancer, kidney cancer or ureteral cancer, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, gliomas and pituitary adenomas, or a combination of one or more cancers.

[0090] The composition of the present invention can be used to treat or prevent skin, respiratory tract (including throat, trachea, bronchi, lungs), digestive system (esophagus, stomach, intestines), urinary system (bladder, urethra) or blood system (including blood vessels and blood vessels together with important organs such as heart, liver, spleen, kidney, bone marrow, brain) infections caused by pathogenic microorganisms through direct contact, breathing and the like. The pathogenic microorganisms can be gram-positive bacteria, gram-negative bacteria, fungi, mycoplasmas, chlamydia. The composition of the present invention is applicable to, but not limited to, infections caused by the following bacteria: Pseudomonas (such as Pseudomonas aeruginosa PA), Staphylococcus, methicillin-resistant Staphylococcus aureus (abbreviated as MRSA), Streptococcus (including Streptococcus pneumoniae), Escherichia coli, Enterococcus faecalis, Klebsiella, Serratia, Haemophilus, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellularis, etc.

[0091] The present invention uses the following technologies:

[0092] The present invention performs modification work on two cell lines: macrophage cell line RAW264.7 and human mononuclear cell THP-1, and uses flow cytometry to screen co-incubation conditions such as incubation feed ratio, incubation temperature and incubation time through co-incubation means.

[0093] Particle size, particle size distribution, and encapsulation efficiency are important characterization parameters of preparations. The present invention uses dynamic light scattering to determine the particle size distribution and charge of the nano-preparation, and uses a laser particle size analyzer to determine the particle size and span of the microparticles; ultracentrifugation and gel column chromatography are selected to determine the encapsulation of the drug in the preparation.

[0094] The present invention uses nanoparticle tracking analysis (NTA), that is, using a laser light source to irradiate the preparation dispersion, recording the Brownian motion of particles with scattered light, and calculating the particle size through the Stokes-Einstein equation, thereby accurately calculating the number of preparation particles in a unit volume, and defining the ratio of the number of cells to the number of preparation particles as the cell / preparation incubation feed ratio.

[0095] The present invention uses a laser confocal microscope and a scanning electron microscope to examine the surface modification of macrophages / monocytes.

[0096] The present invention uses different fluorescent probes to achieve the labeling of macrophages / monocytes and tumor cells: HCT-116 cells are labeled with GFP, and RAW264.7 / THP-1 cells are labeled with DID. The labeled tumor cells are co-cultured with RAW264.7 cells and THP-1 cells, respectively. A flow cytometer is used to record cells that are positive for both fluorescent probes, and the positive rate is calculated. This is defined as the phagocytic rate, which is used as an evaluation indicator for the ability of macrophages / monocytes to phagocytize tumor cells.

[0097] The present invention adopts CCK-8 method and neutral red uptake experiment to examine the effect of the preparation on the proliferation activity and phagocytic performance of macrophages and monocytes, and examines whether the preparation modification can interfere with the normal functions of the above cells.

[0098] The present invention uses transwell experiment and flow cytometry to examine the changes in cell migration ability and phagocytic ability caused by the modification of the preparation on macrophages / monocytes. The superoxide secretion of macrophages is quantitatively detected at OD (450nm) by WST-1 Superoxide Assay Kit as an evaluation index of the intracellular clearance ability of macrophages / monocytes.

[0099] The present invention adopts agarose gel method to construct a tumor sphere model, which is co-incubated with the cell composition provided by the present invention, and the growth inhibition ability of the tumor sphere is reflected by the changes in the morphology and volume of the tumor sphere.

[0100] The present invention selects the bactericidal curve as an evaluation index of in vitro antibacterial activity.

[0101] The present invention uses commercially available human colon adenocarcinoma cells (HCT116) loaded with fluorescent substances (luciferase LUC2) to successfully construct a nude mouse model of colon cancer in situ, uses a preparation to encapsulate fluorescent probes, and administers them to nude mice. The in vivo tumor targeting of the cell composition is evaluated by the change in fluorescence intensity in the tumor-bearing nude mice. In addition, the in vivo anti-tumor activity of the cell composition constructed by the present invention is investigated by the change in tumor volume.

[0102] The present invention uses MRSA and PA to construct a mouse skin abscess model and a pneumonia model, respectively, and adopts the technology of the present invention to construct macrophages / monocytes modified with a drug-loaded preparation, which are administered intravenously to model mice, and live bacteria are counted in the abscess site and the lungs as an indicator of the in vivo anti-infection activity of the cell composition.

[0103] Compared with the prior art, the present invention achieves the following technical effects: (1) The preparation particles provided by the present invention have uniform particle size and narrow particle size distribution, and can stably encapsulate drugs; (2) The carrier preparation designed by the present invention achieves the effect of "killing two birds with one stone". On the one hand, the preparation with surface modified WGA molecules enhances the original functions of macrophages / monocytes (chemotaxis, phagocytosis and clearance). On the other hand, the preparation plays a carrier function to achieve effective loading of various substances, such as anti-tumor drugs, contrast agents and diagnostic reagents. (3) The present invention regulates the cell composition by controlling the number of preparation particles, incubation temperature, and incubation time, and the preparation method is simple and easy to master and implement; (4) The immune cell-loaded preparation provided by the present invention can accurately and efficiently deliver drugs to tumors or infected lesions through cellular immune recognition; (5) The living cells used in the present invention not only have efficient tissue targeting but also exert their own functions, producing the non-specific immune recognition, phagocytosis, and clearance effects of macrophages / monocytes and the dual-strike effect of drugs; (6) The present invention adopts cell surface modification instead of phagocytosis to reduce the adverse effects of intracellular degradation of drugs caused by phagocytosis and facilitate the release of drugs; (7) The technology provided by the present invention produces good effects in anti-tumor and anti-infection, and serves as a reference for the diagnosis and treatment of other diseases, and has a demonstration effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0105] Figure 1 The dynamic light scattering particle size distribution diagram of Examples 1-3 and Comparative Examples 1-3 (a: Example 1; b: Example 2; c: Example 3; d: Comparative Example 1; e: Comparative Example 2; f: Comparative Example 3).

[0106] Figure 2 The graphs show the uptake of the preparations (Examples 1-8, Comparative Examples 1-8) by RAW264.7 cells (a) and THP-1 monocytic cells (b).

[0107] Figure 3 These are laser confocal microscopy images of RAW264.7 cells co-incubated with the examples and control examples at different temperatures (a: Example 1; b: Example 2; c: Example 3; d: Example 4; e: Control Example 1; f: Control Example 4).

[0108] Figure 4 These are scanning electron micrographs of the distribution of the examples on the surface of RAW264.7 cells and THP-1 cells (a: Example 1; b: Example 2; c: Example 3; d: Example 4; e: Example 5; f: Example 6; g: Example 7; h: Example 8; i: Control Example 1; j: Control Example 4; k: Control Example 6; l: Control Example 7).

[0109] Figure 5 The effects of the preparations (Examples 9-14) on the proliferation activity of RAW264.7 cells and THP-1 cells.

[0110] Figure 6 The modification of the preparations (Examples 9-14, Comparative Examples 9-10) on RAW264.7 cells and THP-1 cells caused changes in the number of cell migration.

[0111] Figure 7 The effects of modified preparations (Examples 9-14, Control Examples 9-10) on the ability of RAW264.7 cells (a, c) and THP-1 cells (b, d) to phagocytose HCT-116 cells and MRSA.

[0112] Figure 8 The distribution of MRSA in RAW264.7 cells was observed using a confocal microscope (a: Example 9; b: Example 10; c: Example 11; d: Control Example 9).

[0113] Fig. 9Modification of the preparations (Examples 9-14, Comparative Examples 9-10) on RAW264.7 cells (a) and THP-1 cells (b) caused changes in the expression of intracellular peroxides.

[0114] Fig.10 The modification of the preparations (Examples 9-14, Control Examples 9-10) affects the intracellular bactericidal ability (Figure a: MRSA; Figure b: PA) of RAW264.7 cells (a) and THP-1 cells (b).

[0115] Fig.11 The targeting properties of RAW264.7 cells and THP-1 cells modified by the formulations (Examples 15 and 16) in nude mice (a: tumor targeting; b: abscess targeting).

[0116] Fig.12 The morphology (a) and volume changes (b) of HCT-116 tumor spheres were observed under fluorescence microscopy within 5 days.

[0117] Fig.13 The killing curves of RAW264.7 cells and THP-1 cells modified by the drug-loaded formulations (Examples 21-26) (Figure a: MRSA; Figure b: PA).

[0118] Fig.14 In vivo antibacterial activity of RAW264.7 cells and THP-1 cells modified with drug-loaded formulations (Examples 21-26). DETAILED DESCRIPTION

[0119] The following detailed descriptions are all illustrative and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0120] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. When the terms "comprise" and / or "include" are used in this specification, it indicates that there are features, steps, operations, devices, components and / or combinations thereof.

[0121] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0122] 1. Preparation of micro-nanocarriers and determination of their physicochemical properties

[0123] 1.1 Preparation

[0124]

[0125] According to the prescription in the above table, coumarin 6, phospholipids, cholesterol and other lipid materials were weighed into an eggplant-shaped bottle, and an appropriate amount of chloroform was added to dissolve them. The organic solvent was removed by evaporation under reduced pressure until a uniform lipid film was formed on the inner surface of the eggplant-shaped bottle. 5 ml of PBS preheated at 37° C. was added for hydration. Then, Example 1 and Control Example 1 were treated with probe ultrasound (power of 300 W) for 10 minutes, and Example 2 and Control Example 2 were not treated with ultrasound.

[0126]

[0127] Preparation method of Example 3: According to the above prescription, PLGA, WGA-PEG5k-PLA5k, and coumarin 6 were weighed and dissolved in 10 ml of acetone, and slowly dripped into 200 ml of 1% w / v poloxamer 188 aqueous solution with slow stirring, and the acetone was evaporated and removed by continuous stirring. High-speed centrifugation (speed of 20,000 rpm) was combined with water washing to remove unencapsulated substances, and the centrifugation and water washing operations were repeated alternately for 3 times. The nanoparticles were dispersed in isotonic sterile PBS (pH 7.2) for use.

[0128] According to the prescription in the above table, nanoparticles using PEG5k-PLA5k instead of WGA functional material were prepared, which is Control Example 3.

[0129]

[0130] Example 4 Preparation method: According to the above prescription, coumarin 6, PLGA, and WGA-PEG2k-DSPE were weighed and dissolved in 8 ml of dichloromethane as the oil phase; PVA was weighed and dissolved in water for injection as the aqueous phase. The aqueous phase was added to the organic phase, and probe ultrasound (power of 100 W) was used to prepare O / W colostrum, and all organic solvents were evaporated by magnetic stirring at room temperature, and centrifugation and water washing were performed alternately to remove extra-particle substances, and dispersed with an appropriate amount of 5% mannitol solution, and stored at 4°C for standby use.

[0131] According to the prescription in the above table, PEG2k-PLA2k was used to replace the WGA functional material, and the microparticles were prepared in the same manner to obtain Control Example 4.

[0132]

[0133] Preparation method of Example 5 and Control Example 5: Weigh lipid materials (phospholipids, cholesterol, WGA functional material) and coumarin 6 according to the prescription in the above table, dissolve in an appropriate amount of chloroform, and spread the film by rotary evaporation according to the preparation method of Example 1. Add isotonic sodium chloride solution for hydration, use a high-pressure homogenizer (80 MPa, cycle 10 times) to homogenize, pass the membrane, and store at 4°C for later use.

[0134]

[0135] Example 6 Preparation method: SPC, cholesterol, WGA-PEG2k-DSPE, triolein, and coumarin 6 were weighed according to the above prescription and dissolved in 2 ml chloroform as the oil phase; 2 ml 3% sucrose solution was taken as the inner water phase, the oil phase and the inner water phase were mixed, high-speed shearing (rotation speed of 15000 rpm) for 15 minutes, and ultrasonic (power of 300 W) for 7 minutes to prepare a stable W / O type colostrum, which was quickly transferred into a 3.3% L-lysine solution, and continued to be sheared (rotation speed of 3000 rpm) to form a W / O / W emulsion, and the emulsion was quickly transferred to 20 ml 5% sucrose aqueous solution, magnetically stirred, and the temperature was controlled at 35-40°C. At the same time, nitrogen was continuously blown to the liquid surface to remove all organic reagents, and centrifugation and washing were used alternately for purification, and finally 5% sucrose aqueous solution was used for resuspending. The preparation method of comparative example 6 was the same as that of example 6, and PEG2k-DSPE was used to replace the WGA functional material in the prescription.

[0136]

[0137] Preparation method of Example 7 and Control Example 7: Coumarin 6 and various lipid materials were weighed according to the prescription in the above table, dissolved with appropriate amount of methanol, placed in an eggplant-shaped bottle and rotary evaporated to remove the solvent, spread into a uniform film, added PBS (pH 7.4) for hydration, vortexed for 5 minutes, filtered with a 0.22 μm microporous filter membrane, and stored at 4°C for later use.

[0138]

[0139] Preparation method of Example 8: According to the above prescription, PLGA and DSPC were weighed and dissolved in 1ml of dichloromethane as the oil phase; 12mg of ammonium bicarbonate was weighed and dissolved in 0.2ml of water for injection as the water phase, the oil phase was placed in an ice bath (~4°C), the water phase was added dropwise, and the colostrum was prepared by ultrasound (power of 50W) for 2 minutes, 5ml of 4% PVA solution (containing WGA-PEG2k-DSPE) was added, and high-speed shear (speed of 16000rpm) was performed for 7 minutes to prepare a double emulsion. 10ml of water for injection was added for dilution, and the organic solvent was evaporated by magnetic stirring at room temperature. Centrifuge at 5000rpm for 5 minutes, wash the precipitate with water for injection, and repeat the centrifugation and washing process alternately three times each. The precipitate was mixed with an appropriate amount of isotonic PBS, stored at 4°C for standby use. The preparation method of Control Example 8 was the same as that of Example 8, and PEG2k-DSPE replaced the WGA functional material.

[0140] 2. Determination of physical and chemical properties

[0141] The particle size and Zeta potential of the nanoformulation were measured using a ZetasizerNano ZS laser particle size analyzer. The laser beam wavelength of the instrument was set to 633 nm, the angle between the incident light and the scattered light beam was 90°, and the measurement temperature was set to 25°C.

[0142]

[0143] The average particle size and span of the micron preparation were measured using a Mastersizer 2000. The results are shown in the following table: Average particle size D[3,2](μm) Span Example 2 1.42 1.555 Example 4 2.02 1.202 Example 6 3.35 1.244 Example 8 6.47 1.158 Comparative Example 2 1.88 1.750 Comparative Example 4 2.16 1.632 Comparative Example 6 3.83 1.196 Comparative Example 8 2.31 1.366

[0144] The above data show that the average particle size of the unilamellar liposomes of the present invention is between 130 and 400 nm, the polydispersity coefficient is less than 0.2, and the Zeta potential is between -1 and -3 mV; the average particle size of the multilamellar and multivesicular liposomes is between 1 and 6 μm, and the span is between 1.1 and 1.7.

[0145] The particle concentrations of the examples and the control examples were measured using a Nanosight NS300 nanoparticle tracking analyzer (NTA). The results are shown in the following table:

[0146] 2. Cell modification

[0147] 2.1 Modification of RAW264.7 cells

[0148] RAW264.7 cells (mouse mononuclear macrophage leukemia cells, purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences) were cultured at 37°C and 5% CO 2 In this environment, the cells were passaged and cultured using DMEM high-glucose complete medium (containing 10% FBS and 1% penicillin-streptomycin double antibody).

[0149] A RAW264.7 cell suspension in the logarithmic growth phase was taken and incubated with Examples 1-4 and Control Examples 1-4, respectively (the incubation conditions are shown in the following table), and the number of cells / number of preparation particles was used as the incubation feed ratio (respectively 1:100, 1:500, 1:1000, 1:2500, 1:5000, 1:10000, 1:20000, 1:50000), incubated for different times (as shown in the following table), the culture medium was discarded, the cells were washed with PBS and resuspended, and passed through a 300-mesh cell sieve. The cells were collected into a flow tube, and the fluorescence intensity (excitation wavelength E x and emission wavelength E mare set to 463nm and 506nm respectively).

[0150] Modification conditions Example 1, Comparative Example 1 4°C, 2 hours Example 2, Comparative Example 2 18℃, 20min Example 3, Comparative Example 3 20℃, 30min Example 4, Comparative Example 4 32°C, 1.5 hours The test results are as attached Figure 2 As shown in a, the fluorescence intensity of RAW264.7 cells gradually increased with the increase of incubation ratio. The fluorescence intensity of all preparations modified with WGA was significantly stronger than that of the group without WGA, indicating that WGA enhanced the recognition of the preparations by cells.

[0151] 2.2 Modification of THP-1 monocytic cell line

[0152] THP-1 cells (human acute monocytic leukemia cells, purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in RPMI-1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin double antibody solution) at 37°C and 5% CO 2 Cultivated in environment.

[0153] Take THP-1 mononuclear cell suspension in logarithmic growth phase (cell density is 1×10 6 / ml), each preparation (Example 5-8, Control Example 5-8) was added respectively, incubated according to the conditions described in the following table, centrifuged and washed with PBS to remove the culture medium, resuspended with PBS and passed through a 300-mesh cell sieve, and the cells were collected in a flow tube for detection by flow cytometry.

[0154] The results are attached Figure 2 As shown in b, the fluorescence intensity of THP-1 cells gradually increased with the increase of incubation ratio. All preparations modified with WGA caused the increase of cell fluorescence intensity to be significantly higher than that of other preparations.

[0155]

[0156] 2.3 Laser confocal microscopy observation of RAW264.7 cell modification

[0157] The RAW264.7 cell suspension (2.5×10 5 The cells were inoculated into confocal microplates at 37°C and 5% CO 2Incubate in the environment for 24 hours, replace the culture medium with PBS, pre-balance at 4°C and 37°C for 20 minutes, discard the PBS solution, add each preparation to the small dish for co-incubation (the incubation feed ratio of cell number / liposome number is about 1:5000), and co-incubate according to the incubation conditions described in "2.1 Modification of RAW264.7 cells", add CMAC cytoplasmic fluorescent dye and DiL cell membrane fluorescent dye to stain the cells. After co-incubation, discard the supernatant, wash twice with 1ml PBS, add 2ml sealing slides, and observe using a laser confocal microscope (the excitation wavelength of CMAC is E x and emission wavelength E m 353nm and 466nm respectively, DiL's E x / E m is 549 / 565nm, E of coumarin 6 x / E m is 463 / 506nm).

[0158] Depend on Figure 3 It can be seen that after the RAW264.7 cell line was incubated with Examples 1-4, Control Example 1, and Control Example 4 at 37°C, more preparations were co-localized on the cell membrane, further confirming that the particles can be effectively attached to the surface of RAW264.7 cells. In contrast, Control Example 1 and Control Example 4 without WGA were less distributed on the cell surface and inside.

[0159] 2.4 Scanning electron microscopy observation of cell modification

[0160] RAW264.7 cells (5×10 5 / ml), inoculated on cell slides, and incubated at 37°C, 5% CO 2 Incubate in an environment for 24 hours. After replacing the culture medium with PBS, add each preparation (Example 1-4, Control Example 1 and Control Example 4) to the slide at a feed ratio of 1:5000 according to the number of cells / number of preparation particles, and co-incubate according to the conditions described in "2.1 Modification of RAW264.7 cells" and "2.2 Modification of THP-1 mononuclear cell line". After the incubation, gently wash the slide twice with PBS, add pre-cooled 2.5% glutaraldehyde fixative and place it in a 4°C refrigerator for overnight fixation. Replace the fixative with PBS, pre-cool 1% osmium acid at 4°C, soak with PBS, dehydrate with acetone / isoamyl acetate (1:1), dehydrate with a series of concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%) of gradient ethanol aqueous solution, and observe under an electron microscope (SU8020 field emission scanning electron microscope, JEOL) after gold spraying.

[0161] THP-1 monocytes (1×10 6Cells / ml) were placed in an EP tube, centrifuged at 1300rpm for 3 minutes to discard the supernatant, and each preparation (Examples 5-8, Control Example 6 and Control Example 7) was added respectively, the cell / liposome incubation feed ratio was 1:2500, and co-incubation was performed according to the incubation conditions described in "2.1" and "2.2". After the incubation, the supernatant was removed by centrifugation at 3000rpm for 2 minutes, PBS was added for washing and the supernatant was removed, and the cell pellet was obtained by centrifugation at 3000rpm, fixed with glutaraldehyde, and stored in a refrigerator at 4°C overnight. The subsequent scanning electron microscope sample processing and observation were performed according to the above operation.

[0162] The results are as follows Figure 4 As shown, it can be clearly observed that each example is connected to the cell surface, while the control example is almost not distributed on the cell surface.

[0163] 3. Effects of formulation modification on cell viability and cell function

[0164]

[0165] Example 9 Preparation method: According to the prescription in the above table, the lipid material (including WGA functional material) was weighed and dissolved in 2 ml of a mixed solvent of dichloromethane / ether (3:1, V / V), 0.2 ml of a 5% glucose solution was added, and high-speed shearing (rotation speed of 10000 rpm) was performed for 10 minutes to form a W / O emulsion; the emulsion was mixed with a 0.5% PVA 5% glucose solution, and a W / O / W double emulsion was prepared by shearing (rotation speed of 4000 rpm); nitrogen was introduced to remove dichloromethane and ether, and finally resuspended with an appropriate amount of 5% glucose solution to obtain a multivesicular liposome suspension.

[0166]

[0167] Preparation method of Example 10: According to the prescription in the above table, polymer materials (PLGA, WGA-PEG-PLA) were weighed respectively, dissolved in an appropriate amount of dichloromethane as the oil phase, and the oil phase was dropped into the PVA solution. The O / W emulsion was prepared by high-speed shearing (rotation speed of 10000 rpm) for 15 minutes, and the organic solvent was evaporated to remove at room temperature, and stored at a low temperature of 4°C for use.

[0168]

[0169] Preparation method of Example 11: Weigh the lipid material in the above table and dissolve it in an appropriate amount of anhydrous ethanol. Slowly and uniformly drip the solution into an appropriate amount of deionized water, stirring while dripping. After the dripping is completed, stir at 37°C (speed of 400 rpm) for 1 hour to evaporate and remove the ethanol, ultrasonically treat with a probe (power of 250 W) for 5 minutes, and filter with a 0.45 μm microporous filter membrane. The preparation method of Control Example 9 is the same as that of Example 11, and PEG2k-DSPE is used in the prescription instead of WGA functional material.

[0170]

[0171] Preparation method of Example 12: According to the above table, lipid materials (phospholipids, cholesterol, WGA functional material) were weighed, dissolved in 25 ml of chloroform to prepare an oil phase, PBS (pH 7.4) was added as an aqueous phase, and colostrum was prepared by ultrasonic oscillation. The organic solvent was removed by vacuum in a rotary evaporator to form a gel, and liposomes were prepared by further rotary evaporation. The environment was placed at 60 to -80°C for 5 freeze-thaw cycles. The preparation method of Control Example 10 was the same as that of Example 12, except that the WGA functional lipid material was not included in the prescription.

[0172]

[0173] The preparation method of Example 13 is a thin film dispersion method. According to the prescription in the above table, lipid materials (phospholipids, cholesterol, WGA-PEG2k-DSPE) are weighed and dissolved with appropriate amount of chloroform. The organic solvent is removed by vacuum using a rotary evaporator to form a uniform thin film on the wall of the round-bottom bottle. PBS is added for hydration, and probe ultrasound (power 300 W) is used for 10 minutes.

[0174]

[0175] Preparation method of Example 14: polymer materials (PLGA, WGA-PEG-PLA) were weighed respectively according to the prescription in the above table, dissolved in an appropriate amount of dichloromethane as the oil phase, and poloxamer 188 was weighed separately according to the prescription and dissolved in water for injection as the water phase; the oil phase was dropped into the water phase, and high-speed shearing (rotation speed of 22000rpm) was used for 15 minutes to form an O / W emulsion, the organic solvent was evaporated to remove at room temperature, and stored at a low temperature of 4°C for use.

[0176] The particle size, polydispersity coefficient, and Zeta potential of Examples 9-11 and Comparative Example 9 were measured using the above-mentioned dynamic light scattering method. The results are shown in the following table:

[0177] The average particle size and span of Examples 12-14 and Comparative Example 10 were measured using a laser particle size analyzer. The results are shown in the following table: Average particle size D[3,2](μm) Span Example 12 6.33 1.680 Embodiment 13 2.25 1.756 Embodiment 14 2.52 1.314 Comparative Example 10 7.21 1.232

[0178] 3.1 Cell proliferation activity assessment

[0179] The CCK-8 method was used to investigate the effects of the preparations on the proliferation activity of RAW264.7 cells and THP-1 cells. RAW264.7 cells in the logarithmic growth phase were seeded in 96-well culture plates and incubated at 37°C and 5% CO 2The cells were cultured in an environment and the supernatant was discarded. Each preparation (Example 9-11) was added according to different cell number / preparation particle number ratios, with 6 replicates per group. The same number of RAW264.7 cells was added as a blank control group. The cells were cultured at 37°C and 5% CO. 2 After incubation for 24 hours at room temperature, the supernatant was discarded, and CCK-8 diluent was added to each well. After incubation for 30 minutes, the absorbance of each well was measured at a wavelength of 450 nm using an enzyme reader to calculate the cell viability.

[0180] THP-1 cells in the logarithmic growth phase were inoculated in a 96-well culture plate, and preparations (Examples 12-14) with different incubation and feeding ratios were added according to the above method. The same number of THP-1 cells was used as a blank control group, and the absorbance was measured and the cell viability was calculated using the above method.

[0181] Results (such as Figure 5 As shown in the figure, when the incubation ratio reaches 1:50000, the cell viability of macrophages RAW264.7 and THP-1 under the action of each embodiment exceeds 90%, indicating that the cytotoxicity of embodiments 1-8 is low and the cell safety is good.

[0182] 3.2 Evaluation of cell chemotaxis function.

[0183] Transwell chamber (24 wells) was used to study the changes in cell migration ability of RAW264.7 cells and THP-1 cells after the preparations were modified. After the bottom membrane of the upper chamber of the Transwell was rinsed with PBS, 1% bovine serum albumin solution was added to the lower and upper chambers of the Transwell, respectively, and incubated at 37°C for 1 hour.

[0184] HCT-116 (human colon cancer cell line) single cell suspension in logarithmic growth phase was inoculated into 24-well culture plates (1×10 5 / well), at 37°C, 5% CO 2 Incubate at room temperature for 24 hours.

[0185] The single cell suspension of RAW264.7 in the logarithmic growth phase was inoculated into the upper chamber of the Transwell (5×10 4 / well), 37°C, 5% CO 2 The cells were allowed to adhere to the wall and grow by environmental sedimentation. After 4 hours, Examples 9-11 (cell number / preparation particle number 1:500) and Control Example 9 were added respectively. A blank control group without any preparation was set up. The incubation conditions were shown in the following table, and macrophages modified with the preparations were obtained respectively.

[0186]

[0187] After incubation, the chamber was washed with PBS and transferred to a 24-well plate seeded with HCT-116 cells. After incubation for 24 hours, the cells in the transwell chamber were washed with PBS, fixed with methanol, and stained with crystal violet solution. After drying, the membrane was placed under a microscope for observation and the cells that migrated under the chamber membrane were counted.

[0188] The results of cell migration showed that after HCT-116 tumor cells were inoculated in the lower chamber, the number of RAW264.7 cells that migrated increased significantly, indicating that HCT-116 cells can enhance the migration of RAW264.7 cells. Figure 6 As shown in Figure a, there is no significant difference in the number of RAW264.7 cells in the blank group compared with the control example 9@RAW264.7, indicating that the preparation without WGA has no significant effect on the migration of RAW264.7 cells to HCT-116 cells. After the RAW264.7 cells are modified with the WGA preparation (Examples 9-11), the number of migration of RAW264.7 cells is significantly increased, indicating that the macrophage surface modification technology disclosed in the present invention has a significantly enhanced chemotactic (tumor cell) ability.

[0189] THP-1 cells in the logarithmic growth phase were treated with the same method as RAW264.7 cells and inoculated into the upper chamber of Transwell (5×10 4 / well), and co-incubated with Examples 12-14 to modify THP-1 (specific conditions are shown in the table above), and THP-1 modified with a preparation not containing WGA (Control Example 10) and a blank group of unmodified THP-1 were set at the same time. The detection was carried out according to the above method.

[0190] preparation Incubation conditions Example 12 10℃, 3 hours Embodiment 13 12℃, 50min Embodiment 14 26℃, 30 min Comparative Example 10 32°C, 3 hours

[0191] The results are as follows Figure 6 As shown in Figure b, Examples 12-14 can significantly increase the number of THP-1 cells migrating toward tumor cells HCT-116, and are significantly higher than the control group without WGA preparation modification (Control Example 10).

[0192] 3.3 Evaluation of modified cell phagocytic function

[0193] 3.3.1 Effects of formulation modification on macrophage phagocytosis of tumor cells

[0194] Take the RAW264.7 cell suspension modified according to the method of "3.2" (Examples 9-11, Control Example 9) and the unmodified RAW264.7 cell suspension, and incubate them with DiD dye at 37°C and 5% CO 2The cells were incubated in a dark environment for 20 minutes for staining, and the supernatant was discarded by centrifugation to obtain DiD-labeled RAW264.7 cells, which were inoculated with GFP-labeled HCT-116 cells in a 12-well culture plate and incubated at 37°C and 5% CO 2 After rinsing with PBS, digesting with trypsin, terminating digestion, blowing cells, centrifuging, washing with PBS, resuspending with PBS, and passing through a cell sieve, the intracellular fluorescence intensity (E of DiD) was detected by flow cytometry. x and E m 644nm and 665nm respectively, and the E x and E m The double positive rate was measured as the phagocytosis rate.

[0195] The results are as follows Figure 7 As shown in a, compared with the blank control group and control example 9, the modifications of Examples 9-11 significantly improved the phagocytic ability of RAW264.7 cells on HCT-116 cells.

[0196] Take the THP-1 cell suspension modified by the method of "3.2" (Examples 12-14, Control Example 10), label the THP-1 cells with DID according to the above method, and modify them with a preparation without WGA (Control Example 10) and a preparation containing WGA (Examples 12-14), and then incubate them with GFP-labeled HCT-116 cells to measure the phagocytic rate.

[0197] result Figure 7 As shown in b, the modification of THP-1 cells by Examples 12-14 significantly increased the phagocytic ability of THP cells to tumor cells.

[0198] 3.3.2 Modified macrophage phagocytosis of pathogens

[0199] The modified RAW264.7 cells (Examples 9-11, Control Example 9) and unmodified RAW264.7 cells (as a control) were inoculated in 6-well culture plates (1×10 6 / ml), cultured until the confluence reached 80%-90%, added FITC-labeled methicillin-resistant Staphylococcus aureus MRSA and continued to culture for 1.5 hours, washed with pre-cooled PBS to replace the culture medium, added trypsin to digest the cells, centrifuged and washed with PBS for 3 times, and detected the intracellular fluorescence intensity (E x 494nm, E m is 520nm).

[0200] The results are as follows Figure 7As shown in c, the RAW264.7 cells modified in Examples 9-11 showed a higher ability to phagocytose MRSA, which was significantly stronger than the RAW264.7 cells modified in Control Example 9.

[0201] The modified THP-1 cells (Examples 12-14, Control Example 10) and unmodified THP-1 cells were operated according to the above method for phagocytosis of MRSA by RAW264.7 cells, and the intracellular fluorescence intensity was detected by flow cytometry.

[0202] The results are as follows Figure 7 As shown in Figure d, the THP-1 cells modified in Examples 12-14 significantly enhanced the phagocytosis of MRSA, which was stronger than that of the control example 10.

[0203] 3.3.3 Intracellular distribution of pathogens after phagocytosis by modified macrophages

[0204] Take the preparations (Examples 9-11, Control Example 9), label them with FITC respectively, and set aside; modify the RAW264.7 cell suspension according to the method of "3.2", label them with organelle fluorescent dye, and inoculate them on laser confocal plates (the number is 1×10 6 / ml), cultured overnight, replaced with preheated PBS, added with Hoechst 33258 (blue, nuclear marker) labeled MRSA and continued to culture. After 1.5 hours, the culture medium was replaced with precooled PBS, and each cell was observed with a laser confocal microscope.

[0205] The results are as follows Figure 8 As shown, the control group (reference example 9) has a small amount of blue fluorescence, indicating that unmodified macrophages can also engulf a small amount of MRSA, and more MRSA is engulfed in the cells of each example, indicating that the macrophages modified by the example engulf MRSA, and some MRSA are distributed in lysosomes, which is conducive to the clearance function of macrophages. In addition, some MRSA co-localizes with the preparation, indicating that the preparation is in close contact with MRSA, which is conducive to the further function of the drug encapsulated in the preparation.

[0206] 3.4 Evaluation of cell clearance ability

[0207] 3.4.1 Intracellular peroxides

[0208] Take the macrophages modified according to the method of "3.2" (Examples 9-11, Control Example 9), and set up a control group. Add superoxide detection solution for color development. After continuing to incubate in a cell culture incubator for 1 hour, use an enzyme marker to measure the absorbance at 450nm.

[0209] The results are as follows Fig. 9As shown in a, each example can significantly increase the expression of peroxide in RAW264.7 cells, which is significantly higher than that of control example 9.

[0210] Take THP-1 cells modified according to the method of "3.2" (Examples 12-14, Control Example 10), and detect the expression of intracellular peroxide according to the above method.

[0211] The results are as follows Fig. 9 As shown in FIG. 1B , Examples 12-14 can significantly increase the expression of intracellular peroxide in THP-1 cells, which is significantly higher than that in Control Example 10.

[0212] 3.4.2 Intracellular killing curve

[0213] RAW264.7 cells modified according to the method of "3.2" (Examples 9-11, Control Example 9) of each preparation were plated in 96-well plates (5×10 5 cells / ml), and RAW264.7 cells without preparation modification were set as control group, cultured until the confluence reached 80%-90%, washed with PBS to replace the culture medium, and added MRSA serum-free complete medium. 2 The culture was continued under the same conditions, and the culture medium was replaced by PBS after washing at different time points. The lysis solution was added and pipetted repeatedly, and then the cells were diluted successively and the live bacteria were counted.

[0214] The results are as follows Fig.10 As shown in a, the number of viable cells in Examples 9-11 is significantly lower than that in Control Example 9, indicating that the macrophages modified by the preparations (Examples 9-11) have good intracellular bactericidal ability.

[0215] THP-1 cells modified by the method "3.2" of each preparation (Examples 12-14, Control Example 10) were incubated with Pseudomonas aeruginosa (abbreviated as PA) according to the above operation, and THP-1 cells not modified by the preparation were set as the Control group, and the number of intracellular live bacteria was determined respectively.

[0216] The results are as follows Fig.10 As shown in b, the number of viable cells in Examples 12-14 was significantly lower than that in Control Example 10, indicating that the macrophages modified by the preparations (Examples 12-14) had good intracellular bactericidal ability.

[0217] 3.5 In vivo targeting

[0218]

[0219] Example 15 Preparation method: Weigh lipid materials (including WGA functional lipid materials) according to the prescription in the above table, dissolve them in an appropriate amount of chloroform, prepare a chloroform solution of DiR separately, measure an appropriate amount of volume (equivalent to approximately 0.2 mgDiR) and add it, mix well, transfer to an eggplant-shaped bottle, evaporate the organic solvent by rotary evaporation, form a uniform thin film on the wall of the eggplant-shaped bottle, add PBS to hydrate, vortex and oscillate to obtain.

[0220]

[0221] Example 16 Preparation method: PLGA, WGA functional materials, and DiR were weighed according to the prescription in the above table and dissolved in a dichloromethane / acetone mixed solvent (volume ratio of 5:1), which were slowly added to a 1% TPGS aqueous solution, and the organic solvent was evaporated and removed by magnetic stirring. After centrifugation, water washing, and resuspension, a microparticle suspension was obtained.

[0222] The particle sizes of the preparations of Examples 15-16 were measured using a laser particle size analyzer. The results are shown in the following table: Average particle size D[3,2](μm) Span Embodiment 15 2.1 1.425 Example 16 4.4 1.776

[0223] The encapsulation efficiency of the embodiment was measured by fluorescence method, and the results were as follows: Encapsulation rate (%) Embodiment 15 92.3 Example 16 88.6

[0224] 3.5.1 Tumor targeting evaluation

[0225] BALB / c nude mice (male, 20 g, 6-8 weeks old) were inoculated with luciferase-labeled human colon cancer cells HCT116-LUC2 in situ in the cecum to establish an orthotopic tumor model.

[0226] Take Examples 15 and 16, and incubate them with RAW264.7 cells and THP-1 cells respectively according to the table below to obtain modified cells.

[0227] Modification conditions name Embodiment 15 6°C, 2.2 hours Example 15 @ RAW264.7 Example 16 14°C, 3 hours Example 16@THP-1

[0228] After the abdomen of the nude mice was significantly swollen, the nude mice were randomly divided into four groups: (1) Sterile sodium chloride injection; (2) free DiR group; (3) Example 15 @ RAW264.7; (4) Example 16@THP-1. Four groups containing the same amount of DiR were injected into the tail vein, and the fluorescence changes of nude mice were monitored using a small animal live imaging device at different times. After 24 hours, the nude mice were anesthetized and killed, and the tumors were immediately removed and placed in a small animal live imaging device to observe the fluorescence intensity of the isolated organs (excitation and emission wavelengths: 745 / 810 nm).

[0229] The results showed that the cells modified in Examples 15 and 16 reached and accumulated in the tumor site in a relatively short period of time, and the accumulation in the tumor site increased with the prolongation of administration time until reaching the maximum at 24 hours. Fig.11 As shown in a, compared with the DiR group, the modifications of Examples 15 and 16 significantly increased the tumor targeting ability of the cells.

[0230] 3.5.2 Abscess targeting evaluation

[0231] BALB / c nude mice (male, 20 g, 6-8 weeks old) were subcutaneously injected with MRSA liquid (1×10 10 CFU) to prepare a skin abscess model. 24 hours after inoculation, 150 μl PBS, free DiR, Example 15@RAW264.7 and Example 16@THP-1 were injected intravenously. The fluorescence enrichment of the abscess site was observed at different times using a small animal in vivo imager. The mice were dissected at 96 hours to observe the fluorescence of the internal organs. Finally, the fluorescence intensity of the abscess site was analyzed by software.

[0232] The results are as follows Fig.11 As shown in b, the cells modified by Examples 15 and 16 respectively showed stronger fluorescence intensity at the abscess site, indicating that the cells modified by the formulations can be targeted and accumulated at the abscess site.

[0233] 4. Preparation of drug-loaded formulations and in vitro property determination

[0234] 4.1 Preparation of drug-loaded preparations.

[0235]

[0236] Example 17 Preparation method: According to the above prescription, the lipid material was weighed and dissolved in 15 ml of chloroform. The resulting solution was placed in a 50 ml beaker. 5 ml of oxaliplatin solution (solvent is 5% glucose solution) with a concentration of 4 mg / ml was dripped into the beaker with a syringe. Ultrasonic oscillation was performed for 5 minutes (power was 200 W) to obtain a white emulsion. The organic solvent was removed by rotary evaporation at 40°C. After a white gel was formed, the oxaliplatin liposome suspension was obtained by further rotary evaporation. The liposomes were granulated by high-pressure homogenization combined with microporous membrane (pore size 0.22 μm) filtration, and the liposome suspension was prepared by freeze-thaw method (60~-80, repeated 5 times).

[0237]

[0238] Example 18 Preparation method: DSPC, WGA-PEG2k-DSPE, and cholesterol were weighed according to the above prescription, dissolved in chloroform, and a liposome suspension containing TEA (pH 6.5) containing sucrose octasulfate X salt in the aqueous phase was prepared by a thin film dispersion method. The liposome solution was passed through a dextran gel column (G75) and eluted with a mixed solvent containing 4.05 mg / ml HEPES and 8.42 mg / ml NaCl to remove TEA and sucrose octasulfate X salt; the liposome was incubated with a solution containing irinotecan hydrochloride (solvent is HEPES+NaCl) at a temperature of 60-70°C to actively load irinotecan, and then free irinotecan and other components were removed by a dextran gel column, and finally the pH value of the solution was adjusted to 7.3. The preparation method of Control Example 11 was the same as that of Example 18, except that the prescription did not contain WGA-PEG2k-DSPE.

[0239]

[0240] Example 19 Preparation method: According to the above table, lipid DLIN-MC3-DMA, cholesterol, EPC, WGA-PEG2k-DSPE and DPPE-PEG2k were weighed and dissolved in anhydrous ethanol; oxaliplatin was weighed and dissolved in 20mM citric acid buffer (pH4) to form an aqueous solution. The lipid solution and the aqueous solution were mixed in a ratio of 1:3 (ethanol: aqueous solution) using a microfluidic mixer, and concentrated using an Amicon ultracentrifugal filter membrane to obtain oxaliplatin lipid nanoparticles.

[0241]

[0242] Example 20 Preparation method: According to the above table, lipids sodium cholesterol sulfate, HSPC, DOTAP, HSPC, WGA-PEG2k-DSPE and DMPG-PEG2k were weighed and dissolved in anhydrous ethanol; irinotecan was weighed and dissolved in 20mM citric acid buffer (pH4) to form an aqueous solution. The lipid solution and the aqueous solution were mixed in a ratio of 1:3 (ethanol: aqueous solution) using a microfluidic mixer, and concentrated using an Amicon ultracentrifugal filter membrane to obtain irinotecan lipid particles.

[0243]

[0244] Example 21 Preparation method: According to the above table, lipid material and clarithromycin were weighed and dissolved in chloroform to prepare a lipid solution, and the organic solvent was removed by rotary evaporation using a thin film dispersion method to form a uniform film, which was hydrated with PBS and prepared by a high-pressure homogenizer combined with a microporous filter membrane. The preparation method of Control Example 12 was the same as that of Example 21, except that the prescription did not contain the WGA functional material.

[0245]

[0246] Preparation method of Example 22: Weigh the lipid material according to the above prescription, add 2 ml of ether / chloroform mixed solvent (volume ratio of 1:1) to dissolve, as the oil phase; prepare 2 ml of 8 mg / ml vancomycin hydrochloride solution (using 5% glucose solution as solvent) as the inner water phase, mix the oil phase and the inner water phase and vortex for 2 minutes, and high-speed shear (speed of 22000 rpm) to prepare W / O type colostrum. Quickly transfer it into 5 ml of glucose (4%, W / V) solution containing 40 mM L-lysine (as the outer water phase), shear (speed of 3000 rpm) to prepare a double emulsion, and continuously introduce nitrogen into the liquid surface under magnetic stirring conditions to remove the organic solvent, so as to obtain a vancomycin cationic lipid suspension.

[0247]

[0248] Example 23 Preparation method: Weigh PLGA, WGA-PEG2k-DSPE and clarithromycin according to the prescription in the above table, dissolve in 8 ml of acetone, add dropwise 40 ml of 0.5% (W / V) PVA solution, prepare colostrum by ultrasound (power of 250 W), and remove the organic solvent by natural evaporation to obtain colostrum.

[0249]

[0250] Example 24 Preparation method: According to the above table, PLGA and WGA functional materials were weighed and dissolved in dichloromethane / acetone (volume ratio of 3:1) as the oil phase; in addition, vancomycin was weighed and dissolved in 5% sucrose aqueous solution to form an aqueous phase. O / W emulsion was prepared using probe ultrasound (power of 250W), which was added to 1% poloxamer 188 solution to prepare W / O / W emulsion, and the organic solvent was evaporated at room temperature to obtain PLGA microspheres. The preparation of control example 13 refers to the preparation method of example 24, and PEG2k-DSPE is used in the prescription instead of WGA-PEG2k-DSPE.

[0251] Embodiment 25 Comparative Example 14

[0252] Example 25 Preparation method: DEPC, DPPG, WGA-PEG2k-DSPE, cholesterol, and amikacin base were weighed according to the above prescription, dissolved with 10 ml of chloroform / ether (volume ratio of 5:1), added to 2 ml of 5% sucrose aqueous solution, and emulsified and dispersed using probe ultrasound (power of 250 W) under ice-water bath conditions to form a stable emulsion; the organic solvent was removed by evaporation under reduced pressure, and an amikacin lipid suspension was prepared using a freeze-thaw method (60--80° C., repeated 5 cycles). The preparation of Control Example 14 refers to the preparation method of Example 25, except that the prescription does not contain WGA-PEG2k-DSPE.

[0253]

[0254] The preparation method of Example 26 and Control Example 15 (WGA-PEG2k-DSPE is not contained in the prescription): the lipid material is weighed according to the prescription in the above table and dissolved in dichloromethane as the oil phase; polymyxin is weighed and dissolved in 1 ml of 5% trehalose as the water phase; the oil phase is dripped into the water phase, and a probe ultrasound (power of 300W) process is used for 15 minutes to prepare a W / O emulsion, and the emulsion is added to a solution containing PVA and L-lysine (the solvent is a 5% trehalose solution), and a shearing process (speed of 6000rpm) is used to prepare a W / O / W double emulsion. Nitrogen is blown in to remove the organic solvent to obtain a polymyxin lipid suspension.

[0255] 4.2 Determination of particle size and zeta potential

[0256] The particle size and polydispersity coefficient were measured using a dynamic light scattering instrument. The results are as follows:

[0257] The average particle size and span of the preparation were measured using a laser particle size analyzer, and the results are as follows:

[0258] 4.3 Determination of encapsulation efficiency

[0259] The encapsulation rate of drugs in liposomes was determined by centrifugal ultrafiltration. The encapsulation rate of drugs in microparticles was determined by high-speed centrifugation. The encapsulation rate was calculated by the formula: encapsulation rate = encapsulated drug amount / drug dosage × 100%.

[0260]

[0261] 5. Modification of cells by drug-loaded formulations

[0262] 5.1 Modification of RAW264.7 cells by drug-loaded formulations

[0263]

[0264] 5.2 Modification of THP-1 cells by drug-loaded formulations

[0265]

[0266] 5.3 Determination of drug loading in cells

[0267] The above examples (17-26) and control examples (11-15) were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, an appropriate volume of cell lysate was added to the cell pellet, vortexed thoroughly, and allowed to stand at 4°C for 30 minutes. Four times the volume of acetonitrile was added for protein precipitation and drug extraction, allowed to stand at 4°C for 30 minutes, vortexed at 1500 rpm for 5 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken for HPLC or microplate detection. The results are shown in the following table:

[0268] 6. In vitro efficacy evaluation of drug-loaded preparations modified cells

[0269] 6.1 Tumor sphere growth inhibition experiment

[0270] The tumor sphere model was used to investigate the growth inhibition of macrophages on tumor spheres. The agarose gel method was used to prepare multicellular tumor spheres with a diameter of approximately (400-600 μm).

[0271] HCT-116 cell suspension was added to a 96-well culture plate pre-coated with agarose gel and incubated at 37°C and 5% CO 2 Incubate in the environment for 2 to 3 days, and successfully construct HCT-116 tumor cell sphere model in each well. When the diameter of the tumor sphere increases to 400 μm, the examples 17-20 and the control example 11 (cell number 2×10 5 The growth status of the tumor spheres was observed using an inverted fluorescence microscope on days 0, 1, 2, 3, 4, and 5, and the major diameter a and minor diameter b of the tumor spheres were measured. The volume of the tumor spheres was calculated according to the following formula, and the volume change rate of the tumor spheres was calculated according to the formula.

[0272] V=0.5×a×b2 R=V i / V 0 ×100%b 2 Where V is the volume of the tumor cell sphere, a is the major diameter, and b is the minor diameter. R is the volume change rate of the tumor sphere, V i is the volume of the tumor sphere on day i, V 0 is the volume of the tumor sphere on day 0.

[0273] The results are as follows Fig.12 As shown, the volume of the tumor spheres in the control group gradually increased over time within 5 days of culture. All examples significantly inhibited the growth of tumor spheres and were better than control example 11, indicating that the examples disclosed in the present invention have good tumor penetration efficacy.

[0274] 6.2 Sterilization curve drawing

[0275] RAW264.7 cells in the logarithmic growth phase were plated in 96-well culture plates (5×10 5 cells / ml) and cultured until the confluence reached 80%-90%. The culture medium was washed away with PBS and MRSA (1×10 7 CFU) of serum-free complete medium. Add each group (the preparations of Examples 21-24 and the cell groups modified respectively, the cell groups modified by Control Examples 12-13 as controls, and the drug concentrations of each group are kept consistent), continue culturing in the cell box, take the supernatant at each time point for stepwise dilution, and count the viable bacteria. Add 20 μL of RIPA lysis buffer, repeatedly pipette and dilute by doubling, and count the (intracellular) viable bacteria again. The total number of bacteria at each time point = the number of bacteria in the supernatant + the number of bacteria in the cells.

[0276] THP-1 cells in the logarithmic growth phase were taken and the in vitro bactericidal efficacy of the preparation groups of Examples 25-26 and their modified cell groups against PA was investigated according to the RAW264.7 bactericidal curve experimental method, and the cell group modified in Control Examples 12-13 was used as a control. The total number of bacteria was determined according to the above method.

[0277] The results are as follows Fig.13 As shown, each embodiment significantly reduced the total number of bacteria and was significantly better than the control example, indicating that the embodiments disclosed in the present invention have excellent in vitro anti-infection efficacy.

[0278] 7. In vivo evaluation of drug-loaded formulations modified cells

[0279] 7.1 In vivo antitumor evaluation

[0280] A nude mouse model of colon cancer was prepared by in situ injection of HCT116-LUC2 cells, and the mice were randomly divided into 4 groups, with 8 mice in each group. The dosing regimen was: sterile sodium chloride injection (control group), Example 18, Example 20, and Control Example 11 (the dose was 20 mg / kg). The drugs were injected into the tail vein of nude mice every 2 days, for a total of 6 times, and the dosing cycle was 30 days. After 6 doses of drugs, they were observed for 2 weeks. Finally, all nude mice were killed, and the tumor tissues were isolated for subsequent experiments. The body weight and in vitro tumor weight of the nude mice were recorded, and the formula "Tumor inhibition rate (IRT%) = (W 实 -W 对照 ) / W 对照× 100%" to calculate the tumor inhibition rate.

[0281]

[0282] 7.2 In vivo anti-infection evaluation

[0283] 7.2.1 Evaluation of Anti-abscess Infection

[0284] 90 ICR mice (20 g, half male and half female) were randomly divided into 9 groups after being raised for 7 days: PBS group, modified cell group (Example 21@RAW264.7, Example 22@RAW264.7, Example 23@THP-1, Example 24@THP-1, Control Example 12@RAW264.7, Control Example 13@THP-1), free clarithromycin group and free vancomycin group. Each group was subcutaneously injected with MRSA bacterial solution in the right upper limb of mice to prepare an abscess model. After 24 hours, the above groups were injected through the tail vein (keeping the drug dose consistent, 0.2 ml was injected in the PBS group). After 24 hours, the abscess tissue was taken, homogenized, and the number of live bacteria was counted by successive dilution and plate coating.

[0285] The results showed that compared with the PBS group, both the free clarithromycin and free vancomycin groups showed significant antibacterial efficacy. Fig.14 As shown in a and 14b, under the action of Examples 21-24, the number of live bacteria in the abscess tissue was significantly lower than that in the control preparation group (Control Example 12@RAW264.7, Control Example 13@THP-1) and the respective free drug groups, indicating excellent anti-abscess infection efficacy.

[0286] 7.2.2 Evaluation of anti-pneumonia infection 70 ICR mice (20 g, half male and half female) were pre-fed for 7 days and randomly divided into 7 groups: PBS group, free amikacin group, free polymyxin group, and cell test group (Example 25@RAW264.7, Example 26@THP-1, Control Example 14@RAW264.7, Control Example 15@THP-1). Each group of mice was anesthetized by intraperitoneal injection of sodium pentobarbital solution. After fixation, Pseudomonas aeruginosa solution was slowly dripped into one nasal cavity to prepare a pneumonia model. After 24 hours, the above groups were injected through the tail vein (the drug dosage was kept consistent, and 0.2 ml was injected in the PBS group). After 24 hours, the lung tissue was dissected out and homogenized, and the number of live bacteria was counted by successive dilution and plate coating.

[0287] The results showed that compared with the PBS group, both the free clarithromycin and free vancomycin groups showed significant antibacterial efficacy. Fig.14 As shown in c and 14d, under the action of cells modified in Examples 25 and 26, the number of live bacteria in lung tissue was significantly lower than that in the control preparation group and the free drug group, indicating excellent anti-lung infection efficacy.

[0288] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered cell composition, comprising: (1) Any of monocytes and macrophages; (2) A preparation located on the surface of the monocytes or macrophages.

2. The composition according to claim 1, wherein The cells are monocytes.

3. The composition according to claim 1, wherein The cells are macrophages.

4. The preparation according to claim 1, characterized in that The preparations are liposomes, lipid nanoparticles, and PLGA nanoparticles / microparticles.

5. The preparation according to claim 4, characterized in that The liposome, lipid nanoparticle, PLGA nanoparticle / microparticle contain specific recognition molecules, drugs, and carrier materials.

6. The specific recognition molecule according to claim 5, characterized in that The specific molecule is a polymer containing lectin, specifically selected from wheat germ agglutinin and polyethylene glycol-phosphatidylethanolamine conjugate (WGA-PEG-DSPE), wheat germ agglutinin and polyethylene glycol-polylactic acid copolymer conjugate (WGA-PEG-PLA).

7. The drug according to claim 5, characterized in that The drugs are anti-tumor chemotherapy drugs, anti-infective drugs, and fluorescent probes, wherein the anti-tumor chemotherapy drugs are any one or more of alkylating agents, purine and pyrimidine analogs and derivatives, topoisomerase inhibitors, antifolates, taxanes, angiogenesis inhibitors, anti-tumor monoclonal antibodies, and anti-tumor antibiotics; specifically selected from any one or more of irinotecan and oxaliplatin. The anti-infective drugs are penicillins, cephalosporins, β-lactamase inhibitors, aminoglycosides, macrolides, tetracyclines, sulfonamides, quinolones, furans, antifungals, nitroimidazoles, amides, and glycopeptides, specifically selected from any one or more of clarithromycin, vancomycin, amikacin, and polymyxin. The fluorescent probes are fluorescein probes, inorganic ion fluorescent probes, fluorescent quantum dots, cyanines Cy, fluoroboryl pyrrole BODI PY, and rhodamine, specifically selected from any one or more of coumarin 6, DiI, DiO, DiD, DiR, and Cy7.

8. The composition according to claim 1, characterized in that The preparation method of the composition comprises the following steps: (1) constructing a preparation, introducing cell-specific recognition molecules, and obtaining nanoparticles / microparticles that can be specifically recognized by cells; (2) Co-incubating the nanoparticles / microparticles prepared in step (1) with cells to obtain an engineered cell composition.

9. The engineered cell composition according to claim 8, characterized in that: In the preparation method, the ratio of the number of nanoparticles / microparticles to the number of cells is 1:100-50000, the incubation temperature is 2-32° C., and the incubation time is 10-180 minutes.

10. The engineered cell composition according to claim 1, characterized in that: The composition is used for the diagnosis and treatment of tumors or infections. Anti-tumor applications are specifically selected from colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, osteosarcoma, bladder cancer, lung cancer, cervical cancer, and malignant glioma. Anti-infection applications are specifically selected from skin, respiratory tract, urinary tract, and blood infections caused by pathogens.

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