CN116509818A - Low-toxicity fat-soluble nanoparticle composition and preparation method and application thereof - Google Patents

Low-toxicity fat-soluble nanoparticle composition and preparation method and application thereof Download PDF

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Publication number
CN116509818A
CN116509818A CN202310416659.4A CN202310416659A CN116509818A CN 116509818 A CN116509818 A CN 116509818A CN 202310416659 A CN202310416659 A CN 202310416659A CN 116509818 A CN116509818 A CN 116509818A
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oil
fat
soluble
drug
cancer
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申辽
全东琴
王永安
杨军
骆媛
隋昕
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Academy of Military Medical Sciences AMMS of PLA
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Academy of Military Medical Sciences AMMS of PLA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a low-toxicity fat-soluble nanoparticle composition, and a preparation method and application thereof. The invention prepares the lipid-soluble nanoparticle composition capable of loading the gene medicine by using the phospholipid and the cholesterol-PEG 2 k-transferrin, and the lipid-soluble nanoparticle composition has the advantages of large gene medicine carrying capacity, high encapsulation efficiency, good stability, strong safety, good universality and capability of delivering various gene medicines; the carrier material has simple production process and is easy to develop pertinently; can be used for treating blood tumor and solid tumor.

Description

Low-toxicity fat-soluble nanoparticle composition and preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and relates to a low-toxicity fat-soluble nanoparticle composition, and a preparation method and application thereof.
Background
Gene therapy is the introduction of exogenous genes into recipient cells by gene transfer techniques to treat diseases caused by gene defects. Because the exogenous gene is easy to be digested and degraded by biological enzyme in the cell, the expression level of the coded protein is reduced, and the effect of gene therapy is affected. The gene vector can effectively protect the gene medicine in the gene therapy process, and is the key of the gene therapy. Gene vectors are largely classified into viral vectors and nonviral vectors. The transfection efficiency of the virus vector is high, but the vector is a living virus, so that the virus vector has the defects of high immunogenicity, high toxicity, limited number of carried genes and the like, and the production has poor controllability and doubtful safety, so that the application of the virus vector in the biological field is limited to a certain extent. Patients are frequently afflicted with cancer after years of treatment with viral vector gene therapy, despite remission of disease symptoms. Therefore, non-viral vectors are becoming an important issue.
The fat-soluble nanoparticle is used as a non-viral vector, and can make up for the defects of the viral vector. Moreover, the fat-soluble nanoparticle has the advantages of simple preparation, easy surface modification, easy mass production and the like, and is an ideal material for constructing a gene vector. Nanotechnology has wide application in the field of gene drug delivery, and attracts great attention. The nanomaterial has the following advantages as a gene vector: 1) The preparation is simple and easy to synthesize; 2) The size is small, and the tissue gap of the organism is easy to pass through; 3) Is easy to modify in multiple functions, improves biocompatibility, reduces immune response of organisms and achieves higher expression efficiency.
Although the non-viral class of vectors is of a large variety, it is summarized that the nature is mainly of different types of nanoparticles. There are a number of problems with nanoparticles: 1) Special materials are required, and are expensive, only a few materials can achieve stable gene delivery, and most materials are limited by foreign patents; 2) The carrying capacity of the gene medicine is not high, and the delivery effect is unstable; 3) The complex design and verification are required to be carried out again aiming at the gene medicines with different molecular weights and types, so that the gene medicines have no good universality; 4) Most of the delivery vehicles are cationic delivery vehicles at present, so that cytotoxicity is high and safety is doubtful.
Therefore, how to improve the drug carrying capacity, encapsulation efficiency, stability, versatility and safety of the liposoluble nanoparticles is a problem to be solved.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide a lipid-soluble nanoparticle composition loaded with a gene drug and a preparation method thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a lipid-soluble nanoparticle comprising a surfactant and/or a functional molecular modification material.
Further, the surfactant comprises a phospholipid.
Further, the phospholipid comprises one or more of a natural phospholipid, a semisynthetic phospholipid or a synthetic phospholipid.
Further, the natural phospholipids include soybean phospholipids, egg yolk phospholipids, behenoyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, ditrans oleoyl phosphatidylcholine, dilauroyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, monopalmitoyl phosphatidylcholine, monostearoyl choline, egg yolk phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, dimyristoyl phosphatidylserine, soybean phosphatidylserine, distearoyl phosphatidylserine, dipalmitoyl phosphatidylserine, dimyristoyl phosphatidylserine, egg yolk phosphatidyl sphingomyelin, distearoyl sphingomyelin, dipalmitoyl phosphatidylinositol, dipalmitoyl phosphatidic acid, or dipyristoyl phosphatidate.
Further, the semisynthetic phospholipids include hydrogenated phospholipids.
Further, the synthetic phospholipid comprises DEPC, DOPC, DPPC, DSPC or DMPC.
Further, the amount of the phospholipid is selected from 70 to 99%.
Further, the amount of the phospholipid is selected from 80% -90%.
Further, the functional molecule-modifying material includes one or more of a protein, a nucleic acid, and a compound.
Further, the protein comprises one or more of cholesterol-PEG-transferrin, DSPE-PEG-transferrin, phytolectin, and transmembrane peptide.
Further, the protein is cholesterol-PEG-transferrin.
Further, the cholesterol-PEG-transferrin includes one or more of cholesterol-PEG 2 k-transferrin, cholesterol-PEG 3 k-transferrin, cholesterol-PEG 4 k-transferrin, cholesterol-PEG 5 k-transferrin.
Further, the cholesterol-PEG-transferrin is cholesterol-PEG 2 k-transferrin.
Further, the amount of cholesterol-PEG 2 k-transferrin is selected from 1-30%.
Further, the amount of cholesterol-PEG 2 k-transferrin is selected from 10-20%.
Further, the nucleic acid comprises a transferrin nucleic acid aptamer.
Further, the fat-soluble nanoparticle further includes an organic solvent and water.
Further, the organic solvent includes one or more of methanol, ethanol, hexylene glycol, propylene glycol, dipropylene glycol, glycerol, acetonitrile, butanone, 1-trifluoroethane, hexafluoroisopropanol, ethyl acetate, carbon tetrachloride, butanol, dibutyl ether, diethyl ether, cyclohexylamine, methylene chloride, hexane, butyl acetate, diisopropyl ether, benzene, dipentyl ether, chloroform, heptane, tetrachloroethylene, toluene, hexadecane, dimethylformamide, N-methylpyrrolidone, dimethylacetamide, tetrahydrofuran, glycerol, dioxane.
Further, the organic solvent is ethanol.
In a second aspect the present invention provides a lipid soluble nanoparticle composition comprising the lipid soluble nanoparticle of the first aspect of the invention and a drug.
Further, the medicines comprise genetic medicines and chemical medicines.
Further, the drug is a genetic drug.
Further, the gene medicine comprises DNA and RNA medicine.
Further, the DNA drugs include double-stranded DNA, single-stranded DNA drugs; the RNA drugs include double-stranded RNA and single-stranded RNA drugs.
Further, the double-stranded DNA includes a plasmid.
Further, the single-stranded DNA comprises a DNA aptamer.
Further, the single stranded RNA includes RNA nucleic acid aptamer, mRNA, ncRNA, or antisense oligonucleotide ASO.
Further, the ncrnas include miRNA, siRNA, shRNA, saRNA, sgRNA, piRNA, lncRNA, circRNA or other regulatory RNAs.
Further, the gene drug includes a plasmid drug or an mRNA drug.
Further, the plasmid drugs include plasmids, gene sequences encoding tag proteins, and/or genes of interest.
Further, the mRNA drug includes mRNA and/or a gene sequence encoding a tag protein.
Further, the plasmids include pQE-12, pUC-series, pBluescript, pET-series expression vectors, pCRTOPO, pJOE, pBACKBONE, pBBR-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-N-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry vector system, pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV-dhfr, pIDD 35, okayama-Berg cDNA expression vectors pcDV1, pRc/CMV, pcDNA1, pcDNA3, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pE-10, pTriEx-Hygro, pCINeo, pUC, pMB1, pSC101, EU1, pBEU2, pDF41, pDF42, pBR322, dTomTomDNA1 or pcDNA3.1.
Further, the plasmid was ptdTomN 1 or pCDNA3.1.
Further, the tag includes a fluorescent protein.
Further, the fluorescent protein comprises one or more of GFP, EGFP, mFruit protein, dsRed series fluorescent protein, OFP and YFP.
Further, the fluorescent protein includes GFP or EGFP.
Further, the genes of interest include WT1, MUC1, LMP2, EGFRvIII, her2, p53, NY-ESO-1, PSMA, GD2, CEA, gp100, PR1, PSA, hTERT, ephA, PAP, ML-IAP, AFP, epCAM, ERG, NA, PAX3, MYCN, rhoC, TRP-2, GD3, mesothelin, PSCA, CYP1B1, PLAC1, GM3, BORIS, globoH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, PAX5, OY-TES1, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, tie 2, page4, VEGFR2, MAD-CT-1, FAP, PDGFR-B, or MAD-CT-2.
Further, the plasmid drug is pCDNA3.1-EGFP or ptdToma-N1.
Further, the mRNA drug is mRNA-GFP.
Further, the fat-soluble nanoparticle composition also includes a liquid nonaqueous solvent.
Further, the liquid nonaqueous solvent comprises one or more of vegetable oil, animal oil and alkane liquid solvents.
Further, the vegetable oil includes nut oil, fennel oil, soybean oil, almond oil, corn oil, olive oil, peanut oil, almond oil, walnut oil, cashew oil, rice bran oil, poppy seed oil, cotton seed oil, canola oil, sesame oil, coconut oil, linseed oil, cinnamon oil, clove oil, nutmeg oil, coriander oil, lemon oil, orange oil, safflower oil, cocoa butter, palm oil, palm kernel oil, sunflower oil, rapeseed oil, or castor oil.
Further, the animal oil includes whale oil, fish oil, musk oil or marten oil.
Further, the alkane liquid solvent includes glycerol dioleate, glycerol tripropionate, glycerol tributyrate, polyoxyethylene oil derivatives, medium chain triglycerides, oleic acid, arachidonic acid, hydrogenated oil, squalene, or glycerol monooleate.
Further, the liquid nonaqueous solvent includes medium chain triglycerides, soybean oil, corn oil, squalene, oleic acid, or arachidonic acid.
In a third aspect, the present invention provides a method of preparing a fat-soluble nanoparticle composition, the method comprising the steps of:
1) Preparing the liposoluble nanoparticle of the first aspect of the invention;
2) Preparing a lyophilized powder using the lipid-soluble nanoparticle and the drug of the second aspect of the present invention;
3) The lipid-soluble nanoparticle composition is prepared using the lyophilized powder and the liquid nonaqueous solvent described in the second aspect of the present invention.
Further, the method for preparing the fat-soluble nanoparticle composition in the step 1) comprises an extrusion method, a reverse evaporation method, a carbon dioxide supercritical method, an ethanol injection method, a high-pressure homogenization method, a microfluidic method and/or a film dispersion method.
Further, the ethanol injection method is to prepare a crude suspension by using the surfactant and/or the functional molecule modification material described in the first aspect of the present invention, and treat the crude suspension by using an ultrasonic device to obtain the fat-soluble nanoparticles.
Further, the ultrasonic device is a probe type ultrasonic device.
Further, the high-pressure homogenization method is to prepare a crude suspension by using the surfactant and/or the functional molecule-modified material described in the first aspect of the present invention, and treat the crude suspension using a high-pressure homogenizer to obtain the fat-soluble nanoparticles.
Further, the method for preparing the crude suspension comprises dissolving the surfactant and/or the functional molecule-modifying material described in the first aspect of the present invention in the organic solvent described in the second aspect of the present invention, followed by adding purified water to disperse to obtain the crude suspension.
Further, the microfluidic method obtains the liposoluble nanoparticle by treating the surfactant and/or the functional molecule-modifying material described in the first aspect of the present invention with a microfluidic device.
Further, the microfluidic method obtains an organic solution by dissolving the surfactant and/or functional molecule-modifying material described in the first aspect of the present invention in the organic solvent described in the second aspect of the present invention, and then fills the obtained organic solution into a syringe, another syringe is filled with water, and the obtained organic solution is treated using a microfluidic device to obtain the lipid-soluble nanoparticle.
Further, the water is enzyme-free water.
Further, the flow rate ratio of the organic solvent to water of the microfluidic method is selected from 1:9 to 3:7.
Further, the Zeta potential of the fat-soluble nanoparticle is selected from-5 to-30 mV.
Further, the Zeta potential of the fat-soluble nanoparticle is selected from-7.54 to-29.27 mV.
Further, the thin film dispersion method is to obtain the fat-soluble nanoparticles by treating the surfactant and/or the functional molecule-modifying material described in the first aspect of the present invention with a rotary evaporator and an ultrasonic cell disruptor.
Further, the thin film dispersion method is to obtain fat-soluble nanoparticles by dissolving the surfactant and/or functional molecule-modifying material described in the first aspect of the present invention in the organic solvent, spin-drying the organic solvent using a rotary evaporator and forming a film, then adding a purified water redispersing material, and treating the dispersing material using an ultrasonic cell breaker.
Further, the particle size range of the fat-soluble nanoparticle is selected from 50 to 1000nm.
Further, the particle size range of the fat-soluble nanoparticle is selected from 80-1000 nm.
Further, the average particle diameter of the fat-soluble nanoparticle was 122.1nm.
Further, the method for preparing the freeze-dried powder in the step 2) comprises the steps of mixing the fat-soluble nano particles and the medicine to obtain a mixed solution, and drying the mixed solution to obtain the freeze-dried powder.
Further, the weight ratio of the drug to the liposoluble nanoparticles is selected from 1:10 to 1:50000.
Further, the weight ratio of the drug to the liposoluble nanoparticles is selected from 1:500 to 1:50000.
Further, the drying method comprises one or more of a freeze drying method, an atmospheric drying method, a reduced pressure drying method, a vacuum drying method and a microwave drying method.
Further, the drying method is a freeze-drying method.
Further, the freeze-drying procedure includes a prefreezing procedure, sublimation drying, and analytical drying.
Further, the packaging material of the mixed solution comprises a penicillin bottle, a freeze-drying ampoule and a freeze-drying bubble cap.
Further, the freeze-dried powder is preserved by nitrogen protection, sealing and light-shielding at low temperature.
Further, the method for preparing the fat-soluble nanoparticle composition of step 3) includes dissolving the lyophilized powder using the liquid nonaqueous solvent to obtain the fat-soluble nanoparticle composition.
Further, the use method of the fat-soluble nanoparticle composition is to perform local injection or cell transfection after being uniformly mixed with a buffer solution.
Further, the weight ratio of the liquid nonaqueous solvent to the freeze-dried powder is selected from 5:1 to 1000:1.
In a fourth aspect the present invention provides a pharmaceutical composition comprising 1) a liposoluble nanoparticle according to the first aspect of the invention, a liposoluble nanoparticle composition according to the second aspect of the invention or a liposoluble nanoparticle composition prepared by the method according to the third aspect of the invention and/or 2) a pharmaceutically acceptable carrier.
The fifth aspect of the present invention provides the use of the liposoluble nanoparticle according to the first aspect of the present invention, the liposoluble nanoparticle composition according to the second aspect of the present invention, the liposoluble nanoparticle composition prepared by the method according to the third aspect of the present invention or the pharmaceutical composition according to the fourth aspect of the present invention for the preparation of a product for the treatment of a tumor.
Further, the tumor is selected from the group consisting of hematological tumors, solid tumors.
Further, the solid tumor includes prostate cancer, bladder cancer, liver cancer, head and neck cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, meningioma, pancreatic cancer, multiple squamous cell carcinoma, oral cancer, esophageal cancer, colorectal cancer, breast cancer, medulloblastoma, nasopharyngeal cancer, thymus cancer, lymphoid malignancy, fibrosarcoma, myxosarcoma, or melanoma.
Further, the hematological neoplasm includes acute leukemia, chronic leukemia, polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, or myelodysplasia.
A sixth aspect of the present invention provides a method for optimizing a prescription process of a fat-soluble nanoparticle according to the first aspect of the present invention, a fat-soluble nanoparticle composition according to the second aspect of the present invention, and a fat-soluble nanoparticle composition prepared by the method according to the third aspect of the present invention, wherein the method for optimizing a prescription comprises: the method adopts the encapsulation efficiency, the particle size of the fat-soluble nanoparticle composition, the drug delivery capacity and the placement stability of the fat-soluble nanoparticle composition as indexes to change one or more of the surfactant types, the functional molecule modification material types, the liquid nonaqueous solvent types, the organic solvent types, the component proportions and the preparation method of the fat-soluble nanoparticle composition.
The invention has the advantages and beneficial effects that:
the invention provides a low-toxicity fat-soluble nanoparticle composition, which is a non-viral gene delivery carrier, wherein the carrier material of the fat-soluble nanoparticle composition is a conventional material, and the production process is simple and is easy to develop in a targeted manner; the liposoluble nanoparticle composition has the advantages of large gene medicine carrying capacity, high encapsulation efficiency, good stability, strong safety, good universality and capability of delivering various gene medicines. The liposoluble nanoparticle composition can be used for treating blood tumor and solid tumor.
Drawings
FIG. 1 is a statistical graph of particle size distribution of a fat-soluble nanoparticle composition prepared by an ethanol injection method;
FIG. 2 is a graph showing the effect of red fluorescent protein expression, wherein 1A is a bright field photograph and 1B is a red fluorescent photograph;
FIG. 3 is a graph showing the results of measuring the inhibition rate of cell growth by CCK-8;
FIG. 4 is a graph showing the effect of a flow cell detection microfluidic method for preparing a lipid-soluble nanoparticle composition for cell transfection;
FIG. 5 is a graph showing the effect of flow cytometry on gene drug delivery efficiency.
Detailed Description
According to the invention, through extensive and intensive research, a low-toxicity fat-soluble nanoparticle composition is prepared by using phospholipid and cholesterol-PEG 2 k-transferrin, and the prepared fat-soluble nanoparticle composition is an oily solution and can be uniformly mixed with a certain buffer solution for local injection or cell transfection operation.
In the present invention, the term "fat-soluble nanoparticle" refers to a particle comprising a plurality of (i.e., more than one) fat-soluble molecules physically bound to each other by intermolecular forces. The term "liposoluble nanoparticle composition" refers to a composite drug-loaded particle formed by self-assembly using "liposoluble nanoparticles" with a drug (e.g., a gene drug) to be delivered. The liposoluble nanoparticles and liposoluble nanoparticle compositions of the invention can be used for a variety of purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells in vitro and in vivo, thereby inducing expression of a desired protein or inhibiting expression of a target gene. Accordingly, embodiments of the present invention provide methods of treating or preventing diseases and disorders in a subject in need thereof by contacting the subject with a liposoluble nanoparticle composition encapsulating or associated with a suitable therapeutic agent, wherein the liposoluble nanoparticle composition comprises one or more liposoluble nanoparticles described herein.
In the present invention, the term "surfactant" refers to a material that reduces the surface tension of a liquid and the interfacial tension between two liquids to make them more readily diffusible. Examples of suitable surfactants include, but are not limited to, nonionic, ionic (anionic or cationic) or zwitterionic (or amphoteric in which the head of the surfactant contains two oppositely charged groups) surfactants. Examples of anionic surfactants include, but are not limited to, those based on sulfate, sulfonate, or carboxylate anions, such as perfluorooctanoate (PFOA or PFO), alkylbenzenesulfonate, soap, fatty acid salts, or alkyl sulfate salts, such as Perfluorooctane Sulfonate (PFOs), sodium Dodecyl Sulfate (SDS), ammonium lauryl sulfate, or sodium dodecyl ether sulfate (SLES). Examples of cationic surfactants include, but are not limited to, those based on quaternary ammonium cations, such as alkyl trimethylammonium, including cetyltrimethylammonium bromide (CTAB, also known as cetyltrimethylammonium bromide), cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), or benzethonium chloride (BZT). Examples of zwitterionic surfactants include, but are not limited to, dodecyl betaine, cocamidopropyl betaine, or cocoamphoglycinate. Examples of nonionic surfactants include, but are not limited to, alkyl poly (ethylene oxide), alkylphenol poly (ethylene oxide), copolymers of poly (ethylene oxide) and poly (propylene oxide) (commercially known as poloxamers or poloxamines), alkyl polyglucosides (including octyl glucoside and decyl maltoside), fatty alcohols (including cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, or polysorbates (including tween 20, tween 80), dodecyl dimethyl amine oxide, or phospholipids. In a specific embodiment of the invention, the surfactant is a phospholipid.
In the present invention, the term "phospholipid" refers to any of a number of lipids comprising diglycerides, phosphate groups, and simple organic molecules such as choline. Examples of phospholipids include, but are not limited to, one or more of natural phospholipids, semisynthetic phospholipids or synthetic phospholipids; preferably, the natural phospholipids include soybean phospholipids, egg yolk phospholipids, behenyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, ditrans oleoyl phosphatidylcholine, dilauroyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, monopalmitoyl phosphatidylcholine, monostearoyl choline, egg yolk phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dimyristoyl phosphatidylserine, distearoyl phosphatidylserine, ditolyphosphatidylserine, dimyristoyl phosphatidylserine, distearoyl sphingomyelin, dipalmitoyl phosphatidylinositol, ditolyphosphatidylinositol, dimyristoyl phosphatidylinositol, dimyristoyl phosphatidate or dimyristoyl phosphatidic acid; preferably, the semisynthetic phospholipid comprises a hydrogenated phospholipid; preferably, the synthetic phospholipid comprises DDPC, DEPC, DOPC, DPPC, DRPC, DLPC, DMPC, DSPC, POPC or DMPC.
In the present invention, the term "functional molecular modification material" includes any molecular modification material having biologically desirable properties. Examples of functional molecular modifying materials include, but are not limited to, one or more of proteins, nucleic acids, or compounds; preferably, the proteins include, but are not limited to, cholesterol-PEG-transferrin, DSPE-PEG-transferrin, antigens, antibodies, lectins or transmembrane peptides; preferably, the nucleic acids include, but are not limited to, DNA, RNA, transferrin nucleic acid aptamers; preferably, the compounds include, but are not limited to, carbohydrates; in a specific embodiment of the invention, the functional molecule-modifying material is cholesterol-PEG-transferrin; preferably, the cholesterol-PEG-transferrin includes, but is not limited to, cholesterol-PEG 2K-transferrin, cholesterol-PEG 3K-transferrin, cholesterol-PEG 4K-transferrin or cholesterol-PEG 5K-transferrin; more preferably, the cholesterol-PEG-transferrin is cholesterol-PEG 2 k-transferrin.
In the present invention, the term "nonaqueous solvent" or "liquid nonaqueous solvent" refers to a solvent that is not based on water. The term "nonaqueous solvent" includes, but is not limited to, anhydrous solvents. In other words, the non-aqueous solvent may contain a trace amount of water. Preferably, the amount of water is less than 5vol. -%, then 2% vol. -%, 1% vol. -%, more preferably less than 0.5vol. -%, less than 0.1vol. -%, less than 0.01vol. -% or less than 0.001vol. -%. In particular non-polar solvents, solvents having a dipole moment smaller than that of water and hydrophobic solvents, for example solvents which are hardly mixed with water or which are not mixed with water at all. The term "nonaqueous solvent" as used herein includes, but is not limited to, vegetable oils, animal oils or paraffinic liquid solvents; preferably, the vegetable oil comprises nut oil, fennel oil, soybean oil, almond oil, corn oil, olive oil, peanut oil, almond oil, walnut oil, cashew oil, rice bran oil, poppy seed oil, cotton seed oil, canola oil, sesame oil, coconut oil, linseed oil, cinnamon oil, clove oil, nutmeg oil, coriander oil, lemon oil, orange oil, safflower oil, cocoa butter, palm oil, palm kernel oil, sunflower oil, rapeseed oil, castor oil; preferably, the animal oil comprises whale oil, fish oil, musk oil, and mink oil; preferably, the alkane liquid solvent comprises glycerol dioleate, glycerol tripropionate, glycerol tributyrate, polyoxyethylene oil derivatives, medium Chain Triglycerides (MCT), oleic acid, arachidonic acid, hydrogenated oil, squalene, glycerol monooleate (type 40), and mixtures thereof; in a specific embodiment of the invention, the liquid nonaqueous solvent is medium chain triglycerides, soybean oil, corn oil, squalene, oleic acid or arachidonic acid.
The term "organic solvent" is known in the art and refers to a carbon-based material commonly used in the chemical industry that is capable of dissolving or dispersing one or more materials. In general, organic solvents are more lipophilic or hydrophobic than water. Thus, their logP values are typically greater than zero. In accordance with the present invention, organic solvents relate to unsubstituted hydrocarbon solvents such as paraffinic, aliphatic and aromatic hydrocarbons and their heteroatom-containing derivatives, such as oxygen (alcohols, ketones, glycol esters), halogens (e.g., carbon tetrachloride), nitrogen (e.g., DMF, dimethylformamide and acetonitrile) or sulfur (e.g., DMSO, dimethyl sulfoxide). Commonly used organic solvents include, but are not limited to, methanol, ethanol, hexanediol, propylene Glycol (PG), glycerol, acetonitrile, butanone, 1-Trifluoroethane (TFE), hexafluoroisopropanol (HFIP), ethyl acetate, carbon tetrachloride, butanol, dibutyl ether, diethyl ether, cyclohexylamine, methylene dichloride (dichloromethane), hexane, butyl acetate, diisopropyl ether, benzene, dipentyl ether (dipentyl ether), chloroform, heptane, tetrachloroethylene, toluene, hexadecane, dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethylacetamide (DMA), tetrahydrofuran (THF), glycerol, dioxane, and mixtures thereof. In a specific embodiment of the present invention, the organic solvent is ethanol.
In the present invention, the term "drug" refers to a compound, peptide, nucleic acid, or other entity administered to a subject to elicit a desired biological response. Examples of drugs include, but are not limited to, genetic drugs, chemical drugs; preferably, the medicament further comprises a salt, solvate, isomer, active metabolite or combination of medicaments. The term "genetic drug" relates to any DNA or RNA structure or nucleic acid sequence that can be used to treat or prevent a disease or disorder in a subject. Examples of chemical agents include, but are not limited to, antibiotics, anticancer agents, anesthetics, analgesics, hormones, antidiabetic agents, and metabolic disorder agents, such as cefazolin, metronidazole, bupivacaine, lidocaine, buprenorphine, paclitaxel, and docetaxel.
Gene drugs used in the present invention include, but are not limited to, DNA, RNA drugs. Preferably, the DNA drug comprises double-stranded DNA, single-stranded DNA drug; preferably, the RNA drug comprises a double-stranded RNA, single-stranded RNA drug. Preferably, the double-stranded DNA comprises a plasmid; the single-stranded DNA comprises a DNA aptamer; preferably, the single stranded RNA comprises RNA nucleic acid aptamer, mRNA, ncRNA, or antisense oligonucleotide ASO; preferably, the ncrnas comprise miRNA, siRNA, shRNA, saRNA, sgRNA, piRNA, lncRNA, circRNA or other regulatory RNAs; in a specific embodiment of the invention, the genetic drug comprises a plasmid drug or an mRNA drug; further, the plasmid medicament comprises a plasmid, a gene sequence encoding a tag protein and/or a target gene; the mRNA drug includes mRNA and/or a gene sequence encoding a tag protein.
In the present invention, the term "plasmid" refers to a nucleic acid molecule that may be present in a microorganism (either naturally or by introduction into the microorganism), physically separate from the chromosome(s) of the microorganism, and independent of chromosome(s) replication. As mentioned above, plasmids are typically double-stranded circular DNA molecules, but may also be linear DNA molecules and/or RNA molecules. Plasmids exist in a size range of about 1kb to greater than 2 Mb. Plasmids are also present in copy numbers/cells ranging from low to high copy numbers. The plasmid may be modified to include the gene of interest, a tag.
Non-limiting examples of plasmids include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen), pCRTOPO (Invitrogen), pJOE, pBACKBONE, pBBR-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-N-EK, pESP-1, pOP13CAT, E-027 pCSAK-Cherry (L45 a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-V2 neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV-dhfr, pIDD 35, okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCBRL), pGEMHE (Promega), pLX, pSNTE-cloning, pS-92, pSG5 (Stratagene), pSO-2 neo, pSO-1, pS-35, pS-1, pS-101, pS-dTb, pS-1, pS-pYZ-2, pS-pF, pS-pXF-101, pS-pXp-2, pS-pXp-pXF (Invitrogen). In a specific embodiment of the invention, the plasmid is ptdTomO-N1 or pCDNA3.1.
In the present invention, genes of interest include, but are not limited to, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05, WT1 (Wilmm's tumor antigen 1), MUC1, LMP2 (latent membrane protein from early-Barn virus), EGFRvIII, her2, non-mutant p53, NY-ESO-1, PSMA (prostate specific membrane antigen), GD2, CEA (carcinoembryonic antigen) Melan A/MART1, ras mutant, gp100, mutant p53, protease 3 (PR 1), BCR-Abl breakpoint, tyrosinase, survivin, PSA (prostate-specific antigen), hTERT (human telomerase), sarcoma translocation breakpoint, ephA2, PAP (prostaacid phosphatase), ML-IAP (ML-apoptosis inhibitor), AFP (alpha fetoprotein), epCAM (epithelial cell adhesion molecule), ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3 (paired box 3), androgen receptor, cyclin B1, polysialic acid, MYCN-myc, rhoC, TRP-2 (tyrosinase-related protein 2), MYCN-myc, GD3, fucose GM1, mesothelin, PSCA (prostate stem cell antigen), CYP1B1 (cytochrome P4501B 1), PLAC1 (placenta-specific 1), GM3, BORIS (imprinted site-regulated factor-like protein), tn (N-acetylgalactosamine linked to serine or threonine by glycosidic bond), globoH, ETV6-AML, NY-BR-1, RGS5 (G protein signal transduction regulator 5), SART3 (squamous cell carcinoma antigen recognized by T cell 3), STn (sialylated Tn antigen), carbonic anhydrase IX, PAX5 (paired box 5), OY-TES1, sperm protein 17, LCK (P56 form), hmw maa (high molecular weight melanoma-associated antigen), AKAP-4 (a-kinase anchored protein 4), SSX2 (synovial sarcoma breakpoint gene 2), XAGE1 (x antigen 1), B7H3, legumain, tie 2, mage 2, pagr 2, vascular endothelial growth factor (vascular endothelial growth factor 2), human tumor-2 (human tumor-derived factor 2), human tumor-derived tumor-2 (human tumor-derived antigen), and Fos-related antigen 1. Preferably, the plasmid-insertable gene includes WT1, MUC1, LMP2, EGFRvIII, her2, p53, NY-ESO-1, PSMA, GD2, CEA, gp100, PR1, PSA, hTERT, ephA2, PAP, ML-IAP, AFP, epCAM, ERG, NA, PAX3, MYCN, rhoC, TRP-2, GD3, mesothelin, PSCA, CYP1B1, PLAC1, GM3, BORIS, globoH, ETV-AML, NY-BR-1, RGS5, SART3, STn, PAX5, OY-TES1, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legman, tie 2, page4, VEGFR2, MAD-CT-1, FAP, PDGFR-B or MAD-CT-2.
In the present invention, the term "tag" refers to a chemical moiety that is a nucleotide, oligonucleotide or polynucleotide that, when substituted for or added to another sequence, provides additional utility to that sequence or imparts a useful property, particularly in its detection or isolation. Tags for use in the present invention include, but are not limited to, GFP, EGFP, mFruit protein, dsRed-series fluorescent protein, OFP or YFP; preferably, the tag is GFP or EGFP.
In a specific embodiment of the invention, the plasmid drug is pCDNA3.1-EGFP or ptdTomO-N1; the mRNA drug is mRNA-GFP.
In the present invention, the term "fat-soluble nanoparticle" refers to a complex or structure having an internal environment isolated from an external environment by a continuous encapsulating lipid layer. In the context of the present disclosure, the expression "encapsulated lipid layer" may denote a single layer lipid membrane (e.g. found on micelles (micelles) or reverse micelles), a bilayer lipid membrane (e.g. found on liposomes) or any multi-layer membrane formed from single and/or bilayer lipid membranes. The encapsulated lipid layer is typically a single layer, bilayer or multilayer over its entire circumference, but it is contemplated that other conformations may be possible such that the layer has a different configuration over its circumference. The fat-soluble nanoparticle may contain other vesicle structures in its internal environment (i.e., it may be polycystic). The term "liposoluble nanoparticle" includes a variety of different types of structures including, but not limited to, micelles, inverse micelles, unilamellar liposomes, multilamellar liposomes and polycystic liposomes, provided that the particle size limitations described herein are met.
The fat-soluble nanoparticles can take on a variety of different shapes, and the shape can be changed at any given time (e.g., after drying). Typically, the fat-soluble nanoparticles are spherical or substantially spherical structures. "substantially spherical" means that the fat-soluble nanoparticle is nearly spherical, but may not be perfectly spherical. Other shapes of the fat-soluble nanoparticle include, but are not limited to, oval, oblong (oblong), square, rectangular, triangular, cuboid, crescent, diamond, cylinder, or hemispherical shape. Any regular or irregular shape may be formed. Furthermore, if a single liposoluble nanoparticle is multi-encapsulated, it may comprise different shapes. For example, the outer shape may be oblong or rectangular, while the inner shape may be spherical.
The lipid-soluble nanoparticles are formed from a monolayer lipid film, a bilayer lipid film, and/or a multilayer lipid film. The lipid membrane is composed and formed mainly of lipids, but may also comprise other components. For example, and without limitation, the lipid membrane may include stabilizing molecules to help maintain the size and/or shape of the fat-soluble nanoparticles. Any stabilizing molecule known in the art may be used as long as it does not negatively affect the ability of the liposoluble nanoparticle to be used in the methods of the present disclosure.
In the present invention, the term "suspension" refers to a liquid dispersion system formed by dispersing solid particles in a liquid dispersion medium, including but not limited to water.
In the present invention, the term "dispersed" refers to distributing or suspending a discontinuous solid phase within a continuous liquid phase such that the discontinuous solid phase does not coalesce over a useful period of time (e.g., minutes, hours, or days).
In the present invention, the term "lyophilization" refers to the process of highly dispersing a membrane material such as a phospholipid in an aqueous solution, lyophilizing, and then dispersing in an aqueous medium (optionally containing a drug) to form liposomes.
In the present invention, the term "buffer" means a pharmaceutically acceptable buffer. The term "buffer" includes those reagents that maintain the pH of the solution, e.g., in an acceptable range, and includes, but is not limited to histidine,(tris (hydroxymethyl) aminomethane), citrate, succinate, glycolate, and the like, as described herein. Generally, a "buffer" as used herein has a pKa and buffering capacity suitable for a pH range of about 5-7, preferably about 5.5-6.5, e.g., about pH 6.
In the present invention, the term "transfection" is used to denote any method capable of inserting the liposoluble nanoparticle composition described in the present invention into a cell. Transfection techniques include, but are not limited to: transformation, electroporation, microinjection, lipofection, adsorption, infection, or protoplast fusion.
In the present invention, the term "pharmaceutical composition" refers to a formulation of one or more of the active ingredients described in the present invention with other chemical components, such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of an active ingredient (e.g., a therapeutically active agent according to any of the respective embodiments of the invention, with or without a lipid-soluble nanoparticle composition) to a subject.
In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to a subject when administered in the intended manner, and does not abrogate the activity and properties of the liposomes in the composition (e.g., their ability to reduce the coefficient of friction of a surface, as described in any of the corresponding embodiments of the invention). Non-limiting examples of carriers are: propylene glycol, brine, emulsions, and mixtures of organic solvents with water, as well as solids (e.g., powders) and gas carriers. In some embodiments of any of the embodiments described herein, the composition comprises an aqueous carrier that is a pharmaceutically acceptable carrier, e.g., wherein the composition is a solution. In the present invention, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient of the present invention or to increase shelf life stability. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and salts and various types of starches, cellulose derivatives, gelatin, vegetable oils, EDTA, EGTA, poly-L-lysine, polyethylenimine, pimelic bromide, polyethylene glycol and other polyanions or monoanions. The pharmaceutical composition may advantageously take the form of a foam, an aerosol or a gel. In some embodiments of any of the corresponding embodiments described herein, the pharmaceutical composition further comprises a water-soluble biopolymer, such as a polypeptide and/or polysaccharide. The polymer may be ionic or nonionic.
The pharmaceutical compositions of the invention may also be used in combination with other oncology agents, and other therapeutic compounds may be administered simultaneously with the primary active ingredient, even in the same composition. Other therapeutic compounds may also be administered alone in separate compositions or in dosages different from the primary active ingredient.
In the present invention, the term "tumor" or "cancer" refers to the growth or proliferation of any abnormal cell or tissue in an animal. As used herein, the term "cancer" or "tumor" encompasses both solid tumors and hematological tumors, and also encompasses malignant, premalignant, and benign growths, such as dysplasia.
Non-limiting examples of solid tumors include prostate cancer, bladder cancer, liver cancer, head and neck cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, meningioma, pancreatic cancer, multiple squamous cell carcinoma, oral cancer, esophageal cancer, colorectal cancer, breast cancer, medulloblastoma, nasopharyngeal cancer, thymus cancer, lymphoid malignancy, fibrosarcoma, myxosarcoma, or melanoma.
Non-limiting examples of hematological neoplasms include acute leukemia, chronic leukemia, polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, or myelodysplasia.
The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. Simple modifications of the invention in accordance with the essence of the invention are all within the scope of the invention as claimed.
Example 1 preparation of fat-soluble nanoparticle composition by ethanol injection
1. Experimental materials
1) Reagent: phospholipid (injection grade, shenyang Tianfeng biopharmaceutical Co., ltd.), cholesterol-PEG 2 k-transferrin (electrophoresis purity, sian natural biotechnology Co., ltd.), purified water (homemade), medium chain triglycerides (pharmaceutical grade, fuling North Asia medical oil Co., ltd.), ptdToma-N1 plasmid (injection grade, beijing village biological gene technologies Co., ltd.), absolute ethanol (analytical purity, beijing Co., ltd.);
2) Instrument: ultrasonic cell disruptor (JY 92-IID, manufacturer Nanjing New Chen Biotechnology Co., ltd.), fluorescence microscope (CKX 53FL, manufacturer Olympus);
3) Culture medium: DMEM high sugar medium, 10% fetal bovine serum, 1% penicillin-streptomycin (diabody);
4) Cell lines: hepG2 cell line.
2. Experimental method
1) Preparation of fat-soluble nanoparticles: 0.9g of phospholipid, 0.1g of cholesterol-PEG 2 k-transferrin was weighed out and dissolved well in 5ml of absolute ethanol, followed by addition to 45ml of purified water to give a crude suspension. The suspension was treated with a probe-type ultrasonic device to obtain a semitransparent dispersion of lipid-soluble nanoparticles.
2) And uniformly mixing 0.1g of the fat-soluble nanoparticle dispersion liquid with 2 mug of ptdTommao-N1 plasmid, immediately freeze-drying to remove water, and preserving the obtained product under nitrogen protection, sealing and keeping at a low temperature in a dark place.
3) The above lyophilized powder (medium chain triglyceride: lyophilized powder=20:1, w/w) was sufficiently dissolved by using medium chain triglyceride to obtain a fat-soluble nanoparticle composition, which was stored in a sealed manner in a dark place. The average particle size of the fat-soluble nanoparticle composition was measured.
4) 1ml of the fat-soluble nanoparticle composition is taken, 1ml of PBS is used for fully mixing, vortex dispersion is carried out for 5min, and the emulsion is obtained and then mixed with 9ml of DMEM high-sugar culture medium (containing 10% of fetal calf serum and 1% of diab) for standby.
5) HepG2 cells were cultured and inoculated into a sterile 6-well plate, and after complete adherence, the emulsion prepared in appropriate amount 4) was added, at 37℃with 5% CO 2 After culturing in a cell-specific incubator for 48 hours, the results of protein expression were observed using a fluorescence microscope.
3. Experimental results
The measurement results of the average particle diameter of the fat-soluble nanoparticle composition are shown in FIG. 1, and the result shows that the average particle diameter of the fat-soluble nanoparticle composition is 122.1nm and the particle size distribution index (PDI) is 0.10.
The results of the fluorescent microscope observation of the protein expression are shown in FIG. 2, and the results show that the growth state of the HepG2 cell is good, which indicates that the plasmid carrying the red fluorescent protein sequence is successfully transfected into the HepG2 cell and successfully expressed.
Example 2 preparation of fat-soluble nanoparticle composition by high pressure homogenization
1. Experimental materials
1) Reagent: phospholipid (injection grade, shenyang Tianfeng biopharmaceutical Co., ltd.), cholesterol-PEG 2 k-transferrin (electrophoretic purity, XIAN Hao Biotech Co., ltd.), purified water (homemade), soybean oil (analytical purity, shanghai Michelia Biotechnology Co., ltd.), ptdToma-N1 plasmid (injection grade, beijing bang Biotechnology Co., ltd.), absolute ethanol (analytical purity, beijing Co., ltd.);
2) Instrument: CCK-8 kit (500T, manufacturer next san Jose Biotechnology (Shanghai) Co., ltd.), ultrasonic cell disruptor (JY 92-IID, manufacturer Nanjing New Chen Biotechnology Co., ltd.), enzyme-labeled instrument (iMark, manufacturer Berle Life medicine products (Shanghai) Co., ltd.);
3) Culture medium: DMEM high sugar medium, 10% fetal bovine serum, 1% penicillin-streptomycin (diabody);
4) Cell lines: hepG2 cell line.
2. Experimental method
1) Preparation of fat-soluble nanoparticles: 0.7g of phospholipid, 0.3g of cholesterol-PEG 2 k-transferrin was weighed out and dissolved well in 5ml of absolute ethanol, followed by addition to 45ml of purified water to give a crude suspension. The suspension was treated with a high pressure homogenizer to give a translucent dispersion of lipid-soluble nanoparticles.
2) Taking 0.25g of fat-soluble nanoparticle dispersion liquid, uniformly mixing with 0.5 mgptdTomO-N1 plasmid, immediately freeze-drying to remove water, filling nitrogen for protection, sealing and preserving at low temperature in a dark place.
3) The above lyophilized powder (soybean oil: lyophilized powder=1000:1, w/w) was sufficiently dissolved with soybean oil to obtain a fat-soluble nanoparticle composition, which was stored in a sealed manner in a dark place.
4) 1ml of the fat-soluble nanoparticle composition is taken, 1ml of DMEM high-sugar culture medium (containing 10% of fetal calf serum and 1% of double antibody) is used for fully mixing, and vortex dispersion is carried out for 5min, so as to obtain emulsion for standby.
5) HepG2 cells were cultured and inoculated into sterile 96-well plates, after complete adherence, 0.2ml of the emulsion obtained in 4) was added to the first well and diluted stepwise with a 2-fold concentration gradient, 5% CO at 37℃were added 2 Continuous culture in incubator for cells 24After 48, 72h, the cell activity was measured using CCK-8 kit, and the cell growth inhibition was calculated in parallel with 4 experiments.
3. Experimental results
The experimental results are shown in figure 3, and the results show that the cell growth inhibition rate of the experimental group is mostly negative, and compared with that of the untreated group, after the cells are treated by the fat-soluble nanoparticle composition, the number of the cells is increased to a certain extent, so that the materials used in the invention are nontoxic materials, and the high-dose addition does not have cytotoxicity, but can accelerate the growth of the cells; therefore, the safety of the invention is very high.
EXAMPLE 3 microfluidic preparation of fat-soluble nanoparticle compositions
1. Experimental materials
1) Reagent: phospholipid (injection grade, manufacturer's Shenyang Tianfeng biopharmaceutical Co., ltd.), cholesterol-PEG 2 k-transferrin (electrophoresis purity, manufacturer's Sian Hao Biotechnology Co., ltd.), purified water (self-made), medium chain triglycerides (pharmaceutical grade, manufacturer's Kaolin North Asia medicinal oil Co., ltd.), mRNA-GFP (injection grade, manufacturer's brocade Biotechnology (Beijing) Co., ltd.), absolute ethanol (analytically pure, manufacturer's Beijing Co., ltd.);
2) Instrument: high precision two-channel syringe pump (Fusion 4000-X, chemyx, usa), microfluidic Nano-fabrication chip (Y, micro-fluidics technology inc.), laser particle size analyzer (Nano-ZS 90, malvern instruments inc., uk), flow cytometer (FACSAria, BD, usa);
3) Culture medium: DMEM high sugar medium, 10% fetal bovine serum, 1% penicillin-streptomycin (diabody);
4) Cell lines: HEK293T cell line.
2. Experimental method
1) Preparation of fat-soluble nanoparticles: weighing a certain amount of phospholipid and cholesterol-PEG 2 k-transferrin, fully dissolving in a certain amount of absolute ethyl alcohol, and observing the solution property. And filling the obtained solution into a medical injector, taking a sterile and enzyme-free injector to fill enzyme-free water, preparing a fat-soluble nanoparticle dispersion liquid by using a microfluidic method, wherein the flow rates of the two are subjected to process fumbling by using different ratios, and measuring the particle size distribution and zeta potential of the obtained fat-soluble nanoparticles by using a laser particle size analyzer.
2) And uniformly mixing 4g of the fat-soluble nanoparticle dispersion liquid with 100 mug of mRNA-GFP, immediately freeze-drying to remove water, protecting the obtained product by low nitrogen filling, sealing and preserving at low temperature in a dark place.
3) The above lyophilized powder (medium chain triglyceride: lyophilized powder=5:1, w/w) was sufficiently dissolved by using medium chain triglyceride to obtain a fat-soluble nanoparticle composition, which was stored in a sealed manner in a dark place.
4) 1ml of the fat-soluble nanoparticle composition is taken, 1ml of DMEM high-sugar culture medium (containing 10% of fetal calf serum and 1% of double antibody) is used for fully mixing, and vortex dispersion is carried out for 5min, so as to obtain emulsion for standby.
5) HEK293T cells were cultured and inoculated into a sterile 6-well plate, and after complete adherence, the appropriate amount of emulsion 4) was added, at 37℃with 5% CO 2 After culturing in a cell-specific incubator for 48 hours, the green fluorescent expression was measured using a flow cytometer. In the flow cytometry plot, FL1H represents cells stained with stain A-V.
3. Experimental results
The results are shown in tables 1, 2, 3, 4 and 4, and the results show that when the cholesterol-PEG 2 k-transferrin dosage is more than 30% (w/w), the ethanol solution has high turbidity degree, forms suspension, is not suitable for continuous preparation, and therefore the dosage is between 1 and 30%; the microfluidic technology can successfully prepare the fat-soluble nano-particles required by the invention, and the particle size of the obtained fat-soluble nano-particles is about 80-1000 nm, and all the fat-soluble nano-particles carry negative electricity; the dosage of cholesterol-PEG 2 k-transferrin is in the range of 1-30%, the flow rate ratio of ethanol to water is in the range of 1:9-3:7, mRNA delivery containing GFP fragment markers can be realized, and the protein can be expressed successfully. The amount of cholesterol-PEG 2 k-transferrin is different, the transfection efficiency is different to a certain extent, and the optimal range is 10-20%.
Table 1 formulation ratio and ethanol solution Properties
cholesterol-PEG 2 k-transferrin amount/g Phospholipid content/g Property of ethanol solution
0.01 0.99 Clarifying the solution
0.1 0.9 Clarifying the solution
0.2 0.8 Semitransparent solution
0.3 0.7 Semitransparent solution
0.4 0.6 Cloudiness, obvious precipitation after standing
0.5 0.5 Cloudiness, obvious precipitation after standing
TABLE 2 average particle diameter of liposoluble nanoparticles prepared at different flow ratios
TABLE 3 preparation of the Zeta potential of the liposoluble nanoparticles at different flow Rate ratios
TABLE 4 flow cytometry detection of the transfection expression Effect of the fat-soluble nanoparticle compositions obtained in different proportions
cholesterol-PEG 2 k-transferrin dosage Ethanol: water flow speed ratio Average fluorescence intensity
1％ 3:7 38.03±12.19
10％ 3:7 82.76±23.75
20％ 2:8 95.16±18.08
30％ 1:9 69.45±25.66
Example 4 preparation of fat-soluble nanoparticle composition by thin film dispersion
1. Experimental materials
1) Reagent: phospholipids (injection grade, manufacturer's Shenyang Tianfeng biopharmaceutical Co., ltd.), cholesterol-PEG 2 k-transferrin (electrophoretic purity, manufacturer's Sian Hao Biotechnology Co., ltd.), purified water (homemade), glycerol dioleate, medium chain triglycerides (pharmaceutical grade, manufacturer's Kagaku Beijing Asian medicinal oil Co., ltd.), soybean oil (analytical purity, manufacturer's Shanghai Michelin Biotechnology Co., ltd.), corn oil (analytical purity, manufacturer's Shanghai Michelin Biotechnology Co., ltd.), squalene (analytical purity, shanghai Abin Biotechnology Co., ltd.), oleic acid (analytical purity, shanghai Abin Biotechnology Co., ltd.), pCDNA3.1-EGFP plasmid (injection grade, manufacturer's Beijing Azu Beijing GmbH Biotechnology Co., ltd.), absolute ethanol (analytical purity, manufacturer's Beijing Guangdong Fine chemical Co., ltd.);
2) Instrument: rotary evaporator (RE-2002, manufacturer Shanghai kohlamy biosciences), ultrasonic cell disruption instrument (JY 92-IID, manufacturer Nanjing Xinchen biosciences).
2. Experimental method
1) Preparation of fat-soluble nanoparticles: 0.9g of phospholipid, 0.1g of cholesterol-PEG 2 k-transferrin and the like are weighed, fully dissolved in 5ml of absolute ethyl alcohol, the absolute ethyl alcohol is dried into a film by spin drying by using a rotary evaporator, purified water is added, then the material is redispersed, and an ultrasonic cell disruption instrument is used for preparing fat-soluble nanoparticle dispersion liquid.
2) And (3) uniformly mixing 40g of the fat-soluble nanoparticle dispersion liquid with 2mg of pCDNA3.1-EGFP plasmid, immediately freeze-drying to remove water, and preserving the obtained product under nitrogen protection, sealing and low-temperature light-shielding.
3) The above lyophilized powder was sufficiently dissolved using a certain amount of solvent (solvent: lyophilized powder = 20:1, w/w), the dissolution effect was observed.
3. Experimental results
The results are shown in Table 5, and show that as the amount of cholesterol-PEG 2 k-transferrin increases, the turbidity of the lipid nanoparticle oil dispersion also increases, indicating that the dispersibility thereof decreases. Wherein, when the addition amount of cholesterol-PEG 2 k-transferrin is 1-30%, medium chain triglyceride, soybean oil, corn oil, squalene and arachidonic acid can be selected as a dispersion solvent; when the addition amount of cholesterol-PEG 2 k-transferrin is 1-20%, the glycerol dioleate and oleic acid can be selected as a dispersion solvent; when the amount of cholesterol-PEG 2 k-transferrin added is more than 20%, the glycerol dioleate and oleic acid cannot well disperse the lipid nanoparticles, and are not suitable for use as a dispersing solvent.
TABLE 5 turbidity evaluation Table for different nonaqueous solvents
Turbidity score: the turbidity increases from 1 to 5, 1 is clear and transparent, and 5 is opaque and turbid.
Example 5 comparison of fat-soluble nanoparticle compositions prepared by different methods
1. Control group preparation method:
1) Preparing an oil phase: adding a certain amount of medium chain triglyceride, soybean phospholipid, cholesterol and DSPE-PEG2 k-transferrin into a test tube, and dissolving in water bath at 80 ℃;
2) Preparing an aqueous phase: dissolving mRNA and glycerol in water;
3) And (3) mixing and extruding: adding the oil phase into the water phase, shearing for 30min at 10000r/min, and ultrasonically crushing by a probe; repeatedly squeezing the mixture with 0.45 μm and 0.22 μm filter membrane to obtain nanoemulsion.
2. Experimental group preparation method
1) Preparation of fat-soluble nanoparticles: the amount of phospholipids and cholesterol-PEG 2 k-transferrin were weighed as shown in table 6 and lipid-soluble nanoparticle dispersions were prepared by different methods.
2) After 10g of the lipid-soluble nanoparticle dispersion liquid is uniformly mixed with 2mg of GFP-mRNA plasmid, the mixture is immediately frozen and dried to remove water, and the obtained product is filled with nitrogen for protection, sealed and stored at a low temperature in a dark place.
3) The above lyophilized powder (solvent: lyophilized powder=20:1, w/w) was sufficiently dissolved with a certain amount of solvent for use.
Table 6 prescription design and Process selection
3. Encapsulation efficiency experiment:
1) Control group: the sample was treated with a 30kd ultrafiltration centrifuge tube, free mRNA and liposomes were separated, the mRNA concentration in the filtrate was determined, and the content was calculated.
2) Experimental group: after pbs=1:9 mixing and vortexing, samples were treated using a 30kd ultrafiltration centrifuge tube, free mRNA and liposomes were isolated, mRNA concentration in the filtrate was determined, and content was calculated.
4. Delivery capability verification experiment
HUH-7 cells were cultured in vitro and inoculated into 6-well plates after they had grown to logarithmic growth phase. The inoculation density was 20 ten thousand/well. After the adhesive is completely adhered, adding proper amount of liposoluble nanoparticle composition at 37deg.C and 5% CO 2 After culturing in a cell-specific incubator for 48 hours, the average fluorescence intensity was measured using a flow cytometer.
5. Stability test
The control and experimental samples containing mRNA were prepared according to the above method, placed in a constant temperature environment of 4℃and 20℃and treated with a 30kd ultrafiltration centrifuge tube after 24 hours, free mRNA was isolated, the concentration of mRNA in the filtrate was measured, and the content was calculated.
6. Experimental results
The encapsulation efficiency test results are shown in table 7, and the results show that the encapsulation efficiency of the test group is 80.62 +/-2.35% -94.80 +/-2.30%, and the encapsulation efficiency of the control group is 4.71+/-0.59% -11.98 +/-1.43%, which indicates that the encapsulation efficiency of the test group is far higher than that of the control group.
The results of the delivery capacity verification experiments are shown in fig. 5 and table 8, and the results show that the average fluorescence intensity of the experimental group is between 22.3+/-2.47 and 57.63 +/-11.47, and the average fluorescence intensity of the control group is between 5.28+/-0.88 and 5.28+/-0.88, so that the gene delivery efficiency of the experimental group is far higher than that of the control group.
The stability test results are shown in table 9, and the results show that the drug stability of the test group is significantly higher than that of the control group.
Table 7 encapsulation efficiency comparison results
Grouping Control group (mean+ -SD, n=3) Experiment group (mean+ -SD, n=3)
Prescription 1 11.98±1.43％ 94.80±2.30％
Prescription 2 8.65±0.89％ 91.61±1.32％
Prescription 3 7.20±0.89％ 90.50±1.22％
Prescription 4 6.52±0.69％ 85.88±3.17％
Table 8 delivery capability vs. results
Table 924h content stability comparison results
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A lipid-soluble nanoparticle, characterized in that the lipid-soluble nanoparticle comprises a surfactant and/or a functional molecule-modifying material;
preferably, the surfactant comprises a phospholipid;
Preferably, the phospholipid comprises one or more of a natural phospholipid, a semisynthetic phospholipid or a synthetic phospholipid;
preferably, the natural phospholipids include soybean phospholipids, egg yolk phospholipids, behenyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, ditrans oleoyl phosphatidylcholine, dilauroyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, monopalmitoyl phosphatidylcholine, monostearoyl choline, egg yolk phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, distearoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dimyristoyl phosphatidylserine, distearoyl phosphatidylserine, ditolyphosphatidylserine, dimyristoyl phosphatidylserine, distearoyl sphingomyelin, dipalmitoyl phosphatidylinositol, ditolyphosphatidylinositol, dimyristoyl phosphatidylinositol, dimyristoyl phosphatidate or dimyristoyl phosphatidic acid;
Preferably, the semisynthetic phospholipid comprises a hydrogenated phospholipid;
preferably, the synthetic phospholipid comprises DEPC, DOPC, DPPC, DSPC or DMPC;
preferably, the amount of said phospholipids is selected from 70% to 99%;
preferably, the amount of said phospholipids is selected from 80% to 90%;
preferably, the functional molecule-modifying material comprises one or more of a protein, a nucleic acid, a compound;
preferably, the protein comprises one or more of cholesterol-PEG-transferrin, DSPE-PEG-transferrin, phytolectin, transmembrane peptide;
preferably, the protein is cholesterol-PEG-transferrin;
preferably, the cholesterol-PEG-transferrin comprises one or more of cholesterol-PEG 2 k-transferrin, cholesterol-PEG 3 k-transferrin, cholesterol-PEG 4 k-transferrin, cholesterol-PEG 5 k-transferrin;
preferably, the cholesterol-PEG-transferrin is cholesterol-PEG 2 k-transferrin;
preferably, the cholesterol-PEG 2 k-transferrin is used in an amount selected from 1% to 30%;
preferably, the cholesterol-PEG 2 k-transferrin is used in an amount selected from 10% to 20%;
preferably, the nucleic acid comprises a transferrin nucleic acid aptamer;
Preferably, the fat-soluble nanoparticle further comprises an organic solvent and water;
preferably, the organic solvent comprises one or more of methanol, ethanol, hexylene glycol, propylene glycol, dipropylene glycol, glycerol, acetonitrile, butanone, 1-trifluoroethane, hexafluoroisopropanol, ethyl acetate, carbon tetrachloride, butanol, dibutyl ether, diethyl ether, cyclohexylamine, dichloromethane, hexane, butyl acetate, diisopropyl ether, benzene, dipentyl ether, chloroform, heptane, tetrachloroethylene, toluene, hexadecane, dimethylformamide, N-methylpyrrolidone, dimethylacetamide, tetrahydrofuran, glycerol, dioxane;
preferably, the organic solvent is ethanol.
2. A lipid-soluble nanoparticle composition comprising the lipid-soluble nanoparticle of claim 1 and a drug;
preferably, the drug comprises a genetic drug, a chemical drug;
preferably, the drug is a genetic drug;
preferably, the genetic medicament comprises a DNA, RNA medicament;
preferably, the DNA drug comprises double-stranded DNA, single-stranded DNA drug; the RNA drug comprises double-stranded RNA and single-stranded RNA drugs;
Preferably, the double-stranded DNA comprises a plasmid;
preferably, the single-stranded DNA comprises a DNA aptamer;
preferably, the single stranded RNA comprises RNA nucleic acid aptamer, mRNA, ncRNA or antisense oligonucleotide ASO;
preferably, the ncrnas comprise miRNA, siRNA, shRNA, saRNA, sgRNA, piRNA, lncRNA, circRNA or other regulatory RNAs;
preferably, the genetic drug comprises a plasmid drug or an mRNA drug;
preferably, the plasmid medicament comprises a plasmid, a gene sequence encoding a tag protein and/or a gene of interest;
preferably, the mRNA drug comprises mRNA and/or a gene sequence encoding a tag protein;
preferably, the plasmid comprises pQE-12, pUC-series, pBluescript, pET-series expression vectors, pCRTOPO, pJOE, pBACKBONE, pBBR-MCS-series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-N-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry vector system, pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV-dhfr, pIDD 35, okayama-Berg cDNA expression vectors pcDV1, pRc/CMV, pcDNA1, pcDNA3, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pUC, pMB1, pSC101, pBEU1, pBEU2, pDF41, pDF42, pBR322, ptoma-N1 or DNpcDNA3.1;
Preferably, the plasmid is ptdTomO-N1 or pCDNA3.1;
preferably, the tag comprises a fluorescent protein;
preferably, the fluorescent protein comprises one or more of GFP, EGFP, mFruit protein, dsRed series fluorescent protein, OFP and YFP;
preferably, the fluorescent protein comprises GFP or EGFP;
preferably, the gene of interest comprises WT1, MUC1, LMP2, EGFRvIII, her2, p53, NY-ESO-1, PSMA, GD2, CEA, gp100, PR1, PSA, hTERT, ephA2, PAP, ML-IAP, AFP, epCAM, ERG, NA, PAX3, MYCN, rhoC, TRP-2, GD3, mesothelin, PSCA, CYP1B1, PLAC1, GM3, BORIS, globoH, ETV-AML, NY-BR-1, RGS5, SART3, STn, PAX5, OY-TES1, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legum, tie2, page4, VEGFR2, MAD-CT-1, FAP, PDGFR-B or MAD-CT-2;
preferably, the plasmid drug is pCDNA3.1-EGFP or ptdToma-N1;
preferably, the mRNA drug is mRNA-GFP;
preferably, the fat-soluble nanoparticle composition further comprises a liquid nonaqueous solvent;
preferably, the liquid nonaqueous solvent comprises one or more of vegetable oil, animal oil, alkane liquid solvents;
Preferably, the vegetable oil comprises nut oil, fennel oil, soybean oil, almond oil, corn oil, olive oil, peanut oil, almond oil, walnut oil, cashew oil, rice bran oil, poppy seed oil, cotton seed oil, canola oil, sesame oil, coconut oil, linseed oil, cinnamon oil, clove oil, nutmeg oil, coriander oil, lemon oil, orange oil, safflower oil, cocoa butter, palm oil, palm kernel oil, sunflower oil, rapeseed oil, or castor oil;
preferably, the animal oil comprises whale oil, fish oil, musk oil or mink oil;
preferably, the alkane liquid solvent comprises glycerol dioleate, glycerol tripropionate, glycerol tributyrate, polyoxyethylene oil derivatives, medium chain triglycerides, oleic acid, arachidonic acid, hydrogenated oil, squalene or glycerol monooleate;
preferably, the liquid nonaqueous solvent comprises medium chain triglycerides, soybean oil, corn oil, squalene, oleic acid or arachidonic acid.
3. A method of preparing a fat-soluble nanoparticle composition, the method comprising the steps of:
1) Preparing the fat-soluble nanoparticle of claim 1;
2) Preparing a lyophilized powder using the lipid-soluble nanoparticle and the drug of claim 2;
3) A fat-soluble nanoparticle composition is prepared using the lyophilized powder and the liquid nonaqueous solvent of claim 2.
4. A method according to claim 3, wherein the method for preparing the liposoluble nanoparticles in step 1) comprises extrusion, reverse evaporation, supercritical carbon dioxide, ethanol injection, high pressure homogenization, microfluidic and/or thin film dispersion;
preferably, the ethanol injection method is to prepare a crude suspension by using the surfactant and/or the functional molecule modification material as defined in claim 1, and treat the crude suspension by using ultrasonic equipment to obtain fat-soluble nanoparticles;
preferably, the ultrasonic device is a probe type ultrasonic device;
preferably, the high pressure homogenization method is to prepare a crude suspension by using the surfactant and/or functional molecule modification material as set forth in claim 1, and treat the crude suspension with a high pressure homogenizer to obtain the fat-soluble nanoparticles;
preferably, the preparation method of the crude suspension comprises dissolving the surfactant and/or functional molecule modification material in the organic solvent in the claim 2, and then adding purified water for dispersion to obtain the crude suspension;
Preferably, the microfluidic method obtains the liposoluble nanoparticles by treating the surfactant and/or functional molecular modification material of claim 1 with a microfluidic device;
preferably, the microfluidic method obtains an organic solution by dissolving the surfactant and/or functional molecule-modifying material of claim 1 in the organic solvent of claim 2, and then fills the obtained organic solution into a syringe, fills water into another syringe, and uses a microfluidic device to process to obtain the fat-soluble nanoparticles;
preferably, the water is enzyme-free water;
preferably, the flow rate ratio of the organic solvent to water of the microfluidic method is selected from 1:9 to 3:7;
preferably, the Zeta potential of the fat-soluble nanoparticle is selected from-5 to-30 mV;
preferably, the Zeta potential of the fat-soluble nanoparticle is selected from-7.54 to-29.27 mV;
preferably, the thin film dispersion method is to obtain fat-soluble nanoparticles by treating the surfactant and/or functional molecule-modifying material described in claim 1 with a rotary evaporator, an ultrasonic cell disruptor;
preferably, the thin film dispersion method is to obtain liposoluble nanoparticles by dissolving the surfactant and/or functional molecule-modifying material of claim 1 in the organic solvent, spin-drying the organic solvent using a rotary evaporator and forming a film, then adding purified water redispersing material, and treating the dispersing material using an ultrasonic cell disruptor;
Preferably, the particle size of the fat-soluble nanoparticles is selected from 50-1000 nm;
preferably, the particle size of the fat-soluble nanoparticles is selected from 80-1000 nm;
preferably, the average particle size of the fat-soluble nanoparticles is in the range of 122.1nm.
5. A method according to claim 3, wherein the method for preparing a lyophilized powder in step 2) comprises mixing the fat-soluble nanoparticles and the drug to obtain a mixed solution, and drying the mixed solution to obtain a lyophilized powder;
preferably, the weight ratio of the drug to the liposoluble nanoparticles is selected from 1:10 to 1:50000;
preferably, the weight ratio of the drug to the liposoluble nanoparticles is selected from 1:500 to 1:50000;
preferably, the drying method comprises one or more of freeze drying method, normal pressure drying method, reduced pressure drying method, vacuum drying method and microwave drying method;
preferably, the method of drying is a freeze-drying method;
preferably, the freeze-drying procedure comprises a prefreezing procedure, sublimation drying and analytical drying;
preferably, the packaging material of the mixed solution comprises a penicillin bottle, a freeze-drying ampoule and a freeze-drying bubble cap;
preferably, the freeze-dried powder is preserved under nitrogen protection and sealing at low temperature and in a dark place.
6. A method according to claim 3, wherein the method of preparing a fat-soluble nanoparticle composition of step 3) comprises dissolving the lyophilized powder using the liquid nonaqueous solvent to obtain a fat-soluble nanoparticle composition;
preferably, the fat-soluble nanoparticle composition is used by local injection or cell transfection after being uniformly mixed with a buffer solution;
preferably, the weight ratio of the liquid nonaqueous solvent to the freeze-dried powder is selected from 5:1 to 1000:1.
7. A pharmaceutical composition comprising 1) the liposoluble nanoparticle of claim 1, the liposoluble nanoparticle composition of claim 2, or the liposoluble nanoparticle composition prepared by the method of any one of claims 3-6, and/or 2) a pharmaceutically acceptable carrier.
8. Use of the liposoluble nanoparticle of claim 1, the liposoluble nanoparticle composition of claim 2, the liposoluble nanoparticle composition prepared by the method of any one of claims 3-6, or the pharmaceutical composition of claim 7 in the preparation of a product for treating a tumor.
9. The use according to claim 8, wherein the tumor is selected from the group consisting of hematological tumors, solid tumors;
Preferably, the solid tumor comprises prostate cancer, bladder cancer, liver cancer, head and neck cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, meningioma, pancreatic cancer, multiple squamous cell carcinoma, oral cancer, esophageal cancer, colorectal cancer, breast cancer, medulloblastoma, nasopharyngeal carcinoma, thymus cancer, lymphoid malignancy, fibrosarcoma, myxosarcoma, or melanoma;
preferably, the hematological neoplasm comprises acute leukemia, chronic leukemia, polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia or myelodysplasia.
10. A method of optimizing the prescription process of the liposoluble nanoparticle according to claim 1, the liposoluble nanoparticle composition according to claim 2, or the liposoluble nanoparticle composition prepared by the method according to any one of claims 3-6, comprising: the method adopts the encapsulation efficiency, the particle size of the fat-soluble nanoparticle composition, the drug delivery capacity and the placement stability of the fat-soluble nanoparticle composition as indexes to change one or more of the surfactant types, the functional molecule modification material types, the liquid nonaqueous solvent types, the organic solvent types, the component proportions and the preparation method of the fat-soluble nanoparticle composition.
CN202310416659.4A 2023-04-18 2023-04-18 Low-toxicity fat-soluble nanoparticle composition and preparation method and application thereof Pending CN116509818A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070065432A1 (en) * 1999-02-22 2007-03-22 Synergene Therapeutics, Inc. Antibody fragment-targeted immunoliposomes for systemic gene delivery
WO2014046630A1 (en) * 2012-09-19 2014-03-27 Keskin, Dilek Tumor targeted liposomal drug delivery system
CN104684546A (en) * 2012-06-07 2015-06-03 哈佛大学校长及研究员协会 Nanotherapeutics for drug targeting
CN110960688A (en) * 2018-09-30 2020-04-07 复旦大学 Low-toxicity bionic nano system for improving curative effect of pancreatic cancer and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070065432A1 (en) * 1999-02-22 2007-03-22 Synergene Therapeutics, Inc. Antibody fragment-targeted immunoliposomes for systemic gene delivery
CN104684546A (en) * 2012-06-07 2015-06-03 哈佛大学校长及研究员协会 Nanotherapeutics for drug targeting
WO2014046630A1 (en) * 2012-09-19 2014-03-27 Keskin, Dilek Tumor targeted liposomal drug delivery system
CN110960688A (en) * 2018-09-30 2020-04-07 复旦大学 Low-toxicity bionic nano system for improving curative effect of pancreatic cancer and preparation method thereof

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