CN109549934B - Preparation method of polysaccharide-based lipid nanoparticles - Google Patents
Preparation method of polysaccharide-based lipid nanoparticles Download PDFInfo
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Abstract
The invention discloses a preparation method of polysaccharide-based lipid nanoparticles, which relates to the field of nanoparticle preparation, and is characterized in that the polysaccharide-based nanoparticles are obtained by polymerization crosslinking reaction of water-soluble polysaccharide, monomers and a crosslinking agent, then the polysaccharide-based nanoparticles are used as a framework to induce lipid micromolecules to self-assemble on the surface of the lipid micromolecules to form the polysaccharide-based lipid nanoparticles, and the polysaccharide-based lipid nanoparticles are further mixed with genes to prepare gene-loaded polysaccharide-based lipid nanoparticles; or the polysaccharide-based nanoparticles and the drug are mixed to prepare drug-loaded polysaccharide-based nanoparticles, then the drug-loaded polysaccharide-based nanoparticles are formed by taking the drug-loaded polysaccharide-based nanoparticles as a framework to induce the lipid small molecules to self-assemble on the surface of the drug-loaded polysaccharide-based nanoparticles, and the drug-loaded polysaccharide-based nanoparticles and the gene are further mixed to prepare the drug-loaded and gene-loaded polysaccharide-based lipid nanoparticles. The preparation method is green, pollution-free, high in yield, efficient and convenient to synthesize, the prepared nanoparticles are uniform in particle size and high in transfection efficiency, are not easy to pump out by tumor cells, and have wide application prospects in the field of tumor treatment.
Description
Technical Field
The invention relates to the field of nanoparticle preparation, in particular to a preparation method of polysaccharide-based lipid nanoparticles.
Background
Chemotherapy is the most common method of tumor treatment, but it suffers from the following disadvantages: when chemical drugs are used, drug resistance is easy to generate in tumor cells, and the drug resistance of tumors is one of the important reasons for failure of tumor chemotherapy; meanwhile, because the selectivity of chemical drugs to tumors is not strong, the chemical drugs not only have the effect of killing tumor cells, but also can cause great damage to normal tissues. Therefore, how to overcome the drug resistance of the tumor and reduce the side effects of tumor chemotherapy is an important issue facing the current tumor treatment.
The nanoparticles can be passively targeted to tumor sites through an EPR effect due to the specific nanoscale of the nanoparticles, so that the nanoparticles are the first choice for antitumor drug carriers. The nano particles can enter cells through cell membranes, can load and protect chemotherapeutic drugs or genes to enter the cells and be released in the cells, and improves the delivery efficiency and bioavailability. Therefore, the nano-scale particles are regarded as one of powerful means for controlling the release of the antitumor drugs and achieving the targeted enrichment of the antitumor drugs at tumor sites. Therefore, the construction of the nano-carrier capable of efficiently transporting the anti-tumor drugs by designing an appropriate way becomes a key link in tumor treatment.
The prior art shows that tumor drug resistance is mostly related to the overexpression of P-glycoprotein. P-glycoprotein is located on the cell membrane and is a common molecular pump for protecting cells from invasion of foreign harmful molecules, however, over-expression of P-glycoprotein in tumor cells makes most drugs or carriers difficult to enter cells or easy to be "pumped out" of cells to reduce the retention time in cells, and the treatment effect is seriously reduced.
Therefore, those skilled in the art have made efforts to develop a method for preparing polysaccharide-based lipid nanoparticles, by which polysaccharide-based lipid nanoparticles that enter tumor cells more efficiently and are not easily pumped out can be obtained.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to prepare a nanoparticle that is more efficient for entering tumor cells and is not easily pumped out.
To achieve the above objects, the present invention provides a method for preparing polysaccharide-based lipid nanoparticles, which, in one embodiment, comprises the steps of:
1.1 preparing water solution of water-soluble polysaccharide, adding an initiator under the protection of inert gas, and uniformly stirring;
1.2 adding a monomer or a monomer dissolved in a solvent, and uniformly stirring;
1.3 adding a cross-linking agent, and carrying out polymerization and cross-linking reaction under the protection of inert gas;
1.4 after the reaction is finished, carrying out dialysis treatment, and carrying out freeze drying to obtain polysaccharide-based nanoparticles;
1.5 dissolving amphiphilic lipid molecules in a solvent, removing the solvent by rotary evaporation, adding water, and performing ultrasonic treatment to obtain lipid molecule suspension;
1.6 preparing the polysaccharide-based nanoparticles obtained in the step 1.4 into an aqueous solution, mixing the aqueous solution with the lipid molecule suspension obtained in the step 1.5, and performing ultrasonic treatment to obtain polysaccharide-based lipid nanoparticles; or preparing the polysaccharide-based nanoparticles obtained in the step 1.4 into an aqueous solution, mixing the aqueous solution with a medicinal solution, oscillating, adding water, oscillating, performing ultrafiltration and centrifugation, washing with water to obtain drug-loaded polysaccharide-based nanoparticles, preparing the drug-loaded polysaccharide-based nanoparticles into an aqueous solution, mixing the aqueous solution with the lipid molecule suspension obtained in the step 1.5, and performing ultrasonic treatment to obtain the drug-loaded polysaccharide-based lipid nanoparticles.
Further, the method also comprises the following steps:
1.7 preparing the polysaccharide-based lipid nanoparticles or drug-loaded polysaccharide-based lipid nanoparticles obtained in the step 1.6 into aqueous solution, respectively mixing the aqueous solution with the gene aqueous solution, oscillating and standing to obtain the gene-loaded polysaccharide-based lipid nanoparticles or drug-loaded and gene-loaded polysaccharide-based lipid nanoparticles.
Further, the water-soluble polysaccharide in step 1.1 is one or more of dextran, aminodextran, chitosan, hydroxymethyl chitosan, carboxypropyl chitosan, chitosan oligosaccharide, alginic acid, water-soluble starch, carboxymethyl dextran, carboxymethyl cellulose, hyaluronic acid, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.
Further, the initiator in step 1.1 is one or more of potassium persulfate, ammonium persulfate, benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, di-tert-butyl peroxide, dicumyl peroxide, tert-butyl peroxybenzoate, tert-butyl peroxypivalate, methyl ethyl ketone peroxide, cyclohexanone peroxide, diisopropyl peroxydicarbonate, dicyclohexyl peroxydicarbonate, cerium ammonium nitrate, nickel perchlorate hexahydrate, or radioactive rays.
Further, the monomer in step 1.2 is one or more of acrylic acid, methacrylic acid, ethyl acrylate, methyl acrylate, tert-butyl acrylate, N-isopropylacrylamide, glycidyl methacrylate, styrene and oligo-ethylene glycol methyl ether methacrylate.
Further, the cross-linking agent in step 1.3 is one or more of diallyl disulfide, L-cystine bisacrylamide, cysteamine, or bis (2-methacryloyloxyethyl) disulfide.
Further, the amphiphilic lipid molecule in step 1.5 is one or more of phosphatidylcholine phospholipid, 1-palmitoyl-2-stearoyl lecithin, soybean phospholipid, dipalmitoylphosphatidylethanolamine-pegylated phospholipid, distearoylphosphatidylethanolamine-pegylated phospholipid, phosphatidylserine phospholipid, phosphatidylglycerol phospholipid, glycerophosphatidic phosphatide, lysophospholipid, phosphatidylethanolamine phospholipid, (2, 3-dioleoyl-propyl) -trimethylamine and 1, 2-dioleyloxy-N, N-dimethyl-3-aminopropane.
Further, the drug in step 1.6 is one or more of paclitaxel, daunorubicin, doxorubicin, daunorubicin, mitoxantrone, aclacinomycin, homoharringtonine, vincristine, vindesine, etoposide, teniposide, prednisone, dexamethasone, mechlorethamine hydrochloride, cyclophosphamide, melphalan, lomustine, methotrexate, fluorouracil, mercaptopurine, mitomycin, pingyangmycin, vinorelbine, hydroxycamptothecin, and etoposide.
Further, the gene in step 1.7 is one or more of plasmid, siRNA, microRNA, piRNA, circlerRNA and lncRNA.
Furthermore, the invention also provides polysaccharide-based lipid nanoparticles prepared by any one of the methods.
The method provided by the invention comprises the steps of firstly carrying out polymerization and crosslinking reaction on water-soluble polysaccharide, a monomer and a crosslinking agent to obtain polysaccharide-based nanoparticles, and then using the polysaccharide-based nanoparticles as a framework to induce lipid small molecules to self-assemble on the surfaces of the lipid small molecules to form the polysaccharide-based lipid nanoparticles. The polysaccharide is a biological macromolecule with good biocompatibility, has functional groups such as hydroxyl, amino and the like, and is easy to carry out chemical modification; the phospholipid molecules have chemical structures similar to lipid molecules in cell membranes, and artificially synthesized or artificially modified phospholipid-like amphiphilic small molecules have the advantages of natural phospholipids and the capability of combining genes or better cell membrane fusion capability, and nanoparticles formed by the phospholipid molecules can load the genes or can enter cells more easily. Therefore, the lipid molecule layer formed on the surface of the polysaccharide-based lipid nanoparticle provided by the invention has a structure similar to that of a cell membrane, and can more easily penetrate through the cell membrane to enter cells and reduce the pumping-out effect of P-glycoprotein on the nano-carrier by using the lipid molecule layer as a gene and chemotherapeutic drug carrier, so that more chemotherapeutic drugs are retained in the cells, and the killing effect on drug-resistant cells is further enhanced. Preferably, the polysaccharide-based lipid nanoparticle is loaded with the small interfering RNA (siRNA-MDR1) of the multidrug resistance gene (MDR1) of the tumor cell, the expression of the multidrug resistance gene (MDR1) of the tumor cell is knocked down after the polysaccharide-based lipid nanoparticle enters the tumor cell, and the expression quantity of P-glycoprotein is further reduced, so that the tumor resistance is reduced at the gene level, namely, the polysaccharide-based nanoparticle with the surface modified by lipid is used for loading the antitumor gene such as siRNA-MDR1, and the loaded drug is combined with gene therapy while chemotherapy, so that the polysaccharide-based lipid nanoparticle is an important means for overcoming the tumor resistance by synergistically utilizing various methods. The polysaccharide-based lipid nanoparticles can be applied to the field of tumor treatment and can reduce the drug resistance of tumors and the side effects in the treatment process.
The preparation method provided by the invention is green, pollution-free, high in yield, efficient and convenient in synthesis, the prepared nanoparticles are uniform in particle size, about 100nm in size, high in transfection efficiency, and wide in application prospect in the aspect of tumor treatment.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a TEM image of polysaccharide-based lipid nanoparticles according to a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of the process for preparing polysaccharide-based lipid nanoparticles according to a preferred embodiment of the present invention;
FIG. 3 is a statistical chart of cell viability assay results after drug-loaded and genetically modified LPS nanoparticles intervene in OVCar-3 paclitaxel-resistant cells of ovarian cancer in accordance with a preferred embodiment of the present invention;
FIG. 4 is a diagram showing the results of flow cytometry after intervention of ovarian cancer OVCar-3 paclitaxel resistant cells by using gene-loaded polysaccharide-based lipid nanoparticles according to a preferred embodiment of the present invention;
FIG. 5 is a diagram showing the result of detecting the expression of drug-resistant genes by Western blotting after intervention of ovarian cancer OVCar-3 paclitaxel-resistant cells by using gene-loaded polysaccharide-based lipid nanoparticles according to a preferred embodiment of the present invention;
FIG. 6 is a diagram showing the result of the fluorescent quantitative PCR detection of the expression of drug-resistant genes after intervention of ovarian cancer OVCar-3 paclitaxel-resistant cells by using the gene-loaded polysaccharide-based lipid nanoparticles according to a preferred embodiment of the present invention.
Detailed Description
The technical content of the invention is further explained by the following embodiments: the following examples are illustrative and not intended to be limiting, and are not intended to limit the scope of the invention. The test methods used in the examples described below are, unless otherwise specified, generally carried out under conventional conditions or conditions recommended by the manufacturer. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The gene sources and/or functions and cell sources referred to in the following examples are illustrated below:
Fam-siRNA: purchased from jima pharmaceutical technology ltd, shanghai, Fam is a green fluorophore excited by blue light with an excitation wavelength of 480nm and an emission wavelength of 520nm, and used for observing positive transfected cells under a fluorescence microscope;
siRNA-MDR 1: small interfering RNA (RNA) which is purchased from Shanghai Jima pharmaceutical technology GmbH and codes a multi-drug resistance gene (MDR1) can be used for verifying the transfection and expression efficiency by detecting the expression level of MDR1 of positive transfected cells;
human ovarian cancer OVCar-3 paclitaxel resistant cells were constructed by the permissive topic group of Jinshan Hospital, affiliated with the university of double denier, patent No.: ZL 201410708515.7; human lung carcinoma A549 paclitaxel resistant cells were purchased from Jiangsu Kai-based biotechnology GmbH; human colon cancer HCT-8 paclitaxel resistant cells were purchased from Jiangsu Kai-based biotechnology, Inc.; human leukemia cell K562 doxorubicin-resistant cells were purchased from Jiangsu Kai-ji Biotechnology, Inc.; human ovarian cancer A2780 taxol-resistant cells were purchased from Hunan Fenghui Biotech Limited; the MCF-7 paclitaxel resistant cells of human breast cancer are purchased from Hu nan Fenghui biotechnology limited; human liver cancer Bel fluorouracil-resistant cells were purchased from Jiangsu Kai-based biotechnology, Inc.; human breast cancer MCF-7 doxorubicin-resistant cells were purchased from Jiangsu Kai Bio-technology GmbH; human colon cancer HCT-8 vinblastine drug-resistant cells were purchased from Kyoho Kai Biotechnology, Inc.; human breast cancer MCF-7 doxorubicin-resistant liposome drug-resistant cells were purchased from Hu Nanfeng Hui Biotech Ltd.
The preparation process of polysaccharide-based lipid nanoparticles shown in the following examples is shown in fig. 2, and comprises the steps of firstly carrying out polymerization and crosslinking reaction on water-soluble polysaccharide, monomers and a crosslinking agent to obtain polysaccharide-based nanoparticles, then using the polysaccharide-based nanoparticles as a framework to induce lipid small molecules to self-assemble on the surfaces of the polysaccharide-based lipid nanoparticles to form polysaccharide-based lipid nanoparticles, and further mixing the polysaccharide-based lipid nanoparticles with genes to prepare gene-loaded polysaccharide-based lipid nanoparticles; or the polysaccharide-based nanoparticles and the drug are mixed to prepare drug-loaded polysaccharide-based nanoparticles, then the drug-loaded polysaccharide-based nanoparticles are formed by taking the drug-loaded polysaccharide-based nanoparticles as a framework to induce the lipid small molecules to self-assemble on the surface of the drug-loaded polysaccharide-based nanoparticles, and the drug-loaded polysaccharide-based nanoparticles and the gene are further mixed to prepare the drug-loaded and gene-loaded polysaccharide-based lipid nanoparticles.
Example 1:
dissolving 0.5 g of glucan (with the weight-average molecular weight of 40,000Da) in 25 mL of water, heating (the temperature is not more than 90 ℃) and stirring until the glucan is dissolved, cooling to 30 ℃, introducing nitrogen for 1 hour, adding a dilute nitric acid solution containing 0.06 g of ammonium ceric nitrate under the protection of nitrogen, stirring and mixing uniformly, adding 0.08 mL of methyl acrylate after 5 minutes, stirring and mixing uniformly, adding 0.015 g of diallyl disulfide after half an hour, reacting for 4 hours, dialyzing for three days after the reaction is finished to obtain a glucan nanoparticle aqueous solution, freezing and drying to obtain polysaccharide-based nanoparticles, and partially dissolving the polysaccharide-based nanoparticles in water to obtain an aqueous solution with the concentration of 45mg/mL for subsequent experiments.
Example 2:
dissolving 0.25 g of hydroxymethyl dextran in 25 ml of water, heating (the temperature is not more than 90 ℃) and stirring until the hydroxymethyl dextran is dissolved, cooling the temperature to 30 ℃, introducing nitrogen for 1 hour, adding a dilute nitric acid solution containing 0.04 g of ammonium ceric nitrate under the protection of nitrogen, stirring and mixing uniformly, adding 0.07 ml of methyl acrylate after 5 minutes, stirring and mixing uniformly, adding 0.012 g of diallyl disulfide after half an hour, and reacting for 4 hours. And dialyzing for three days after the reaction is finished to obtain a hydroxymethyl glucan nano particle aqueous solution, and freeze-drying to obtain the polysaccharide-based nano particles. Part of the extract was dissolved in water to give an aqueous solution with a concentration of 45mg/mL for subsequent experiments.
Example 3:
dissolving 0.5 g of dextran (with the weight-average molecular weight of 40,000Da) in 25 ml of water, heating (the temperature is not more than 90 ℃) and stirring until the dextran is dissolved, cooling to 30 ℃, introducing nitrogen for 1 hour, adding an aqueous solution containing 0.05 g of nickel perchlorate hexahydrate under the protection of nitrogen, stirring and mixing uniformly, adding 0.08 ml of methyl acrylate after 5 minutes, stirring and mixing uniformly, adding 0.015 g of diallyl disulfide after half an hour, and reacting for 4 hours. Dialyzing for three days after the reaction is finished to obtain glucan nano particle water solution, and freeze-drying to obtain polysaccharide-based nano particles.
Example 4:
dissolving 0.5 g of hydroxymethyl dextran in 25 ml of water, heating (the temperature is not more than 90 ℃) and stirring until the hydroxymethyl dextran is dissolved, cooling the temperature to 30 ℃, introducing nitrogen for 1 hour, adding an aqueous solution containing 0.05 g of nickel perchlorate hexahydrate under the protection of the nitrogen, stirring and mixing uniformly, adding 0.08 ml of glycidyl methacrylate after 5 minutes, stirring and mixing uniformly, adding 0.022 g of methylene bisacrylamide after half an hour, and reacting for 4 hours. And dialyzing for three days after the reaction is finished to obtain a hydroxymethyl glucan nano particle aqueous solution, and freeze-drying to obtain the polysaccharide-based nano particles.
Example 5:
dissolving 0.5 g of glucan (with the weight-average molecular weight of 40,000Da) in 25 ml of water, heating (the temperature is not more than 90 ℃) and stirring until the glucan is dissolved, cooling to 30 ℃, introducing nitrogen for 1 hour, adding a dilute nitric acid solution containing 0.06 g of ammonium ceric nitrate under the protection of nitrogen, stirring and mixing uniformly, adding 0.10 ml of glycidyl methacrylate after 5 minutes, stirring and mixing uniformly, adding 0.015 g of diallyl disulfide after half an hour, and reacting for 4 hours. Dialyzing for three days after the reaction is finished to obtain glucan nano particle aqueous solution, freeze-drying to obtain polysaccharide-based nano particles, and partially dissolving the glucan-based nano particles in water to obtain aqueous solution with the concentration of 45mg/mL for subsequent experiments.
Example 6:
dissolving 0.25 g hydroxypropyl chitosan in 25 ml water, heating (the temperature is not more than 90 ℃) and stirring until the hydroxypropyl chitosan is dissolved, cooling the temperature to 30 ℃, introducing nitrogen for 1 hour, adding a dilute nitric acid solution containing 0.04 g ammonium ceric nitrate under the protection of nitrogen, stirring and mixing uniformly, adding 0.07 ml methyl acrylate after 5 minutes, stirring and mixing uniformly, adding 0.012 g diallyl disulfide after half an hour, and reacting for 4 hours. And dialyzing for three days after the reaction is finished to obtain hydroxypropyl chitosan nanoparticle aqueous solution, and freeze-drying to obtain polysaccharide-based nanoparticles.
Example 7:
dissolving 0.5 g of hydroxymethyl chitosan in 25 ml of water, heating (the temperature is not more than 90 ℃) and stirring until the hydroxymethyl chitosan is dissolved, cooling the temperature to 30 ℃, introducing nitrogen for 1 hour, adding an aqueous solution containing 0.05 g of nickel perchlorate hexahydrate under the protection of the nitrogen, stirring and mixing uniformly, adding 0.08 ml of methyl acrylate after 5 minutes, stirring and mixing uniformly, adding 0.015 g of diallyl disulfide after half an hour, and reacting for 4 hours. And dialyzing for three days after the reaction is finished to obtain hydroxymethyl chitosan nanoparticle aqueous solution, and freeze-drying to obtain the polysaccharide-based nanoparticles.
Example 8:
dissolving 0.5 g of glucan in 25 ml of water, heating (the temperature is not more than 90 ℃) and stirring until the glucan is dissolved, cooling the temperature to 30 ℃, introducing nitrogen for 1 hour, adding an aqueous solution containing 0.05 g of nickel perchlorate hexahydrate under the protection of the nitrogen, stirring and mixing uniformly, adding 0.08 ml of methacrylic acid after 5 minutes, stirring and mixing uniformly, adding 0.022 g of methylene bisacrylamide after half an hour, and reacting for 4 hours. Dialyzing for three days after the reaction is finished to obtain glucan nano particle water solution, and freeze-drying to obtain polysaccharide-based nano particles.
Example 9:
50 mg dioleoyl phosphatidylethanolamine (DOPE) is dissolved in 200 microliters of chloroform, 25 microliters of 10 mg/ml dimethyl sulfoxide solution of Cy5.5-N-hydroxysuccinimide ester (Cy5.5-NHS) is added, vortex oscillation is carried out for 5 hours, and DOPE-Cy5.5-NHS solution is obtained, wherein the Cy5.5-N-hydroxysuccinimide ester is a red fluorescent dye, the maximum absorption wavelength is 678 nanometers, the maximum emission wavelength is 695 nanometers, and the solution is used for observing and positioning the polysaccharide-based lipid nanoparticles under a laser confocal microscope. 4 ml of chloroform was added to the DOPE-Cy5.5-NHS solution, rotary evaporated at 40 deg.C, the solvent was evaporated off, 5ml of DEPC water was added, and sonication was carried out at 150W for 10 minutes to obtain a lipid suspension of 10 mg/ml.
0.5 ml of 45mg/ml polysaccharide-based nanoparticles obtained in example 1 is added into 100 microliters of 10 mg/ml lipid suspension, and the mixture is subjected to 150 watts of ultrasound for 10 minutes to obtain polysaccharide-based lipid nanoparticles, wherein the obtained polysaccharide-based lipid nanoparticles are observed to be of a regular spherical structure through a transmission electron microscope, and the particle size is about 100 nanometers.
Example 10:
50 mg of (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP) was dissolved in 4 ml of chloroform, rotary evaporated at 40 ℃ and the solvent was evaporated off, 5ml of DEPC water was added and sonicated at 150W for 10 minutes to give a 10 mg/ml lipid suspension.
0.5 ml of 45mg/ml polysaccharide-based nanoparticles obtained in example 1 is added into 100 microliters of 10 mg/ml lipid suspension, and the mixture is subjected to 150 watts of ultrasound for 10 minutes to obtain polysaccharide-based lipid nanoparticles, wherein the obtained polysaccharide-based lipid nanoparticles are observed to be of a regular spherical structure through a transmission electron microscope, and the particle size is about 100 nanometers.
Example 11:
50 mg dioleoyl phosphatidylethanolamine (DOPE) and 50 mg (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP) were dissolved in 4 ml chloroform, rotary evaporated at 40 deg.C, the solvent was evaporated, 5ml DEPC water was added, and sonication was performed at 150W for 10 minutes to obtain a lipid suspension of 10 mg/ml.
0.5 ml of the 45mg/ml polysaccharide-based nanoparticles obtained in example 1 is added into 100 microliters of 10 mg/ml lipid suspension, and the mixture is subjected to ultrasonic treatment at 150 watts for 10 minutes to obtain the polysaccharide-based lipid nanoparticles, wherein the obtained polysaccharide-based lipid nanoparticles are observed to have a regular spherical structure through a transmission electron microscope, and the particle size is about 100 nanometers.
Example 12:
50 mg Dioleoylphosphatidylethanolamine (DOPE) was dissolved in 200. mu.L of chloroform, and 25. mu.L of a 10 mg/mL solution of Cy5.5-N-hydroxysuccinimide ester (Cy5.5-NHS) in dimethyl sulfoxide was added thereto, followed by vortex shaking for 5 hours to obtain a DOPE-Cy5.5-NHS solution. 50 mg of (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP) was dissolved in 4 ml of chloroform, mixed well with DOPE-Cy5.5-NHS solution, rotary evaporated at 40 ℃ and the solvent evaporated, 5ml of DEPC water was added and sonicated at 150W for 10 min to give a 10 mg/ml lipid suspension.
0.5 ml of 45mg/ml polysaccharide-based nanoparticles obtained in example 1 was added to 100. mu.l of 10 mg/ml lipid suspension, and subjected to 150W sonication for 10 minutes to obtain polysaccharide-based lipid nanoparticles. As shown in figure 1, the polysaccharide-based lipid nanoparticles obtained in the system have a regular spherical structure as observed by a transmission electron microscope, and the particle size is about 100 nanometers.
Example 13:
mixing 2.5 mg/mL paclitaxel ethanol solution with 375 mg/mL aqueous solution of polysaccharide-based nanoparticles obtained in example 1 in the same volume, shaking for 4 hours, slowly adding 15 mL of water into 1mL mixed solution, shaking while adding water, continuously shaking for 2 hours, performing ultrafiltration centrifugation by using a 100K ultrafiltration centrifuge tube, then adding 5mL of water for cleaning, performing ultrafiltration centrifugation, and repeating for 3 times to obtain the paclitaxel-loaded polysaccharide-based nanoparticles.
Example 14:
mixing 2.5 mg/ml paclitaxel ethanol solution with 375 mg/ml aqueous solution of polysaccharide-based nanoparticles obtained in example 2 in the same volume, shaking for 4 hours, slowly adding 60 ml water into 4 ml mixed solution, shaking while adding water, continuously shaking for 2 hours, performing ultrafiltration centrifugation by using a 100K ultrafiltration centrifugal tube, then adding 60 ml water for cleaning, performing ultrafiltration centrifugation, and repeating for three times to obtain the paclitaxel-loaded polysaccharide-based nanoparticles.
Example 15:
the paclitaxel-loaded polysaccharide-based nanoparticles obtained in example 14 were prepared into a 45mg/ml aqueous solution, and the solution was mixed with the 10 mg/ml liposome suspension prepared in example 9 in a mass ratio of 5: 1, mixing and carrying out ultrasonic treatment at 150 watts for 10 minutes to obtain the paclitaxel-loaded polysaccharide-based lipid nanoparticles.
Example 16:
the paclitaxel-loaded polysaccharide-based nanoparticles obtained in example 13 were prepared into a 45mg/ml aqueous solution, and the weight ratio of the aqueous solution to the 10 mg/ml liposome suspension prepared in example 10 was 8: 1, mixing and carrying out ultrasonic treatment at 150 watts for 10 minutes to obtain the paclitaxel-loaded polysaccharide-based lipid nanoparticles.
Example 17:
the paclitaxel-loaded polysaccharide-based nanoparticles obtained in example 14 were prepared into a 45mg/ml aqueous solution, and the weight ratio of the aqueous solution to the 10 mg/ml liposome suspension prepared in example 11 was 6: 1, mixing and carrying out ultrasonic treatment at 150 watts for 10 minutes to obtain the paclitaxel-loaded polysaccharide-based lipid nanoparticles.
Example 18:
the paclitaxel-loaded polysaccharide-based nanoparticles obtained in example 13 were prepared into a 45mg/ml aqueous solution, and the weight ratio of the aqueous solution to the 10 mg/ml liposome suspension prepared in example 12 was 10: 1, mixing and carrying out ultrasonic treatment at 150 watts for 10 minutes to obtain the paclitaxel-loaded polysaccharide-based lipid nanoparticles.
Example 19:
the paclitaxel-loaded polysaccharide-based lipid nanoparticles obtained in example 18 were prepared into 38 mg/ml aqueous solution, and mixed with siRNA-MDR1 with working concentration of 20 μmol/l in a mass ratio of 300: 1, mixing, vortexing and shaking for 20 seconds, and standing for 30 minutes to obtain the polysaccharide-based lipid nanoparticles loaded with paclitaxel and siRNA-MDR 1.
Example 20:
treating human ovarian cancer OVCar-3 with Taxus cuspidataAlcohol cells were plated in 6-well plates, the confluence of the cells was 40-60%, and the polysaccharide-based lipid nanoparticles obtained in example 12 were prepared in 38 mg/ml aqueous solution in exchange for opti-MEM medium, and mixed with Fam-siRNA at a working concentration of 20 μmol/l in a volume ratio of 2: 1 mixing, vortex shaking for 20 seconds, standing for 30 minutes to obtain Fam-siRNA-loaded polysaccharide-based lipid nanoparticles, adding the Fam-siRNA-loaded polysaccharide-based lipid nanoparticles into a 6-hole plate, wherein the final concentration of the Fam-siRNA is 50 nanomole/liter, and CO is added2Culturing for 4 hours at 37 ℃ in an incubator, discarding the culture medium, washing with PBS once, digesting with pancreatin containing EDTA, centrifugally collecting cells, washing with PBS twice, detecting the endocytosis efficiency and transfection efficiency of the nanoparticles with a flow cytometer, and obtaining the result shown in FIG. 4, wherein the endocytosis efficiency of Fam-siRNA-loaded polysaccharide-based lipid nanoparticles is>99％。
Example 21:
human ovarian carcinoma OVCar-3 paclitaxel-resistant cells are paved in a 6-well plate, the cell confluency is 40-60%, the opti-MEM culture medium is changed, the polysaccharide-based lipid nanoparticles obtained in the example 12 are prepared into 38 mg/ml aqueous solution, and the volume ratio of the aqueous solution to Fam-siRNA with the working concentration of 20 micromole/L is 2: 1 mixing, vortex shaking for 20 seconds, standing for 30 minutes to obtain Fam-siRNA-loaded polysaccharide-based lipid nanoparticles, adding the Fam-siRNA-loaded polysaccharide-based lipid nanoparticles into a 6-hole plate, wherein the final concentration of the Fam-siRNA is 50 nanomole/liter, and CO is added2Culturing for 4 hours at 37 ℃ in an incubator, fixing paraformaldehyde, dyeing DAPI, and observing the distribution condition of the nano-carrier and the Fam-siRNA in cells by using a laser confocal microscope. The results show that the nanoparticles are mainly distributed in the cytoplasm, and the Fam-siRNA has distribution in the cytoplasmic nucleus.
Example 22:
human ovarian carcinoma OVCar-3 paclitaxel-resistant cells were plated in 6-well plates, the confluency of the cells was 40-60%, the opti-MEM medium was changed, the polysaccharide-based lipid nanoparticles obtained in example 12 were prepared into 38 mg/ml aqueous solution, and the volume ratio of the aqueous solution to the siRNA-MDR1 at a working concentration of 20. mu.M was 2: 1 mixing, vortex shaking for 20 seconds, standing for 30 minutes to obtain polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1, adding into a 6-well plate, wherein the final concentration of siRNA-MDR1 is 50 nanomole/liter, and adding reagent into a control group
2000 (ex Invitrogen), operating procedures refer to the instructions; after 24 hours, the RPMI-1640 medium containing 10% serum is replaced, the culture is continued for 24 hours, and the expression level of the drug-resistant gene MDR1 is detected by a Western blot method (Western blot) and a fluorescence quantitative PCR (Q-PCR) respectively. As shown in fig. 5 and fig. 6, the expression level of MDR1 in the experimental group of polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1 was reduced by more than 80% by Western blot (Western blot), while the expression level of MDR1 in the control group was not significantly reduced; the expression level of MDR1 of the polysaccharide-based lipid nanoparticle experiment group carrying siRNA-MDR1 is reduced by more than 80 percent through fluorescent quantitative PCR (Q-PCR) detection, and the expression level of MDR1 of the control group is not obviously reduced.
Example 23:
human ovarian cancer OVCar-3 paclitaxel-resistant cells are paved in a 96-well plate, the number of the cells is 1 ten thousand per well, the opti-MEM culture medium is changed the next day, paclitaxel-loaded polysaccharide nanoparticles obtained in example 13 are added into the 96-well plate according to the concentration of 0.5-5 micromoles/liter of paclitaxel, paclitaxel injection (purchased from Taiji group Sichuan Taiji pharmaceutical Co., Ltd.) with the concentration of 0.5-5 micromoles/liter is added into a control group, RPMI-1640 culture medium containing 10% serum is changed after 24 hours, the culture is continued for 48 hours, and the cytotoxicity of the nanoparticles is detected by using a CCK-8 kit. The half-inhibitory concentration (IC50) of paclitaxel carried OVCar-3 drug-resistant cells of the paclitaxel polysaccharide nanoparticle group is 1.8 micromole/liter, and the half-inhibitory concentration (IC50) of paclitaxel in the control group is 2.3 micromole/liter.
Example 24:
the OVCar-3 drug-resistant cells of human ovarian cancer were plated in 96-well plates with 1 million cells per well, the Opti-MEM medium was changed the next day, the paclitaxel-loaded polysaccharide-lipid nanoparticles obtained in example 18 were added to the 96-well plates at a concentration of 0.5-5. mu.M paclitaxel, paclitaxel injection at a concentration of 0.5-5. mu.M was added to the control group, RPMI-1640 medium containing 10% serum was changed after 24 hours, the culture was continued for 48 hours, the cytotoxicity of the nanoparticles was measured with CCK-8 kit, and the paclitaxel half inhibitory concentration (IC50) of the OVCar-3 drug-resistant cells of the paclitaxel-loaded polysaccharide-lipid nanoparticle group was 1.2. mu.M and the paclitaxel half inhibitory concentration (IC50) of the control group was 2.3. mu.M.
Example 25:
human ovarian carcinoma OVCar-3 paclitaxel-resistant cells were plated in 96-well plates at 1 ten thousand cells/well, the opti-MEM medium was changed the next day, the polysaccharide-lipid nanoparticles loaded with paclitaxel and siRNA-MDR1 obtained in example 19 were added to the 96-well plates at concentrations of paclitaxel 0.1-5. mu.M and siRNA-MDR 150. mu.M, paclitaxel injection at a concentration of 0.1-5. mu.M was added to the control group, RPMI-1640 medium containing 10% serum MI was changed after 24 hours, culture was continued for 48 hours, and the cytotoxicity of the nanoparticles was examined using CCK-8 kit. The half-inhibitory concentration of paclitaxel (IC50) of OVCar-3 drug-resistant cells of polysaccharide lipid nanoparticle group loaded with paclitaxel and siRNA-MDR1 was measured to be 0.6. mu.M, and the half-inhibitory concentration of paclitaxel (IC50) of control group was measured to be 2.3. mu.M.
Example 26:
the human lung cancer A549 taxol-resistant cells are paved in a 96-well plate, the number of the cells is 1 ten thousand per hole, the opti-MEM culture medium is changed the next day, the polysaccharide lipid nanoparticles loaded with the taxol and the siRNA-MDR1 obtained in the example 19 are added into the 96-well plate according to the concentration of 0.5-5 micromole/liter of the taxol and 150 nanomole/liter of the siRNA-MDR, the taxol injection with the concentration of 0.5-5 micromole/liter is added into a control group, the RPMI-1640 culture medium containing 10% serum is changed after 24 hours, the culture is continued for 48 hours, and the cytotoxicity of the nanoparticles is detected by using a CCK-8 kit. Paclitaxel and siRNA-MDR 1-loaded polysaccharide lipid nanoparticle group A549 drug-resistant cells were measured to have a paclitaxel half-inhibitory concentration (IC50) of 1.2 micromoles/liter and a paclitaxel half-inhibitory concentration (IC50) of 3.5 micromoles/liter in the control group.
Example 27:
the drug-resistant cells of human colon cancer HCT-8 paclitaxel are paved in a 96-well plate, the number of the cells is 1 ten thousand per well, the opti-MEM culture medium is changed the next day, the polysaccharide lipid nanoparticles loaded with paclitaxel and siRNA-MDR1 obtained in example 19 are added into the 96-well plate according to the concentration of 0.5-5 micromole/liter of paclitaxel and 150 nanomole/liter of siRNA-MDR, the paclitaxel injection with the concentration of 0.5-5 micromole/liter is added into a control group, the RPMI-1640 culture medium containing 10% serum is changed after 24 hours, the culture is continued for 48 hours, and the cytotoxicity of the nanoparticles is detected by using a CCK-8 kit. Paclitaxel and siRNA-MDR 1-loaded polysaccharose lipid nanoparticle group HCT-8 drug-resistant cells were measured to have a paclitaxel half inhibitory concentration (IC50) of 0.9 micromoles/liter. The control group had a semi-inhibitory concentration (IC50) of paclitaxel of 1.6. mu.M.
Example 28:
dispersing human leukemia cells K562 adriamycin-resistant cells in opti-MEM medium and paving the cells in a 6-well plate according to 40 ten thousand per well, preparing the polysaccharide-based lipid nanoparticles obtained in example 12 into 38 mg/ml aqueous solution, and mixing the aqueous solution with siRNA-MDR1 with the working concentration of 20 micromoles/liter according to the volume ratio of 2: 1 mixing, vortex shaking for 20 seconds, standing for 30 minutes to obtain polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1, adding into a 6-well plate, wherein the final concentration of siRNA-MDR1 is 50 nanomole/liter, and adding reagent into a control group
2000, the operation steps refer to the specification; after 24 hours, the suspension cells are centrifuged, the RPMI-1640 culture medium containing 10% serum is replaced, the culture is continued for 24 hours, and the expression level of the drug-resistant gene MDR1 is detected by a Western blot method (Western blot) and a fluorescence quantitative PCR (Q-PCR) respectively. Western blot and fluorescent quantitative PCR results show that the expression level of the polysaccharide-based lipid nanoparticle experiment group MDR1 carrying the siRNA-MDR1 is reduced by over 80 percent, and the expression level of the control group MDR1 is reduced by 60 percent.
Example 29:
the paclitaxel-resistant cells of human ovarian cancer A2780 were plated in a 96-well plate, the number of the cells was 1 ten thousand per well, the opti-MEM medium was changed the next day, and the polysaccharide-lipid nanoparticles loaded with paclitaxel and siRNA-MDR1 obtained in example 19 were added to the 96-well plate at a paclitaxel half inhibitory concentration (IC50) of 1.6. mu.M per liter in the control group at a concentration of 0.5-5. mu.M and 150. mu.M of siRNA-MDR. Adding 0.5-5 micromole/liter concentration of paclitaxel injection into the control group, changing RPMI-1640 culture medium containing 10% serum after 24 hours, continuing culturing for 48 hours, and detecting the cytotoxicity of the nanoparticles by using a CCK-8 kit. Paclitaxel and siRNA-MDR 1-loaded polysaccharose lipid nanoparticle group A2780 drug-resistant cells were tested to have a paclitaxel half inhibitory concentration (IC50) of 0.9 micromoles/liter. The control group had a semi-inhibitory concentration (IC50) of paclitaxel of 1.8. mu.M.
Example 30:
the MCF-7 paclitaxel resistant cells of human breast cancer are paved in a 96-well plate, the number of the cells is 1 ten thousand per well, the opti-MEM culture medium is changed the next day, the polysaccharide lipid nanoparticles loaded with paclitaxel and siRNA-MDR1 obtained in example 19 are added into the 96-well plate according to the concentration of 0.5-5 micromole/liter of paclitaxel and 150 nanomole/liter of siRNA-MDR, the paclitaxel injection with the concentration of 0.5-5 micromole/liter is added into a control group, the RPMI-1640 culture medium containing 10% serum is changed after 24 hours, the culture is continued for 48 hours, and the cytotoxicity of the nanoparticles is detected by using a CCK-8 kit. Paclitaxel and siRNA-MDR 1-loaded polysaccharose lipid nanoparticle group MCF-7 drug-resistant cells were measured to have a paclitaxel half inhibitory concentration (IC50) of 0.8 micromoles/liter. The control group had a semi-inhibitory concentration (IC50) of paclitaxel of 1.6. mu.M.
Example 31:
dispersing the Bel fluorouracil drug-resistant cells of the human liver cancer in an opti-MEM culture medium, paving the cells in a 6-well plate according to 40 ten thousand per well, preparing the polysaccharide-based lipid nanoparticles obtained in the example 12 into 38 mg/ml aqueous solution, and mixing the aqueous solution with siRNA-MDR1 with the working concentration of 20 micromoles per liter according to the volume ratio of 2: 1 mixing, vortex shaking for 20 seconds, standing for 30 minutes to obtain polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1, adding into a 6-well plate, wherein the final concentration of siRNA-MDR1 is 50 nanomole/liter, and adding reagent into a control group
2000, the operation steps refer to the description. After 24 hours, the suspension cells are centrifuged, the RPMI-1640 culture medium containing 10% serum is replaced, the culture is continued for 24 hours, and the expression level of the drug-resistant gene MDR1 is detected by a Western blot method (Western blot) and a fluorescence quantitative PCR (Q-PCR) respectively. Western blot and fluorescent quantitative PCR results show that the expression level of MDR1 is reduced by more than 80%, and the expression level of MDR1 in a control group is reduced by 60%.
Example 32:
dispersing human breast cancer MCF-7 adriamycin resistant cells in opti-MEM medium by 40 ten thousandOne per well was plated in a six-well plate, and the polysaccharide-based lipid nanoparticles obtained in example 12 were prepared as an aqueous solution of 38 mg/ml, mixed with siRNA-MDR1 at a working concentration of 20 μmol/l in a volume ratio of 2: 1 mixing, vortex shaking for 20 s, standing for 30 min to obtain polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1, adding into 6-well plate, with siRNA-MDR1 final concentration of 50 nanomole/L, suspending cells after 24 hr, centrifuging, changing RPMI-1640 culture medium containing 10% serum, adding reagent into control group
2000, the operation steps refer to the description. The culture is continued for 24 hours, and the expression level of the drug-resistant gene MDR1 is detected by Western blotting (Western blot) and fluorescent quantitative PCR (Q-PCR) respectively. Western blot and fluorescent quantitative PCR results show that the expression level of MDR1 is reduced by more than 80%, and the expression level of MDR1 in a control group is reduced by 60%.
Example 33:
dispersing human colon cancer HCT-8 vinblastine-resistant cells in opti-MEM medium and spreading the cells in a six-well plate at 40 ten thousand/well, preparing the polysaccharide-based lipid nanoparticles obtained in example 12 into 38 mg/ml aqueous solution, mixing the aqueous solution with siRNA-MDR1 with working concentration of 20 micromole/L according to the volume ratio of 2: 1 mixing, vortex shaking for 20 s, standing for 30 min to obtain polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1, adding into 6-well plate, with siRNA-MDR1 final concentration of 50 nanomole/L, suspending cells after 24 hr, centrifuging, changing RPMI-1640 culture medium containing 10% serum, adding reagent into control group
2000, the operation steps refer to the description. And (3) continuously culturing for 24 hours, respectively detecting the expression level of the drug-resistant gene MDR1 by using a Western blot method (Western blot) and a fluorescent quantitative PCR (Q-PCR), wherein the Western blot and fluorescent quantitative PCR results show that the expression level of the MDR1 is reduced by over 80 percent, and the expression level of the MDR1 in a control group is reduced by 60 percent.
Example 34:
dispersing human breast cancer MCF-7 doxorubicin-resistant liposome drug-resistant cells in opti-MEM medium according to 40%Ten thousand per well were plated in a six-well plate, and the polysaccharide-based lipid nanoparticles obtained in example 12 were prepared as an aqueous solution of 38 mg/ml, mixed with siRNA-MDR1 at a working concentration of 20 μmol/l in a volume ratio of 2: 1 mixing, vortex shaking for 20 seconds, standing for 30 minutes to obtain polysaccharide-based lipid nanoparticles loaded with siRNA-MDR1, adding into a 6-well plate, wherein the final concentration of siRNA-MDR1 is 50 nanomole/liter, and adding reagent into a control group
2000, the operation steps refer to the description. And after 24 hours, suspending the cells, centrifuging, replacing an RPMI-1640 culture medium containing 10% serum, continuously culturing for 24 hours, and detecting the expression level of the drug-resistant gene MDR1 by using a Western blot method (Western blot) and a fluorescence quantitative PCR (Q-PCR) respectively, wherein the Western blot and the fluorescence quantitative PCR result show that the expression level of the MDR1 is reduced by more than 80%, and the expression level of the MDR1 in a control group is reduced by 60%.
Example 35:
spreading human ovarian cancer OVCar-3 paclitaxel resistant cells into a 96-well plate, wherein the number of the cells is 1 ten thousand per well, changing the opti-MEM culture medium the next day, and respectively adding: the polysaccharide-lipid nanoparticles loaded with paclitaxel (final concentration of 5 micromoles/liter) obtained in example 18, the polysaccharide-lipid nanoparticles loaded with paclitaxel (final concentration of 5 micromoles/liter) and siRNA-MDR1 (final concentration of 50 nanomoles/liter) obtained in example 19, the polysaccharide-lipid nanoparticles (final concentration of 0.65 g/liter as empty carrier negative control) obtained in example 12, opti-MEM medium (as blank control), RPMI-1640 medium containing 10% serum after 24 hours, culturing for 48 hours, and detecting the cell viability of the nanoparticles by using CCK-8 kit, the results are shown in FIG. 3, the cell viability of the empty carrier treated group is not significantly different from that of the control, the cell viability of the experiment group loaded with paclitaxel-lipid nanoparticles is 43% of that of the blank control group, and the cell viability of the polysaccharide-lipid nanoparticles loaded with paclitaxel and siRNA-MDR1 is 29% of that of the blank control group, the drug-loaded polysaccharide lipid nanoparticle plays a significant role in inhibiting drug-resistant cells, and meanwhile, the small interfering RNA loaded with a drug-resistant gene (MDR1) further improves the inhibition effect on cell viability by reducing the expression level of the small interfering RNA.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (4)
1. A preparation method of polysaccharide-based lipid nanoparticles is characterized by comprising the following steps:
1.1 preparing water solution of water-soluble polysaccharide, adding an initiator under the protection of inert gas, and uniformly stirring; the water-soluble polysaccharide is glucan, and the initiator is ammonium ceric nitrate;
1.2 adding a monomer or a monomer dissolved in a solvent, and uniformly stirring; the monomer is methyl acrylate;
1.3 adding a cross-linking agent, and carrying out polymerization and cross-linking reaction under the protection of inert gas; the cross-linking agent is diallyl disulfide;
1.4 after the reaction is finished, carrying out dialysis treatment, and carrying out freeze drying to obtain polysaccharide-based nanoparticles;
1.5 dissolving amphiphilic lipid molecules in a solvent, removing the solvent by rotary evaporation, adding water, and performing ultrasonic treatment to obtain lipid molecule suspension; the amphiphilic lipid molecule is one or two of dioleoyl phosphatidylethanolamine (DOPE) and (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP);
1.6 preparing the polysaccharide-based nanoparticles into an aqueous solution, mixing the aqueous solution with the lipid molecule suspension, and performing ultrasonic treatment to obtain polysaccharide-based lipid nanoparticles; or preparing the polysaccharide-based nanoparticles into an aqueous solution, mixing the aqueous solution with a medicinal solution, oscillating, adding water, oscillating, ultrafiltering and centrifuging, washing with water to obtain drug-loaded polysaccharide-based nanoparticles, preparing the obtained drug-loaded polysaccharide-based nanoparticles into an aqueous solution, mixing the aqueous solution with the lipid molecule suspension, and performing ultrasonic treatment to obtain drug-loaded polysaccharide-based lipid nanoparticles;
1.7 preparing the polysaccharide-based lipid nanoparticles or the polysaccharide-based lipid nanoparticles carrying drugs into aqueous solution, mixing the aqueous solution with gene aqueous solution respectively, oscillating and standing to obtain the polysaccharide-based lipid nanoparticles carrying genes or polysaccharide-based lipid nanoparticles carrying drugs and genes.
2. A method for preparing the polysaccharide-based lipid nanoparticles of claim 1, wherein the drug in step 1.6 is one or more of paclitaxel, daunorubicin, doxorubicin, daunorubicin, mitoxantrone, aclacinomycin, homoharringtonine, vincristine, vindesine, teniposide, prednisone, dexamethasone, mechlorethamine hydrochloride, cyclophosphamide, melphalan, lomustine, methotrexate, fluorouracil, mercaptopurine, mitomycin, pingyangmycin, vinorelbine, hydroxycamptothecin, and etoposide.
3. The method for preparing polysaccharide-based lipid nanoparticles according to claim 1, wherein the gene in step 1.7 is one or more of plasmid, siRNA, microRNA, pirRNA, circleRNA and lncRNA.
4. Polysaccharide-based lipid nanoparticles prepared by the method of any one of claims 1 to 3.
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