CN113368257A

CN113368257A - Preparation method of nanoparticle composition delivery system

Info

Publication number: CN113368257A
Application number: CN202110626366.XA
Authority: CN
Inventors: 不公告发明人
Original assignee: Lanxi Lishun Biological Co ltd
Current assignee: Lanxi Lishun Biological Co ltd
Priority date: 2019-05-22
Filing date: 2019-05-22
Publication date: 2021-09-10
Also published as: CN110279661A; CN113384531A

Abstract

The invention provides a preparation method of a nanoparticle composition delivery system, which belongs to the field of nano biological materials and comprises the steps of preparing an anti-tumor drug-loaded cationic liposome from a cationic liposome raw material and an anti-tumor drug by a film dispersion method; mixing the anti-tumor drug-loaded cationic liposome and GAPDH-siRNA by adopting a mixing incubation method to prepare a nanoparticle composition delivery system; wherein the cationic liposome raw material comprises 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose and dioleoyl phosphatidylethanolamine. The delivery system prepared by the preparation method of the nanoparticle composition delivery system provided by the invention has the advantages of high encapsulation efficiency, good stability, large drug and gene loading capacity, small particle size change, high cell uptake efficiency and rate and the like.

Description

Preparation method of nanoparticle composition delivery system

The application is a divisional application of Chinese patent application with application date of 2019, 05 and 22, application number of 201910428250.8, and invented name of 'a nanoparticle composition delivery system, a preparation method and application thereof'.

Technical Field

The invention belongs to the field of nano biological materials, and relates to a preparation method of a nanoparticle composition delivery system.

Background

The development mechanism of tumors is very complex, usually the malignant proliferation and apoptosis disorder of cells is caused by abnormal activation of some protooncogenes and inactivation of cancer suppressor genes or apoptosis related genes, and the complexity makes the effective treatment of tumors difficult to achieve breakthrough.

The combined use of chemical drugs and therapeutic genes with different mechanisms of action can not only improve the therapeutic effect synergistically, but also increase the genetic disorders required to be overcome by cancer cell mutation and delay the adaptability of tumor cells to drugs. Meanwhile, the problem of multi-drug resistance of cancer chemotherapeutic drugs can be hopefully solved, so in recent years, a lot of basic researches shift the focus to the construction of a combined delivery system of the chemotherapeutic drugs and therapeutic genes. Currently, common strategies for constructing drug and gene co-delivery systems include: the hydrophobic drug and the gene are simultaneously wrapped in the core through micelle, or the drug is bonded on a cationic carrier material and then compressed with the gene to obtain a co-delivery system. These strategies can load drugs and genes into the same nano delivery system, have good drug and gene synergistic therapeutic effect, but have many disadvantages, such as complex assembly process, use of organic solvents, difficulty in controlling the loading ratio of drugs and genes, and the like.

Chinese invention patent CN 103179952B discloses nanoparticle-based tumor targeted drug delivery, providing an aqueous tumor targeting liposome nanoparticle composition comprising an aqueous dispersion of liposome nanoparticles. The nanoparticles preferably encapsulate an anticancer chemotherapeutic agent, which may be added to a preformed liposome composition or may be incorporated into the liposome during liposome formation. Liposomal nanoparticles comprising legumain-targeting lipids mixed with one or more other micelle or vesicle-forming lipid materials, the composition being in the form of nanoparticle liposomes dispersed in an aqueous carrier. Preferred tumor-targeting liposome nanoparticle compositions comprise (a) a legumain-targeting lipid component, (b) a zwitterionic lipid component; (c) an amino-substituted lipid component; (d) a neutral lipid component and (e) a polyethylene glycol-conjugated lipid component. The legumain-targeting lipid component comprises a hydrophobic lipid moiety covalently linked to a legumain-binding moiety.

Disclosure of Invention

The invention aims to provide a preparation method of a nanoparticle composition delivery system, and the prepared delivery system has the advantages of high encapsulation efficiency, good stability, large drug and gene loading capacity, small particle size change, high cell uptake efficiency, high uptake rate and the like, effectively reduces the interaction between the delivery system and protein in serum, improves the cell transfection efficiency under the action of the serum, prolongs the circulation time in vivo, and is beneficial to the delivery system to a specific target position and the uptake of cells, thereby playing a role in curative effect.

The technical scheme adopted by the invention for realizing the purpose is as follows:

there is provided a method of making a nanoparticle composition delivery system comprising the steps of:

preparing the cationic liposome raw material and the anti-tumor drug into the anti-tumor drug-loaded cationic liposome by adopting a film dispersion method;

mixing the anti-tumor drug-loaded cationic liposome and GAPDH-siRNA by adopting a mixing incubation method to prepare a nanoparticle composition delivery system;

the cationic liposome raw material comprises 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose and dioleoyl phosphatidylethanolamine;

the molar ratio of 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose and dioleoyl phosphatidylethanolamine in the cationic liposome raw material is 1:0.8-1.2:0.2-0.5: 2.8-3.2.

The preparation method of the liposome comprises a reverse evaporation method, an ethanol injection method, a film dispersion method and the like, wherein ethanol in the ethanol injection method is not easy to remove and is not suitable for large-scale preparation; the reverse evaporation method is suitable for water-soluble drugs. The preparation method loads the medicine in the cationic liposome, and simultaneously the cationic lipid can compress the siRNA to form the liposome/siRNA compound with positive electricity.

One of the problems of cancer is multidrug resistance of tumor, wherein drug-resistant cells are mostly in hypoxic environment, ATP required for tumor cell survival in the hypoxic environment is mainly derived from glycolysis, most multidrug resistance proteins belong to ATF (atom transfer factor) binding type transporter families, and the efflux of paclitaxel molecules is also dependent on the supply of ATP, so that the glycolysis of tumor cells under the control of hypoxic condition can inhibit the initiation of the function of the drug-resistant proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a key role in glycolysis, and can be highly expressed in a low-oxygen environment, and GAPDH-siRNA can inhibit the expression of GAPDH so as to achieve the effect of gene therapy. Cationic Liposome (CL) is one of the most studied drug and siRNA co-delivery vehicles at present. The cationic liposome is used as a co-carrier of the drug and the siRNA, wherein the anti-tumor drug is loaded in the liposome, the cationic lipid can compress the siRNA to form a positively charged liposome and siRNA compound, and the assembly process is simple. The membrane material, proportion and dosage of the liposome play a key role in stability and encapsulation efficiency, and long-chain lipid can enhance the cohesive force between bimolecular layers, promote membrane stability and reduce drug leakage. Unlike other phospholipids, the head of dioleoyl phosphatidylethanolamine is a relatively hydrophobic primary amine group with small volume, and the two hydrophobic ends are longer C18 unsaturated fatty chains, so that the special structure can be used for gene transfection, and can wrap insoluble drugs and promote lysosome escape. The head of the 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride is a hydrophilic quaternary ammonium group, and the volume is large. The 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride is inserted into dioleoyl phosphatidylethanolamine, so that the formation of phospholipid bilayers is facilitated, the hydrophilic capacity of the cationic liposome is improved, in addition, a large amount of surface positive charges can be provided, the viscosity is enhanced, the probability of fusion and flocculation among liposomes is reduced, and the physical stability of the liposome is improved. According to the invention, plant sterol and diacetone-D-galactose which are used as raw materials of the cationic liposome can be embedded into dioleoyl phosphatidylethanolamine to hinder the interaction force between adjacent molecules of the dioleoyl phosphatidylethanolamine, so that the rigidity and the fluidity of the liposome are favorably adjusted, the entrapment rate and the stability of the liposome are improved, the bending constant and the curvature free energy of a bilayer membrane are reduced, the double-layer membrane structure of the cationic liposome is stabilized, a nanoparticle composition delivery system tends to form a smaller radius, and the uptake rate of cells to the nanoparticle composition delivery system is improved; on the other hand, the positive charge quantity of the cationic liposome can be increased to reach a higher potential, so that a stronger electrostatic repulsion effect is generated between the delivery systems, the fusion and aggregation of the delivery systems are avoided, the particle size change of the prepared delivery system is smaller, the stability is high, and the increase of the positive charge quantity enables the liposome to carry more GAPDH-siRNA, so that the drug-loading capacity of the delivery system is increased; in addition, the combination degree of the liposome and the antitumor drug, especially paclitaxel, can be improved, so that the drug loading rate of the antitumor drug in a delivery system is improved, and the cell uptake efficiency is finally improved. The invention adopts plant sterol and diacetone-D-galactose to replace the cholesterol, can reduce the harm of the cholesterol to human bodies, and particularly can improve the medication safety of patients with overhigh cholesterol.

The delivery system prepared by the invention has the advantages of high encapsulation efficiency, good stability, large drug loading amount, small particle size change, high cellular uptake efficiency, high uptake rate and the like, can realize the synchronization of pharmacokinetics of the anti-tumor drug and the GAPDH-siRNA, can simultaneously deliver the anti-tumor drug and the GAPDH-siRNA into cells, ensures that the anti-tumor drug and the GAPDH-siRNA take effect in a synergistic way in the same cells, and improves the anti-cancer effect.

In some embodiments, the mass ratio of the antineoplastic drug to dioleoyl phosphatidylethanolamine in the liposome starting material is 1: 6-7. In a certain range, the drug encapsulation efficiency is increased along with the reduction of the mass ratio of the drug to the lipid, dioleoyl phosphatidylethanolamine is used as the main lipid component, and when the mass of the drug is larger than that of the dioleoyl phosphatidylethanolamine, partial coagulation occurs after hydration; when the ratio of the medicine to the dioleoyl phosphatidylethanolamine is 1:6-7, the encapsulation efficiency is higher; the increase of the dosage of dioleoyl phosphatidylethanolamine is continued, the increase of the encapsulation efficiency is slow, the hydration time is prolonged, and the difficulty is increased.

In some embodiments, the mass ratio of cationic liposome to GAPDH-siRNA is 18-22: 1. The size of the particle size and the number of surface charges of the complex can affect the final transfection efficiency during liposomal delivery of siRNA. When the mass of the cationic liposome is low, the structure of the compound is loose, the particle size is large, the structure is unstable, and the siRNA binding rate is not high. With the increase of the mass of the cationic liposome, the liposome adsorbs siRNA to form a multi-layer liposome structure, the particle size of the complex is small, the siRNA binding rate is obviously improved, positive charges are uniformly distributed on the surface, and the complex is favorable for being combined with a cell membrane with negative charges and being absorbed into cells; when the mass of the cationic liposome is too high, the combination of siRNA and multilayer lipid is very tight, the particle size is gradually increased, but the nonspecific ion interaction between the compound and cells is accelerated by excessive positive charges distributed on the surface, and cytotoxicity is caused.

In some embodiments, the anti-tumor drug is paclitaxel. The P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) families of the tumor have an exclusion effect on paclitaxel drugs, the nanoparticle composition delivery system can simultaneously deliver GAPDH-siRNA and paclitaxel into cells, inhibit the expression of GAPDH, further reduce ATP generated by glycolysis, eliminate drug resistance and improve the sensitivity of tumor cells to paclitaxel.

In some embodiments, the surface of the cationic liposome is modified with alkyl glycoside and guanidinoacetic acid. The cationic liposome has poor targeting specificity, so that the gene transfer rate is low, but the surface of the structure of the cationic liposome is easy to modify, and the surface modification is carried out by using nonionic surfactants, namely alkyl glycoside and glycocyamine, so that the loading capacity and the transfer rate of the gene can be improved by influencing the physicochemical properties of the cationic liposome, on one hand, the positive charge quantity carried by the cationic liposome can be further improved, the +/-charge ratio is further improved, the adsorption effect of electrostatic force between the cationic liposome and GAPDH-siRNA is effectively improved, more GAPDH-siRNA enters the liposome, and finally the loading capacity of the GAPDH-siRNA in a delivery system is improved; on the other hand, an effective hydration layer can be formed on the surface of the cationic liposome, so that the interaction between the delivery system and protein in serum is effectively reduced, the cell transfection efficiency under the action of serum is improved, the in vivo circulation time is prolonged, the degradation of enzyme can be effectively protected, the cell uptake efficiency of the delivery system is improved, and the delivery system is favorable for reaching a specific target position, so that the curative effect is exerted.

In some embodiments, the nanoparticle composition delivery system has an average particle size of 159.1 to 193.7 nm. The nano-particles with the particle size of less than 200nm have the following advantages: can reduce phagocytosis of human reticuloendothelial system; can improve the targeting property of the medicament, improve the curative effect and reduce the toxicity; the gaps between the endothelial cells of the capillary vessels are more than 200nm, and the endothelial cells can be absorbed by cell tissues when passing through the capillary vessels; is beneficial to the transportation and storage of the medicine.

In some embodiments, the alkylglycoside has a hydrophilic-hydrophobic equilibrium value of 4.5 to 5.2. The alkyl glycoside performs surface modification on the cationic liposome, and influences the uptake of cells by acting on the surface hydrophobicity of the cationic liposome and the permeability of cell membranes; the cell surface has a hydration layer and hydrophilic glycoprotein, when the hydrophobicity is too strong, the liposome can be prevented from approaching the cell surface, and when the hydrophobicity is too low, the cell adhesion and endocytosis are not facilitated. The alkyl glycoside within the range is used for modifying the surface of the cationic liposome, so that the endocytosis of a delivery system into cells is facilitated.

The invention has the beneficial effects that:

1) the delivery system prepared by the invention uses the same carrier to load the anti-tumor drug and the GAPDH-siRNA for combined administration, has the advantages of high encapsulation rate, good stability, large drug-loading rate, small particle size change, high cellular uptake efficiency, high uptake rate and the like, can realize the synchronization of the pharmacokinetics of the anti-tumor drug and the GAPDH-siRNA, can simultaneously send the anti-tumor drug and the GAPDH-siRNA into cells, ensures that the anti-tumor drug and the GAPDH-siRNA take effect in a synergistic way in the same cell, and improves the anti-cancer effect;

2) according to the invention, alkyl glycoside and glycocyamine are used for modifying the surface of the cationic liposome, so that more GAPDH-siRNA enters the inside of the liposome, and an effective hydration layer can be formed on the surface of the cationic liposome, the interaction between a delivery system and protein in serum is effectively reduced, the cell transfection efficiency under the action of the serum is improved, the in vivo circulation time is prolonged, meanwhile, the degradation of resistance enzyme can be effectively protected, the gene loading capacity and the transfer rate are finally improved, the delivery system is favorable for being taken up to a specific target position and cells, and the curative effect is exerted.

Drawings

FIG. 1 is a schematic diagram of gel electrophoresis of GAPDH siRNA-paclitaxel liposome prepared by the method of the present invention;

FIG. 2 is a schematic diagram of the encapsulation efficiency of GAPDH siRNA-paclitaxel liposome prepared by the present invention;

FIG. 3 is a schematic diagram of the drug loading of GAPDH siRNA-paclitaxel liposome prepared by the present invention;

FIG. 4 is a schematic diagram showing the cell transfection effect of GAPDH siRNA-paclitaxel liposome prepared by the present invention;

FIG. 5 is a diagram showing the gene transfection efficiency of GAPDH siRNA-paclitaxel liposome prepared by the present invention;

FIG. 6 is a schematic representation of the cytotoxicity of cationic liposomes of the invention;

FIG. 7 is a graph showing the change in tumor volume following administration of different therapeutic agents;

FIG. 8 is a graph showing tumor mass and tumor inhibition rate of different therapeutic agents after the end of administration of the different therapeutic agents;

FIG. 9 is a graph showing the body weight changes of tumor-bearing nude mice after administration of GAPDH siRNA-paclitaxel liposome prepared by the present invention.

Detailed Description

The application provides a preparation method of a nanoparticle composition delivery system, which comprises the steps of preparing an antitumor drug cationic liposome by adopting a film dispersion method; the antitumor drug and GAPDH-siRNA co-carried cationic liposome compound is prepared by adopting a mixed incubation method. The prepared nanoparticle composition delivery system can improve the sensitivity of antitumor drugs, and thus can be used for treating cancers.

By utilizing a tumor-bearing nude mouse model, a nanoparticle composition delivery system is shown, namely, the antitumor drug and the GAPDH-siRNA co-carried cationic liposome complex are injected once by tail vein every other day, and after 12 times of administration, the nanoparticle composition delivery system has a very high-efficient antitumor effect. GAPDH-siRNA can control the expression of GAPDH-siRNA in tumor to be always at a lower level, and indirectly eliminates or reduces the hypoxia drug resistance of tumor cells to drugs, thereby fully playing the role of anti-tumor drugs.

Accordingly, the present application provides a method for preparing a nanoparticle composition delivery system as described herein, comprising preparing cationic liposomes of an antitumor drug using a thin film dispersion method; the antitumor drug and GAPDH-siRNA co-carried cationic liposome compound is prepared by adopting a mixed incubation method.

Many embodiments are described herein in the context of methods of making nanoparticle composition delivery systems. Those of ordinary skill in the art will realize that the following detailed description of the embodiments is illustrative only and is not intended to be in any way limiting. Other embodiments will be readily suggested to those skilled in the art, given the benefit of this disclosure. References herein to "an embodiment" or "an example" mean that the embodiment of the invention so described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Furthermore, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

In the interest of clarity, not all of the routine features of the implementations or methods described herein are shown and described. It will of course be appreciated that in the development of any such actual implementation, numerous implementation-specific decisions should be made to achieve the specific goals, such as compliance with administration, treatment, and subject-related constraints, and that these specific goals will vary from one implementation to another and from one user to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

The present application provides, in some embodiments, a method of making a nanoparticle composition delivery system, comprising:

preparing the antitumor drug cationic liposome by adopting a film dispersion method: dissolving cationic liposome raw material and antitumor drug in anhydrous ethanol, placing the solution in eggplant-shaped bottle, performing rotary evaporation under reduced pressure in water bath condition of 40-44 deg.C (such as 40 deg.C, 41 deg.C, 42 deg.C, 43 deg.C, 44 deg.C, etc.), vacuumizing for about 1-1.3h (such as 1h, 1.1h, 1.2h, 1.3h, etc.) to remove residual solvent, adding RNase-free water into the bottle, and hydrating at 40-42 deg.C (such as 40 deg.C, 41 deg.C, 42 deg.C, etc.) for 30-35min (such as 30min, 31min, 32min, 33min, 34min, 35min, etc.), extruding with 450-480nm (such as 450nm, 451nm, 452nm, 453nm, 454nm, 456nm, 458nm, 460nm, 462nm, 464nm, 465nm, 466nm, 467nm, 468nm, 472nm, 471nm, 478nm, 476nm, 479nm, 480nm, etc.) filter membrane once, then extruding the mixture 5-6 times through a filter membrane of 200-220nm (such as 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm and 120-140nm (such as 120nm, 121nm, 122nm, 123nm, 124nm, 125nm, 126nm, 127nm, 128nm, 129nm, 130nm, 131nm, 132nm, 133nm, 134nm, 135nm, 136nm, 137nm, 138nm, 139nm and 140 nm) by using a liposome extruder, for example, 5 times or 6 times, storing at 2-4 deg.C, such as 2 deg.C, 3 deg.C, 4 deg.C, preparing liposome by reverse evaporation, ethanol injection, and thin film dispersion, wherein ethanol is not easily removed, and are not suitable for large-scale preparation; the reverse evaporation method is suitable for water-soluble drugs, and the film dispersion method is easy to operate and enlarge production.

Preparing an antitumor drug and GAPDH-siRNA co-loaded cationic liposome complex by adopting a mixed incubation method: dissolving GAPDH-siRNA in RNase-free water according to a certain proportion, mixing the anti-tumor drug-loaded cationic liposome with GAPDH-siRNA, standing at room temperature for 30-35min (such as 30min, 31min, 32min, 33min, 34min, 35min, etc.), and obtaining the anti-tumor drug and GAPDH-siRNA co-loaded cationic liposome compound. The preparation of the antitumor drug and siRNA co-loaded cationic liposome compound is a self-assembly process of the cationic liposome and siRNA, and the process is simple.

The cationic liposome raw material comprises 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose and dioleoyl phosphatidylethanolamine; in the cationic liposome raw material, the molar ratio of 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose and dioleoyl phosphatidylethanolamine is 1:0.8-1.2:0.2-0.5:2.8-3.2, preferably, 1:0.82:0.22:2.8, 1:0.82:0.25:2.81, 1:0.85:0.30:2.83, 1:0.9:0.35:2.84, 1:0.94:0.38:2.85, 1:1:0.4:2.86, 1:1:0.4:2.87, 1:1:0.4:2.88, 1:1.05:0.42:2.89, 1:1.08:0.45:2.89, 1:1: 1.1: 0.9: 2.88, 1:1: 1.05:0.42:2.89, 1:1: 1.08:0.45: 2.06, 1: 1.9: 0.45: 1.43, 1:1.15: 1.6, 1: 2.06, 1.8: 2.8, 1.8: 2.8, 1.8, 1.6, 1: 2.6, 1.6, 1.9: 2.6, 1.6, 1. One of the problems of cancer is multidrug resistance of tumor, wherein drug-resistant cells are mostly in hypoxic environment, ATP required for tumor cell survival in the hypoxic environment is mainly derived from glycolysis, most multidrug resistance proteins belong to ATF (atom transfer factor) binding type transporter families, and the efflux of paclitaxel molecules is also dependent on the supply of ATP, so that the glycolysis of tumor cells under the control of hypoxic condition can inhibit the initiation of the function of the drug-resistant proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a key role in glycolysis, and can be highly expressed in a low-oxygen environment, and GAPDH-siRNA can inhibit the expression of GAPDH so as to achieve the effect of gene therapy. Cationic Liposome (CL) is one of the most studied drug and siRNA co-delivery vehicles at present. The cationic liposome is used as a co-carrier of the drug and the siRNA, wherein the anti-tumor drug is loaded in the liposome, the cationic lipid can compress the siRNA to form a positively charged liposome and siRNA compound, and the assembly process is simple. The membrane material, proportion and dosage of the liposome play a key role in stability and encapsulation efficiency, and long-chain lipid can enhance the cohesive force between bimolecular layers, promote membrane stability and reduce drug leakage. Unlike other phospholipids, the head of dioleoyl phosphatidylethanolamine is a relatively hydrophobic primary amine group with small volume, and the two hydrophobic ends are longer C18 unsaturated fatty chains, so that the special structure can be used for gene transfection, and can wrap insoluble drugs and promote lysosome escape. The head of the 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride is a hydrophilic quaternary ammonium group, and the volume is large. The 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride is inserted into dioleoyl phosphatidylethanolamine, so that the formation of phospholipid bilayers is facilitated, the hydrophilic capacity of the cationic liposome is improved, in addition, a large amount of surface positive charges can be provided, the viscosity is enhanced, the probability of fusion and flocculation among liposomes is reduced, and the physical stability of the liposome is improved. According to the invention, plant sterol and diacetone-D-galactose which are used as raw materials of the cationic liposome can be embedded into dioleoyl phosphatidylethanolamine to hinder the interaction force between adjacent molecules of the dioleoyl phosphatidylethanolamine, so that the rigidity and the fluidity of the liposome are favorably adjusted, the entrapment rate and the stability of the liposome are improved, the bending constant and the curvature free energy of a bilayer membrane are reduced, the double-layer membrane structure of the cationic liposome is stabilized, a nanoparticle composition delivery system tends to form a smaller radius, and the uptake rate of cells to the nanoparticle composition delivery system is improved; on the other hand, the positive charge quantity of the cationic liposome can be increased to reach a higher potential, so that a stronger electrostatic repulsion effect is generated between the delivery systems, the fusion and aggregation of the delivery systems are avoided, the particle size change of the prepared delivery system is smaller, the stability is high, and the increase of the positive charge quantity enables the liposome to carry more GAPDH-siRNA, so that the drug-loading capacity of the delivery system is increased; in addition, the combination degree of the liposome and the antitumor drug, especially paclitaxel, can be improved, so that the drug loading rate of the antitumor drug in a delivery system is improved, and the cell uptake efficiency is finally improved. The invention adopts plant sterol and diacetone-D-galactose to replace the cholesterol, can reduce the harm of the cholesterol to human bodies, and particularly can improve the medication safety of patients with overhigh cholesterol. The delivery system has the advantages of high encapsulation efficiency, good stability, large drug-loading rate, small particle size change, high cellular uptake efficiency, high uptake rate and the like, can realize the synchronization of pharmacokinetics of the anti-tumor drug and the GAPDH-siRNA, can simultaneously deliver the anti-tumor drug and the GAPDH-siRNA into cells, ensures that the anti-tumor drug and the GAPDH-siRNA take effect in a synergistic way in the same cells, and improves the anti-cancer effect.

In some embodiments, the mass ratio of the antineoplastic drug to dioleoylphosphatidylethanolamine in the cationic liposome starting material is 1:6-7, preferably, e.g., 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, etc. In a certain range, the drug encapsulation efficiency is increased along with the reduction of the mass ratio of the drug to the lipid, dioleoyl phosphatidylethanolamine is used as the main lipid component, and when the mass of the drug is larger than that of the dioleoyl phosphatidylethanolamine, partial coagulation occurs after hydration; when the ratio of the medicine to the dioleoyl phosphatidylethanolamine is 1:6-7, the encapsulation efficiency is higher; the increase of the dosage of dioleoyl phosphatidylethanolamine is continued, the increase of the encapsulation efficiency is slow, the hydration time is prolonged, and the difficulty is increased.

In some embodiments, the mass ratio of cationic liposome to GAPDH-siRNA is 18-22:1, preferably, e.g., 18:1, 18.1:1, 18.2:1, 18.3:1, 18.4:1, 18.5:1, 18.6:1, 18.7:1, 18.8:1, 18.9:1, 19:1, 19.1:1, 19.2:1, 19.3:1, 19.4:1, 19.5:1, 19.6:1, 19.7:1, 20:1, 20.2:1, 20.3:1, 20.5:1, 20.8:1, 21:1, 21.1:1, 21.2:1, 21.4:1, 21.7:1, 21.8:1, 22:1, etc. The size of the particle size and the number of surface charges of the complex can affect the final transfection efficiency during liposomal delivery of siRNA. When the mass of the cationic liposome is low, the structure of the compound is loose, the particle size is large, the structure is unstable, and the siRNA binding rate is not high. With the increase of the mass of the cationic liposome, the liposome adsorbs siRNA to form a multi-layer liposome structure, the particle size of the complex is small, the siRNA binding rate is obviously improved, positive charges are uniformly distributed on the surface, and the complex is favorable for being combined with a cell membrane with negative charges and being absorbed into cells; when the mass of the cationic liposome is too high, the siRNA is tightly combined with the multilayer lipid, the particle size is gradually increased, but the nonspecific ion interaction between the compound and the cells is accelerated by the excessive positive charges distributed on the surface, and the cytotoxicity is caused.

In some embodiments, the antineoplastic agent provided is paclitaxel. The P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) families of the tumor have an exclusion effect on paclitaxel drugs, the nanoparticle composition delivery system can simultaneously deliver GAPDH-siRNA and paclitaxel into cells, inhibit the expression of GAPDH, further reduce ATP generated by glycolysis, eliminate drug resistance and improve the sensitivity of tumor cells to paclitaxel.

In some embodiments, the surface of the cationic liposome is modified with alkyl glycoside and guanidinoacetic acid. The cationic liposome has poor targeting specificity, so that the gene transfer rate is low, but the surface of the structure of the cationic liposome is easy to modify, and the surface modification is carried out by using nonionic surfactants, namely alkyl glycoside and glycocyamine, so that the loading capacity and the transfer rate of the gene can be improved by influencing the physicochemical properties of the cationic liposome, on one hand, the positive charge quantity carried by the cationic liposome can be further improved, the +/-charge ratio is further improved, the adsorption effect of electrostatic force between the cationic liposome and GAPDH-siRNA is effectively improved, more GAPDH-siRNA enters the liposome, and finally the loading capacity of the GAPDH-siRNA in a delivery system is improved; on the other hand, an effective hydration layer can be formed on the surface of the cationic liposome, so that the interaction between the liposome and protein in serum is effectively reduced, the cell transfection efficiency under the action of serum is improved, the circulation time in vivo is prolonged, the degradation of enzyme can be effectively protected against, the cell uptake efficiency of a delivery system is improved, and the delivery system is favorable for reaching a specific target position, so that the curative effect is exerted.

In some embodiments, the nanoparticle composition delivery system has an average particle size of 159.1-193.7nm, preferably, e.g., 159.1, 159.4, 159.7, 159.9, 160.5, 161, 161.4, 161.7, 161.8, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 193.7, and the like. The nano-particles with the particle size of less than 200nm have the following advantages: can reduce phagocytosis of human reticuloendothelial system; can improve the targeting property of the medicament, improve the curative effect and reduce the toxicity; the gaps between the endothelial cells of the capillary vessels are more than 200nm, and the endothelial cells can be absorbed by cell tissues when passing through the capillary vessels; is beneficial to the transportation and storage of the medicine.

In some embodiments, the alkylglycoside has a hydrophilic-hydrophobic equilibrium value of 4.5 to 5.2, preferably, e.g., 4.5, 4.51, 4.52, 4..53, 4.54, 4.55, 4.56, 4.57, 4.58, 4.59, 4.6, 4.61, 4.63, 4.64, 4.67, 4.68, 4.69, 4.7, 4.72, 4.74, 4.75, 4.78, 4.8, 4.85, 4.86, 4.9, 4.92, 4.93, 4.95, 4.96, 4.98, 5, 5.01, 5.02, 5.04, 5.05, 5.08, 5.1, 5.11, 5.12, 5.13, 5.14, 5.17, 5.18, 5.19, 5.2, etc. The alkyl glycoside performs surface modification on the cationic liposome, and influences the uptake of cells by acting on the surface hydrophobicity of the cationic liposome and the permeability of cell membranes; the cell surface has a hydration layer and hydrophilic glycoprotein, when the hydrophobicity is too strong, the liposome can be prevented from approaching the cell surface, and when the hydrophobicity is too low, the cell adhesion and endocytosis are not facilitated. The alkyl glycoside within the range is used for modifying the surface of the cationic liposome, so that the endocytosis of a delivery system into cells is facilitated.

In some embodiments according to any of the above embodiments, the nanoparticle composition delivery system has a tumor inhibition rate of not less than 94.8%.

The present invention is further described in detail with reference to the following examples:

example 1:

a method of making a nanoparticle composition delivery system:

preparing the antitumor drug cationic liposome by adopting a film dispersion method: dissolving 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose, dioleoyl phosphatidylethanol and dioleoyl phosphatidylethanolamine with the molar ratio of 1:0.8:0.2:2.8-3.2 in absolute ethyl alcohol, wherein the mass ratio of the taxol to the dioleoyl phosphatidylethanolamine is 1:6, and alkyl glycoside and guanidinoacetic acid with the hydrophilic-hydrophobic equilibrium value of 4.5, putting the solution into a solanaceous bottle, performing reduced pressure rotary evaporation under the water bath condition to form a uniformly distributed lipid membrane, removing residual solvent, adding RNase-free water, hydrating and extruding.

Preparing GAPDH siRNA-paclitaxel liposome by adopting a mixed incubation method: dissolving Cy 3-labeled GAPDH-siRNA in RNase-free water, mixing the cationic liposome and Cy 3-labeled GAPDH-siRNA in a mass ratio of 18:1, standing at room temperature to obtain Cy 3-labeled GAPDH siRNA-paclitaxel liposome, wherein the average particle diameter is 159.1-163nm, and the zeta potential is 36.5-38.1 mV.

Example 2:

a method of making a nanoparticle composition delivery system:

preparing the antitumor drug cationic liposome by adopting a film dispersion method: dissolving 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose, dioleoyl phosphatidyl ethanol and taxol with the mass ratio of 1:6.6 to dioleoyl phosphatidyl ethanolamine of 1:1.05:0.32:2.89, dissolving alkyl glycoside and guanidinoacetic acid with the hydrophilic-hydrophobic balance value of 4.8 in absolute ethyl alcohol, putting the solution into a solanaceous bottle, performing reduced pressure rotary evaporation under the water bath condition to form a uniformly distributed lipid membrane, removing residual solvent, adding RNase-free water, hydrating and extruding.

Preparing GAPDH siRNA-paclitaxel liposome by adopting a mixed incubation method: dissolving Cy 3-labeled GAPDH-siRNA in RNase-free water, mixing the cationic liposome and Cy 3-labeled GAPDH-siRNA in a mass ratio of 19.3:1, standing at room temperature to obtain Cy 3-labeled GAPDH siRNA-paclitaxel liposome, wherein the average particle diameter is 161.1-165nm, and the zeta potential is 38.6-40.5 mV.

Example 3:

a method of making a nanoparticle composition delivery system:

preparing the antitumor drug cationic liposome by adopting a film dispersion method: dissolving 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose, dioleoyl phosphatidyl ethanol and taxol with the mass ratio of 1:7 to dioleoyl phosphatidyl ethanolamine of 1:1.2:0.5:3.2, dissolving alkyl glycoside and guanidinoacetic acid with the hydrophilic-hydrophobic balance value of 5.2 in absolute ethanol, putting the solution into a solanaceous bottle, performing reduced pressure rotary evaporation under the water bath condition to form a uniformly distributed lipid membrane, removing residual solvent, adding RNase-free water, hydrating and extruding.

Preparing GAPDH siRNA-paclitaxel liposome by adopting a mixed incubation method: dissolving Cy 3-labeled GAPDH-siRNA in RNase-free water, mixing the cationic liposome and Cy 3-labeled GAPDH-siRNA in a mass ratio of 22:1, standing at room temperature to obtain Cy 3-labeled GAPDH siRNA-paclitaxel liposome, wherein the average particle diameter is 179-183.7nm, and the zeta potential is 35.2-37.6 mV.

The method for measuring the particle size and zeta potential of the GAPDH siRNA-paclitaxel liposome comprises the following steps: GAPDH siRNA-paclitaxel liposome with a certain proportion is dispersed in double distilled water, the volume is adjusted to 1mL, the particle size and the Zeta potential of the GAPDH siRNA-paclitaxel liposome are measured by a Malveren Zetasizer3000HS, water is selected as a medium according to parameters, the measurement temperature is 25 ℃, and the balance time is 2 min.

The GAPDH siRNA-paclitaxel liposomes prepared in example 1, example 2 and example 3 all have an average particle size of less than 200nm and a Zeta potential of positive charge, and under the conditions of example 2, the average particle size is the smallest, the Zeta potential is 38.6-40.5mV, and the dynamic stability is high.

Example 4:

no paclitaxel was added and the rest was identical to example 2.

Comparative example 1:

no alkyl glycoside was added for surface modification, and the remainder was identical to example 2.

Comparative example 2:

no glycocyamine was added for surface modification, the remainder being identical to example 2.

Comparative example 3:

no surface modification was carried out with the addition of alkyl glycoside and guanidinoacetic acid, the remainder being identical to example 2.

Comparative example 4:

the starting material for the cationic liposomes contained no phytosterols, the remainder being identical to that of example 2.

Comparative example 5:

the starting material for the cationic liposome did not contain diacetone-D-galactose, and the remainder was identical to that of example 2.

Comparative example 6:

the starting material for the cationic liposome did not contain phytol and diacetone-D-galactose, and the remainder was identical to that of example 2.

Comparative example 7:

the raw materials for the cationic liposome comprise 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, cholesterol and dioleoyl phosphatidylethanol, and the rest is completely the same as that in example 2.

Comparative example 8:

2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, cholesterol and dioleoyl phosphatidylethanol were used as the cationic liposome starting material, and the remainder was identical to that of example 4.

Test example 1:

agarose gel electrophoresis:

the binding ability of the cationic liposome to GAPDH-siRNA was examined by agarose gel electrophoresis.

0.45g agarose was placed in an Erlenmeyer flask, 20mL of 1 XTAE electrophoresis buffer was added, heated to complete dissolution, cooled to 60 ℃, added with 2. mu.L Goldview and gently shaken. Pouring the gel solution into an electrophoresis sample application groove, inserting a sample application comb, fully cooling the gel, then pulling out the gel, and putting the gel into an electrophoresis groove filled with 1 XTAE electrophoresis buffer solution. Adding the 6 × loading buffer into a sample to be detected, uniformly mixing, and loading, wherein the electrophoresis conditions are as follows: voltage 95V, time 12 min. The gel was photographed using a gel imaging system. The results are shown in FIG. 1.

Example 2, comparative examples 1-7 GAPDH siRNA-paclitaxel liposomes were prepared according to different mass ratios of cationic liposomes and Cy3 labeled GAPDH-siRNA, respectively. As can be seen from fig. 1, the brightness of electrophoretic band of GAPDH siRNA-paclitaxel liposome at the same position was significantly reduced compared to free siRNA, and the cationic liposome was most fully bound to free GAPDH siRNA under the conditions of experimental example 2. The results of comparative example 2 and comparative examples 1-3 show that the loading of GAPDH-siRNA in a delivery system can be effectively improved only when the surface of the cationic liposome is jointly modified by alkyl glycoside and glycocyamine; comparing the results of example 2 and comparative examples 4-6, it can be seen that the cationic liposome raw materials including phytosterol, diacetone-D-galactose can make the liposome carry more GAPDH-siRNA; comparing the results of example 2 and comparative example 7, it can be seen that phytosterol, diacetone-D-galactose, was used in place of cholesterol, enabling the liposomes to carry more GAPDH-siRNA.

Test example 2:

determination of encapsulation efficiency:

1) GAPDH-siRNA content determination in liposomes

99 μ L of the prepared GAPDH siRNA-paclitaxel liposome solution is taken, 10% Triton X-1001 μ L of demulsifier is added, vortex mixing is carried out, 100 μ L of HBS is added for dilution, uniform mixing is carried out, and 150 μ L of the mixture is added into a black 96-well plate. Standard curve solutions were prepared in the same manner. In this method, Cy3 is used mainly as a fluorescent label, and therefore the excitation wavelength used for the measurement is 550nm and the emission wavelength is 570 nm.

2) Determination of paclitaxel content in liposomes

The prepared solution of GAPDH siRNA-paclitaxel liposome was diluted with 9.8mL of methanol in 200 μ L, subjected to water bath at room temperature and sonication for 10min, filtered through a 0.45 μm filter membrane, and the paclitaxel content in the cationic liposome was measured by HPLC (acetonitrile: water 60:40, flow rate 1.0mL/min, wavelength 227nm, column temperature 25 ℃, sample size 10 μ L).

3) Calculation of Liposome encapsulation efficiency and drug Loading

The calculation formula is as follows:

the entrapment rate is the content of the drug in the liposome/the total amount of the drug added x 100%;

the drug loading is the content of the drug in the liposome/the total mass of the cationic liposome multiplied by 100%.

The results are shown in FIGS. 2 and 3.

As can be seen from FIG. 2 and FIG. 3, the GAPDH-siRNA encapsulation efficiency in examples 1-3 of the present invention is above 69%, and the drug loading is above 0.28%; the entrapment rate of the paclitaxel is more than 92%, and the drug loading rate is more than 1.91%. Wherein, under the condition of the example 2, the encapsulation rate and the drug loading rate of the GAPDH-siRNA and the encapsulation rate and the drug loading rate of the paclitaxel are optimal. Comparative example 2 and comparative examples 4-6, the encapsulation efficiency and drug loading of example 2 are superior to those of comparative examples 4-6, which shows that the raw materials plant sterol and diacetone-D-galactose for cationic liposome can improve the encapsulation efficiency and drug loading of the delivery system.

Test example 3:

detection of GAPDH siRNA-paclitaxel liposome in vitro cell uptake:

taking out human cervical cancer cells (Hela) frozen in liquid nitrogen tank, recovering, resuspending the cells, resuspending in RPMI-1640 culture medium containing 10% fetal calf serum, and culturing at 37 deg.C under 5% CO₂And (5) standing and culturing in an incubator.

Hela cells at 1.5X 10 per well⁴Was seeded in 12-well plates at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator. The original medium was removed, PBS was added to rinse the cells 2 times, free Cy3-GAPDH-siRNA and GAPDH siRNA-paclitaxel liposome were diluted in serum-free RPMI-1640 medium and added to the corresponding wells, respectively, and Lipofectamin-2000 was used as a positive control, with a final Cy3-GAPDH-siRNA concentration of 20nM and a volume of 1mL per well. Incubate at 37 ℃ for 4h, wash the cells 3 times with PBS, place under a confocal microscope for observation and taking pictures. PBS was removed, cells were digested with 0.25% trypsin and then collected in a flow tube, PBS was added, centrifugation was performed at 1500rpm/min for 5min, the supernatant was removed,repeat 2 times. 200 μ L of PBS was added to each tube to resuspend the cells. 10000 cells were collected using free Cy3-GAPDH-siRNA as a negative control and detected by flow cytometry. Examples 1, 2 and 3 were surface-modified with alkylglycosides having different hydrophilic-hydrophobic equilibrium values, respectively, to prepare GAPDH siRNA-paclitaxel liposomes, which were mixed with Hela cells and then detected by a flow cytometer. The results are shown in FIG. 4.

As can be seen from fig. 4, fluorescence appeared in all of examples 1, 2 and 3, indicating that the GAPDH siRNA-paclitaxel liposomes prepared in all of examples 1, 2 and 3 could be efficiently taken up by Hela cells, and the fluorescence intensity was highest under the conditions of example 2, i.e., when the alkylglycoside value was 4.8, and almost corresponded to positive control Lipo 2000.

Test example 4:

determination of the effect of GAPDH siRNA-paclitaxel liposome-mediated transfection of cells in vitro:

approximately 7500 cells were seeded into each well of a 96-well plate, and the density of cells per well was controlled between 30% and 50% for transfection experiments based on cell growth rate. After 24h, 50. mu.L of Opti-MEM reduced serum medium was taken to dilute GAPDH siRNA to make a negative control formulation of free siRNA.

After aspirating the culture medium from the 96-well plate, GAPDH siRNA-paclitaxel liposomes at different concentrations of 10nM, 25nM, 50nM and a negative control preparation at the corresponding concentrations were added to the corresponding wells for transfection. After 4 hours the preparation was aspirated off, and fresh medium was added for further incubation. After 48 hours, the culture solution is removed by suction, cell lysate in the GAPDH enzyme activity assay kit is added, the incubation is carried out for 20min at the temperature of 4 ℃, and then the cell lysate is blown up and down by a pipette gun for 4 to 5 times. mu.L of sample was taken per well into a black 96-well plate and 90. mu.L KDalert was added^TMThe Master mixed luminescent agent is placed in a multifunctional microplate reader, the fluorescence change value which increases along with the time extension is measured under the room temperature condition, the excitation wavelength is 550nm, and the emission wavelength is 600 nm.

Determination of total protein: and adding 10 mu L of the sample cracked in each well into a 96-well plate, adding 90 mu L of RIPA for dilution by 10 times, adding a color developing agent in the BCA kit, incubating for 1h at 37 ℃, placing into an enzyme-linked immunosorbent assay, and measuring the light absorption value at 562 nm. The calibration curve was calculated according to the above method using bovine serum albumin as a standard. The total protein amount of the sample was calculated. To avoid errors due to differences in the total protein per well, the change in fluorescence corresponding to GAPDH enzymatic activity was converted to Δ FLU/mg protein based on the total protein measured. The results are shown in FIG. 5.

As can be seen from fig. 5, when the GAPDH siRNA-paclitaxel liposome prepared in example 2 was administered, the expression level of GAPDH in Hela cells was significantly decreased compared to the negative control group; when the GAPDH siRNA-paclitaxel liposome prepared in comparative examples 1-3 was administered, the expression level of GAPDH in Hela cells was somewhat decreased compared to the negative control group, but was significantly higher compared to example 2, and thus, example 2 had better gene transfection ability. The results show that the surface of the cationic liposome is modified by the alkyl glycoside and the glycocyamine, so that the cell transfection efficiency under the action of serum can be improved, the cell uptake efficiency of a delivery system is further improved, and the delivery system is facilitated to reach a specific target position, so that the curative effect is exerted.

Test example 5:

determination of cytotoxicity of cationic liposomes:

collecting Hela cells in logarithmic growth cycle, digesting with 0.25% trypsin, centrifuging to remove supernatant, adding DMEM culture solution containing no serum, resuspending, counting, inoculating 150 μ L of DMEM culture solution containing no serum at 4500 cell density in 96-well plate, standing at 37 deg.C and 5% CO₂The incubator was incubated overnight. After the cells reach 60-80% fusion, 50 μ L of GAPDH siRNA liposome with different concentrations is added, meanwhile, a blank serum-free DMEM medium is set as a blank zero-adjusting group, untransfected cells are set as a negative control group, the cells are cultured for 24h, 20 μ L of MTT with the concentration of 5mg/mL is added into each well, and the cells are incubated for 4h at 37 ℃. The supernatant was removed, 150 μ L of DMSO was added to each well to dissolve formazan crystals, the absorbance value at 490nm was measured with a microplate reader over 20min, and the survival rate of Hela was calculated according to the following formula and repeated 3 times.

Cell viability ═ 100% (sample absorbance value-absorbance value for blank zero set)/(absorbance value for negative control set-absorbance value for blank zero set) ×

The results are shown in FIG. 6.

As can be seen from FIG. 6, both example 4 and comparative example 8 showed a certain cytotoxicity to Hela cells, and in comparative example 8, cationic liposomes prepared by substituting cholesterol for carbazole and administered with the prepared GAPDH siRNA liposomes, the survival rate of Hela cells was lower than that of example 4, and the cationic liposomes of comparative example 8 showed a greater cytotoxicity.

Test example 6:

evaluation of in vivo efficacy of GAPDH siRNA-paclitaxel liposome:

1) establishment of tumor-bearing nude mouse model

Taking Hela cells which well grow in logarithmic phase, digesting the cells by 0.1% trypsin, adding DMEM cell culture solution to stop digestion, centrifuging, removing the cell culture solution, resuspending by serum-free DMEM cell culture solution, counting, centrifuging and collecting the cells. Adding PBS at 4 deg.C to make it uniformly dispersed until its concentration is 1 × 10⁷When cells/mL, the cells are stored in an ice box. Firstly, the skin of a mouse is disinfected by iodophor, 0.2mL of Hela cells are inoculated to the left armpit of a nude mouse by subcutaneous injection, and an animal model of the nude mouse with human cervical carcinoma (Hela) tumor is established.

2) Dosing regimens

Approximately 3 weeks after inoculation, the tumor volume in tumor-bearing nude mice grew to approximately 100mm³Groups were randomized, 6 mice per group, for a total of 5 groups. The GAPDH siRNA-paclitaxel liposome prepared in example 2 of the present invention was injected into the tail vein with normal saline, paclitaxel liposome, or the like. The administration dose of GAPDH siRNA was 75nM/kg and the administration dose of paclitaxel was 3mg/kg, 1 time every other day for 12 times.

3) Evaluation index of in vivo drug efficacy

After the first dose, the major and minor diameters of the tumor were measured daily, the tumor volume was calculated according to the following formula, and a tumor volume growth curve was plotted. The results are shown in FIG. 7.

Tumor volume (square of short diameter x long diameter)/2

After the last administration, all tumor-bearing nude mice were weighed, sacrificed, tumor tissues were taken out and weighed, and the tumor inhibition ratio was calculated according to the following formula using a physiological saline group as a control group. The results are shown in FIG. 8.

Tumor inhibition rate-weight of tumor tissue in control group-weight of tumor tissue in treatment group/weight of tumor tissue in control group

4) Evaluation of safety

The body weight of tumor-bearing nude mice was measured every day from the first administration, a change curve of body weight was plotted, and the change of body weight of tumor-bearing nude mice after administration was evaluated. The results are shown in FIG. 9.

As can be seen from fig. 7 and 8, GAPDH siRNA-paclitaxel liposome has significant anti-tumor efficiency, after administration, the tumor tissue of tumor-bearing nude mice becomes very small, the tumor inhibition rate is as high as 94.8%, while paclitaxel liposome only inhibits the growth of tumor to a certain extent, and the tumor inhibition rate is only 72.4%.

As can be seen from FIG. 9, after 12 times of continuous intravenous administration of GAPDH siRNA-paclitaxel liposome, the tumor-bearing nude mice showed no significant weight loss and no significant systemic toxicity, compared with the normal saline control group.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims

1. A method of making a nanoparticle composition delivery system, comprising the steps of:

1) preparing the cationic liposome raw material and the anti-tumor drug into the anti-tumor drug-loaded cationic liposome by adopting a film dispersion method;

2) mixing the anti-tumor drug-loaded cationic liposome and GAPDH-siRNA by adopting a mixing incubation method to prepare a nanoparticle composition delivery system;

the molar ratio of 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride, phytosterol, diacetone-D-galactose and dioleoyl phosphatidylethanolamine in the cationic liposome raw material is 1:0.8-1.2:0.2-0.5: 2.8-3.2;

the mass ratio of the antitumor drug to the dioleoyl phosphatidylethanolamine in the cationic liposome raw material is 1: 6-7;

the mass ratio of the cationic liposome to the GAPDH-siRNA is 18-22: 1;

the surface of the cationic liposome is modified by alkyl glycoside and glycocyamine.

2. A method of making a nanoparticle composition delivery system as in claim 1, wherein: the nanoparticle composition delivery system has an average particle size of 159.1 to 193.7 nm.

3. A method of making a nanoparticle composition delivery system as in claim 1, wherein: the hydrophilic-hydrophobic balance value of the alkyl glycoside is 4.5-5.2.