Nanosphere composition as well as preparation method and application thereof

Abstract

The invention provides a nanosphere composition as well as a preparation method and application thereof. The nanosphere composition comprises a lipid and an inert gas; wherein the lipid is composed of a non-ionizable lipid A and an ionizable lipid B, and the nanosphere has an average particle size of 100 to 300 nm. The nanosphere composition is prepared through an extrusion method, and the nanosphere composition has wide application potential in the fields of drug delivery, gene therapy, vaccination and the like as a drug delivery carrier.

Description

Technical Field

[0001] The invention belongs to the field of nano materials and relates to a nano ball composition and a preparation method and application thereof. Background Art

[0002] Nanospheres are an important drug delivery carrier. Due to their unique physical and chemical properties, they have broad application potential in drug delivery, gene therapy, vaccination and other fields. In recent years, with the advancement of nanotechnology and biotechnology, the research on nanospheres has developed rapidly and has become a hot topic in pharmaceutical and biomedical research. However, although nanospheres have shown great application prospects in laboratory and preclinical studies, their promotion in clinical applications still faces many challenges, among which the monodispersity of particle size is a key factor.

[0003] The particle size of nanospheres has a direct impact on their in vivo distribution, cellular uptake, drug release rate, and biocompatibility. Generally, nanospheres with a particle size less than 200 nanometers can better avoid the reticuloendothelial system (RES), thereby prolonging blood circulation time and improving drug targeting. However, if the particle size distribution range of nanospheres is too wide, it may lead to uneven drug release, reduced targeting, and increased immune system clearance. Therefore, the monodispersity of particle size is crucial to the stability and efficacy of nanospheres.

[0004] Current research shows that the particle size distribution of nanospheres is not only restricted by the preparation method, but also by the parameter control of the extrusion process. Extrusion is a widely used particle size control technology. By extruding the membrane material solution through membranes with different pore sizes, the particle size of the nanospheres can be effectively controlled. However, the selection of extrusion pressure, the size of the membrane pore size, the number of extrusions and other parameters are directly related to the particle size distribution of the final product. Unfortunately, although the monodispersity of nanospheres can be continuously improved by strategies such as reducing the pore size of the filter membrane and multiple extrusion iterations, its relatively complex condition control and high cost of use limit its large-scale industrial application.

[0005] In summary, the research in the field of nanospheres is in a rapid development stage, and the control of particle size monodispersity has become one of the key issues restricting its clinical transformation. Only through continuous process improvement and parameter optimization can this challenge be overcome and nanocarrier technology can play a greater potential in clinical applications. Summary of the invention

[0006] The present invention proposes a novel nanosphere composition and its preparation method and use, aiming to solve the problem that the monodispersity of nanosphere particle size is difficult to control in the prior art. By adopting a nanosecond pulse electric field control strategy, the present invention can achieve precise control of the nanosphere particle size, thereby improving the stability, targeting and clinical application value of the nanosphere.

[0007] In order to meet the necessary conditions for lipid charge, the present invention selects ionizable lipids as an important component of the membrane material. Due to its ionizable property, ionizable lipids are common amphiphilic materials in the current field of gene delivery. Based on this, the inventors found that by applying a nanosecond pulse electric field to a dispersed solution of ionizable lipids, the electrical properties of the lipid solution can be significantly improved, which is specifically manifested as a change in zeta potential. Furthermore, the inventors found that by adjusting the molar ratio of non-ionizable lipids to ionizable lipids, the electrical properties of the solution can be further controlled, thereby accurately affecting the particle size distribution of the nanospheres after extrusion.

[0008] In order to achieve the above objectives, the present invention first provides a nanosphere composition, which includes lipids and an inert gas. Specifically, the lipids are composed of non-ionizable lipids A and ionizable lipids B. The nanospheres have an average particle size of 100 to 300 nm.

[0009] The relative standard deviation (RSD) of the nanospheres of the present invention is less than 10%, and more preferably less than 8%.

[0010] Preferably, the phase transition temperature range of non-ionizable lipid A and ionizable lipid B is 0-70°C. Preferably, the molar ratio of non-ionizable lipid A to ionizable lipid B is (2.8-4.6): (1-1.5), preferably (3-4): 1. Such a design of more A and less B helps to improve the stability of the nanospheres, and the particle size distribution of the nanospheres can be regulated by the molar ratio of non-ionizable lipid to ionizable lipid.

[0011] Preferably, in order to enhance the stability of the nanospheres, the non-ionized lipid A of the present invention is selected from non-ionized lipids with a phase transition temperature range of 0-60°C, and more preferably 0-55°C. Non-ionized lipids with a phase transition temperature range of 0-50°C are further preferred. Non-ionized lipid A is, for example, one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), etc. Such non-ionized lipids generally have good biocompatibility and low toxicity, and are very suitable for drug delivery carriers, and have been found to enhance the in vivo transfection efficiency of genes. In addition, due to the low phase transition temperature of lipid A, it can achieve extrusion of nanospheres only at room temperature, which provides a prerequisite for encapsulating temperature-sensitive enzymes, proteins, genes and other drugs.

[0012] Preferably, in order to enhance the controllability of the particle size of the nanospheres, the ionizable lipid B of the present invention is selected from ionizable lipids with a phase transition temperature range of 0-60°C, more preferably 0-55°C. More preferably, ionizable lipids with a phase transition temperature range of 0-50°C are selected. Ionizable lipids B include one or more of tertiary amine lipids: 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol hydrochloride (DC-Chol), secondary amine lipids: (3β-[N-(N'-methylaminoethane)carbamoyl]cholesterol (MC-Chol), primary amine lipids: (3β-[N-(aminoethane)carbamoyl]cholesterol (AC-Chol), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), etc. The notable feature of this type of ionizable lipids is that they all have ionizable groups, namely various amine groups. Under the regulation of nanosecond pulsed electric fields, such groups can undergo an orderly protonation process, making it possible to regulate the charge load of lipids by the voltage of nanosecond pulsed electric fields. In addition, due to the low phase transition temperature of lipid B, it can achieve nanosphere extrusion only at room temperature, which provides a prerequisite for encapsulating temperature-sensitive enzymes, proteins, genes and other drugs.

[0013] In a specific embodiment, the ionizable lipid B is selected from a lipid having a phase transition temperature range of 0-60°C and an amine group, wherein the amine group is selected from one or more of a tertiary amine group, a secondary amine group, a primary amine group and / or dimethylammonium.

[0014] In a specific embodiment, the non-ionizable lipid A is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), the ionizable lipid B is (3β-[N-(N'-methylaminoethane)carbamoyl]cholesterol (MC-Chol), and the molar ratio of lipid A to lipid B is (2.8-4.6): (1-1.5), preferably (3-4): 1.

[0015] In a specific embodiment, the non-ionizable lipid A is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the ionizable lipid B is 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol hydrochloride (DC-Chol), and the molar ratio of lipid A to lipid B is (2.8-4.6):(1-1.5), preferably (3-4):1.

[0016] In a specific embodiment, the non-ionizable lipid A is a composition of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the molar ratio of DSPC to DOPE is (1-9): (1-9), the ionizable lipid B is 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol hydrochloride (DC-Chol), (3β-[N-(N'-methylaminoethane)carbamoyl]cholesterol (MC-Chol), (3β-[N-(aminoethane)carbamoyl]cholesterol (AC-Chol) or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and the molar ratio of lipid A to lipid B is (2.8-4.6): (1-1.5), preferably (3-4): 1.

[0017] The inert gas selected by the present invention is one or more organic fluorides with a relative dielectric constant of less than 3, preferably less than 2.5. It is further preferred that the relative dielectric constant is less than 2.2. The inert gas is preferably one or more of perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane. It is further preferred that it is one or more of perfluoropropane, perfluorobutane, and perfluoropentane. These gases can form a stable structure inside the nanosphere by replacing air during high-pressure extrusion, further enhancing the stability and targeting of the nanosphere. Inert gases play a very important role in the preparation of nanospheres. Organic fluorides with low dielectric constants can reduce charge accumulation and prevent agglomeration between nanospheres, thereby ensuring the monodispersity of nanospheres. In addition, the chemical inertness of these gases ensures their stability in the in vivo environment, and will not react adversely with biological molecules, ensuring the biocompatibility and safety of the nanospheres.

[0018] The present invention adopts a particle size control strategy based on nanosecond pulse electric field in the preparation process of nanospheres. Traditional nanosphere preparation methods often rely on physical extrusion processes, but the uniformity of particle size is difficult to guarantee. The present invention applies a 10kV-100kV nanosecond pulse electric field in the extrusion equipment, and utilizes the charge repulsion effect under the action of the electric field to accurately control the charge of the ionizable lipid B, thereby limiting the particle size growth of the nanospheres during the self-assembly process. This method significantly improves the monodispersity of the nanospheres, simplifies the preparation process, and reduces costs.

[0019] The preparation method of the nanosphere composition of the present invention comprises the step of applying a nanosecond pulse electric field to a dispersed solution comprising ionizable lipids, wherein the nanosecond pulse electric field has a pulse width of 10 to 100 ns and an amplitude of 10 kV-100 kV. The dispersed solution may comprise non-ionizable lipids.

[0020] The preparation method of the nanosphere composition of the present invention specifically includes the following three steps.

[0021] 1) Prepare lipid solution.

[0022] Preferably, when preparing the lipid solution, lipid A and lipid B (according to a set molar ratio) are dispersed in a dispersant (solvent) to obtain a uniform lipid solution (material C). Preferably, the dispersant is deionized water. Preferably, the lipid concentration of the lipid solution is 1-2 mg / ml.

[0023] Preferably, the dispersant is 2-5 ml, and the total mass of lipids is controlled within the range of 2-6 mg. More preferably, the dispersant is 2-4 ml, and the total mass of lipids is controlled within the range of 3-5.5 mg.

[0024] Preferably, the present invention selects a molar ratio of lipid A to lipid B of (3-4):1, a dispersant of 2-3 ml, and a total lipid mass range of 3-5 mg, which can further improve the yield.

[0025] 2) Transfer the lipid solution to the extrusion equipment.

[0026] Preferably, the extrusion device is a high-pressure extrusion device, and the extrusion device has a microporous / nanoporous membrane. The lipid solution (material C) is transferred to the high-pressure extrusion device having a microporous / nanoporous membrane. The pore size of the membrane is 600 to 1500 nm, more preferably 800-1000 nm.

[0027] 3) Applying nanosecond pulsed electric fields in the extrusion equipment.

[0028] The amplitude of the nanosecond pulse electric field applied in the present invention is 10kV-100kV. Preferably, during the extrusion process, a pressure of 100-1000psi is applied while applying a 10kV-100kV nanosecond pulse electric field to extrude the suspension D. Preferably, the pulse width of the nanosecond pulse electric field is 10 to 100 ns.

[0029] The introduction of nanosecond pulse electric field is the core innovation of the present invention. Specifically, when positive pulse excitation occurs, the pulse electric field applies positive charge to the solution, causing a large amount of H + , thereby achieving controllable protonation of lipids, and ultimately causing controlled ionization of ionizable lipids. The present invention finds that by regulating the applied voltage and time of the nanosecond pulse electric field, precise regulation of the zeta potential of the lipid solution can be achieved. Preferably, the application time of the nanosecond pulse electric field is 5 to 30 minutes.

[0030] Preferably, before applying the nanosecond pulse electric field, the inert gas of the present invention is continuously blown into the extrusion device to replace the original air. During the extrusion process, the inert gas is continuously blown in to replace the original air, which is to prevent air from entering the core of the nanosphere and affecting the stability of the nanosphere.

[0031] In the preparation method of the present invention, the extrusion step can be optionally repeated 1-3 times. After extrusion, nanospheres E with uniform particle size can be obtained. Subsequently, the nanospheres can be further purified by static or centrifugal separation.

[0032] The nanospheres of the present invention may optionally encapsulate drugs or not encapsulate drugs during the above preparation process. The half-life of the nanospheres of the present invention may reach 100-150 minutes. The nanospheres of the present invention have a longer half-life and higher stability.

[0033] In one embodiment, the nanospheres of the present invention do not encapsulate drugs during the preparation process. After the nanospheres are prepared, the drugs are loaded on the nanospheres. The prepared nanospheres have a zeta potential of -70 to 70 mV, and more preferably, the absolute value of the zeta potential is 20-70 mV. The nanospheres have a strong loading capacity for drugs.

[0034] In a preferred embodiment, the absolute value of the zeta potential of the nanospheres prepared by the present invention is 20-70 mV, and the half-life is 100-150 min.

[0035] In one embodiment, the nanospheres of the present invention can encapsulate drugs during the preparation process to obtain nanospheres encapsulated with drugs. Preferably, before blowing the inert gas of the present invention, in the extrusion device, an appropriate amount of encapsulated drugs, such as small molecule drugs, proteins, plasmids, etc., are added to the lipid solution (material C) of step 2). The significant advantage of the present invention is that it has good drug compatibility. Whether it is common small molecule drugs (doxorubicin, paclitaxel, platinum, etc.) or cutting-edge macromolecular drugs such as proteins and genes, efficient loading of target drugs can be achieved based on the water-in-oil / oil-in-water system.

[0036] In a specific embodiment, the preparation method of the nanosphere composition of the present invention may include the following steps: 1) dissolving lipid A and lipid B in a solvent to obtain material C, wherein the lipid concentration in material C is 1-2 mg / ml; 2) transferring the material C to a high pressure extrusion device having a microporous / nanoporous membrane with a pore size of 600 to 1500 nm; 3) Optionally, adding or not adding an appropriate amount of encapsulated drugs, such as small molecule drugs, proteins, plasmids, etc., to the above material C; 4) continuously blowing an inert gas composition into the high-pressure extrusion device to replace the original air; 5) applying a 10 kV-100 kV nanosecond pulse electric field to the above solution and applying a pressure of 500-1000 psi to extrude the solution to obtain a suspension D; the pulse width of the nanosecond pulse electric field is 10 to 100 ns, and the application time is 5 to 30 min; 6) Repeat steps 2)-5) once to obtain nanosphere E; 7) Allow to stand or centrifuge.

[0037] The nanosphere composition prepared by the present invention is used as a drug delivery carrier and has wide application potential in the fields of drug delivery, gene therapy, vaccination, etc.

[0038] In summary, the beneficial effect of the present invention is that by applying a nanosecond pulse electric field in the extrusion solution, the charge control of the ionizable lipids can be easily achieved, thereby utilizing the principle of mutual repulsion of like charges to limit the particle size distribution during the self-assembly process, thereby ultimately achieving controllable monodispersity control of the nanospheres. DETAILED DESCRIPTION

[0039] The embodiments described in the present invention are only a part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0040] Unless otherwise specified, the materials used in the following examples were of analytical grade.

[0041] Example 1 1) Dissolve 4 mg of DSPC and MC-Chol with a molar ratio of 3.5:1 in 3 ml of deionized water solvent to obtain a lipid solution; 2) The above solution was transferred to a thin film high-pressure extrusion device with a pore size of 1 µm; 3) Continuously blow perfluoropropane into the high-pressure extrusion equipment to replace the original air; 4) applying a 10 kV nanosecond pulse electric field to the above solution and applying a pressure of 1000 psi to extrude the solution to obtain a suspension; the pulse width of the nanosecond pulse electric field is 10 ns and the application time is 5 min; 5) Repeat the extrusion step once to obtain nanospheres; 6) Allow to stand or centrifuge.

[0042] The nanospheres obtained in this example are denoted as E1.

[0043] Example 2 1) Dissolve 4 mg of DSPC and MC-Chol with a molar ratio of 4.6:1 in 3 ml of deionized water solvent to obtain a lipid solution; 2) The above solution was transferred to a thin film high-pressure extrusion device with a pore size of 1 µm; 3) Continuously blow perfluoropropane into the high-pressure extrusion equipment to replace the original air; 4) applying a 10 kV nanosecond pulse electric field to the above solution and applying a pressure of 1000 psi to extrude the solution to obtain a suspension; the pulse width of the nanosecond pulse electric field is 10 ns and the application time is 5 min; 5) Repeat the extrusion step once to obtain nanospheres; 6) Allow to stand or centrifuge.

[0044] The nanospheres obtained in this example are denoted as E2.

[0045] Example 3 1) Dissolve 4 mg of DOPE and DC-Chol with a molar ratio of 3:1 in 3 ml of deionized water to obtain a lipid solution; 2) The above solution was transferred to a thin film high-pressure extrusion device with a pore size of 1 µm; 3) Continuously blow perfluoropropane into the high-pressure extrusion equipment to replace the original air; 4) applying a 50 kV nanosecond pulse electric field to the above solution and applying a pressure of 100 psi to extrude the solution to obtain a suspension; the pulse width of the nanosecond pulse electric field is 20 ns and the application time is 30 min; 5) Repeat the extrusion step once to obtain nanospheres; 6) Allow to stand or centrifuge.

[0046] The nanospheres obtained in this example are denoted as E3.

[0047] Example 4 1) Dissolve 4 mg of phospholipids with a molar ratio of 4:1 (DSPC:DOPE molar ratio = 5:1) and AC-Chol in 3 ml of deionized water solvent to obtain a lipid solution; 2) The above solution was transferred to a thin film high-pressure extrusion device with a pore size of 1 µm; 3) Continuously blow perfluoropropane into the high-pressure extrusion equipment to replace the original air; 4) applying a 100 kV nanosecond pulse electric field to the above solution and applying a pressure of 800 psi to extrude the solution to obtain a suspension; the pulse width of the nanosecond pulse electric field is 100 ns and the application time is 20 min; 5) Repeat the extrusion step once to obtain nanospheres; 6) Allow to stand or centrifuge.

[0048] The nanospheres obtained in this example are denoted as E4.

[0049] Example 5 This example was carried out according to the method of Example 1, except that DSPC was not added in this example, and 4 mg of MC-Chol was dissolved in 3 ml of deionized water solvent. The nanospheres obtained in this example were denoted as E5.

[0050] Example 6 This example was carried out according to the method of Example 1, except that DSPC was replaced by a combination of DSPC and DOPE, and the molar ratio of DSPC:DOPE was 5:1. The nanospheres obtained in this example were denoted as E6.

[0051] Comparative Example 1 1) Dissolve 4 mg of DSPC and MC-Chol with a molar ratio of 3.5:1 in 3 ml of deionized water solvent to obtain a lipid solution; 2) The above solution was transferred to a thin film high-pressure extrusion device with a pore size of 1 µm; 3) Continuously blow perfluoropropane into the high-pressure extrusion equipment to replace the original air; 4) Without applying nanosecond pulse electric field, pressurizing to 1000 psi, and extruding to obtain suspension; 5) Repeat the extrusion step once to obtain nanospheres; 6) Allow to stand or centrifuge.

[0052] The nanospheres obtained in this comparative example are denoted as F1.

[0053] Comparative Example 2 1) Dissolve 4 mg of DSPC and MC-Chol in a molar ratio of 1:1 in 3 ml of deionized water to obtain a lipid solution; 2) The above solution was transferred to a thin film high-pressure extrusion device with a pore size of 1 µm; 3) Continuously blow perfluoropropane into the high-pressure extrusion equipment to replace the original air; 4) applying a 10 kV nanosecond pulse electric field to the above solution and applying a pressure of 1000 psi to extrude the solution to obtain a suspension; the pulse width of the nanosecond pulse electric field is 10 ns and the application time is 5 min; 5) Repeat the extrusion step once to obtain nanospheres; 6) Allow to stand or centrifuge.

[0054] The nanospheres obtained in this comparative example are recorded as F2.

[0055] Comparative Example 3 This comparative example was carried out in accordance with the method of Example 1, except that MC-Chol was not added in this comparative example, and 4 mg of DSPC was dissolved in 3 ml of deionized water solvent. The nanospheres obtained in this comparative example were recorded as F3.

[0056] Comparative Example 4 This comparative example was carried out according to the method of Example 5, except that no nanosecond pulse electric field was applied. The nanospheres obtained in this comparative example were denoted as F4.

[0057] The nanospheres of the embodiments of the present invention and the comparative examples were tested as follows: The average particle size and particle size distribution were measured using dynamic light scattering (DLS).

[0058] In the present invention, the average particle size is a volume-weighted average particle size.

[0059] The test method of nanosphere zeta potential is based on the zetasizer equipment of Malvern Pana and uses the standard protocol to measure the zeta potential of nanospheres at room temperature.

[0060] Half-life determination: 1) Inject 1 mg of nanospheres modified with fluorescent dyes (near-infrared dyes such as DiR or Cy7) into nude mice; 2) Use the small animal in vivo imaging system to quantitatively analyze the fluorescence metabolic intensity decay of nude mice; 3) By comparing the changes in signal intensity at different time points, the elimination curve of nanospheres in vivo can be drawn; 4) Use the imaging data to draw a decay curve of fluorescence or bioluminescence intensity over time, and fit the curve according to the exponential decay formula:

[0061] Where C(t) is the signal intensity at a certain time point, C0 is the initial signal intensity, and k is the elimination rate constant. 1 / 2 It can be calculated by the following formula:

[0062] The test results of the nanospheres of Examples 1-6 of the present invention and Comparative Examples 1-4 are shown in the following table.

[0063] Table 1.

[0064]

[0065] As can be seen from Table 1, the monodispersity of the nanospheres is well controlled in Examples 1-6 of the present invention, and the RSD of the average particle size of the nanospheres is small, which means that the introduction of the nanosecond pulse electric field increases the positive charge of the lipid solution, and effectively reduces the average particle size of the nanospheres, while narrowing the range of particle size distribution. The zeta potential of the nanospheres is suitable, and the loading capacity of the drugs is strong. Moreover, the half-life of the nanospheres in the embodiments of the present invention is relatively long, which is significantly improved compared to the comparative example.

[0066] Further, in order to verify the loading efficiency of the drug. The present invention adds 1 mg of plasmid carrying the mRNA gene to the nanosphere E1 obtained in Example 1 and the nanosphere F1 obtained in Comparative Example 1 before the air replacement step, and measures the plasmid content in the nanosphere by a standard qPCR method. The results show that after the nanosphere E1 is loaded with the plasmid, the loading efficiency is about 93%, compared with the drug loading efficiency of about 71% after the F1 is loaded with the plasmid. Therefore, nanosecond pulsed electric fields can significantly enhance the encapsulation efficiency of drugs.

Claims

1. A nanosphere composition, characterized in that: The nanosphere composition comprises lipids and an inert gas; wherein the lipids are composed of non-ionizable lipids A and ionizable lipids B, and the nanospheres have an average particle size of 100 to 300 nm.

2. The nanosphere composition according to claim 1, characterized in that: Preferably, the inert gas is one or more organic fluorides with a relative dielectric constant less than 3; preferably, the relative standard deviation RSD of the nanospheres is <10%.

3. A nanosphere composition, characterized in that: The nanosphere composition comprises ionizable lipids, and the preparation method of the nanosphere composition comprises the step of applying a nanosecond pulse electric field to a dispersed solution comprising the ionizable lipids, wherein the nanosecond pulse electric field has a pulse width of 10 to 100 ns and an amplitude of 10 kV-100 kV.

4. The nanosphere composition according to claim 3, characterized in that: The nanosphere composition comprises non-ionized lipids, and the dispersed solution comprises non-ionized lipids.

5. The nanosphere composition according to claim 3, characterized in that: The relative standard deviation RSD of the nanospheres is <10%.

6. The nanosphere composition according to any one of claims 1 to 5, characterized in that: The phase transition temperature range of the ionizable lipid is 0-60°C, preferably 0-55°C.

7. A method for preparing a nanosphere composition, characterized in that: The nanosphere composition is prepared by an extrusion method, which comprises the following steps: (1) Preparing lipid solution; (2) Transferring the lipid solution to the extrusion device; (3) Applying a nanosecond pulse electric field in the extrusion equipment.

8. The preparation method according to claim 7, characterized in that: Preferably, the amplitude of the applied nanosecond pulse electric field is 10kV-100kV; preferably, the pulse width of the nanosecond pulse electric field is 10 to 100 ns; preferably, the application time of the nanosecond pulse electric field is 5 to 30 min; preferably, the concentration of the lipid solution is 1-2 mg / ml.

9. A nanosphere composition, characterized in that: The nanosphere composition is prepared according to the preparation method according to claim 7 or 8.

10. Use of the nanosphere composition according to any one of claims 1 to 6 and 9 in preparing a drug delivery carrier.

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