CN109700782B

CN109700782B - High-drug-loading-rate disulfiram nanoparticles and application thereof in tumor prevention and treatment

Info

Publication number: CN109700782B
Application number: CN201910170699.9A
Authority: CN
Inventors: 王向涛; 刘彪; 敖惠; 路丽康; 李好文
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2019-03-07
Filing date: 2019-03-07
Publication date: 2021-07-20
Anticipated expiration: 2039-03-07
Also published as: CN109700782A

Abstract

The invention provides a disulfiram nanoparticle which comprises disulfiram and a stabilizer, wherein the stabilizer comprises one or more of a phospholipid material, vitamin E-polyethylene glycol succinate, methoxy polyethylene glycol-polycaprolactone, methoxy polyethylene glycol-polylactide, poloxamer P188, sodium oleate and hydroxypropyl-beta-cyclodextrin, or a combination of the phospholipid material and benzyl. The hydrophilic part of the stabilizer in the disulfiram nanoparticles forms a hydrophilic outer layer, the lipophilic part of the stabilizer forms a fat-soluble core to wrap the disulfiram drug, so that the water-insoluble disulfiram drug can be uniformly dispersed in a water phase in a nano-sized particle form, the problems of insolubility and difficult drug delivery are solved, and the encapsulated drug can be protected from being rapidly degraded. The disulfiram nanoparticles are used as an active ingredient, and can be prepared into various clinically common dosage forms by a modern preparation process, so that various clinical requirements including tumor prevention and treatment are met.

Description

High-drug-loading-rate disulfiram nanoparticles and application thereof in tumor prevention and treatment

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a disulfiram nanoparticle with high drug loading and application thereof in tumor prevention and treatment.

Background

Disulfiram (DSF), formula C₁₀H₂₀N₂S₄296.5, a member of the dithiocarbamate family, is widely clinically used as an anti-alcoholism drug (Hamidreza Fasehe, Ardeshir Ghamzadeh, Kamran Alimoghaddam, et al.A. comprehensive cytological Evaluation of Disufiram Encapsulated PLGA nanoparticules MCF-7Cells [ J]IJHOSCR,2017,11: 16-21.). In recent years, it has been found by researchers that DSF exhibits strong cytotoxicity against many types of cancers, including rectal cancer, melanoma, brain glioma, breast cancer, and prostate cancer (Morrison BW, Doudican NA, Patel KR, et al]Melanoma Res,2010,20: 11-20.). The anti-cancer effects of DSF in hematological malignancies and solid tumors have been demonstrated in preclinical studies (Robinson TJ, Pai M, Liu JC, et al, high-throughput assays as a potential therapeutic for triple-negative breast cells: interaction with IQ mole-containing factors [ J].Cell Cycle,2013,12:3013-3024；Mohammad Najlah,Zahima Ahmed,Mohammed Iqbal,et al.Development and characterisation of disulfirm-loaded PLGA nanoparticle for the treatment of non-small cell lung cancer[J]Eur J Pharm Biopharm,2017,112:224-].Expert Opin Inv Drug,2009,18:957-971；Hamidreza F,Rassoul D,Ardeshir G,et al.Delivery of disulfiram into breast cancer cells using folate-receptor-targeted PLGA-PEG nanoparticles:in vitro and in vivo investigations[J].J Nanobiotecg,2016,14:32；Cvek,B.Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-likr cells[J]BJC,2013,108: 993), but poor solubility in water and short half-life in blood, etc., limit the full exploitation of DSF antitumor activity. Therefore, there is a need to find a drug delivery system that addresses the solubility of DSF and promotes its distribution to tumor tissues.

Disulfiram is very unstable in solution, and nano encapsulation can keep the solid state form of many unstable drugs and is relatively isolated from factors causing instability of the drugs from the outside, so that the stability of the drugs is improved; the nanoparticle has high apparent solubility, good stability and small drug particle size, and more importantly, the suspension with the particle size less than 200nm can be easily enriched in tumor tissues through high permeability and retention (EPR) effects, so that the nano preparation is a feasible scheme for solving the solubility and stability of DSF.

Disclosure of Invention

The invention aims to provide disulfiram nanoparticles, DSF nanoparticles (DSF-NPs) prepared by using soybean lecithin (SPC) and Tocopheryl Polyethylene Glycol Succinate (TPGS) as combined carriers can obviously improve drug loading, have good stability and excellent anti-tumor effect, can be used for preparing anti-tumor drugs, simplifies the preparation method, solves the problem of DSF intravenous injection administration, and is expected to improve the distribution of the drugs in tumor tissues based on the EPR effect so as to improve the in-vivo anti-tumor drug effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a disulfiram nanoparticle which comprises disulfiram and a stabilizer, wherein the stabilizer comprises one or more of vitamin E-polyethylene glycol succinate (TPGS), methoxypolyethylene glycol phospholipid (mPEG-DSPE), soybean lecithin (SPC), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk lecithin (HEPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), methoxypolyethylene glycol-polycaprolactone (mPEG-PCL), methoxypolyethylene glycol-polylactide (mPEG-PLA), poloxamer P188, sodium oleate and hydroxypropyl-beta-cyclodextrin, or a combination of a phospholipid material and benzyl.

Preferably, the stabilizer is a combination of phospholipid material (soy lecithin and soy lecithin, egg yolk lecithin, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine) and vitamin E-polyethylene glycol succinate.

Preferably, the mass ratio of the disulfiram to the stabilizer is 1: (0.1-10).

Preferably, the particle size of the disulfiram nanoparticles is 10-1000 nm.

The invention provides a preparation method of the disulfiram nanoparticles in the technical scheme, which comprises the following steps:

mixing disulfiram, a stabilizer, an organic solvent and water to obtain a precursor solution;

removing the organic solvent in the precursor solution to obtain disulfiram nanoparticle suspension;

and removing water in the disulfiram nanoparticle suspension to obtain the disulfiram nanoparticles.

Preferably, the concentration of disulfiram in the precursor solution is 0.1-200 mg/mL; the volume ratio of the organic solvent to the water is 1: (1-100).

Preferably, the organic solvent is a first organic solvent, or a mixture of a first organic solvent and a second organic solvent; the first organic solvent comprises one or more of methanol, ethanol, acetone, dimethyl sulfoxide and N, N-dimethylformamide, and the second organic solvent comprises one or more of ethyl acetate, dichloromethane and trichloromethane.

The invention provides an application of the disulfiram nanoparticles prepared by the technical scheme or the preparation method in the technical scheme in the preparation of antitumor drugs.

Preferably, the content of the disulfiram nanoparticles in the antitumor drug is 5-95%.

Preferably, the dosage form of the antitumor drug is a solid dosage form, a semisolid dosage form, a liquid dosage form or a gas dosage form.

The invention provides a disulfiram nanoparticle which comprises disulfiram and a stabilizer. The hydrophilic part of the stabilizer in the disulfiram nanoparticles provided by the invention forms a hydrophilic outer layer, and the lipophilic part of the stabilizer forms a fat-soluble core to wrap the disulfiram drug in the stabilizer, so that the water-insoluble disulfiram drug can be uniformly dispersed in a water phase in a nano-sized particle form, and the problems of insolubility and difficult drug administration are solved; the disulfiram nanoparticles are used as an active ingredient, and can be prepared into various clinically common dosage forms by a modern preparation process, so that various clinical medication requirements are met.

Drawings

FIG. 1 is a graph showing the particle size distribution of disulfiram nanoparticles in a DSF-NPs dispersion prepared in example 44;

fig. 2 is a transmission electron microscope morphology chart of the disulfiram nanoparticles prepared in example 3 (fig. 2A) and example 44 (fig. 2B);

FIG. 3 is a graph showing the particle size change of the disulfiram nanoparticles prepared in example 3 (FIG. 3A) and example 44 (FIG. 3B) during incubation with various physiological media at 37 ℃ for 8 h;

FIG. 4 shows the degradation curves of the disulfiram nanoparticles prepared in example 3 (FIG. 4A) and example 44 (FIG. 4B) in pure water

FIG. 5 is a graph showing the degradation curves of the disulfiram nanoparticles prepared in example 3 (FIG. 5A) and example 44 (FIG. 5B) in pure water and PBS containing 10% fetal bovine serum

Fig. 6 is a graph of the in vitro release behavior of the disulfiram nanoparticles prepared in example 3 (fig. 6A) and example 44 (fig. 6B) in PBS buffer containing 1% tween 80;

FIG. 7 is a bar graph of the inhibition of 4T1 cells in breast cancer cells 48h after administration of the disulfiram nanoparticles prepared in example 3 (FIG. 7A) and example 44 (FIG. 7B) (mean. + -. SD, P < 0.05;. P < 0.01;. P < 0.001);

FIG. 8 is a distribution diagram of the DIR fluorescence labeled DSF-SPC nanoparticles in the main organs of 4T1 tumor-bearing mice after intravenous injection for 12 h;

FIG. 9 is a graph of the tumor volume as a function of time in groups of 4T1 tumor-bearing nude mice administered disulfiram nanoparticles prepared in example 3 (FIG. 9A) and example 44 (FIG. 9B);

FIG. 10 is a graph showing the change of body weight with time in 4T1 tumor-bearing nude mice in each group after administration of the disulfiram nanoparticles prepared in example 3 (FIG. 9A) and example 44 (FIG. 9B);

FIG. 11 is a pictorial view of mouse tumor tissues after the pharmacodynamic experiment of DSF-SPC-NPs prepared in example 3 on 4T1 tumor-bearing nude mice is completed (A is oral administration group, B is intravenous injection group);

FIG. 12 is a schematic diagram of mouse tumor tissues after the pharmacodynamic experiment of DSF-SPC-TPGS-NPs prepared in example 44 on 4T1 tumor-bearing nude mice was completed (A is oral administration group, B is intravenous injection group).

Detailed Description

In the present invention, the vitamin E-polyethylene glycol succinate is preferably vitamin E-polyethylene glycol 1000 succinate (TPGS), and the methoxypolyethylene glycol phospholipid is preferably methoxypolyethylene glycol 2000 phospholipid (mPEG)₂₀₀₀-DSPE)。

In the present invention, the mass ratio of disulfiram to stabilizer is preferably 1: (0.1 to 10), more preferably 1: (0.1 to 8), and more preferably 1: (0.5 to 6), most preferably 1: (1-2).

The hydrophilic part of the stabilizer in the disulfiram nanoparticles provided by the invention forms a hydrophilic outer layer, and the lipophilic part of the stabilizer forms a fat-soluble core to wrap the disulfiram drug in the stabilizer, so that the water-insoluble disulfiram drug can be uniformly dispersed in a water phase in a nano-sized particle form, and the problems of insolubility and difficult drug administration are solved; the disulfiram nanoparticles are used as an active ingredient, and can be prepared into various clinically common dosage forms by a modern preparation process, so that various clinical medication requirements are met.

In the invention, the particle size of the disulfiram nanoparticles is preferably 10-1000 nm, more preferably 50-800 nm, further preferably 50-600 nm, and most preferably 60-300 nm.

The method comprises the step of mixing disulfiram, a stabilizer, an organic solvent and water to obtain a precursor solution. In the invention, the concentration of disulfiram in the precursor solution is preferably 0.1-200 mg/mL, more preferably 1-100 mg/mL, and most preferably 5-60 mg/mL. In the present invention, the organic solvent is preferably a first organic solvent, or a mixture of a first organic solvent and a second organic solvent; the first organic solvent preferably comprises one or more of methanol, ethanol, acetone, dimethyl sulfoxide and N, N-dimethylformamide, and the second organic solvent preferably comprises one or more of ethyl acetate, dichloromethane and trichloromethane. In the present invention, the volume ratio of the organic solvent to water is preferably 1: (1 to 100), more preferably 1: (2-20).

The present invention preferably divides the stabilizer into a water-soluble stabilizer and a water-insoluble stabilizer according to the water solubility of the stabilizer. According to the invention, the mixing mode of each material is preferably selected according to the water solubility of the stabilizer, and particularly, when the stabilizer is one or more water-insoluble stabilizers, the water-insoluble stabilizer and disulfiram are preferably dissolved in an organic solvent to obtain an organic phase, and the organic phase is dropwise added into water to obtain a precursor solution; when the stabilizer is one or more water-soluble stabilizers, the water-soluble stabilizers are preferably dissolved in water to obtain a water phase, disulfiram is dissolved in an organic solvent to obtain an organic phase, and the organic phase is dropwise added into the water phase to obtain a precursor solution; when the stabilizer is a mixture of one or more water-soluble stabilizers and one or more water-insoluble stabilizers, the invention preferably dissolves the water-soluble stabilizers in water to obtain an aqueous phase, dissolves the water-insoluble stabilizers and disulfiram in an organic solvent to obtain an organic phase, and dropwise adds the organic phase to the aqueous phase to obtain a precursor solution.

The dropping rate of the organic phase is not particularly limited in the present invention, and may be a dropping rate known to those skilled in the art, specifically, a dropping rate. In the present invention, the dropping process of the organic phase is preferably performed under ultrasonic conditions; the ultrasound is not particularly limited in the present invention, and the ultrasound technical solution well known to those skilled in the art can be adopted. In the invention, the ultrasonic temperature is preferably 12-60 ℃, and more preferably 25-40 ℃.

After the precursor solution is obtained, the organic solvent in the precursor solution is removed to obtain the disulfiram nanoparticle suspension. The method for removing the organic solvent in the precursor solution is not particularly limited in the present invention, and a technical scheme for removing the solvent, which is well known to those skilled in the art, may be adopted, specifically, centrifugation or reduced pressure rotary evaporation.

The method for removing the disulfiram nanoparticle suspension is not specially limited, and a technical scheme for removing the solvent, which is well known to a person skilled in the art, is adopted, specifically, the disulfiram nanoparticle suspension is evaporated to dryness or freeze-dried under reduced pressure in the actual use process, and the disulfiram nanoparticle suspension is converted into a solid form by treatment such as freeze-drying and the like, so that the disulfiram nanoparticle suspension is more convenient to store and transport.

In the invention, the content of the disulfiram nanoparticles in the antitumor drug is preferably 5-95%, more preferably 8-80%, and further preferably 40-50%. The dosage form of the antitumor drug is not particularly limited, and the dosage form known to those skilled in the art can be adopted.

In the present invention, the dosage form of the antitumor drug preferably includes a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form, and more preferably includes, but is not limited to, a tablet, a pellet, a dragee, a capsule, a suppository, a cream, an ointment, an aerosol, a powder, an emulsion, a suspension, a syrup, an injection or other pharmaceutical dosage forms suitable for rectal, intranasal, pulmonary, intravaginal, external (topical), oral or parenteral (including subcutaneous, implant, intravenous and intramuscular) administration.

In the present invention, the injection preferably comprises an aqueous dispersion medium, and the aqueous dispersion medium is preferably a physiological isotonic system prepared from a high concentration of sodium chloride, glucose or phosphate buffer solution to physiological saline (0.9% sodium chloride), 5% glucose or phosphate buffer solution (PBS solution).

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of disulfiram nanoparticles

Preparation example 1.SPC as vehicle, acetone at a drug loading ratio of 1:1, concentration of 1mg/ml

5mg disulfiram and 5mg SPC are weighed and dissolved in 600 muL acetone, slowly dripped into 5ml deionized water at room temperature under 250W ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 187.2 +/-4.2 nm and the PDI value is 0.272 +/-0.013 measured by a Malvern particle size analyzer.

Preparation example 2.SPC as vehicle, DMSO at a loading ratio of 1:1, concentration of 1mg/ml

Weighing 5mg disulfiram and 5mg SPC, dissolving in 800 μ L DMSO, slowly dripping into 5ml deionized water at room temperature under 250W ultrasound, centrifuging at 13000rpm to remove organic solvent, precipitating, and dissolving again with ultrasound, wherein the average particle diameter is 551.5 + -37.60 nm and the PDI value is 0.591 + -0.064 as measured by Malvern particle size analyzer.

Preparation example 3 SPC as vehicle, acetone + ethanol, drug loading ratio 1:1, concentration 1mg/ml

Weighing 5mg disulfiram to dissolve in 200 mu L of acetone, weighing 5mg SPC to dissolve in 200 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under 250W ultrasound, continuing the ultrasound for 10min, performing reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer and a potentiometer to be 162.6 +/-5.64 nm, wherein the PDI value is 0.214 +/-0.014, and the surface potential is-19.6 mV.

Preparation example 4 SPC as vehicle, acetone + ethanol, drug loading ratio 1:2, concentration 1mg/ml

Weighing 5mg disulfiram to dissolve in 200 mu L of acetone, weighing 10mg SPC to dissolve in 200 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer and a potentiometer to be 219.7 +/-5.7 nm, the PDI value to be 0.212 +/-0.016 and the surface potential to be-7.73 mV.

Preparation example 5 SPC as vehicle, acetone + ethanol, drug Loading ratio 1:1, concentration 1mg/ml

Weighing 5mg disulfiram to dissolve in 100 mu L of acetone, weighing 5mg SPC to dissolve in 100 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under the stirring of 800rpm, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring by a Malvern particle size analyzer and a potentiometer to obtain the average particle size of 186.5 +/-3.12 nm, the PDI value of 0.201 +/-0.016 and the surface potential of-17.6 mV.

Preparation example 6 SPC as vehicle, acetone + ethanol, 2:1, concentration 1mg/ml

Weighing 10mg disulfiram to dissolve in 100 mu L of acetone, weighing 5mg SPC to dissolve in 100 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 10ml deionized water at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle diameter by a Malvern particle diameter instrument and a potentiometer to be 226.2 +/-5.79 nm, the PDI value to be 0.455 +/-0.026 and the surface potential to be-20 mV.

Preparation example 7 SPC as vehicle, acetone + ethanol, drug Loading ratio 3:1, concentration 1mg/ml

Weighing 15mg disulfiram to dissolve in 200 mu L of acetone, weighing 5mg SPC to dissolve in 200 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 15ml deionized water at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size of 351.8 +/-56.15 nm, the PDI value of 0.404 +/-0.046 and the surface potential of-22.6 mV by a Malvern particle size analyzer and a potentiometer.

Preparation example 8 SPC as vehicle, acetone + ethanol, drug Loading ratio 3:1, concentration 1mg/ml

Weighing 15mg disulfiram to dissolve in 200 mu L of acetone, weighing 3mg SPC to dissolve in 100 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 15ml deionized water at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle diameter by a Malvern particle size analyzer to be 122.7 +/-3.051 nm and the PDI value to be 0.271 +/-0.010.

Preparation example 9 yolk lecithin EPC as vehicle, acetone at a 1:1 loading ratio, at a concentration of 1mg/ml

Weighing 5mg disulfiram and 5mg EPC, dissolving in 600 μ L acetone, slowly dripping into 5ml deionized water at room temperature under 250W ultrasound, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring average particle diameter 153.9 + -3.1 nm and PDI value 0.198 + -0.011 with a Malvern particle diameter instrument.

Preparation example 10 EPC as vehicle, acetone + ethanol, drug load ratio 1:1, concentration 1mg/ml

Weighing 5mg disulfiram to dissolve in 200 mu L acetone, weighing 5mg EPC to dissolve in 200 mu L ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 5ml deionized water at room temperature under 250W ultrasound, continuing ultrasound for 2min, performing reduced pressure rotary evaporation at 40 ℃ to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer to be 142.6 +/-4.14 nm and the PDI value to be 0.137 +/-0.012.

Preparation example 11 EPC as vehicle, acetone + ethanol, vehicle ratio 5:1, concentration 1mg/ml

Weighing 15mg disulfiram to dissolve in 300 mu L of acetone, weighing 3mg EPC to dissolve in 300 mu L of ethanol, mixing the two uniformly, slowly dripping the mixture into 15ml deionized water at room temperature under 250W ultrasound, continuing ultrasound for 2min, carrying out reduced pressure rotary evaporation at 40 ℃ to remove the organic solvent, and measuring the average particle size of 297.3 +/-8.31 nm and the PDI value of 0.278 +/-0.062 by a Malvern particle size analyzer.

Preparation example 12 EPC as vehicle, acetone + ethanol, drug load ratio 10:1

Weighing 30mg disulfiram to dissolve in 3mL of acetone, weighing 3mg of EPC to dissolve in 2mL of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 30mL of deionized water at room temperature under 250W of ultrasound, continuing the ultrasound for 2min, performing reduced pressure rotary evaporation at 40 ℃ to remove the organic solvent, and measuring the average particle size of 386.5 +/-9.76 nm and the PDI value of 0.381 +/-0.085 by a Malvern particle size analyzer.

Preparation example 13 Hydrogenated Soybean Phospholipids (HSPC) as vehicle, acetone + ethanol at a loading ratio of 1:2, concentration of 1mg/ml

Weighing 5mg disulfiram to dissolve in 200 mu L of acetone, weighing 10mg HSPC to dissolve in 200 mu L of ethanol, mixing the two uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer to be 235.9 +/-6.3 nm and the PDI value to be 0.235 +/-0.023.

Preparation example 14 Hydrogenated Soybean Phospholipids (HSPC) as vehicle, acetone + ethanol at a loading ratio of 1:5, concentration of 1mg/ml

Weighing 5mg disulfiram, dissolving in 400 muL acetone, weighing 15mg HSPC, dissolving in 400 muL ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 5ml deionized water at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size of 178.5 +/-8 nm and the PDI value of 0.186 +/-0.016 by a Malvern particle size analyzer.

Preparation example 15 hydrogenated egg yolk lecithin (HEPC) as vehicle, acetone + ethanol at a drug loading ratio of 1:1, concentration of 1mg/ml

Weighing 5mg disulfiram to dissolve in 200 mu L of acetone, weighing 5mg HEPC to dissolve in 200 mu L of ethanol, mixing the two solutions uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under 250W ultrasound, performing reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer to be 245.1 +/-5.7 nm and the PDI value to be 0.246 +/-0.023.

Preparation example 16 Distearoylphosphatidylcholine (DSPC) as vehicle, acetone + ethanol, drug load ratio 1:1, concentration 1mg/ml

Weighing 5mg disulfiram to dissolve in 300 mu L of acetone, weighing 5mg DSPC to dissolve in 300 mu L of ethanol, mixing the two uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under 250W ultrasound, decompressing and rotary evaporating to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer to be 252.3 +/-5.3 nm and the PDI value to be 0.227 +/-0.022.

Preparation example 17 Dioleoylphosphatidylcholine (DOPC) as vehicle, acetone + ethanol in a drug-to-drug ratio of 1:1 at a concentration of 1mg/ml

Weighing 5mg disulfiram to dissolve in 200 mu L of acetone, weighing 5mg DOPC to dissolve in 200 mu L of ethanol, mixing the two uniformly, slowly dripping the mixture into 5ml of deionized water at room temperature under 250W ultrasound, decompressing and rotationally evaporating to remove the organic solvent, wherein the average particle size is 245.1 +/-5.7 nm measured by a Malvern particle sizer, and the PDI value is 0.246 +/-0.023.

Preparation example 18 TPGS as vehicle, acetone at a loading ratio of 1:1, concentration of 1mg/ml

Weighing 5mg disulfiram and 5mg TPGS, dissolving in 200 μ L acetone, slowly dripping into 5ml deionized water at room temperature under 250W ultrasound, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring average particle diameter 159.1 + -1.82 nm, PDI value of 0.130 + -0.020 and surface potential-25.3 mV by Malvern particle size analyzer and potentiometer.

Preparation example 19 TPGS as vehicle, acetone at a loading ratio of 1:1, concentration of 1mg/ml, aqueous phase was instilled into organic phase

Weighing 5mg disulfiram and 5mg TPGS, dissolving in 200 μ L acetone, slowly dripping 5ml deionized water into the acetone solution at room temperature under 250W ultrasound, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring average particle diameter 379.4 + -2.13 nm and PDI value of 0.286 + -0.098 by Malvern particle sizer.

Preparation example 20 TPGS as vehicle, acetone at a loading ratio of 1:9, concentration of 1mg/ml

5mg of disulfiram and 45mg of TPGS are weighed and dissolved in 500 microliter of acetone, the mixture is slowly dripped into 5ml of deionized water at room temperature under 250W ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 385.3 +/-45.95 nm and the PDI value is 439 +/-0.046 measured by a Malvern particle size analyzer.

Preparation example 21 TPGS as vehicle, acetone at a loading ratio of 1:9, concentration of 1mg/ml, aqueous phase was instilled into organic phase

5mg disulfiram and 45mg TPGS are weighed and dissolved in 500 microliter of acetone, 5ml deionized water is slowly dripped into the acetone solution at room temperature under 250W ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 127.8 +/-7.12 nm and the PDI value is 0.206 +/-0.013 measured by a Malvern particle sizer.

Preparation example 22.P188 as a vehicle, acetone at a drug-to-vehicle ratio of 1:1 at a concentration of 1mg/ml, aqueous phase was dropped into organic phase

Weighing 5mg disulfiram and 5mg P188 and dissolving in 200 mu L acetone, slowly dripping 5ml deionized water into the acetone solution at room temperature under 250W ultrasound, carrying out reduced pressure rotary evaporation to remove the organic solvent, wherein the average particle size is 465.3 +/-29.81 nm, the PDI value is 0.052 +/-0.058 and the potential is-13.7 mV as measured by a Malvern particle size analyzer and a potentiometer.

Preparation example 23.P188 as a vehicle, acetone at a drug-to-vehicle ratio of 1:9 at a concentration of 1mg/ml, aqueous phase was dropped into organic phase

5mg disulfiram and 45mg of P188 are weighed and dissolved in 500 mul of acetone, 5ml of deionized water is slowly dripped into the acetone solution at room temperature under 250W of ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 343.6 +/-11.86 nm and the PDI value is 0.194 +/-0.016 measured by a Malvern particle sizer.

Preparation example 24.P188 as carrier, acetone at a loading ratio of 1:9, concentration of 1mg/ml

5mg of disulfiram and 45mg of P188 are weighed and dissolved in 500 microliter of acetone, the mixture is slowly dripped into 5ml of deionized water at room temperature under 250W ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 643.6 +/-17.39 nm and the PDI value is 0.212 +/-0.044 measured by a Malvern particle size analyzer.

Preparation example 25 PCL2000-mPEG2000 as vehicle, acetone at a drug loading ratio of 1:1 at a concentration of 1mg/ml

5mg disulfiram and 5mg PCL2000-mPEG2000 are weighed and dissolved in 500 muL acetone, slowly dripped into 5ml deionized water under the ultrasonic condition of room temperature and 250W, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 833.8 +/-21.46 nm and the PDI value is 0.922 +/-0.164 measured by a Malvern particle size analyzer.

Preparation example 26 PCL4800-mPEG3000 as vehicle, acetone at a drug loading ratio of 1:9, at a concentration of 1mg/ml

5mg of disulfiram and 45mg of PCL4800-mPEG3000 are weighed and dissolved in 500 mu L of acetone by ultrasonic, slowly dripped into 5ml of deionized water at room temperature under 250W of ultrasonic, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 153.4 +/-68.19 nm and the PDI value is 0.247 +/-0.076 measured by a Malvern particle size analyzer.

Preparation example 27 PCL4800-mPEG3000 as vehicle, acetone at a drug loading ratio of 1:9, and ultrasonic dropwise adding the aqueous phase into the organic phase

Weighing 5mg disulfiram and 45mg PCL4800-mPEG3000, ultrasonically dissolving in 500 mu L acetone, slowly dripping 5ml deionized water into the acetone solution at room temperature under 250W ultrasonic, performing reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle diameter by a Malvern particle size analyzer to be 191.2 +/-1.68 nm and the PDI value to be 0.252 +/-0.012.

Preparation example 28 PLA2000-mPEG2000 as a vehicle, acetone at a drug loading ratio of 1:1 at a concentration of 1mg/ml

5mg disulfiram and 5mg PLA2000-PEG2000 are dissolved in 400 mu L of acetone by ultrasonic, slowly dripped into 5ml of deionized water at room temperature under 250W ultrasonic, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 284.4 +/-9.03 nm, the PDI value is 0.163 +/-0.072 and the surface potential is-0.166 mV as measured by a Malvern particle size analyzer and a potentiometer. After standing for three days, the particle size is 286.1 + -9.16 nm, and the PDI value is 0.213 + -0.049.

Preparation example 29 PLA5000-mPEG5000 as a vehicle, acetone at a drug loading ratio of 1:10 at a concentration of 1mg/ml

5mg of disulfiram and 50mg of PLA5000-PEG5000 are weighed, dissolved in 500 mu L of acetone by ultrasonic waves, slowly dripped into 5ml of deionized water at room temperature under 250W of ultrasonic waves, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 677.2 +/-39.16 nm and the PDI value is 0.963 +/-0.156 measured by a Malvern particle sizer.

Preparation example 30. sodium oleate as carrier, acetone in a drug-to-drug ratio of 1:1

Weighing 5mg disulfiram, dissolving in 0.2mL of acetone, dissolving 5mg of sodium oleate in 5mL of deionized water, dropwise adding the acetone solution into the water phase at room temperature under 250W of ultrasound, continuing to perform ultrasound for 5 minutes, performing rotary evaporation under reduced pressure to remove the organic solvent, filtering through a 0.45-micrometer microporous filter membrane, and measuring the average particle diameter by a Malvern particle diameter instrument of 491.5 +/-28.34 nm, the PDI value of 0.862 +/-0.123 and the surface point of-107 mV.

Preparation example 31 hydroxypropyl-beta-cyclodextrin as vehicle, acetone in a drug-to-drug ratio of 1:9

Weighing 5mg disulfiram to dissolve in 0.5mL of acetone, dissolving 45mg of hydroxypropyl-beta-cyclodextrin in 5mL of deionized water, dropwise adding the acetone solution into the water phase at room temperature under 250W of ultrasound, performing reduced pressure rotary evaporation to remove the organic solvent, filtering the solution through a 0.45 mu m microporous filter membrane, and measuring the average particle size to be 152.3 +/-15.30 nm and the PDI value to be 0.535 +/-0.119 by a Malvern particle size analyzer.

Preparation example 32 hydroxypropyl-beta-cyclodextrin Carrier, acetone in a drug Loading ratio of 1:9, aqueous phase was ultrasonically added dropwise to organic phase

Weighing 5mg disulfiram to dissolve in 0.5mL of acetone, dissolving 45mg of hydroxypropyl-beta-cyclodextrin in 5mL of deionized water, dripping into the acetone solution at room temperature under 250W of ultrasound, performing reduced pressure rotary evaporation to remove the organic solvent, and measuring the average particle size of 750.3 +/-84.26 nm and the PDI value of 0.621 +/-0.034 by a Malvern particle size analyzer.

Preparation example 33 hydroxypropyl-. beta. -cyclodextrin saturated solution and acetone were added dropwise with stirring to a concentration of 1mg/mL

5mg disulfiram is weighed, dissolved in 0.2mL of acetone, dripped into 5mL of saturated hydroxypropyl-beta-cyclodextrin solution under the stirring of 1500rpm, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 304.2 +/-118.3 nm and the PDI value is 0.459 +/-0.033 measured by a Malvern particle size analyzer.

Preparation example 34 saturated hydroxypropyl-. beta. -cyclodextrin solution, acetone, dropwise addition under ultrasound, concentration 1mg/mL

5mg disulfiram is weighed and dissolved in 0.2mL of acetone, the solution is dripped into 5mL of saturated hydroxypropyl-beta-cyclodextrin solution at room temperature under 250W of ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 14.43 +/-7.26 nm and the PDI value is 0.226 +/-0.023 measured by a Malvern particle sizer.

Preparation example 35 saturated hydroxypropyl-. beta. -cyclodextrin solution, acetone, dropwise addition under ultrasound, concentration 4mg/mL

Weighing 20mg disulfiram, dissolving in 0.2mL of acetone, dripping into 5mL of saturated hydroxypropyl-beta-cyclodextrin solution at room temperature under 250W of ultrasound, performing rotary evaporation under reduced pressure to remove the organic solvent, and measuring the average particle size by a Malvern particle size analyzer to be 10.94 +/-3.89 nm and the PDI value to be 0.198 +/-0.008.

Preparation example 36.SPC + DSPE-mPEG2000 as vehicle, acetone + ethanol in a drug-to-drug ratio of 2:1:1, 2mg/ml

Weighing 10mg disulfiram and 5mg DSPE-mPEG2000, ultrasonically dissolving in 400 muL acetone, dissolving 5mg SPC in 200 muL ethanol, mixing, dripping into 5ml deionized water under 250W ultrasound at room temperature, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring average particle diameter by a Malvern particle diameter instrument and a potentiometer to obtain 438.4 +/-9.79 nm, PDI value of 0.218 +/-0.036 and potential of-15.5 mV.

Preparation example 37 SPC + DSPE-mPEG2000 as vehicle, acetone + ethanol in a drug loading ratio of 3:3:1, 1mg/ml

Weighing 9mg disulfiram and 3mg DSPE-mPEG2000, ultrasonically dissolving in 400 muL acetone, dissolving 9mg SPC in 200 muL ethanol, mixing, dripping into 9ml deionized water under 250W ultrasound at room temperature, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring average particle diameter by a Malvern particle diameter instrument and a potentiometer to obtain 426.5 +/-9.12 nm, PDI value of 0.346 +/-0.043 and potential of-1.38 mV.

Preparation example 38.SPC +

58 is carrier, acetone and ethanol, the medicine carrying ratio is 1:1:1, 1mg/ml

Weighing 10mg disulfiram and 10mg

Dissolving 58 mg of SPC in 400 mu L of acetone by ultrasonic wave, dissolving 10mg of SPC in 200 mu L of ethanol, mixing uniformly, dripping into 10ml of deionized water at room temperature under 250W of ultrasonic wave, performing rotary evaporation under reduced pressure to remove the organic solvent, and measuring the average particle diameter by a Malvern particle size analyzer and a potentiometer to be 362.4 +/-24.89 nm, the PDI value to be 0.090 +/-0.018 and the potential to be 1.35 mV.

Preparation example 39.SPC +

58 is carrier, acetone and ethanol, the medicine carrying ratio is 5:5:1, 1mg/ml

Weighing 20mg disulfiram, 4mg

Dissolving 58 mg of SPC in 400 μ L of acetone by ultrasonic wave, dissolving 20mg of SPC in 200 μ L of ethanol, mixing, dripping into 20ml of deionized water at room temperature under 250W of ultrasonic wave, performing rotary evaporation under reduced pressure to remove the organic solvent, wherein the average particle diameter is 338.4 + -4.79 nm and the PDI value is 0.251 + -0.055 measured by a Malvern particle size analyzer.

Preparation example 40.SPC +

S2 is carrier, acetone and ethanol, the medicine carrying ratio is 1:1:1, 1mg/ml

Weighing 10mg disulfiram and 10mg

S2 ultrasonic dissolving in 400 μ L acetone, dissolving SPC 10mg in ethanol 200 μ L, mixing, dripping into deionized water 10ml under ultrasonic wave 250W at room temperature, rotary evaporating under reduced pressure to remove organic solvent, and measuring average particle diameter 630.4 + -21.91 nm and PDI value 0.307 + -0.026 by Malvern particle diameter instrument.

Preparation example 41.SPC +

S2 is carrier, acetone and ethanol, the medicine carrying ratio is 5:5:1, 1mg/ml

Weighing 20mg disulfiram, 4mg

S2 ultrasonic dissolving in 400 μ L acetone, dissolving 20mg SPC in 200 μ L ethanol, mixing, dripping into 20ml deionized water under 250W ultrasonic at room temperature, rotary evaporating under reduced pressure to remove organic solvent, and measuring average particle diameter 775.4 + -34.96 nm and PDI value of 0.445 + -0.078 by Malvern particle diameter analyzer.

Preparation example 42.SPC +

78 is carrier, acetone and ethanol, the medicine carrying ratio is 2:1:1, 1mg/ml

Weighing 10mg disulfiram and 5mg

Dissolving 78 mg of SPC in 400 μ L of acetone by ultrasonic wave, dissolving 10mg of SPC in 200 μ L of ethanol, mixing, dripping into 5ml of deionized water at room temperature under 250W of ultrasonic wave, performing rotary evaporation under reduced pressure to remove organic solvent, wherein the average particle diameter is 743.7 + -13.33 nm and the PDI value is 0.515 + -0.116 as measured by a Malvern particle size analyzer.

Preparation example 43 SPC + TPGS as vehicle, acetone + ethanol, drug load ratio 5:5:1, 1mg/ml

Weighing 20mg disulfiram and 4mg TPGS, ultrasonically dissolving in 400 muL acetone, dissolving 20mg SPC in 200 muL ethanol, mixing, dripping into 20ml deionized water under 250W ultrasound at room temperature, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring average particle size 210 + -1.26 nm and PDI value 0.249 + -0.039 by a Malvern particle size analyzer.

Preparation example 44 SPC + TPGS as vehicle, acetone + ethanol, drug load ratio 24:20:4, 1mg/ml

24mg of disulfiram and 4mg of TPGS are weighed and ultrasonically dissolved in 400 mu L of acetone, another 20mg of SPC is dissolved in 200 mu L of ethanol, the mixture is uniformly mixed and dripped into 24ml of deionized water under the condition of room temperature and 250W ultrasound, the organic solvent is removed by reduced pressure rotary evaporation, and the average particle size is 205.9 +/-2.37 nm and the PDI value is 0.255 +/-0.012 as measured by a Malvern particle size analyzer. After standing for 3 days, the average particle diameter was 159.8. + -. 2.97nm, and the PDI value was 0.350. + -. 0.003.

Preparation 45.SPC + TPGS as carrier, acetone + ethanol, the drug loading ratio is 1:1:1, 1mg/ml

Weighing 10mg disulfiram and 10mg TPGS, ultrasonically dissolving in 400 muL acetone, dissolving 10mg SPC in 200 muL ethanol, mixing, dripping into 10ml deionized water under 250W ultrasound at room temperature, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring with Malvern particle size analyzer to obtain average particle size of 189.9 + -6.82 nm and PDI value of 0.255 + -0.009.

Preparation example 46 SPC + PLA2000-PEG2000 as vehicle, acetone + ethanol, drug load ratio 2:1:1, 2mg/ml

Weighing 10mg disulfiram and 5mg PLA2000-PEG2000, ultrasonically dissolving in 400 muL acetone, dissolving 5mg SPC in 200 muL ethanol, mixing, dripping into 5ml deionized water under 250W ultrasound at room temperature, ultrasonically treating for 1min, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring with Malvern particle size analyzer and potentiometer to obtain average particle size of 265.2 + -1.02 nm, PDI value of 0.168 + -0.055, and surface potential of-0.197 mV.

Preparation example 47.SPC + PLA2000-PEG2000 as vehicle, acetone + ethanol, drug loading ratio 2:1:1, 2mg/ml

Weighing 10mg disulfiram and 5mg PLA2000-PEG2000, ultrasonically dissolving in 400 muL acetone, dissolving 5mg SPC in 200 muL ethanol, mixing, dripping into 5ml deionized water at room temperature under stirring at 800rpm, ultrasonically treating for 1min, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring with Malvern particle size analyzer and potentiometer to obtain the final product with average particle size of 745.1 + -12.85 nm, PDI value of 0.457 + -0.027, and surface potential of-0.301 mV.

Preparation example 48 SPC + PLA2000-PEG2000 vehicle, acetone + ethanol, drug load ratio 10:10:1, 1mg/ml

Weighing 20mg disulfiram and 2mg PLA2000-PEG2000, ultrasonically dissolving in 400 muL acetone, dissolving 20mg SPC in 200 muL ethanol, mixing, ultrasonically dripping into 20ml deionized water at room temperature and 250W, ultrasonically treating for 1min, performing rotary evaporation under reduced pressure to remove organic solvent, and measuring with Malvern particle size analyzer and potentiometer to obtain the final product with average particle size of 197.9 + -1.56 nm, PDI value of 0.199 + -0.011, and surface potential of-0.133 mV.

Preparation example 49 SPC + PLA2000-PEG2000 vehicle, acetone + ethanol, drug load ratio 10:10:1, 1mg/ml

Weighing 20mg disulfiram and 1mg PLA2000-PEG2000, ultrasonically dissolving in 400 muL acetone, dissolving 20mg SPC in 200 muL ethanol, mixing, ultrasonically dripping into 20ml deionized water at room temperature and 250W, ultrasonically treating for 1min, performing reduced pressure rotary evaporation to remove organic solvent, and measuring with Malvern particle size analyzer and potentiometer to obtain the final product with average particle size of 204.8 + -3.90 nm, PDI value of 0.216 + -0.054, and surface potential of-1.99 mV.

Preparation example 50SPC + P188 vector at a drug-to-drug ratio of 2:1:1, 2mg/mL

Weighing 10mg disulfiram and 5mg P188, ultrasonically dissolving in 400 mu L acetone, dissolving 5mg SPC in 200 mu L ethanol, mixing, dropwise adding 5ml deionized water into the mixed organic solution at room temperature under 250W ultrasound, performing reduced pressure rotary evaporation to remove the organic solvent, wherein the average particle diameter is 324.5 +/-13.40 nm as measured by a Malvern particle diameter instrument, and the PDI value is 0.226 +/-0.071.

Preparation 51SPC + P188+ PLA2000-PEG2000 as vehicle: the drug loading ratio is 2:1:1:1, 2mg/ml

Weighing 10mg disulfiram, 5mg P188 and 5mg PLA2000-PEG2000, ultrasonically dissolving in 600 muL acetone, dissolving 5mg SPC in 200 muL ethanol, mixing, dripping 5ml deionized water into the mixed organic solution at room temperature under 250W ultrasound, ultrasonically treating for 1min, and performing rotary evaporation under reduced pressure to remove the organic solvent, wherein the average particle diameter is 273.0 +/-3.20 nm as measured by a Malvern particle diameter instrument, and the PDI value is 0.141 +/-0.029.

Preparation 52SPC + Brij78+ PLA2000-PEG2000 as vehicle: the drug loading ratio is 2:1:1:1, 2mg/ml

Weighing 10mg disulfiram, 5mg P188 and 5mg Brij78, dissolving in 600 muL acetone by ultrasonic, dissolving 5mg SPC in 200 muL ethanol, mixing, dripping 5ml deionized water into the mixed organic solution at room temperature under 250W ultrasonic, performing ultrasonic treatment for 1min, performing rotary evaporation under reduced pressure to remove the organic solvent, and obtaining an average particle size of 121.9 +/-0.64 nm and a PDI value of 0.194 +/-0.025 by a Malvern particle size analyzer.

Example 2 characterization of disulfiram nanoparticles

Referring to the method of preparation example 44, 24mg of disulfiram and 4mg of TPGS are dissolved in 0.5mL of acetone, 20mg of SPC is dissolved in ethanol, the ethanol solution and the acetone solution are mixed uniformly to form an organic phase, the organic phase is dropwise added into 24mL of deionized water at room temperature under the ultrasonic condition of 250W, and the organic solvent is removed by reduced pressure rotary evaporation at 37 ℃ to obtain a disulfiram nanoparticle (DSF-NPs) dispersion system.

The DSF-NPs prepared in this example were milky white and slightly opalescent.

(1) And (3) measuring the drug loading capacity: freeze-drying disulfiram nanoparticles, adding a certain amount of freeze-dried powder (the mass is recorded as W) into a certain volume of acetonitrile (the volume is recorded as V) for dissolving, centrifuging at 13000r/min for 20min, taking supernatant high-performance liquid to measure the mass concentration of disulfiram (the mass is recorded as C), carrying out 3 experiments in parallel, and calculating the Drug Loading Capacity (DLC) of DSF-NPs according to the following equation:

the drug loading rate is c.V/W x 100%

As a result, the average drug loading rates of the DSF-NPs in production examples 5, 18, 19 and 44 were (44.36. + -. 1.09)%, (44.82. + -. 2.25)%, (45.16. + -. 1.87)% and (45.36. + -. 2.09)%, respectively, and the average drug loading rate of the DSF-NPs in production example 8 was (69.76. + -. 3.79)%, which was relatively close to the theoretical value of 75%. The drug loading rate is an important parameter for evaluating the quality of the pharmaceutical preparation, and the higher drug loading rate reduces the potential safety hazard of using a large amount of auxiliary materials and improves the safety of the pharmaceutical preparation.

(2) Observing the form by a transmission electron microscope: diluting DSF-NPs to the concentration of about 100 mug/mL, dripping 6 muL of the DSF-NPs onto a 300-mesh copper net, standing for 5min, sucking excess liquid by using filter paper, standing for 10min at room temperature for airing, dripping 6.0 muL of uranium acetate with the mass concentration of 2% onto the copper net for dyeing for 90s, sucking the excess liquid by using the filter paper, airing naturally at room temperature, and observing the form and size of the disulfiram nanoparticles at the accelerating voltage of 120kV under a transmission electron microscope.

Fig. 2A and fig. 2B are transmission electron microscope (tem) morphology graphs of the disulfiram nanoparticles of preparation example 3(SPC as a carrier) and preparation example 44(SPC and TPGS as carriers), respectively, and the results show that the disulfiram nanoparticles have uniform particle size distribution, are spherical, have a particle size of about 100nm, and are slightly smaller than the particle size measured by the dynamic light scattering method. The reason is that the particle size of the dried disulfiram nanoparticles is detected in a transmission electron microscope, and the hydration radius of the disulfiram nanoparticles is detected by a dynamic light scattering method, so that the detection results of the two methods are inconsistent according to the difference of objects.

(3) Stability study in physiological media

Preparing artificial gastric juice: 1mol/L dilute hydrochloric acid containing 1% pepsin;

preparing artificial intestinal juice: PBS (0.01M) buffer pH 6.8 containing 1% trypsin.

Mixing the prepared DSF-NPs with NaCl solution with the mass concentration of 1.8%, glucose with the mass concentration of 10% and PBS buffer solution (0.02M) with the double concentration in equal volume respectively, mixing with plasma, artificial gastric juice and artificial intestinal juice according to the ratio of 1:4(v/v), incubating for 8h at 37 ℃, measuring the particle size of the disulfiram nanoparticles at a specific time point, and repeating for 3 times for each sample.

Fig. 3A and fig. 3B are graphs of particle size and PDI variation (mean ± SD, n ═ 3) of the disulfiram nanoparticles of preparation example 3(SPC is a carrier) and preparation example 44(SPC and TPGS are carriers) respectively in the incubation process of different physiological media at 37 ℃ for 8h, and the results show that the disulfiram nanoparticles of the two preparation examples do not increase in particle size in the incubation process of 37 ℃ with physiological saline, 5% glucose, PBS, artificial gastric juice, artificial intestinal juice and plasma for 8h, which indicates that the disulfiram nanoparticles can be prepared into isotonic solution for intravenous injection in addition to meeting the stability requirement of oral administration.

(4) Protective effect of nanoparticles on chemical degradation of disulfiram

And (3) degradation protection in pure water, namely dissolving 10mg of disulfiram in 200ul of DMSO, and adding deionized water to dilute to 10mL to prepare 1mg/mL of disulfiram mother liquor. Taking 50ul of disulfiram mother liquor, adding 950ul of methanol, shaking, centrifuging at 13000rpm for 10min, separating supernatant, taking a proper amount of supernatant, and measuring the concentration of disulfiram by using HPLC. The remaining mother liquor was continuously stirred at 37 ℃ and 100rpm, sampled at 0h, 6h, 24h, 48h, 72h, 96h and processed as above to measure the concentration of DSF by HPLC, the percentage of DSF remaining was calculated in comparison with the initial DSF concentration, and the corresponding time was plotted to plot the degradation curve of disulfiram drug in pure water.

The disulfiram nanoparticles in preparation example 3 and preparation example 44 were diluted to 1mg/mL with deionized water, and the degradation curves of the disulfiram nanoparticles in pure water were plotted in the same manner as above.

Degradation protection in PBS containing 10% fetal calf serum, 10mg disulfiram drug is dissolved in 200ul DMSO, diluted to 10ml by PBS containing 10% fetal calf serum, 50ul diluent is added with 950ul methanol, centrifuged at 13000rpm for 10min, supernatant is separated, and a proper amount of initial concentration of disulfiram in the system is measured by HPLC. The remaining dilution was stirred continuously at 37 ℃ and 100rpm, sampled at 0h, 6h, 24h, 48h, 72h, 96h and the same time with 19 volumes of methanol, shaken, centrifuged, the supernatant was taken and the concentration of disulfiram was measured by HPLC, and the degradation curve of DSF in 10% fetal calf serum was plotted as above.

The disulfiram nanoparticles in preparation examples 3 and 44 were prepared into a system with a theoretical concentration of 1mg/mL with PBS containing 10% fetal calf serum, and the degradation curves of the disulfiram nanoparticles in 10% fetal calf serum were drawn by the same method.

As can be seen from the degradation curve of disulfiram in pure water (FIG. 4), the free disulfiram rapidly degrades in water, only 45% remains after 6h, and less than 25% remains after 48 h; while both nanoparticles were able to significantly reduce the decomposition of the encapsulated disulfiram drug, more than 70% of the DSF remained present at 48h (fig. 4A and 4B). As can be seen from the degradation curve of disulfiram in PBS containing 10% fetal calf serum (FIG. 5), 43% remains after 6h and less than 20% remains after 48h of free disulfiram; under the same conditions, 70% disulfiram still exists in the DSF-NPs at 24h, and the DSF-NPs are obviously degraded after 24h (FIG. 5A and FIG. 5B). In conclusion, analysis shows that free disulfiram is quickly explained in a liquid dispersion medium and body fluid, and the stability of the disulfiram can be obviously improved by nanoparticle encapsulation; it is also suggested that the disulfiram nanoparticles should be converted into solid state (e.g. lyophilized) for storage as soon as possible after their preparation.

(5) In vitro drug delivery of dual-theoretical nanoparticles

2mL of the disulfiram nanoparticles of example 3 and example 44 (disulfiram concentration 2 mg. multidot.mL)^-1) Respectively putting into dialysis bags (MWCO: 8000-14000), sealing, soaking in 50mL phosphate buffer PB (0.01 mol. L-1, pH 7.4) containing 1% polysorbate 80, continuously stirring at 37 deg.C for 150 r. min-1, taking 1mL release external liquid at time points of 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120 and 144h, supplementing fresh release medium with the same volume, and replacing 1 time release external liquid every 24 h. Cumulative NSps release was calculated from the increase in DSF in the release sheath, in parallel with 3 experiments. Simultaneously, 10mg of disulfiram raw material drug is precisely weighed, is uniformly dispersed in 10mL of 0.4% sodium carboxymethylcellulose under the assistance of ultrasound to prepare disulfiram suspension, the actual concentration is determined by HPLC, the accumulative release of the disulfiram suspension is determined by adopting the method, and 3 experiments are performed in parallel.

As shown in FIG. 6, it can be seen that the two nanoparticles (DSF-SPC-NPs and DSF-SPC-TPGS) are released relatively rapidly within 24h, the cumulative drug release reaches about 25%, and then the release is slow, and the cumulative release (42.26 + -2.35)%, is 168 h. While DSF suspension (0.4% CMC) releases slowly, the cumulative drug release in 24h is about 13.61%, and the cumulative drug release in 168h is only (15.76 +/-2.48)%.

Example 3 in vitro killing of tumor cells by Thielam nanoparticles

4T1 cells were cultured to log phase and single cell suspensions were prepared at 1X 10⁴The cells were seeded at a density of 150. mu.L/well in 96-well cell culture plates and placed at 37 ℃ in 5% CO₂Culturing overnight in a cell culture box, observing cell adherence under an inverted microscope, discarding supernatant, and adding 150 μ L of DSF-NPs (diluted with incomplete culture medium) and DSF free drug (diluted with incomplete culture medium in DMSO solution) with DSF-NPs concentration of 100, 50, 25, 10, 5, 1, 0.1, 0.05, 0.01 μ g/mL respectivelyDiluting), culturing in incubator for 48 hr, adding 20 μ L MTT (5mg/ml) per well, 37 deg.C, and 5% CO₂After further incubation for 4h, the culture was terminated, the supernatant was discarded, 200. mu.L of DMSO was added to each well, the mixture was shaken, and the absorbance value (DSF) was measured at an absorption wavelength of 570nm using a microplate reader, and the cell survival rate was calculated by the following formula:

cell survival rate-OD value_{Experimental group}OD value_{Control group}×100％

Calculation of IC of cells by GraphPad prism 5 software₅₀Values, statistical differences were analyzed using IBM SPSS Statistics 19 software.

Fig. 7A and 7B are bar graphs of the inhibition rate of disulfiram nanoparticles at different concentrations for 48h after preparation example 3(SPC as a vehicle) and preparation example 44(SPC and TPGS as vehicles), respectively. As can be seen from the figure, the disulfiram nanoparticles and the DMSO solutions of the two preparation examples can inhibit the proliferation of 4T1 cells and show a dose-dependent relationship; however, at all the administration concentrations, the inhibition rate of the two nanoparticles on 4T1 cells is significantly higher than that of DSF free drug) (P<0.01). According to calculation, the IC50 of the disulfiram nanoparticles of preparation examples 3 and 44 on breast cancer 4T1 cells is 1.23 and 1.069 mu g/mL respectively^-1All significantly lower than the IC50 value (5.61. mu.g.mL) of the disulfiram DMSO solution in the corresponding experiment^-1、5.526μg·mL^-1And the P value is respectively less than 0.05 and 0.01), which shows that the nanoparticle has stronger growth inhibition effect on tumor cells after being encapsulated. The protection effect of nanoparticle encapsulation reduces the degradation of DSF in DSF-NPs, and the drug-loaded nanoparticles improve the factor of drug uptake by cells.

Example 4 drug efficacy and in vivo tissue distribution of disulfiram nanoparticles on 4T1 tumor-bearing mice

(1) Preparing the disulfiram nanoparticle marked by the near-infrared probe: referring to preparation example 3, 20mg of disulfiram was dissolved in 0.5ml of acetone, 20mg of SPC and 0.5mg of near-infrared probe Dir were dissolved in 0.5ml of ethanol, the ethanol solution and the acetone solution were mixed uniformly to form an organic phase, the organic phase was added dropwise to 15ml of water at room temperature under an ultrasonic condition of 250W, and the organic solvent was removed by rotary evaporation under pressure at 37 ℃ to obtain a Dir-labeled disulfiram nanoparticle (Dir-DSF-SPC-NPs) dispersion system.

The particle size distribution and the Zeta potential of the Dir-DSF-NPs were determined by Dynamic Light Scattering (DLS) principle using a Zetasizer nano ZS type particle sizer in 3 replicates per sample at 25 ℃.

(2)4T1 tumor-bearing mouse model establishment: culturing 4T1 cells in vitro to logarithmic phase, digesting adherent cells with pancreatin, collecting healthy BABL/c mice (6 weeks old, female), inoculating 0.2mL of 4T1 mammary cancer cell suspension (the concentration of the cell suspension is 8.0 × 10) subcutaneously in right axilla of each mouse⁵Mice), tumor volumes were measured periodically (V ═ ab)²2; a is long side, b is short side), the tumor volume reaches 100mm³And in the left and right, 64 mice with relatively consistent tumor sizes are screened to perform experiments on the DSF-SPC nanoparticles, and 64 mice with relatively consistent tumor sizes are selected to perform experiments on the DSF-SPC-TPGS nanoparticles.

(3) Experimental grouping and administration

DSF-SPC nanoparticle efficacy experiment: the screened tumor-bearing mice were randomly divided into 7 groups of 7 mice, and the mice were divided into groups, except for normal diet, according to intravenous injection (i.v.) once every 2 days and intragastric administration (i.g.) daily for 12 days of the experiment. DSF-SPC nanoparticles were prepared according to example 3.

1)DSF-SPC-NPs 20mg/kg，i.v.

2)DSF-SPC-NPs 10mg/kg，i.v.

3)DSF-SPC-NPs 5mg/kg，i.v.

4)DSF-SPC--NPs 20mg/kg，i.g.

5) DSF suspensions 20mg/kg, i.g. (DSF dispersed in 0.4% sodium carboxymethylcellulose solution)

6) Positive control (broad-spectrum anticancer drug Paclitaxel (PTX) injection sold in market 8mg/kg, i.v.)

7) Negative control (Saline 0.2 mL/tube, i.v.)

DSF-SPC-TPGS nanoparticle efficacy experiment: the screened tumor-bearing mice were randomly divided into 7 groups of 7 mice, and the mice were administered once every 2 days by intravenous injection (i.v.) and daily by gavage (i.g.) except for normal diet. DSF-SPC-TPGS nanoparticles were prepared according to example 44.

1) DSF-SPC-TPGS nanoparticle 20mg/kg, i.v.

2) DSF-SPC-TPGS nanoparticle 10mg/kg, i.v.

3) DSF-SPC-TPGS nanoparticle 5mg/kg, i.v.

4) DSF-SPC-TPGS nanoparticle 20mg/kg, i.g.

7) Negative control (Saline 0.2 mL/tube, i.v.)

(4) And (4) investigation indexes are as follows: observing whether the mice have abnormality or not and death conditions every day; the body weight and tumor volume (V ═ ab) of each group of mice were measured every two days ²2; a is a long side and b is a short side). On day 12, the mice were sacrificed by cervical dislocation, dissected to obtain each group of tumors and the tumor inhibition rate was calculated according to the following formula:

the tumor inhibition rate is (1-tumor weight of administration group/tumor weight of physiological saline) x 100%

Meanwhile, the liver and spleen of the mice are dissected out and weighed, and the liver and spleen indexes of the mice in each group are calculated according to the following formula so as to investigate the damage condition of the liver and the spleen:

liver index ═ liver weight/body weight

Spleen index ═ spleen weight/body weight

(5) Tissue distribution studies and results: in the DSF-SPC nanoparticle efficacy experiment, Dir-labeled DSF-SPC-NPs (the DSF dose is 20mg/kg, and n is 8) are injected into a single tail vein of a high-dose group of tumor-bearing mice at the last administration time, the animals are killed at 24h, and main organs are dissected to take a fluorescent photograph.

Fig. 8 is a distribution diagram of Dir-DSF-SPC-NPs in the major organs of 4T1 tumor-bearing mice (n ═ 7) after 12h administration, and the results show that the concentrations of DSF-SPC-NPs in each tissue are, from high to low: liver, spleen, tumor, kidney, heart, NPs were found to be mainly concentrated in liver, spleen, and tumor in mice. The fluorescence intensity ratio of the fluorescence in the tumor and the liver of the mouse is 20.28 +/-4.37%, which indicates that the NPs have certain passive targeting property and can reach the tumor part through the EPR effect.

(5) Research results of antitumor drug efficacy

Fig. 9A and 9B are graphs showing changes in tumor volume with time in mice in pharmacodynamic studies of DSF-SPC nanoparticles in preparation example 3 and DSF-SPC-TPGS nanoparticles in preparation example 44, respectively, fig. 10A and 10B are graphs showing changes in body weight with time in groups of mice in pharmacodynamic studies of DSF-SPC nanoparticles in preparation example 3 and DSF-SPC-TPGS nanoparticles in preparation example 44, respectively, and fig. 11 and 12 are graphs showing tumor tissues obtained by dissecting groups of mice after the pharmacodynamic studies of DSF-SPC nanoparticles in preparation example 3 and DSF-SPC-TPGS nanoparticles in preparation example 44 are completed, respectively. Tables 1 and 2 show the tumor weight, tumor inhibition rate, liver index, spleen index and other data of each group of mice at the end of pharmacodynamic study of the DSF-SPC nanoparticles in preparation example 3 and the DSF-SPC-TPGS nanoparticles in preparation example 44, respectively.

FIG. 9 shows that the tumor volume in the saline group rapidly increased to 2500mm after two weeks³Almost no antitumor effect was observed with disulfiram physical suspension (fig. 9A).

DSF-SPC-NPs showed significant tumor growth inhibition both in intravenous injection and oral administration (p <0.05 relative to physiological saline). DSF-NPs are orally administered at 20mg/kg per day, and the change of tumor volume with time and the tumor inhibition rate of the DSF-NPs are comparable to that of 8mg/kg paclitaxel injection (every other day administration) (the tumor inhibition rate is 59.03% vs 55.01%). The intravenous injection has better tumor inhibition effect, the tumor inhibition rate of the 20mg/kg dose administered every other day reaches 80.22%, and simultaneously, the injection has good dose-effect relationship (the tumor inhibition rates of medium and low concentration are respectively 75.14% and 66.10%) (Table 1).

TABLE 1 data of the effect of DSF-SPC nanoparticles on antitumor drugs of 4T1 tumor-bearing mice

(P <0.05, P <0.01vs physiological saline group; # P <0.05, # P <0.01vs blank nanoparticle group; # P <0.05, & P <0.01vs paclitaxel injection group; $ P <0.05, $ P <0.01vs5mg/kg DSF-SPC nanoparticle group)

DSF-SPC-TPGS-NPs also showed significant tumor growth inhibition (p <0.05 relative to physiological saline) in both intravenous and oral administrations. DSF-NPs are orally administered at 20mg/kg per day, and the change of tumor volume with time and the tumor inhibition rate of the DSF-NPs are comparable to that of 8mg/kg paclitaxel injection (every other day administration) (the tumor inhibition rate is 57.06% vs 55.01%). The DSF-SPC-TPGS-NPs have better tumor inhibition effect by intravenous injection, the tumor inhibition rate reaches 80% by alternate daily administration of 20mg/kg dose, and simultaneously, the dose-effect relationship is good (the tumor inhibition rates of medium and low concentrations are 74.86% and 69.20%, respectively) (Table 2).

TABLE 2 DSF-SPC-TPGS nanoparticle for anti-tumor data of 4T1 tumor-bearing mice

Tumor weight results area present as mean + -SD, n is 8; p <0.05vs. saline; blank nanoparticles, # # p <0.05 vs; and & p <0.05vs. paclitaxel injection $ $ p <0.05vs.5mg/kg DSF-SPC-TPGS-NPs

From the curve of weight change over time (fig. 10), it can be seen that the average weight of each group of mice was relatively stable and slightly increased throughout the experiment; the data of liver and spleen indexes (Table 1 and Table 2) also show that there is no significant difference (p >0.05) between the groups administered with normal saline, suggesting that the liver and spleen of mice in each group administered with normal saline are not seriously damaged, the DSF-SPC-NPs and DSF-SPC-TPGS-NPs have low toxicity and good biological safety.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The disulfiram nanoparticle is characterized by comprising the following components in percentage by mass: 0.1-5 parts of disulfiram and a stabilizer;

the stabilizer is one or a mixture of more of phospholipid material or vitamin E-polyethylene glycol succinate; the phospholipid material is one or a mixture of more of methoxy polyethylene glycol phospholipid, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, distearoyl phosphatidylcholine and dioleoyl phosphatidylcholine;

the particle size of the disulfiram nanoparticles is 100-1000 nm.

2. A disulfiram nanoparticle according to claim 1, wherein said stabilizer is a mixture of a phospholipid material and vitamin E-polyethylene glycol succinate.

3. A preparation method of disulfiram nanoparticles is characterized by comprising the following steps:

1) weighing the raw materials according to the mass ratio of any one of claims 1 to 2;

2) mixing disulfiram, a stabilizer, an organic solvent and water to obtain a precursor solution;

3) removing the organic solvent in the precursor solution to obtain disulfiram nanoparticle suspension;

4) and removing water in the disulfiram nanoparticle suspension to obtain the disulfiram nanoparticles.

4. The preparation method of the disulfiram nanoparticles according to claim 3, characterized in that the concentration of disulfiram in the precursor solution is 0.1-200 mg/mL;

the solvent is an organic solvent and water with the volume ratio of 1:1 to 100.

5. The method for preparing disulfiram nanoparticles according to claim 4, wherein the organic solvent is a first organic solvent or a mixture of the first organic solvent and a second organic solvent;

the first organic solvent is any one or a mixture of several of methanol, ethanol, acetone, acetonitrile, dimethyl sulfoxide and N, N-dimethylformamide, and the second organic solvent is any one or a mixture of several of ethyl acetate, dichloromethane and trichloromethane.

6. An antitumor drug, which is characterized by comprising 5-95% of the disulfiram nanoparticles as claimed in any one of claims 1-3.

7. The antitumor drug as claimed in claim 6, wherein the dosage form of the antitumor drug is a solid dosage form, a semisolid dosage form, a liquid dosage form or a gas dosage form.