阅读说明：本技术 一种药物及用途和其制备方法 (Medicine, application and preparation method thereof ) 是由 付碧石 李倩 于 2021-01-08 设计创作，主要内容包括：本发明提供了一种药物,包括如下物质作为活性成分：3-溴丙酮酸或其药学上可接受的盐和/或CPI-613或其药学上可接受的盐。本发明还提供该药物在制备具有抗病毒活性的药物中的用途。本发明进一步提供一种3-溴丙酮酸和CPI-613药物联用的复合纳米颗粒剂,本发明还提供该药物联用的复合纳米颗粒剂的制备方法。该药物在单独使用时具有抗病毒活性,两药联合使用具有协同效应,联用时,其抗病毒活性可达到1+1＞2的技术效果；该药物联用用使用复合纳米颗粒剂剂型,药物相容性好,抗病毒活性高；该复合纳米颗粒的制备方法简单易操作,所得复合纳米颗粒剂的两个活性药物成分均匀地包载于PLGA纳米粒中。(The invention provides a medicament, which comprises the following substances as active ingredients: 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof and/or CPI-613 or a pharmaceutically acceptable salt thereof. The invention also provides the application of the medicine in preparing the medicine with antiviral activity. The invention further provides a compound nano granule for combining 3-bromopyruvic acid and CPI-613 medicaments, and a preparation method of the compound nano granule for combining the medicaments. The medicine has antiviral activity when used alone, and the two medicines have synergistic effect when used together, and the antiviral activity can achieve the technical effect of 1+1 > 2 when used together; the medicine combination uses compound nanometer granule formulation, has good drug compatibility and high antiviral activity; the preparation method of the composite nano-particles is simple and easy to operate, and the two active pharmaceutical ingredients of the obtained composite nano-particles are uniformly encapsulated in the PLGA nano-particles.)

1. A medicament, characterized by comprising as active ingredients:

3-bromopyruvic acid or a pharmaceutically acceptable salt thereof and/or

CPI-613 or a pharmaceutically acceptable salt thereof.

2. The medicament of claim 1, wherein the medicament is in a dosage form selected from granules, tablets, pills, capsules and injections.

3. The medicament of claim 1, wherein the medicament is a nanoparticle formulation.

4. The medicament of claim 1, wherein 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof and CPI-613 or a pharmaceutically acceptable salt thereof are used in combination, and the active ingredients are co-formulated or separately formulated for co-administration, simultaneous administration or separate administration.

5. The pharmaceutical combination according to claim 4, in the form of a single dosage unit comprising 4-16 mg of 3-bromopyruvic acid or a pharmaceutically acceptable salt thereof and

4-16 mg of CPI-613 or a pharmaceutically acceptable salt thereof.

6. Use of a medicament as claimed in claim 1 in the manufacture of a medicament having antiviral activity.

7. A process for the preparation of a medicament according to claim 4, comprising the steps of:

step 1): dissolving fat-soluble medicines CPI-613 and PLGA into dichloromethane together to prepare an oil phase solution;

step 2): dissolving a water-soluble drug 3-bromopropanoic acid in water to prepare an aqueous phase solution;

step 3): dropwise adding the water-phase solution obtained in the step 2) into the oil-phase solution obtained in the step 1) under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

step 4): dropwise adding the first emulsion subjected to ultrasonic treatment in the step 3) into a polyvinyl alcohol (PVA) aqueous solution under a vortex condition, and then carrying out ice bath ultrasonic treatment to obtain a second emulsion;

step 5): quickly pouring the second emulsion obtained in the step 4) into a polyvinyl alcohol (PVA) aqueous solution, and accelerating stirring at the temperature of 15-35 ℃ to evaporate an organic solvent dichloromethane in the second emulsion;

step 6): centrifugally collecting the prepared nano particles at a high speed, and washing the nano particles for three times by using deionized water;

step 7): dispersing the nano particles obtained in the step 6) in a trehalose aqueous solution, freeze-drying at-50 to-80 ℃, and storing in a refrigerator at-20 ℃ for later use.

8. The method for preparing the medicine according to claim 7, wherein in the step 1), the mass ratio of the CPI-613 to the PLGA is (1-8): 100.

9. The method for preparing a pharmaceutical according to claim 7, wherein in the step 2), the concentration of 3-BPA in the aqueous solution is (1 to 100). times.10^-3mg/μL。

10. The method of claim 7, wherein in step 4), the final concentration of the first emulsion in the aqueous solution of polyvinyl alcohol (PVA) is 1-5 (v/v)%.

Technical Field

The invention belongs to the technical field of nano-drugs, and particularly relates to a drug, application and a preparation method thereof.

Background

The invasion and immunopathological effects of viruses and their derivatives on susceptible cells are the major causes of normal cytopathic effects. For years, the continuous use of nucleoside antiviral drugs has led to the emergence of drug-resistant virus strains, and the serious toxic and side effects of the drugs also limit the clinical application of the drugs. The effect of antiviral drugs is mainly achieved by inhibiting virus propagation, the virus itself cannot be damaged, otherwise host cells can be damaged, and the number of effective antiviral drugs applied to clinic at present is relatively small, which are reasons for slow development of the drugs.

In recent years, although some progress has been made in the research of medicaments for preventing and treating viral diseases, and medicaments for treating eruptive keratitis and preventing influenza A are found, the search for effective antiviral medicaments is still a current difficult task, and further research on theory and practice is needed.

The research on the treatment for blocking the energy metabolism of cells has become a research hotspot in countries such as America and Japan, wherein 3-bromopyruvate is a small molecular weight alkylating agent, is easy to dissolve in water, and the major target proteins are GADPH and HK-II (hexokinase (HK) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) are key enzymes of glycolysis); 3-bromopyruvic acid has strong affinity to GADPH and HK-II, and can completely block the activities of GADPH and HK-II under the condition of extremely low concentration. The in vivo and in vitro research results show that the compound has strong capability of inhibiting metabolism; foreign studies have simultaneously confirmed that: the 3-bromopyruvate also has the killing effect on tumor stem cells and can reverse the multi-drug resistance of the tumor cells to certain drugs.

However, 3-bromopyruvate, due to its own structural properties, is more affected by the first pass effect for systemic administration in vivo and has a shorter half-life in an aqueous environment. In the experiment, the 3-bromopyruvic acid is also found to be unstable, easy to decompose and easy to lose efficacy in a liquid state. It must be freshly prepared for each use.

On the other hand, after three years of research, the mitochondrial tricarboxylic acid cycle metabolism blocker CPI-613 of the tumor cells enters the clinic in 2013, is specially batched as an orphan drug by the US FDA when a phase II clinical experiment is not completed, is a broad-spectrum antitumor drug, and has indications including solid tumors and blood system tumors, and the phase pharmacological mechanism is as follows: the CPI-613 has a structure similar to that of vitamin B1, and can phosphorylate Pyruvate Dehydrogenase (PDH) E1 alpha after entering cell mitochondria, inactivate the phosphorylated PDH, block the metabolism of acetyl coenzyme A originated from pyruvic acid or fat metabolism and block the energy generation of the pyruvic acid or fat metabolism, and the CPI-613 can also inhibit ketoglutarate dehydrogenase complex (KGDH) and block a glutamine anaplerosis pathway, namely block glutamine from entering the metabolic energy generation of tumor cells.

However, CPI-613 is fat-soluble, easily soluble in organic solvents and hardly soluble in water, so it is difficult to completely dissolve it during use, and it can only be dissolved in organic solvents and then further diluted for use.

Therefore, it is necessary to study the novel use of 3-bromopyruvate and CPI-613, and to change the dosage form and mode of use of 3-bromopyruvate and CPI-613.

Disclosure of Invention

With the development of nanotechnology and the continuous improvement of research progress, the nanotechnology is increasingly paid more attention to the treatment of diseases by virtue of the small size effect and the surface effect of the nanoparticles. The most widely studied is the treatment of tumors by nanoparticles. Especially, PLGA nanoparticles are most widely studied as carriers for transporting drugs. It has excellent biocompatibility, easy degradation in body and other excellent characteristics. Researchers are also gradually applying them to the prevention and treatment of viral infections. For example, nano-drugs are used to carry antiviral drugs, improving the tolerance of drug delivery vehicles. Researchers have also found that certain nanoparticles have direct interference and inhibit viral replication through multivalent mechanisms or block the viral assembly process directly; nanoparticles are also used to construct non-viral delivery systems carrying small interfering rnas (sirnas), to silence gene expression in cell lines, and the like.

Based on the description of the background technology, the two drugs, namely 3-bromopyruvate and CPI-613, can block the cell metabolism of tumors, however, the two drugs, namely 3-bromopyruvate and CPI-613, have not been used for other purposes except for tumor resistance, and the two drugs, namely 3-bromopyruvate and CPI-613, have not been used together to achieve the related research on the antiviral effect, and the researchers do not put forward the research scheme for preparing the antiviral nanoparticles carrying the 3-bromopyruvate and the CPI-613.

The research combines a tumor grape glycolysis specific energy retarder 3-bromopyruvic acid, a mitochondrial aerobic oxidation specific blocker CPI-613 and a nano drug delivery system to prepare a novel composite nanoparticle and explore the antiviral property of the novel composite nanoparticle. An innovative antiviral nano platform is expected to be developed, and the novel antiviral nano platform possibly has good innovation and wide scientific research and clinical application prospects.

The present invention aims to solve at least to some extent one of the technical problems underlying the prior art, and to this end, in a first aspect of the invention, the invention provides a medicament comprising as active ingredients:

3-bromopyruvic acid or a pharmaceutically acceptable salt thereof and/or

CPI-613 or a pharmaceutically acceptable salt thereof.

In the technical scheme of the invention, the dosage form of the medicine is selected from granules, tablets, pills, capsules and injections.

In the technical scheme of the invention, the medicine is nano granules.

In the technical scheme of the invention, 3-bromopyruvic acid or pharmaceutically acceptable salt thereof and CPI-613 or pharmaceutically acceptable salt thereof are combined, and the active ingredients are prepared together or separately for compatible use, simultaneous use or separate use.

In the technical scheme of the invention, the drug combination is in a single dose unit form and comprises 4-16 mg of 3-bromopyruvic acid or pharmaceutically acceptable salt thereof and 4-16 mg of CPI-613 or pharmaceutically acceptable salt thereof.

In a second aspect of the invention, the invention provides the use of a medicament as described above in the manufacture of a medicament having antiviral activity.

In a third aspect of the present invention, the present invention also provides a preparation method of the above-mentioned medicament (composite nanoparticle), comprising the steps of:

step 1): co-dissolving fat-soluble medicine CPI-613 and PLGA in dichloromethane to prepare an oil phase solution;

step 2): dissolving a water-soluble drug 3-bromopropanoic acid in water to prepare an aqueous phase solution;

step 3): dropwise adding the water-phase solution obtained in the step 2) into the oil-phase solution obtained in the step 1) under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

step 4): dropwise adding the first emulsion subjected to ultrasonic treatment in the step 3) into a polyvinyl alcohol (PVA) aqueous solution under a vortex condition, and then carrying out ice bath ultrasonic treatment to obtain a second emulsion;

step 5): quickly pouring the second emulsion obtained in the step 4) into a polyvinyl alcohol (PVA) aqueous solution, and accelerating stirring at the temperature of 15-35 ℃ to evaporate an organic solvent dichloromethane in the second emulsion;

step 6): centrifugally collecting the prepared nano particles at a high speed, and washing the nano particles for three times by using deionized water;

step 7): dispersing the nano particles obtained in the step 6) in a trehalose aqueous solution, freeze-drying at-50 to-80 ℃, and storing in a refrigerator at-20 ℃ for later use.

In the technical scheme of the invention, in the step 1), the mass ratio of CPI-613 to PLGA is (1-8): 100.

In the technical scheme of the invention, in the step 2), the concentration of 3-BPA is (1-100) multiplied by 10^-3mg/. mu.L, preferably, the concentration of 3-BPA is (25-30). times.10^-3mg/μL。

In the technical scheme of the invention, in the step 4), the final concentration of the first emulsion in the polyvinyl alcohol (PVA) aqueous solution is 1-5 (v/v)%.

In the technical scheme of the invention, in the step 4), the ultrasonic parameters are ultrasonic amplitude of 20-50%, each time is 5-20 s, the interval is 1-10 s, and the total ultrasonic time is 0.5-3 min.

In the technical scheme of the invention, in the step 5), the concentration of the second emulsion in the polyvinyl alcohol (PVA) aqueous solution is 0.1-0.5 (v/v)%.

In the technical scheme of the invention, in the step 6), the centrifugation speed is 6000-15000 g, and the centrifugation time is 8-30 min.

The CPI-613 is 6, 8-bis (benzylthio) octanoic acid; 3-BPA is 3-bromopyruvic acid; the CPI-613+3-BPA free drug is a mixture of CPI-613 and 3-BPA as active pharmaceutical ingredients, and the CPI-613+3-BPA composite nano-particles are composite nano-particles which are co-loaded with the CPI-613 and the 3-BPA as active pharmaceutical ingredients. The 3-BPA single-drug nano-particles are PLGA nano-particles only loading 3-BPA as an active ingredient. The CPI-613 single-drug nanoparticles are PLGA nanoparticles loaded with CPI-613 as the active ingredient only.

The invention also provides a preparation method of the CPI-613 single-medicine nano-particles, which comprises the following steps:

step 1): dissolving PLGA and CPI-613 together in dichloromethane to obtain an oil phase solution;

step 2): preparing a secondary aqueous solution to obtain an aqueous solution;

step 3): dropwise adding the water phase solution obtained in the step 2) into the oil phase solution obtained in the step I under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

step 4): under the condition of vortex, dropwise adding the first emulsion obtained in the step 3) after ultrasonic treatment into a polyvinyl alcohol aqueous solution, and immediately placing the mixture into an ice bath for ultrasonic treatment to obtain a second emulsion;

step 5): after the ultrasonic treatment is finished, quickly pouring the second emulsion obtained in the step 4) into a polyvinyl alcohol aqueous solution; accelerating stirring at 15-35 ℃ to evaporate the organic solvent dichloromethane in the second emulsion;

step 6): centrifuging at 12000rpm for 20min, collecting the prepared nanoparticles, and washing with deionized water for three times;

step 7): finally, dispersing the nano particles obtained in the step 6) in the aqueous solution of trehalose again; freeze-drying the nano particles at the temperature of minus 80 to minus 50 ℃ under vacuum, and storing the nano particles in a refrigerator at the temperature of minus 20 ℃ for later use.

The invention also provides a preparation method of the 3-BPA single-drug nano-particles, which comprises the following steps:

step 1): accurately weighing 100mg of PLGA and dissolving the PLGA in 2mL of dichloromethane to obtain an oil phase solution;

step 2): accurately weighing 8mg of 3-bromopyruvic acid, and adding the 3-bromopyruvic acid into 150 mu L of secondary aqueous solution to obtain aqueous phase solution; (the ratio of carrier/drug is 100: 8)

Step 3): dropwise adding the water phase solution obtained in the step 2) into the oil phase solution obtained in the step I under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

step 4): under the condition of vortex, dropwise adding the first ultrasonic emulsion obtained in the step 3) into 4mL of 5 (w/v)% (5g of polyvinyl alcohol/100 mL of water) polyvinyl alcohol aqueous solution, and immediately placing the mixture into an ice bath for ultrasonic treatment to obtain a second emulsion;

step 5): after the ultrasonic treatment is finished, quickly pouring the second emulsion obtained in the step 4) into 0.05 (w/v)% of polyvinyl alcohol (PVA) aqueous solution; accelerating stirring at 15-35 ℃ to evaporate the organic solvent dichloromethane in the second emulsion;

step 6): centrifuging at 12000rpm for 20min, collecting the prepared nanoparticles, and washing with deionized water for three times;

step 7): finally, the nanoparticles obtained in step 6) were redispersed in 1mL trehalose (0.1 g/mL; lyoprotectant) in an aqueous solution; freeze-drying the nano particles at the temperature of minus 80 to minus 50 ℃ under vacuum, and storing the nano particles in a refrigerator at the temperature of minus 20 ℃ for later use.

The invention has the beneficial effects that:

1. the invention provides a medicament, which comprises active ingredients CPI-613 and/or 3-bromopyruvic acid, wherein both medicaments have antiviral activity, the combined treatment of the two medicaments has a synergistic effect, and the antiviral activity can achieve the technical effect that 1+1 is more than 2 when the two medicaments are combined;

2. the invention provides a CPI-613 and 3-bromopyruvic acid combined composite nanoparticle, which is used in a composite nanoparticle formulation for drug combination, and has good drug compatibility and high antiviral activity;

3. the invention provides a preparation method of CPI-613 and 3-bromopyruvic acid composite nano granules, which is simple and easy to operate, and two active pharmaceutical ingredients of the obtained composite nano granules are uniformly encapsulated in PLGA nano granules.

Drawings

FIG. 1 is a transmission electron micrograph of CPI-613+3-BPA composite nanoparticles;

FIG. 2 is a graph showing the results of quantitative detection of CPI-613 in the CPI-613+3-BPA composite nanoparticle;

FIG. 3 is a graph showing the results of quantitative detection of 3-BPA in CPI-613+3-BPA composite nanoparticles;

FIG. 4 is a graph of the results of toxicity testing of the free drug CPI-613, free drug 3-BPA and CPI-613+3-BPA composite nanoparticles against 293 cells;

FIG. 5 is a graph showing the results of verification of antiviral activity of free drug CPI-613, free drug 3-BPA and CPI-613+3-BPA free drug;

FIG. 6 is a chart showing the results of verification of antiviral activity of CPI-613 single-drug nanoparticles, 3-BPA single-drug nanoparticles, and CPI-613+3-BPA composite nanoparticles;

FIG. 7 is a graph showing the results of antiviral concentration screening of CPI-613+3-BPA composite nanoparticles;

FIG. 8 is a graphical representation of the results of comparing the antiviral activity of CPI-613+3-BPA composite nanoparticles to CPI-613+3-BPA free drug.

In the attached drawings, Free 3-BPA is Free 3-BPA; free CPI-613 is Free CPI-613; free 3-BPA + Free CPI-613 is CPI-613+3-BPA Free drug; CPI NPs are CPI-613 single-drug nanoparticles; the BPA NPs are 3-BPA single-drug nanoparticles; the BCP NPs are CPI-613+3-BPA composite nanoparticles.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. Free 3-BPA is Free 3-BPA; free CPI-613 is Free CPI-613; free 3-BPA + Free CPI-613 is CPI-613+3-BPA Free drug; CPI NPs are CPI-613 single-drug nanoparticles; the BPA NPs are 3-BPA single-drug nanoparticles; the BCP NPs are CPI-613+3-BPA composite nanoparticles.

Example 1: preparation of different nanoparticles

(1) The preparation method of the CPI-613 and 3-BPA co-supported composite PLGA nano-particles (CPI-613+3-BPA composite nano-particles) comprises the following steps:

accurately weighing 100mg PLGA and 4mg CPI-613, and dissolving the PLGA and CPI-613 together in 2mL dichloromethane to obtain an oil phase solution;

accurately weighing 4mg of 3-bromopyruvic acid, and adding the 3-bromopyruvic acid into 150 mu L of secondary aqueous solution to obtain aqueous phase solution; (the total carrier/drug ratio is 100: 8, then the two drugs are in different ratios)

Dropwise adding the water-phase solution obtained in the step II into the oil-phase solution obtained in the step I under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

fourthly, under the condition of vortex, dropwise adding the first emulsion obtained in the third step into 4mL of 5 (w/v)% aqueous solution of (5g of polyvinyl alcohol/100 mL of water) of polyvinyl alcohol, and immediately placing the mixture into an ice bath for ultrasonic treatment to obtain a second emulsion;

fifthly, after the ultrasonic treatment is finished, quickly pouring the second emulsion obtained in the step (iv) into 0.05 (w/v)% polyvinyl alcohol (PVA) aqueous solution; accelerating stirring at 15-35 ℃ to evaporate the organic solvent dichloromethane in the second emulsion;

sixthly, centrifuging at 12000rpm for 20min, collecting the prepared nano particles, and washing the nano particles for three times by using deionized water;

seventhly, re-dispersing the nano particles obtained in the step sixthly into 1mL of aqueous solution of trehalose (0.1 g/mL; freeze-drying protective agent); freeze-drying the nano particles at the temperature of between 50 ℃ below zero and 80 ℃ below zero under vacuum, and storing the nano particles in a refrigerator at the temperature of between 20 ℃ below zero for later use.

(2) The preparation method of the CPI-613-loaded PLGA nanoparticles (CPI-613 single-medicine nanoparticles) comprises the following steps:

accurately weighing 100mg PLGA and 8mg CPI-613, and dissolving the PLGA and the CPI-613 together in 2mL dichloromethane to obtain an oil phase solution;

preparing 150 mu L of secondary aqueous solution to obtain aqueous phase solution; (the ratio of carrier/drug is 100: 8)

Dropwise adding the water-phase solution obtained in the step II into the oil-phase solution obtained in the step I under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

fourthly, under the condition of vortex, dropwise adding the first emulsion obtained in the third step after ultrasonic treatment into 4mL of 5 (w/v)% (5g of polyvinyl alcohol/100 mL of water) polyvinyl alcohol aqueous solution, and immediately placing the mixture into an ice bath for ultrasonic treatment to obtain a second emulsion;

fifthly, after the ultrasonic treatment is finished, quickly pouring the second emulsion obtained in the step (iv) into 0.05 (w/v)% polyvinyl alcohol (PVA) aqueous solution; accelerating stirring at 15-35 ℃ to evaporate the organic solvent dichloromethane in the second emulsion;

sixthly, centrifuging at 12000rpm for 20min, collecting the prepared nano particles, and washing the nano particles for three times by using deionized water;

seventhly, re-dispersing the nano particles obtained in the step sixthly into 1mL of aqueous solution of trehalose (0.1 g/mL; freeze-drying protective agent); freeze-drying the nano particles at the temperature of between 50 ℃ below zero and 80 ℃ below zero under vacuum, and storing the nano particles in a refrigerator at the temperature of between 20 ℃ below zero for later use.

(3) The preparation method of the PLGA nano-particles (3-BPA single medicine nano-particles) loaded with 3-BPA comprises the following steps:

accurately weighing 100mg of PLGA and dissolving the PLGA in 2mL of dichloromethane to obtain an oil phase solution;

② accurately weighing 8mg of 3-bromopyruvic acid, adding into 150 mu L of secondary aqueous solution to obtain aqueous phase solution; (the ratio of carrier/drug is 100: 8)

Dropwise adding the water-phase solution obtained in the step II into the oil-phase solution obtained in the step I under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

fourthly, under the condition of vortex, dropwise adding the first emulsion obtained in the third step after ultrasonic treatment into 4mL of 5 (w/v)% (5g of polyvinyl alcohol/100 mL of water) polyvinyl alcohol aqueous solution, and immediately placing the mixture into an ice bath for ultrasonic treatment to obtain a second emulsion;

fifthly, after the ultrasonic treatment is finished, quickly pouring the second emulsion obtained in the step (iv) into 0.05 (w/v)% polyvinyl alcohol (PVA) aqueous solution; accelerating stirring at 15-35 ℃ to evaporate the organic solvent dichloromethane in the second emulsion;

sixthly, centrifuging at 12000rpm for 20min, collecting the prepared nano particles, and washing the nano particles for three times by using deionized water;

seventhly, re-dispersing the nano particles obtained in the step sixthly into 1mL of aqueous solution of trehalose (0.1 g/mL; freeze-drying protective agent); freeze-drying the nano particles at the temperature of between 50 ℃ below zero and 80 ℃ below zero under vacuum, and storing the nano particles in a refrigerator at the temperature of between 20 ℃ below zero for later use.

(4) The preparation method of the PLGA empty carrier nano-particles comprises the following steps:

accurately weighing 100mg of PLGA and dissolving the PLGA in 2mL of dichloromethane to obtain an oil phase solution;

preparing 150 mu L of secondary aqueous solution to obtain aqueous phase solution;

dropwise adding the water-phase solution obtained in the step II into the oil-phase solution obtained in the step I under a vortex condition, and carrying out ultrasonic treatment under an ice bath condition to obtain a first emulsion;

under the condition of vortex, dropwise adding 4mL of the first emulsion obtained in the step (III) after ultrasonic treatment into 5% ((w/v) 5g of polyvinyl alcohol/100 mL of aqueous polyvinyl alcohol solution, and immediately placing the mixture into an ice bath for ultrasonic treatment to obtain a second emulsion;

fifthly, after the ultrasonic treatment is finished, quickly pouring the second emulsion obtained in the step (iv) into 0.05 (w/v)% polyvinyl alcohol (PVA) aqueous solution;

accelerating stirring at 15-35 ℃ to evaporate the organic solvent dichloromethane in the second emulsion;

sixthly, centrifuging at 12000rpm for 20min, collecting the prepared nano particles, and washing the nano particles for three times by using deionized water;

seventhly, re-dispersing the nano particles obtained in the step sixthly into 1mL of aqueous solution of trehalose (0.1 g/mL; freeze-drying protective agent); freeze-drying the nano particles at the temperature of between 50 ℃ below zero and 80 ℃ below zero under vacuum, and storing the nano particles in a refrigerator at the temperature of between 20 ℃ below zero for later use.

And (3) observing the appearance of the CPI-613+3-BPA composite nano-particles synthesized in the step (1) by using a transmission electron microscope. Prior to analysis, the CPI-613+3-BPA composite nanoparticles were subjected to a vacuum platinum-blasting treatment. A transmission electron micrograph of the CPI-613+3-BPA composite nanoparticles is shown in FIG. 1. As can be seen from FIG. 1, the composite nanoparticles have uniform morphology and sizes of about 50-150 nm.

In order to further verify whether two medicines (CPI-613 and 3-BPA) are loaded in the PLGA nanoparticles, the CPI-613 and 3-BPA medicines in the composite nanoparticles are detected by a high performance liquid quantitative method and a pyruvic acid kit quantitative method respectively. The detection results are shown in FIG. 2, wherein FIG. 2 shows the high performance liquid chromatography test results of CPI-613, the characteristic peak with the retention time of 9.287min is the characteristic peak of CPI-613, and the content of CPI-613 is calculated by an external standard method; FIG. 3 is a plot of 3-BPA against background subtraction values obtained by measuring absorbance at different concentrations (we use PLGA nanoparticles coated with CPI-613 as background). Meanwhile, the absorbance value of 3-BPA in the supernatant of the CPI-613+3-BPA composite nanoparticle in the synthesis process is 0.03467 (after background subtraction) by using the subtraction method, the value is substituted into a formula obtained by standard curve within the obtained standard curve range, the concentration of 3-BPA in the supernatant is 72.7 mu g/mL, and the encapsulation rate of 3-BPA in the composite nanoparticle is 16.39% by calculating through the subtraction method. As can be seen from the detection results, the two drugs are uniformly encapsulated in the PLGA nanoparticles.

Example 2: MTT toxicity test of free CPI-613 with different concentrations, free 3-BPA with different concentrations and CPI-613+3-BPA composite PLGA nano-particles with different concentrations on 293 cells

Step 1): 293 cells were seeded in 96-well plates at a cell concentration of 1X 10 per well⁴And then cultured overnight.

Step 2): the next day, cell supernatants were aspirated and cells were washed three times with PBS solution.

Step 3): the cells of the experimental group were added with different concentrations (CPI-613 concentration was 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 160. mu.M) of composite nanoparticles per well (when composite nanoparticles were prepared by the method of (1) composite nanoparticle preparation in example 1, equal amounts of CPI-613 and 3-bromopyruvic acid were used), different concentrations of free CPI-613 (20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 160. mu.M), and different concentrations of free 3-BPA (20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 160. mu.M), and the incubation was continued for 8 hours and 16 hours, respectively.

Step 4): after the pre-designed time point was reached, the drug-containing medium was removed and the cells were washed three times with PBS solution. mu.L of MTT solution (0.5mg/ml) was added to each well.

Step 5): after further incubation for 4h, the medium was removed and 200. mu.L of DMSO solution was added to each well. And placing the pore plate in a shaking box, and shaking at low speed of 150rpm for 15min to completely dissolve the generated purple crystals.

Step 6): the Optical Density (OD) of each well was measured with a multifunctional microplate reader. Untreated cells served as negative controls and 1% Triton X-100 solution (v/v) treated cells served as positive controls, with 5 biological replicates per group.

The results of MTT test with different concentrations of free CPI-613 added, 8h and 16h of culture are shown in FIG. 4a, the results of MTT test with different concentrations of free 3-BPA added, 8h and 16h of culture are shown in FIG. 4b, the results of MTT test with 8h of culture are shown in FIG. 4c, the results of CPI-613+3-BPA composite nanoparticles added, and the results of MTT test with 16h of culture are shown in FIG. 4d, and it can be seen from the figure that the CPI-613+3-BPA composite nanoparticles have no obvious toxic and side effects on 293 cells, and especially have no obvious toxicity when the drug concentration is less than 80 μ M. Therefore, the concentration less than the concentration range is selected as the antiviral drug concentration.

Example 3: verification of antiviral Activity of free CPI-613, free 3-Bromopyruvic acid, CPI-613+ 3-Bromopyruvic acid free drug

The verification of the antiviral activity of free CPI-613, free 3-bromopyruvic acid and CPI-613+ 3-bromopyruvic acid free drugs comprises the following steps:

step 1): 293 cells were seeded in 24-well plates and cultured overnight.

Step 2): the next day, cell supernatants were aspirated and cells were washed three times with PBS solution.

Step 3): preparing a virus suspension: mu.L of vaccinia virus (VACV) suspension containing the luciferase reporter was added to 10 ml DMEM. The prepared virus suspension was then added to a 24-well plate at 400. mu.L per well. Culturing in an incubator for 2 h.

Step 4): after incubation to the set time point, the well plates were charged with free CPI-613 (40. mu.M), free 3-bromopyruvate (40. mu.M), free CPI-613 (40. mu.M) + free 3-bromopyruvate (40. mu.M) (containing equal amounts of CPI-613: 40. mu.M, 3-bromopyruvate: 40. mu.M, respectively)

Step 5): after further incubation for 16h, the drug-containing medium was removed and the cells were washed three times with PBS solution. 50 μ L of TAP lysate was added to each well.

Step 6): after cell lysis, the cells were centrifuged at 15000rpm for 10min, and the supernatant was collected and assayed.

Step 7): the bioluminescence intensity of each well was measured with a multifunctional microplate reader.

The test results are shown in fig. 5, from which it can be seen that the blank medium DMEM has no antiviral activity, both free drugs have a certain antiviral activity at a concentration of 40 μ M, and the antiviral activity is significantly enhanced when both drugs are used simultaneously.

Example 4: verification of antiviral Activity of empty PLGA vectors (Blank PLGA NPs), CPI-613 Single drug nanoparticles, 3-Bromopyruvic acid Single drug nanoparticles and CPI-613+3-BPA composite nanoparticles

Verification of antiviral activity of empty PLGA carriers (Blank PLGA NPs), CPI-613 single-drug nanoparticles, 3-bromopyruvate single-drug nanoparticles and CPI-613+ 3-bromopyruvate composite nanoparticles, comprising the steps of:

step 1): 293 cells were seeded in 24-well plates and cultured overnight.

Step 2): the next day, cell supernatants were aspirated and cells were washed three times with PBS solution.

Step 3): preparing a virus suspension: mu.L of vaccinia virus (VACV) suspension containing the luciferase reporter was added to 10 ml DMEM. The prepared virus suspension was then added to a 24-well plate at 400. mu.L per well. Culturing in an incubator for 2 h.

Step 4): after incubation to a set time point, PLGA carriers (Blank PLGA NPs), CPI-613 single drug nanoparticles (40. mu.M), 3-bromopyruvate single drug nanoparticles (40. mu.M) and CPI-613+3-BPA composite nanoparticles (CPI-613 (40. mu.M) and 3-bromopyruvate (40. mu.M)) were added to the well plate (containing equal amounts of CPI-613: 40. mu.M and 3-bromopyruvate: 40. mu. M, PLGA, respectively, in the same amount as the PLGA content in the composite nanoparticles, and the total drug loading of the composite nanoparticles was 40. mu.M CPI-613.)

Step 5): after further incubation for 16h, the drug-containing medium was removed and the cells were washed three times with PBS solution. 50 μ L of TAP lysate was added to each well.

Step 6): after cell lysis, the cells were centrifuged at 15000rpm for 10min, and the supernatant was collected and assayed.

Step 7): the bioluminescence intensity of each well was measured with a multifunctional microplate reader.

The test results are shown in fig. 6, from which it can be seen that the empty PLGA carrier (DMEM) has no antiviral activity, the CPI-613 single-drug nanoparticles, the 3-bromopyruvate single-drug nanoparticles and the CPI-613+ 3-bromopyruvate composite nanoparticles all have better antiviral activity at a concentration of 40 μ M, and the CPI-613+ 3-bromopyruvate composite nanoparticles have significantly better antiviral activity than the two single-drug nanoparticles.

Example 5: concentration screening of CPI-613+ 3-bromopyruvic acid composite nanoparticle antiviral activity

Concentration screening based on the antiviral activity of the CPI-613+ 3-bromopyruvic acid composite nanoparticle comprises the following steps:

step 1): 293 cells were seeded in 24-well plates and cultured overnight.

Step 2): the next day, cell supernatants were aspirated and cells were washed three times with PBS solution.

Step 3): preparing a virus suspension: mu.L of vaccinia virus (VACV) suspension containing the luciferase reporter was added to 10 ml DMEM. The prepared virus suspension was then added to a 24-well plate at 400. mu.L per well. Culturing in an incubator for 2 h.

Step 4): after incubation to a set time point, CPI-613+ 3-bromopyruvate composite nanoparticles (CPI-613 and 3-bromopyruvate were used in equal amounts when CPI-613 and 3-bromopyruvate were prepared as described in example 1 (1) preparation of CPI-613+ 3-bromopyruvate composite nanoparticles) were added to the well plate to achieve different final CPI-613 gradient concentrations (5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 80. mu.M CPI-613)

Step 5): after further incubation for 16h, the drug-containing medium was removed and the cells were washed three times with PBS solution. 50 μ L of TAP lysate was added to each well.

Step 6): after cell lysis, the cells were centrifuged at 15000rpm for 10min, and the supernatant was collected and assayed.

Step 7): the bioluminescence intensity of each well was measured with a multifunctional microplate reader.

The test results are shown in FIG. 7, from which it can be seen that the antiviral ability of the CPI-613+ 3-bromopyruvate composite nanoparticle increases with the increase of the drug concentration thereof, and that the relative amount of the virus is reduced by half when the CPI-613 content of the CPI-613+ 3-bromopyruvate composite nanoparticle is 20. mu.M. The CPI-613+ 3-bromopyruvic acid composite nano-particle has stronger antiviral property.

Example 6: comparison test of the antiviral activity of the CPI-613+ 3-bromopyruvic acid composite nanoparticle and the antiviral activity of the CPI-613+ 3-bromopyruvic acid free drug (the CPI-613 concentration is 40 mu M; the 3-bromopyruvic acid concentration is 40 mu M)

Comparing the antiviral activity of CPI-613 (40. mu.M) + 3-bromopyruvate (40. mu.M) free drug in example 3 with that of CPI-613+ 3-bromopyruvate complexed nanoparticles (40. mu.M) in example 4, the results are shown in FIG. 8a, where it can be seen that the antiviral ability of CPI-613+ 3-bromopyruvate complexed nanoparticles is superior to that of CPI-613+ 3-bromopyruvate free drug. The reason for this may be that the nanoparticles can enhance the stability of CPI-613 and 3-bromopyruvic acid in the composite use, so that the compatibility is good and the antiviral activity is better.

Example 7: test for comparing antiviral activity of CPI-613+ 3-bromopyruvic acid composite nanoparticle with antiviral activity of CPI-613+ 3-bromopyruvic acid free drug

The antiviral capacity of the CPI-613+ 3-bromopyruvic acid composite nanoparticle is compared with that of a CPI-613+ 3-bromopyruvic acid free drug, and the method comprises the following steps of:

step 1): 293 cells were seeded in 24-well plates and cultured overnight.

Step 2): the next day, cell supernatants were aspirated and cells were washed three times with PBS solution.

Step 3): preparing a virus suspension: mu.L of vaccinia virus (VACV) suspension containing the luciferase reporter was added to 10 ml DMEM. The prepared virus suspension was then added to a 24-well plate at 400. mu.L per well. Culturing in an incubator for 2 h.

Step 4): after incubation to a set time point, CPI-613+ 3-bromopyruvate composite nanoparticles, free CPI-613+ free 3-bromopyruvate (containing equal amounts of CPI-613: 20. mu.M, 3-bromopyruvate: 20. mu.M, and total loading of composite nanoparticles to 20. mu.M CPI-613) were added to the well plate, respectively

Step 5): after further incubation for 16h, the drug-containing medium was removed and the cells were washed three times with PBS solution. 50 μ L of TAP lysate was added to each well.

Step 6): after cell lysis, the cells were centrifuged at 15000rpm for 10min, and the supernatant was collected and assayed.

Step 7): the bioluminescence intensity of each well was measured with a multifunctional microplate reader.

The results are shown in FIG. 8b, which also shows that the antiviral ability of CPI-613+ 3-bromopyruvate composite nanoparticles is superior to that of CPI-613+ 3-bromopyruvate free drug.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.