Self-assembled paclitaxel nano-drug combined with cyclosporin A as well as preparation method and application of self-assembled paclitaxel nano-drug

Abstract

The invention discloses a cyclosporin A combined self-assembly paclitaxel nano-drug and a preparation method and application thereof, and belongs to the field of biological medicine, the cyclosporin A combined self-assembly paclitaxel nano-drug is a nano-particle which is composed of paclitaxel, cyclosporin A and zinc ions and has a regular rod-like morphology, and the particle size of the nano-particle is 1-10 micrometers. The self-assembled paclitaxel nano-drug is prepared by combining cyclosporin A on the basis of a zinc ion coordinated supramolecular self-assembly strategy, so that the stability of the nano-drug can be improved, the toxic and side effects are reduced, the killing effect on drug-resistant tumors is enhanced, the approximate biological safety is retained, and the drug-resistant tumors are prevented from being damaged. The invention provides a new thought for overcoming the current situation of drug resistance of paclitaxel, and has important research value and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a self-assembled paclitaxel nanomedicine combined with cyclosporin A, and a preparation method and application thereof. Background Art

[0002] Paclitaxel is a diterpene alkaloid compound composed of a taxane ring and an N-benzoylphenylisoserine group. It is widely used to treat various malignant tumors such as breast cancer, ovarian cancer and lung cancer. Drug resistance is one of the main reasons for the failure of paclitaxel chemotherapy.

[0003] Overexpression of P-glycoprotein (P-glycoprotein) is one of the main causes of multidrug resistance. P-glycoprotein is a key member of the ABC transporter family, mediating the efflux of various anti-tumor drugs such as doxorubicin and paclitaxel. Cyclosporin A, a cyclic decapeptide, is a commonly used immunosuppressant with strong anti-inflammatory and antifungal effects. As a P-glycoprotein external pump inhibitor, cyclosporin A inhibits P-glycoprotein's transport function after binding to it, or causes conformational changes in the P-glycoprotein molecule, reducing its affinity for drug binding. This can inhibit drug pumping out of tumor cells and play a role in combating multidrug resistance in tumors. Combining it with chemotherapy drugs can significantly improve the efficacy of chemotherapy, but cyclosporin A itself still has disadvantages such as poor oral availability and high nephrotoxicity. Summary of the Invention

[0004] To address the above problems, the present invention utilizes a zinc ion-coordinated supramolecular self-assembly strategy in combination with cyclosporin A to prepare a self-assembled paclitaxel nanodrug. This not only improves the stability of the nanodrug, but also reduces toxic side effects, enhances the killing effect on drug-resistant tumors, and retains similar biosafety. This provides a new approach to overcoming the current drug resistance of paclitaxel, and has important research value and broad application prospects.

[0005] The purpose of the present invention is achieved by adopting the following technical solutions: A self-assembled paclitaxel nanomedicine combined with cyclosporin A is a nanoparticle composed of paclitaxel, cyclosporin A and zinc ions and having a regular rod-like morphology.

[0006] In some preferred embodiments, the average particle size of the nanoparticles is 200 nm-500 nm.

[0007] In some preferred embodiments, the surface potential of the nanoparticles is -(8-10) mV.

[0008] Another object of the present invention is to provide a method for preparing the self-assembled paclitaxel nanodrug combined with cyclosporin A, comprising the following steps: (1) Dissolve paclitaxel in an organic solvent, add cyclosporine A and mix to obtain solution A; dissolve zinc salt in deionized water to obtain solution B; (2) mixing the solution A and the solution B and adjusting the pH to neutral; (3) After the reaction is fully stirred, the self-assembled paclitaxel nanodrug combined with cyclosporin A is obtained by cooling and centrifuging.

[0009] In some preferred embodiments, the molar ratio of the paclitaxel to the zinc ion in the zinc salt is 1:(1-6); the mass ratio of the paclitaxel to the cyclosporin A is 8:(1-4).

[0010] In some preferred embodiments, the organic solvent is DMSO.

[0011] In some preferred embodiments, the mixing volume ratio of the solution A to the solution B is 1:(1-3).

[0012] In some preferred embodiments, the stirring reaction has a stirring rate of 800 rpm-1000 rpm, a reaction temperature of 25° C.-60° C., and a reaction time of 30 min-60 min.

[0013] The present invention also provides an application of the self-assembled paclitaxel nanomedicine combined with cyclosporin A, specifically an application in preparing a drug for treating tumors.

[0014] In some preferred embodiments, the application includes application in the preparation of drugs for inhibiting tumor cell growth in vivo, promoting apoptosis of drug-resistant tumor cells and / or reversing tumor resistance.

[0015] The beneficial effects of the present invention are: The present invention combines zinc ion coordination supramolecular self-assembly technology with the coordinated drug delivery strategy of drug resistance reversal agents to construct a paclitaxel nanodrug with both high stability and anti-resistance function. It has a high drug loading capacity and obvious sustained-release effect, which enhances the killing effect on drug-resistant tumors, can inhibit tumor growth, promote apoptosis of drug-resistant cells and reverse tumor resistance, and has certain advantages in cellular uptake and promoting tumor cell apoptosis. At the same time, the nanodrug has no obvious adverse reactions or organ damage in mice and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is further described with reference to the accompanying drawings. However, the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative effort.

[0017] Figure 1is a TEM image of self-assembled paclitaxel nanomedicine combined with cyclosporine A prepared in Example 1; Figure 2 is a particle size distribution graph of self-assembled paclitaxel nanomedicine combined with cyclosporine A prepared in Example 1; Figure 3 is a surface potential graph of self-assembled paclitaxel nanomedicine combined with cyclosporine A prepared in Example 1; Figure 4 is a graph of the killing effect of paclitaxel nanomedicine at different concentrations on tumor cells; Figure 5 is a graph of the killing effect of paclitaxel nanomedicine at different concentrations on drug-resistant tumor cells; Figure 6 is a graph of the effect of different paclitaxel nanomedicines on inducing apoptosis of drug-resistant tumor cells; Figure 7 is a graph of the inhibitory effect of different paclitaxel nanomedicines on mouse tumors; Figure 8 is the body weight change of mice after being given different paclitaxel nanomedicines. DETAILED DESCRIPTION

[0018] The present application will be further described in conjunction with the following examples.

[0019] The embodiments of the present application relate to a self-assembled paclitaxel nanomedicine combined with cyclosporine A, which is a nanoparticle composed of paclitaxel, cyclosporine A and zinc ions, having a regular rod-like morphology; the preparation method comprises the following steps: (1) Dissolve paclitaxel in an organic solvent, add cyclosporine A to mix, to obtain solution A; dissolve zinc salt in deionized water to obtain solution B; (2) Mix the solution A and solution B, and adjust the pH to neutral; (3) After sufficient stirring and reaction, the self-assembled paclitaxel nanomedicine combined with cyclosporine A is prepared by cooling and centrifugation; In some embodiments, the average particle size of the nanoparticle is 200-500 nm, such as 200 nm, 300 nm, 400 nm, 500 nm; In some embodiments, the surface potential of the nanoparticle is - (8-10) mV, such as -8 mV, -9 mV, -10 mV; In some embodiments, the molar ratio of paclitaxel to zinc ions in the zinc salt is 1: (1-6), such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6; the mass ratio of paclitaxel to cyclosporine A is 8: (1-4), such as 8:1, 4:1, 8:3, 2:1; In some embodiments, the organic solvent is DMSO; In some embodiments, the volume ratio of the solution A to the solution B is 1:(1-3), such as 1:1, 1:2, or 1:3; In some embodiments, the stirring rate of the stirring reaction is 800-1000 rpm, such as 800 rpm, 900 rpm, 1000 rpm, the reaction temperature is 25-60°C, such as 25°C, 30°C, 40°C, 50°C, 60°C, and the reaction time is 30-60 min, such as 30 min, 40 min, 50 min, 60 min.

[0020] Example 1 This embodiment relates to a self-assembled paclitaxel nanodrug combined with cyclosporin A, and the preparation method thereof comprises the following steps: (1) Dissolve 8 mg of paclitaxel monomer in 1 mL of DMSO solvent, and add 2 mg of cyclosporine A (CsA) after dissolution to obtain solution A; dissolve 1.28 mg of zinc chloride in 1 mL of ultrapure water to obtain solution B; (2) adding the solution A to a glass reaction bottle, and then slowly adding the solution B, wherein the molar ratio of paclitaxel to zinc ion is 1:1, and adjusting the pH value of the mixed reaction system to 7 with a 1 mol / L NaOH solution. During the adjustment process, the pH value of the solution is measured in real time with a pH meter; (3) The reaction was stirred in a water bath at 37°C, with a stirring speed of 800 rpm and a reaction time of 30 min. After the reaction was completed, the mixture was subjected to refrigerated centrifugation to obtain the self-assembled paclitaxel nanodrug, which was designated as PTXNDs-CsA.

[0021] Comparative Example A paclitaxel self-assembly drug, the preparation method of which comprises the following steps: (1) Dissolve 8 mg of paclitaxel monomer in 1 mL of DMSO solvent to obtain solution A; dissolve 1.28 mg of zinc chloride in 1 mL of ultrapure water to obtain solution B; (2) adding the solution A to a glass reaction bottle, and then slowly adding the solution B, wherein the molar ratio of paclitaxel to zinc ion is 1:1, and adjusting the pH value of the mixed reaction system to 7 with a 1 mol / L NaOH solution. During the adjustment process, the pH value of the solution is measured in real time with a pH meter; (3) The reaction was stirred in a water bath at 37°C, with a stirring speed of 800 rpm and a reaction time of 30 min. After the reaction was completed, the mixture was subjected to refrigerated centrifugation to obtain the paclitaxel self-assembled drug, which was designated as PTXNDs.

[0022] Experimental example (1) Morphological characterization Take an appropriate amount of PTXNDs-CsA powder sample, dissolve it with ultrapure water and vortex mix, dilute a certain multiple, until the solution is close to clear; take 12 μL of the diluted sample, drop on 100 mesh copper mesh, dry at room temperature overnight, use transmission electron microscope (TEM) to observe, see Figure 1 , are regular and uniform rod-shaped nanoparticles.

[0023] (2) Particle size and potential characterization The particle size and surface potential of PTXNDs-CsA prepared in Example 1 were detected by dynamic light scattering particle size analyzer and Zeta potential analyzer (DLS), and the specific method was as follows: Take 0.5 mg of PTXNDs and PTXNDs-CsA samples as controls, dissolve them in 1 mL of ultrapure water, ultrasonic them to disperse uniformly, and then add them to the sample cell for particle size and Zeta potential determination, see Figures 2-3 The average particle size of the rod-shaped structure of PTXNDs-CsA prepared in Example 1 was 310.2 nm, and the surface potential was -9.33 mV.

[0024] (3) In vitro inhibition activity The in vitro inhibition activity of PTXNDs and PTXNDs-CsA on tumor cells MDA-MB-231 and drug-resistant cells MDA-MB-231 / Taxol was verified, and the specific method was as follows: MDA-MB-231 was seeded in 96-well plates at a density of 5 × 10 3 MDA-MB-231 / Taxol cells were seeded in 96-well plates at a density of 8 × 10 3 After the cells adhered, different concentrations of PTX monomer drug, self-assembled drug PTXNDs or PTXNDs-CsA were added to each well by replacing the medium and continued to be cultured, washed once with PBS buffer after 48 h, added 10% CCK-8 solution in DMEM medium and continued to be incubated, transferred to the enzyme marker to measure the absorbance at 450 nm, calculated the cell survival rate of each group, and evaluated its killing or safety.

[0025] The calculation formula was: cell viability (%) = (absorbance average value of drug group - absorbance average value of blank control group) ÷ (absorbance average value of control group - absorbance average value of blank control group) × 100%. See Figure 4For MDA-MB-231, the effect of PTXNDs-CsA is significantly better than that of the monomer and PTXNDs, and when the drug concentration reaches 20 μg / mL, the cell killing rate can reach about 90%, indicating that the nano-drug has potential in enhancing the efficiency of drug delivery, and the synergistic effect of cyclosporine A enhances the anti-tumor activity of PTXNDs; see attached Figure 5 For MDA-MB-231 / Taxol cells, with the increase of drug concentration, the effect of PTX monomer and PTXNDs in drug-resistant cells is limited, while PTXNDs-CsA shows stronger anti-drug resistance.

[0026] (4) Inducing apoptosis of drug-resistant tumor cells Flow cytometry combined with Annexin V-FITC / PI double staining was used to analyze the apoptosis of cells treated with different drugs.

[0027] MDA-MB-231 / Taxol cells were seeded in a 6-well plate at a density of 5 × 10 5 After the cells adhered, 50 nM of PTX monomer drug, nano-drug PTXNDs and PTXNDs-CsA were added to 3 wells respectively, and after 48 h of incubation, the culture medium and centrifuge tube were collected, the cells in each well were digested and centrifuged, resuspended, and each 100 μL was separately packaged as an experimental tube. 5 μL Annexin V-FITC and 10 μL PI Staining Solution were added to each experimental tube under light-proof conditions, and after gentle mixing, they were further incubated at room temperature for 10-15 min under light-proof conditions. Then 300 μL of 1 × Binding Buffer was added to each tube, mixed well and placed on ice for testing. Within 1 h, the cell clumps were filtered out using a cell filter before being loaded onto the machine; see Figure 6 For MDA-MB-231 / Taxol cells, the proportion of early apoptotic cells in the control group without drug treatment was 4.67%, the proportion of late and necrotic cells was 7.09%, and the total apoptosis rate was 11.76%. The proportion of early apoptotic cells in the PTX monomer drug treatment group was 7.35%, the proportion of late and necrotic cells was 10.30%, and the total apoptosis rate was 17.65%. The proportion of early apoptotic cells in the PTXNDs treatment group was 10.50%, the proportion of late and necrotic cells was 12.80%, and the total apoptosis rate was 23.30%. The proportion of early apoptotic cells in the PTXNDs-CsA treatment group was 13.50%, the proportion of late and necrotic cells was 14.40%, and the total apoptosis rate was 27.90%. This indicates that PTXNDs-CsA has a significant advantage in inducing apoptosis of drug-resistant tumor cells.

[0028] (5) Drug-resistant tumor animal experiment The treatment effects of PTXNDs and PTXNDs-CsA on drug-resistant tumors in mice were verified respectively, and the specific method was as follows: When the growth density of MDA-MB-231 / Taxol cells reached 90%, the cells were digested and centrifuged, resuspended with PBS buffer, diluted, and counted to adjust the cell density to 1 × 10 8 When the growth density of MDA-MB-231 / Taxol cells reached 90%, the cells were digested and centrifuged, resuspended with PBS buffer, diluted, and counted to adjust the cell density to 1 × 10

[0029] Referring to Figures 7-8 In comparison, the tumor growth rate of mice treated with PTXNDs-CsA was slower, and the body weight change was relatively stable, that is, the growth of drug-resistant tumors was significantly inhibited after the introduction of cyclosporine A, and it had good in vivo biological safety, further verifying the superiority of PTXNDs-CsA in reversing tumor drug resistance.

[0030] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A self-assembled paclitaxel nanodrug combined with cyclosporin A, characterized in that: The nanoparticles are composed of paclitaxel, cyclosporine A and zinc ions and have a regular rod-like morphology.

2. The self-assembled paclitaxel nanodrug combined with cyclosporin A according to claim 1, characterized in that: The average particle size of the nanoparticles is 200nm-500nm.

3. The self-assembled paclitaxel nanodrug combined with cyclosporin A according to claim 1, characterized in that: The surface potential of the nanoparticles is -(8-10) mV.

4. The method for preparing a self-assembled paclitaxel nanomedicine combined with cyclosporin A according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) dissolving paclitaxel in an organic solvent, adding cyclosporine A and mixing to obtain solution A; Dissolve zinc salt in deionized water to obtain solution B; (2) mixing the solution A and the solution B and adjusting the pH to neutral; (3) After the reaction is fully stirred, the self-assembled paclitaxel nanodrug combined with cyclosporin A is obtained by cooling and centrifuging.

5. The method for preparing a self-assembled paclitaxel nanodrug combined with cyclosporin A according to claim 4, characterized in that: The molar ratio of the paclitaxel to the zinc ion in the zinc salt is 1:(1-6); the mass ratio of the paclitaxel to the cyclosporin A is 8:(1-4).

6. The method for preparing a self-assembled paclitaxel nanomedicine combined with cyclosporin A according to claim 4, characterized in that: The organic solvent is DMSO.

7. The method for preparing a self-assembled paclitaxel nanomedicine combined with cyclosporin A according to claim 4, characterized in that: The mixing volume ratio of the solution A to the solution B is 1:(1-3).

8. The method for preparing a self-assembled paclitaxel nanodrug combined with cyclosporin A according to claim 4, characterized in that: The stirring reaction has a stirring rate of 800 rpm-1000 rpm, a reaction temperature of 25° C.-60° C., and a reaction time of 30 min-60 min.

9. Use of the self-assembled paclitaxel nanodrug combined with cyclosporin A according to any one of claims 1 to 3 in the preparation of a drug for treating tumors.

10. The use according to claim 9, characterized in that The invention includes application in the preparation of drugs for inhibiting tumor cell growth in vivo, promoting apoptosis of drug-resistant tumor cells and / or reversing tumor resistance.

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