Preparation method of platinum nano-enzyme drug-loaded CX4945 delivery system as well as product and application of platinum nano-enzyme drug-loaded CX4945 delivery system

Abstract

The invention discloses a preparation method of a platinum nano-enzyme drug-loaded CX4945 delivery system as well as a product and application of the platinum nano-enzyme drug-loaded CX4945 delivery system. A platinum nano-enzyme drug-loaded CX4945 nano delivery system can enable CX4945 to be slowly released, so that the half-life period of the drug is prolonged; the compound has POD activity and can regulate the level of active oxygen in cells, amplify oxidative stress pressure of tumor cells and effectively inhibit proliferation of T-ALL cells. Phosphorylation of IKAROS can be reduced through the action of CX4945, the normal function of IKAROS can be recovered again, the inhibition effect of IKAROS on downstream carcinogenic target genes can be better recovered, cooperative killing of T-ALL cells is achieved in combination with ROS generated by platinum nanoparticles, and the treatment effect of the medicine is enhanced.

Description

Technical Field

[0001] The present invention relates to a preparation method, product and application of a platinum nanozyme drug-loaded CX4945 delivery system, belonging to the field of platinum nanozyme drug-loaded delivery systems. Background Art

[0002] Acute leukemia is a highly heterogeneous hematological malignancy, which can be divided into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). ALL can be further classified into acute B-lymphoblastic leukemia (B-ALL) and acute T-lymphoblastic leukemia (T-ALL). With the continuous improvement of treatment technologies, the treatment of T-ALL, especially in adult patients, has not been significantly improved. In particular, targeted therapy for relapsed and refractory T-ALL remains an urgent problem in clinical treatment. At present, the development of new drugs and new materials for the treatment of T-ALL is extremely slow compared with other hematological malignancies. Therefore, it is urgent to develop new, efficient and low-toxic drugs, improve the treatment methods of patients, and prolong the survival of patients.

[0003] The CK2 inhibitor CX4945 drug shows good prospects for the treatment of acute leukemia. It can competitively bind to the CK2α subunit with ATP, prevent their binding, and inhibit the activity of CK2. CK2 can phosphorylate IKAROS, which causes abnormal transcriptional regulatory functions of IKAROS, and then leads to abnormalities in cell cycle, cell sublocalization, DNA binding ability, chromatin remodeling, etc., ultimately causing leukemia. CX4945 can restore its normal function by reducing IKAROS phosphorylation. Our research found that genome-wide DNA binding and gene expression analysis treated with CX4945 showed that IKAROS has a strong binding affinity with the promoters of downstream oncogenic target genes. IKAROS can reduce the expression of downstream oncogenic target genes at the mRNA level and protein level, and shows a dose-dependent relationship with the CX4945 drug concentration. However, as a small molecule drug, CX4945 has low bioavailability and fast degradation rate, thus limiting its wide application in the treatment of hematological malignancies.

[0004] Nanozymes are a class of nanomaterials with excellent enzyme-like catalytic activities. The progress in this field is expected to provide new ideas for solving clinical problems and developing new treatment regimens. Platinum (Pt) nanozymes have been shown to possess multiple enzyme-like activities related to redox pathways, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and oxidase (OXD). Pt nanozymes can mimic OXD activity to catalyze the conversion of glucose into gluconic acid and hydrogen peroxide. In the microenvironment enriched with OXD reaction products, the low pH provided by gluconic acid enables Pt nanozymes to exhibit POD activity, catalyzing the OXD reaction product H2O2 to form highly toxic hydroxyl radicals to amplify the oxidative stress in tumor cells, thereby inhibiting tumor growth. Chemodynamic therapy based on nanozymes is an emerging form of tumor treatment that utilizes catalytic reactions induced by nanozymes to convert endogenous chemicals in tumors (such as H2O2) into reactive oxygen species (ROS) to kill tumor cells. This therapy has minor side effects, requires no external energy (such as light energy and thermal energy), and has a strong inhibitory effect on drug-resistant tumor cells. Therefore, it is considered an effective preclinical malignant tumor treatment strategy. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a preparation method, product, and application of a platinum nanozyme-loaded CX4945 delivery system with low toxicity and side effects and delayed drug metabolism.

[0006] Technical Solution: To solve the above technical problem, the present invention provides a method for preparing a platinum nanozyme-loaded CX4945 delivery system, comprising the following steps: Dissolve the CX4945 drug and poly(lactic-co-glycolic acid)-polyethylene glycol-thiol in tetrahydrofuran, then add it to an aqueous solution of polyvinylpyrrolidone, and perform ice bath and ultrasonic fragmentation; Stir, centrifuge, and filter through a membrane; Then add chloroplatinic acid solution, stir, and remove the excess chloroplatinic acid; Then add sodium borohydride reducing agent, and after the reaction is completed, remove the excess reducing agent to obtain the platinum nanozyme-loaded CX4945 delivery system.

[0007] Among them, the molecular weight of the poly(lactic-co-glycolic acid)-polyethylene glycol is 10K - 12K.

[0008] Among them, the mass ratio of chloroplatinic acid to CX4945 is 1:0.5 - 1, and the mass ratio of sodium borohydride reducing agent to chloroplatinic acid is 1:0.5 - 1.

[0009] Among them, the reaction time after adding chloroplatinic acid is 15 - 30 min.

[0010] Among them, the reduction time using sodium borohydride is 20 - 30 min.

[0011] Among them, the power of the ultrasonic disruption is 120 - 250 W, the working time is 2 - 5 min, the ultrasonic on-time is 5 - 10 s, and the ultrasonic off-time is 2 - 5 s.

[0012] Among them, the stirring and volatilization speed is 300 - 500 rpm, and the stirring time is 2 - 5 h. After the reaction, ultrafiltration is carried out. The molecular weight of the ultrafiltration centrifuge tube is 30 - 100 KDa, the centrifugation time is 8 - 10 min, the centrifugation speed is 4000 - 5000 rpm, and the number of centrifugation washes is 3 - 5 times. After the reaction, ultrafiltration is carried out. The molecular weight of the ultrafiltration centrifuge tube is 30 - 100 KDa, the centrifugation time is 8 - 10 min, the centrifugation speed is 4000 - 5000 rpm, and the number of centrifugation washes is 3 - 5 times. The adsorption reaction time of chloroplatinic acid is 15 - 30 min, and the ultrasonic working frequency is 20 - 30 Hz. The reduction time of sodium borohydride is 20 - 30 min.

[0013] The present invention also provides a platinum nanozyme-loaded CX4945 delivery system prepared by the above method.

[0014] Among them, the platinum nanozyme-loaded CX4945 delivery system contains CX4945 nanoparticles wrapped with poly(lactic-co-glycolic acid)-polyethylene glycol-thiol (CX4945@PLGA-PEG) and platinum nanoparticles connected thereto; the platinum nanoparticles are formed by in-situ reduction of adsorbed platinum ions by the thiols exposed at the outer end of the surface of CX4945@PLGA-PEG.

[0015] Among them, the nano-particle size of the platinum nanozyme-loaded CX4945 delivery system is 70 - 100 nm.

[0016] The present invention also provides the application of the platinum nanozyme-loaded CX4945 delivery system in the preparation of a drug for treating acute leukemia.

[0017] The present invention also provides a drug for treating acute leukemia, which contains the platinum nanozyme-loaded CX4945 delivery system.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0019] 1. The platinum nanozyme-loaded CX4945 nano-delivery system can enable the slow release of CX4945 and prolong the half-life of the drug;

[0020] 2. The platinum nanozyme-loaded CX4945 nano-delivery system has POD activity, can regulate the level of intracellular reactive oxygen species (ROS), amplify the oxidative stress pressure of tumor cells, effectively inhibit the proliferation of T-ALL cells, and is expected to lay a theoretical foundation for solving clinical problems and developing new treatment regimens;

[0021] 3. The platinum nanozyme-loaded CX4945 nano-delivery system can not only reduce the phosphorylation of IKAROS through the action of CX4945 and restore its normal function, better restoring the inhibitory effect of IKAROS on downstream carcinogenic target genes, but also combine with the ROS generated by platinum nanoparticles to achieve synergistic killing of T-ALL cells and enhance the therapeutic effect of the drug.

[0022] 4. Poly(lactic-co-glycolic acid)-polyethylene glycol-thiol (PLGA-PEG-SH), as a coating polymer material, exhibits excellent comprehensive properties. Due to its combination of hydrophilic and hydrophobic characteristics, it can effectively bind to many drugs and effectively delay the drug release rate, prolonging the action time of the drug in the body. In addition, its outstanding biocompatibility enables it to reduce the stimulation to the organism while reducing the occurrence of immune reactions. Brief Description of the Drawings

[0023] Figure 1 Schematic diagram of the preparation mechanism of the platinum nanozyme-loaded CX4945 delivery system CX4945@PLGA-PEG@Pt NPs;

[0024] Figure 2 Morphologies of CX4945@PLGA-PEG@Pt NPs and PLGA-PEG@Pt NPs under transmission electron microscope.

[0025] Figure 3 Particle size distribution diagram of CX4945@PLGA-PEG@Pt NPs;

[0026] Figure 4 Changes in hydrodynamic size (A), PDI (B), and Zeta potential (C) of CX4945@PLGA-PEG@Pt NPs stored at 4°C in distilled water for 7 days;

[0027] Figure 5 Images of the blue oxidation product generated by the catalytic oxidation reaction of TMB by CX4945@PLGA-PEG@Pt NPs, PLGA-PEG@Pt NPs, and CX4945@PLGA-PEG NPs for 10 min and the ultraviolet absorption spectra in the range of 400 nm to 1000 nm;

[0028] Figure 6 Enzymatic catalytic activity assays of CX4945@PLGA-PEG@Pt NPs, PLGA-PEG@Pt NPs, and CX4945@PLGA-PEG NPs at different concentrations;

[0029] Figure 7Hemolysis assay of red blood cells with different concentrations of CX4945@PLGA-PEG@Pt NPs, PLGA-PEG@Pt NPs and CX4945@PLGA-PEG NPs;

[0030] Figure 8 Cell viability of CEM cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG NPs and CX4945@PLGA-PEG@Pt NPs for 48 h;

[0031] Figure 9 Cell viability of CEM cells after incubation with different concentrations of PLGA-PEG@Pt NPs and CX4945@PLGA-PEG@Pt NPs for 48 h;

[0032] Figure 10 Cell viability of MOLT4 cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG NPs and CX4945@PLGA-PEG@Pt NPs for 48 h;

[0033] Figure 11 Cell viability of MOLT4 cells after incubation with different concentrations of PLGA-PEG@Pt NPs and CX4945@PLGA-PEG@Pt NPs for 48 h;

[0034] Figure 12 Cell viability of NALM6 cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG NPs and CX4945@PLGA-PEG@Pt NPs for 48 h;

[0035] Figure 13 Cell viability of NALM6 cells after incubation with different concentrations of PLGA-PEG@Pt NPs and CX4945@PLGA-PEG@Pt NPs for 48 h. Detailed implementation mode

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0037] Example 1 Preparation of platinum nanozyme drug-loaded CX4945 delivery system

[0038] As Figure 1 shown, the specific preparation process is as follows:

[0039] (1) Preparation of CX4945@PLGA-PEG nano-drug

[0040] Accurately weigh 1 mg of CX4945 drug and 20 mg of poly (lactic-co-glycolic acid)-polyethylene glycol-thiol PLGA(10K)-PEG(2K)-SH (Xi'an Ruixi Biotechnology Co., Ltd.; R-PLS-010) (PLGA-PEG-SH), ultrasonically dissolve them in 500 μL of tetrahydrofuran, then add them to 10 mL of an aqueous solution containing 2% polyvinylpyrrolidone (PVP K30), and perform ice bath and ultrasonic fragmentation (parameters of the ultrasonic crusher: working time is 3 min, ultrasonic on time is 10 s, ultrasonic off time is 2 s, power is 195 W). Then transfer it into a 50 mL three-necked flask and mechanically stir at 400 rpm for 4 h. Centrifuge at 4000 rpm for 10 min (ordinary centrifugation to remove larger-sized nano-drugs), centrifuge at 4000 rpm for 10 min (centrifugation with a 100 KD ultrafiltration tube to remove free CX4945 or unexcessive PLGA-PEG and PVPK30), wash 3 times with ddH2O, and filter through a 0.45 μm filter membrane to obtain yellow CX4945@PLGA-PEG nano-drugs.

[0041] (2) In-situ reduction and connection of Pt nanoparticles

[0042] Add 0.5 mL of 10 mg / mL chloroplatinic acid solution to the CX4945@PLGA-PEG system synthesized in step (1), adsorb platinum ions through the thiol groups exposed at the outer end of the polymer material surface, stir at a speed of 200 rpm, react for 20 min, and set the ultrasonic to 220 V with a working frequency of 25 Hz. After the reaction, remove the excessive chloroplatinic acid with a 100 KD ultrafiltration tube and repeat the ultrafiltration 3 times. Then add 5 mg of sodium borohydride reducing agent, stir at a speed of 500 rpm, react for 5 min, and set the ultrasonic to 220 V with a working frequency of 40 Hz. After the reaction, place it on ice for 20 min, and finally remove the excessive reducing agent with a 100 KD ultrafiltration tube to obtain a composite system CX4945@PLGA-PEG@Pt in which Pt nanoparticles are in-situ reduced and formed on the surface of the polymer nanoparticles, filter through a 0.45 μm filter membrane to remove larger particles.

[0043] Example 2 Characterization of the platinum nanozyme drug-loaded CX4945 delivery system

[0044] The solutions of the platinum nanozyme drug-loaded CX4945 delivery system prepared in Example 1 all showed black, with no precipitation observed and relatively uniform dispersion.

[0045] Table 1 Particle size, Zeta potential, and PDI distribution of PLGA-PEG@Pt NPs and CX4945@PLGA-PEG@Pt NPs

[0046]

[0047] Drop CX4945@PLGA-PEG@Pt NPs and PLGA-PEG@Pt NPs onto copper grids respectively, and observe the morphology with a transmission electron microscope. All nanoparticles are spherical ( Figure 2 ), and platinum nanoparticles are adsorbed on the spherical surface. In addition, as shown in Table 1, the particle sizes of both PLGA-PEG@Pt NPs and CX4945@PLGA-PEG@Pt Nps are distributed around 83 nm, and the distribution peak shape is a single peak ( Figure 3 ), indicating that the prepared nanoparticles are relatively uniform and stable. The Zeta potential shows a negative potential, which can inhibit the mutual aggregation between nanoparticles and enhance the stability of the solution. The charge range is -23.8 to -25.5 mV. In addition, the PDI of both nanoparticles is less than 0.2, indicating a uniform particle size distribution. In the research and development of intravenous drug delivery formulations, a uniform particle size distribution is of great significance, which lays a good foundation for the stability of the formulation and the effective exertion of drug efficacy.

[0048] Example 3 Determination of the stability of the CX4945@PLGA-PEG@Pt Nps delivery system

[0049] During the drug delivery process, the stability of the drug delivery system plays a crucial role in ensuring the safety and effectiveness of the drug delivery process. Specifically, for the CX4945@PLGA-PEG@Pt Nps delivery system, it is dispersed in H2O, and then under the environmental conditions of 4 °C, sampling operations are carried out according to the preset sampling time points. After each sampling is completed, the hydrodynamic size ( Figure 4 A), PDI ( Figure 4 B), and Zeta potential ( Figure 4 C) changes of the drug delivery system are detected and analyzed by the DLS (dynamic light scattering) detection method. As Figure 4 shown, the hydrodynamic size diameter of CX4945@PLGA-PEG@Pt NPs in H2O for 7 days is 85.050 ± 5.463 to 82.297 ± 3.314 nm, the PDI is 0.111 ± 0.008 to 0.128 ± 0.011, and the Zeta potential is -22.167 ± 0.416 to 25.533 ± 0.252 mv. It shows that CX4945@PLGA-PEG@Pt NPs can remain stable under the condition of storing at 4 °C for 7 days.

[0050] Example 4 Determination of the peroxidase-like activity of CX4945@PLGA-PEG@Pt Nps

[0051] The reaction system includes the following: (1) 948 μL of acetic acid-sodium acetate buffer solution (pH = 3.6) as the catalytic medium; (2) 32 μL of 1% H2O2; (3) 10 μL of 10 mg / mL TMB as the catalytic substrate; (4) 10 μL of the sample to be tested. After adding the above reaction system, the ultraviolet absorption value of the formed TMB blue product was measured at 652 nm to study the peroxidase-like activity of CX4945@PLGA-PEG@Pt Nps in catalyzing the oxidation of TMB. As Figure 5 shown, CX4945@PLGA-PEG@Pt Nps and PLGA-PEG@Pt Nps have strong peroxidase-like activity, can rapidly catalyze the oxidation of the TMB substrate to generate a blue oxidation product, and have a strong absorption peak at 652 nm wavelength.

[0052] In addition, Figure 6 the enzymatic catalytic activity of the CX4945@PLGA-PEG@Pt Nps delivery system shown increases continuously with the increase of platinum concentration, and PLGA-PEG@Pt Nps also has the same peroxidase-like activity. The above data all indicate that the platinum nanozyme delivery system has good peroxidase-like activity.

[0053] Example 5 Determination of the blood compatibility of CX4945@PLGA-PEG@Pt Nps

[0054] Mouse whole blood was centrifuged at 400 g for 5 min, and pure red blood cells were obtained by washing with PBS solution. Set the sample groups: (1) Positive control: 800 μL of H2O + 200 μL of red blood cells; (2) Negative control: 800 μL of PBS + 200 μL of red blood cells; (3) Experimental group: 800 μL of different concentrations of materials (the Pt concentration in CX4945@PLGA-PEG@Pt Nps and PLGA-PEG@Pt Nps is 5, 25, 50, 100 μg / mL; the CX4945 concentration in CX4945@PLGA-PEG Nps is 5, 25, 50, 100 μg / mL) + 200 μL of red blood cells. After adding the samples, they were incubated at 37 °C for 3 h, then centrifuged at 800 g for 5 min, and 100 μL of the supernatant was taken into a 96-well plate, and the ultraviolet absorption value was measured at 540 nm. The results are as Figure 7 shown, no obvious red blood cell hemolysis phenomenon was observed after the nano-drug acted for 3 h, indicating that the platinum nanozyme delivery system has good biocompatibility.

[0055] Example 6 Killing effect of CX4945@PLGA-PEG@Pt Nps on T-ALL cells

[0056] The CCK8 kit was used to detect the effect of the platinum nanozyme delivery system on the activity of T-ALL cells. CEM cells were cultured until the exponential growth phase, and the density of the cell suspension was adjusted to reach 2×10 4 cells / well and seeded in 96-well plates, with 50 μL of cell suspension added to each well. Then, 50 μL of CX4945, CX4945@PLGA-PEG Nps, PLGA-PEG@Pt Nps, or CX4945@PLGA-PEG@Pt Nps solution with different concentration gradients was added respectively (the equivalent final concentration of CX4945 was set to 0, 1.5, 3, 6, 9 μM, and the final concentration of Pt: 0, 0.35, 0.7, 1.4, 2.1 μg / mL). After mixing evenly, the 96-well plates were placed in an incubator at 37 °C and 5% CO2 for 48 h. After 48 h of drug treatment, the supernatant was removed by centrifugation, and after resuspending with 100 μL of fresh medium, 10 μL of CCK8 detection solution was added and incubated for another 4 h. After incubation, the absorbance was measured at 450 nm. The results are shown in Figure 8 and Figure 9 As shown, after 48 h of drug treatment, the killing ability of CX4945@PLGA-PEG@Pt on CEM cells was significantly enhanced, and the killing ability showed CX4945@PLGA-PEG@Pt > CX4945@PLGA-PEG > CX4945 > PLGA-PEG@Pt. Using the same experimental method to treat MOLT4 cells, as shown in Figure 10 and Figure 11 As shown, after 48 h of drug treatment

[0057] the killing ability of CX4945@PLGA-PEG@Pt on MOLT4 cells was significantly higher than that of other drugs, and the killing ability also showed CX4945@PLGA-PEG@Pt > CX4945@PLGA-PEG > CX4945 > PLGA-PEG@Pt.

[0058] Example 7 Killing effect of CX4945@PLGA-PEG@Pt Nps on B-ALL cells

[0059] According to the method of Example 6, the difference was that the cell number was 3×10 4 cells / well, and the viability of NALM6 cells after incubation with different concentrations of CX4945@PLGA-PEG@Pt, CX4945@PLGA-PEG, CX4945, and PLGA-PEG@Pt for 48 h was detected. The results are shown in Figure 12 and Figure 13As shown, after 48 hours of drug action, the killing ability of CX4945@PLGA-PEG@Pt against NALM6 cells was significantly higher than that of other drugs. Moreover, the killing ability also showed the order of CX4945@PLGA-PEG@Pt > CX4945@PLGA-PEG > CX4945 > PLGA-PEG@Pt.

Claims

1. A method for preparing a platinum nanozyme drug-loaded CX4945 delivery system, characterized in that: The method comprises the following steps: dissolving the CX4945 drug and polylactic acid glycolic acid-polyethylene glycol-thiol in tetrahydrofuran, adding them to a polyvinyl pyrrolidone aqueous solution, ice bathing and ultrasonically crushing; stirring, centrifuging, and filtering the membrane; then adding chloroplatinic acid solution, stirring, and removing excess chloroplatinic acid; then adding sodium borohydride reducing agent, and removing excess reducing agent after the reaction is completed, so as to obtain a platinum nanozyme drug-loaded CX4945 delivery system.

2. The method according to claim 1, characterized in that: The molecular weight of the polylactic acid glycolic acid-polyethylene glycol-thiol group is 10K-12K.

3. The method according to claim 1, characterized in that: The mass ratio of chloroplatinic acid to CX4945 is 1:0.5-1, and the mass ratio of sodium borohydride reducing agent to chloroplatinic acid is 1:0.5-1.

4. The method according to claim 1, characterized in that: The reaction time after adding chloroplatinic acid is 15 to 30 minutes.

5. The method according to claim 1, characterized in that: The reduction time using sodium borohydride is 20 to 30 minutes.

6. The method according to claim 1, characterized in that: The power of the ultrasonic crushing is 120-250W, the working time is 2-5min, the ultrasonic on time is 5-10s, and the ultrasonic off time is 2-5s.

7. A platinum nanozyme-loaded CX4945 delivery system prepared by the method according to any one of claims 1 to 6.

8. The platinum nanozyme drug-loaded CX4945 delivery system according to claim 7, characterized in that: The particle size of the platinum nanozyme-loaded CX4945 delivery system is 70 to 100 nm.

9. Use of the platinum nanozyme-loaded CX4945 delivery system according to claim 7 in the preparation of a drug for treating acute leukemia.

10. A drug for treating acute leukemia, characterized in that: It contains the platinum nanozyme-loaded CX4945 delivery system as described in claim 7.

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