A nano drug delivery system co-loaded with cytarabine and venetoclax, and its preparation method and application

Abstract

The present invention discloses a co-loaded cytarabine and venetoclax nano-drug delivery system and its preparation method and application, belonging to the field of medical technology. The co-loaded cytarabine and venetoclax nano-drug delivery system is composed of human serum albumin, cytarabine derivatives and venetoclax. First, cytarabine is modified, then the disulfide bonds in the albumin are broken with a reducing agent, and finally, the cytarabine derivatives and venetoclax are loaded simultaneously to prepare the co-loaded cytarabine and venetoclax nano-drug delivery system AV‑NP. The AV‑NP prepared by the present invention has a small size and reduction response characteristics, specifically responds to release drugs in cancer cells, and has good biocompatibility; and the preparation method is green, simple, and efficient, achieving the co-delivery of cytarabine and venetoclax, providing a new idea for chemotherapy of leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a nano drug delivery system co-loaded with cytarabine and venetoclax, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Leukemia is a malignant clonal disease of hematopoietic stem cells characterized by enhanced cell self-renewal, uncontrolled proliferation, impaired differentiation, and blocked apoptosis, leading to stagnation in cell development. Despite the emergence of new treatments over the past 20 years, chemotherapy still plays a crucial role in the treatment of leukemia. However, traditional chemotherapy suffers from issues such as short drug circulation time in the body and significant toxic side effects. Therefore, it is particularly important to identify safe drugs or novel drug delivery systems that can effectively kill leukemia cells.

[0004] Venetoclax combined with cytarabine has demonstrated excellent efficacy in the clinical treatment of leukemia, offering new hope for leukemia patients who are unsuitable for intensive induction chemotherapy. However, due to the five-membered sugar ring in the cytarabine (Ara-C) molecular structure, the Ara-C molecule is highly polar and water-soluble, making it easily deaminated and inactivated by deaminases in the body to form the inactive arabinouracil. Consequently, Ara-C has an extremely short plasma half-life after intravenous administration. In contrast to Ara-C's physicochemical properties, Venetoclax (Ven) is highly lipophilic and has good membrane permeability, but its water solubility is poor, requiring a higher dose to enhance bioavailability. Therefore, the development of a safe and effective nanodelivery system that co-loads Ven and Ara-C has important research significance and clinical application value. Summary of the Invention

[0005] In response to the shortcomings of existing technologies for treating leukemia, the present invention provides a co-loaded cytarabine and venetoclax nano-drug delivery system, a preparation method, and an application thereof. The prepared co-loaded cytarabine and venetoclax nano-drug delivery system has the characteristics of high biosafety and targeting, can increase the circulation time of the drug in the body, and significantly improve the killing effect on leukemia cells.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides a nano drug delivery system co-loaded with cytarabine and venetoclax, which is composed of albumin, cytarabine derivatives and venetoclax, wherein the cytarabine derivatives are modified on the albumin, and venetoclax is loaded on the hydrophobic region of the albumin modified with the cytarabine derivatives.

[0008] Preferably, the albumin is human albumin, and the cytarabine derivative is linked to human albumin via a disulfide bond. Human albumin is a nanocarrier commonly used in the research of new drug carriers, and has advantages such as good biocompatibility and long circulation time in the body.

[0009] Preferably, venetoclax is loaded onto the hydrophobic region of albumin via non-covalent bonding.

[0010] Preferably, the cytarabine derivative is a group of 4-acyl-substituted cytarabine derivatives, and its general structural formula is shown in formula (I):

[0011]

[0012] wherein n=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. The cytarabine derivative is formed by linking cytarabine and a fatty acid containing a terminal thiol group via an amide bond.

[0013] The second aspect of the present invention provides a method for preparing the aforementioned nano-drug delivery system co-loaded with cytarabine and venetoclax, comprising the following steps:

[0014] 1) Dispersing albumin into deionized water, adding a surfactant and a reducing agent, stirring at high temperature, and washing to obtain reduced albumin;

[0015] 2) Adding reduced albumin to a cytarabine derivative, ultrasonically dispersing the albumin, and then adding an organic solution containing venetoclax. Ultrasonic dispersing the albumin again, adding an oxidant under stirring at room temperature, stirring, and washing to obtain a nano-drug delivery system co-loaded with cytarabine and venetoclax.

[0016] Preferably, in step 1), the reducing agent is dithiothreitol or reduced glutathione.

[0017] Preferably, the surfactant comprises sodium dodecylbenzenesulfonate.

[0018] Preferably, the mass ratio of albumin, surfactant and reducing agent is 40:2-20:1-20.

[0019] Preferably, in step 1), the high-temperature stirring is stirring at 55-65° C. for 25-35 minutes, more preferably stirring at 60° C. for 30 minutes.

[0020] Preferably, in step 2), the solvent of the organic solution comprises dichloromethane or chloroform. In the presence of the organic solvent, venetoclax is covalently bonded to the hydrophobic region of the albumin modified with the cytarabine derivative in a self-assembly manner.

[0021] In the organic solution, the concentration of venetoclax is 1-10 mg / mL.

[0022] The volume ratio of the organic solution of venetoclax to the reduced albumin is 1:3-6.

[0023] Preferably, the ratio of reduced albumin to cytarabine derivative is: 0.3-0.6 mL: 0.8-1.2 mg, preferably 0.5 mL: 1.0 mg.

[0024] Preferably, the oxidant is hydrogen peroxide, and the volume ratio of the oxidant to the reduced albumin is 1:12-24.

[0025] The present invention uses a reducing agent to break the disulfide bonds in albumin, making it easier for cytarabine derivatives to be modified on albumin through the disulfide bonds, and then uses an oxidant to reconstruct the broken disulfide bonds, thereby successfully modifying the albumin with cytarabine derivatives.

[0026] The present invention's co-loaded cytarabine and venetoclax nanoparticle drug delivery system exhibits excellent biodegradability and biosafety, and effectively kills leukemia cells through the synergistic effects of the cytarabine derivative and venetoclax. Therefore, a third aspect of the present invention provides the use of the co-loaded cytarabine and venetoclax nanoparticle drug delivery system in the preparation of leukemia-related drugs.

[0027] The beneficial effects of the present invention are:

[0028] The present invention links cytarabine to albumin via a disulfide bond, reconstructs the disulfide bond in the albumin using a reducing agent and an oxidizing agent, and simultaneously loads venetoclax into the albumin, achieving co-loading of cytarabine and venetoclax. The prepared co-loaded cytarabine and venetoclax nano-drug delivery system has reduction-sensitive properties and can respond to reduction in leukemia cells, causing structural collapse to release the drugs. It has good biodegradability and biosafety, is small in size, and efficiently kills leukemia cells through the synergistic effect of cytarabine and venetoclax.

[0029] The preparation method of the co-loaded cytarabine and venetoclax nano drug delivery system provided by the present invention can achieve the co-loading of cytarabine and venetoclax, increase the circulation time of the drugs in the body, specifically release them in leukemia cells, reduce the adverse reactions of the drugs, and thus safely and effectively kill leukemia cells. It is green, simple, and efficient, and provides a new idea for chemotherapy of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 This is a transmission electron micrograph of the AV-NP prepared in Example 1 of the present invention;

[0032] Figure 2 This is the particle size distribution diagram of AV-NP prepared in Example 1 of the present invention;

[0033] Figure 3 This is a release curve of AV-NP prepared in Example 1 of the present invention in 10 mM glutathione.

[0034] Figure 4 The inhibition rate of AV-NP on molm13 cells was measured by CCK8 assay;

[0035] Figure 5 This is the drug-time curve of AV-NP in rats. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0037] Example 1

[0038] A method for preparing a nano drug delivery system co-loaded with cytarabine and venetoclax comprises the following steps: (1)

[0040]

[0041] wherein: n=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0042] The preparation method of the cytarabine derivative having the structure of formula (I) comprises the following steps:

[0043] 1) 2 mg of 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate and 4 mg of 3-(tritylthio)propionic acid were dissolved in dimethylformamide. 5 mg of cytarabine and 1 mg of N,N-diisopropylethylamine were added dropwise at 0°C under nitrogen protection. After the addition was complete, the mixture was stirred at room temperature. The reaction solution was slowly poured into water, and the organic phase was extracted with dichloromethane. The organic phase was washed with hydrochloric acid, sodium bicarbonate solution, and sodium chloride solution, respectively, and finally dried, filtered, and dried to obtain a crude product. The crude product was purified by normal phase column chromatography.

[0044] 2) 10 mg of the purified product obtained above was dissolved in a dichloromethane solution containing 10% trifluoroacetic acid, and 6 mg of triethylsilane was added at 0°C. After the reaction was completed, the solvent was dried, diethyl ether was added, stirred, and filtered to obtain the cytarabine derivative having the structure of formula (I).

[0045] (2) Human serum albumin was dispersed in deionized water, sodium dodecylbenzenesulfonate and dithiothreitol were added, and the mass ratio of albumin, surfactant and reducing agent was 40:11:10. The mixture was stirred at 60°C for 30 minutes. The reaction solution was washed several times with an ultrafiltration centrifuge tube to obtain human serum albumin in a reduced state.

[0046] (3) Take 0.5 mL of reduced human serum albumin and add it to 0.5 mL of phosphate buffer saline (PBS), then add 1 mg of cytarabine derivative, ultrasonically disperse for 60 s, add 100 μL of chloroform solution of venetoclax at a concentration of 5 mg / mL, and ultrasonically disperse for another 60 s. Stir at room temperature at a stirring speed of 800 rpm, and at the same time add 25 μL of 3% H2O2 solution, stir for 30 min, centrifuge the ultrafiltration tube and wash with PBS three times, and finally disperse with PBS to obtain the co-loaded cytarabine and venetoclax nano-drug delivery system AV-NP.

[0047] Comparative Example 1

[0048] A method for preparing a nano drug delivery system loaded with cytarabine derivatives, wherein compared with Example 1, venetoclax is not loaded on human serum albumin. The obtained nano drug delivery system loaded with cytarabine derivatives is named A-NP.

[0049] Comparative Example 2

[0050] A method for preparing a nano drug delivery system loaded with venetoclax is disclosed. Compared with Example 1, no cytarabine derivative is modified on human albumin. The obtained nano drug delivery system loaded with venetoclax is named V-NP.

[0051] Application Examples

[0052] The performance of the co-loaded cytarabine and venetoclax nano drug delivery system obtained in the Examples and Application Examples was tested, and the test content and test results are as follows:

[0053] (1) Transmission electron microscopy observation of AV-NP

[0054] The morphology of AV-NPs was characterized by transmission electron microscopy (TEM). Figure 1 As shown, AV-NP is quasi-circular and relatively evenly dispersed. The core of AV-NP is dark in color and surrounded by a light halo, indicating that the hydrophilic cytarabine derivative is mainly on the periphery of AV-NP, and the hydrophobic venetoclax is mainly loaded into the core of AV-NP.

[0055] The particle size of AV-NP was measured using a particle size distribution analyzer. The test results are as follows: Figure 2 As shown, the particle size of AV-NP is about 50 nm and the surface charge is negative.

[0056] (2) Investigation of the redox release performance of AV-NPs

[0057] 1 mL of 1 mg / mL AV-NP was placed in an activated dialysis bag (MWCO 8000-12000 Da), then placed in 20 mL of different release media (PBS, pH 7, without reduced glutathione (GSH), PBS, pH 7, with 10 mM reduced glutathione (GSH)). The mixture was shaken on a (37±0.5)°C incubator. 0.5 mL of release media was removed at 1, 2, 4, 6, 8, 12, and 24 h, and replaced with 0.5 mL of blank release media. Content was determined by liquid chromatography, and time-dependent cumulative release curves were plotted.

[0058] like Figure 3 As shown in the data, in the PBS simulated body fluid without GSH, the drug release was very slow, with less than 20% released in 24 h. However, in the PBS simulated body fluid with 10 mM GSH, the drug release rate increased significantly, with the cumulative release rate in 24 h increasing from 20% to 80%, indicating that AV-NP has reduction-responsive drug release properties.

[0059] (3) In vitro apoptosis-inducing activity of different preparations in leukemia cells

[0060] Molm13 logarithmic phase cell suspension was plated at 5×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate; AV-NP, cytarabine derivative Ara-SH, A-NP, and V-NP were diluted to the required concentration according to the drugs they contained, mixed by pipetting, and added to the 6-well plates respectively. The plates were sealed and placed in a 37°C incubator for 24 hours; the 6-well plates were taken out, and the apoptosis of cells in each group was detected using an apoptosis kit and flow cytometry, and the cell apoptosis rate was calculated.

[0061] Depend on Figure 4 It can be seen that the cell apoptosis induced by cytarabine derivatives Ara-SH monomer and A-NP is low, and V-NP can induce cell apoptosis rate to 37.1%. Under the same conditions, AV-NP has the highest anti-leukemia activity, and the cell apoptosis induced by AV-NP is 52.6%. This shows that loading cytarabine derivatives and venetoclax together into the nanodrug delivery system can significantly improve the in vitro anti-leukemia cell activity.

[0062] (4) Pharmacokinetics of different preparations in rats

[0063] Cytarabine (Ara-C) has an extremely short half-life in vivo, only 10-20 minutes. To evaluate the in vivo circulation time of cytarabine derivatives loaded into AV-NPs, an in vivo pharmacokinetic study in rats was conducted. SD rats were divided into two groups: the Ara-C group and the AV-NP group. Drugs were administered via the tail vein. Blood samples (0.3 ml) were collected at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration. The collected blood samples were placed in heparinized centrifuge tubes and centrifuged at 3000 rpm for 10 minutes. 100 μL of the upper plasma layer was collected, 10 μL of dithiothreitol and trichloroacetic acid solution was added, and the mixture was vortex-mixed for 30 seconds. The mixture was then centrifuged at 14000 rpm for 20 minutes. 20 μL of the supernatant was aspirated and the drug concentration was determined by HPLC.

[0064] like Figure 5 As shown in the results, Ara-C has a very short retention time in rats and is completely eliminated in less than 5 hours. However, AV-NP can significantly prolong the drug's circulation time in the body. The mean retention time (MRT) of AV-NP in the body is 8.75 hours, significantly longer than the mean retention time of Ara-C of 1.07 hours. In addition, the area under the plasma concentration-time curve (AUC) of the AV-NP group was 1693 μg mL -1 h, significantly greater than the AUC of the Ara-C group (55.31 μg mL -1 h). In summary, AV-NP has a longer circulation time and a longer average residence time in the body than Ara-C, which is very beneficial for the drug to reach the lesion site.

[0065] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A nano drug delivery system co-loaded with cytarabine and venetoclax, characterized in that: The invention is composed of albumin, cytarabine derivatives and venetoclax, wherein the cytarabine derivatives are modified on the albumin, and venetoclax is loaded on the hydrophobic region of the albumin modified with the cytarabine derivatives; The albumin is human serum albumin, and the cytarabine derivative is linked to the human serum albumin via a disulfide bond; Venetoclax is loaded non-covalently onto the hydrophobic region of albumin; The cytarabine derivatives are a group of 4-acyl-substituted cytarabine derivatives, and their general structural formula is shown in formula (I): Formula (I) Where: n=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

2. The method for preparing the co-loaded cytarabine and venetoclax nano drug delivery system according to claim 1, characterized in that: The steps include: 1) Disperse albumin in deionized water, add surfactant and reducing agent, stir at high temperature, and wash to obtain reduced albumin; 2) Adding a cytarabine derivative to the reduced albumin, ultrasonically dispersing the albumin, and then adding an organic solution containing venetoclax. Ultrasonic dispersing the albumin again, adding an oxidant under stirring at room temperature, stirring, and washing to obtain a nano-drug delivery system co-loaded with cytarabine and venetoclax.

3. The preparation method according to claim 2, characterized in that In step 1), the reducing agent is dithiothreitol or reduced glutathione; The surfactant includes sodium dodecylbenzenesulfonate; The mass ratio of albumin, surfactant and reducing agent is 40: 2-20: 1-20; In step 1), the high-temperature stirring is stirring at 55-65° C. for 25-35 minutes.

4. The preparation method according to claim 3, characterized in that In step 1), the high-temperature stirring is stirring at 60° C. for 30 minutes.

5. The preparation method according to claim 2, characterized in that In step 2), the solvent of the organic solution is dichloromethane or chloroform; In the organic solution, the concentration of venetoclax is 1-10 mg / mL; The volume ratio of the organic solution of venetoclax to the reduced albumin is 1:3-6.

6. The preparation method according to claim 2, characterized in that The dosage ratio of the reduced albumin to the cytarabine derivative is: 0.3-0.6 mL: 0.8-1.2 mg.

7. The preparation method according to claim 2, characterized in that The dosage ratio of the reduced albumin to the cytarabine derivative is 0.5 mL: 1.0 mg.

8. The preparation method according to claim 2, characterized in that The oxidant is hydrogen peroxide, and the volume ratio of the oxidant to the reduced albumin is 1:12-24.

9. Use of the co-loaded cytarabine and venetoclax nano drug delivery system according to claim 1 in the preparation of drugs for treating leukemia.

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