Nanometer system loaded with anti-tumor drugs of Enberic acid and Oxaliplatin and preparation method of nanometer system

Abstract

The invention relates to the technical field of medicines, in particular to a nano system loaded with anti-tumor drugs, namely Enberic acid and Oxaliplatin, and a preparation method of the nano system. The nano-system is prepared by the following steps: in-situ packaging or synthesizing the empiratic acid and the oxaliplatin, modifying and loading the empiratic acid and the oxaliplatin into a highly ordered crystal porous coordination polymer; the preparation method comprises the following steps: rapidly adding an aqueous solution containing Zn (NO3) 2.6 H2O and oxaliplatin into a methanol solution containing 2-methylimidazole and enroic acid, and carrying out stirring, centrifugation, pure water washing and other treatments to obtain a uniform and stable nano dispersion liquid; then F127 is used for surface modification, and the nanometer system is obtained through stirring, centrifuging, pure water washing, vacuum drying and other operations. The nano system is used for targeting and combined with anti-tumor drug treatment, and the application range and the curative effect of a nano drug delivery therapy in clinical tumor treatment are greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically relates to a nano-system loaded with anti-tumor drugs embelin and oxaliplatin and a preparation method thereof. Background Art

[0002] Colorectal cancer is one of the most common digestive tract malignancies and also one of the main cancer types causing death of cancer patients. At present, adjuvant chemotherapy according to tumor stage after surgical resection is one of the most commonly used treatment strategies. For patients with advanced colorectal cancer that cannot be surgically resected, treatment options include chemotherapy, targeted and immunotherapy. Among them, chemotherapy based on oxaliplatin is the first-line treatment regimen, which is used in combination with 5-fluorouracil (5-FU) and leucovorin (LV) (FOLFOX). The effective rate of treating metastatic colorectal cancer is >50%, and the median survival period is nearly 2 years, occupying a major position in the treatment of patients with advanced colorectal cancer. However, a large proportion of patients receiving chemotherapy will inevitably develop into drug-resistant patients, which greatly affects the treatment effect of advanced colorectal cancer. Therefore, enhancing the anti-tumor response of chemotherapy, expanding the treatment scope of chemotherapy, and developing a drug treatment method with general effectiveness, small toxic and side effects, and high specificity are crucial for the national life and health.

[0003] Embelin is a natural product isolated from the Japanese herb Ardisia, and has attracted attention in the field of cancer research due to its pharmacological properties and non-toxic nature. As an inhibitor of X-linked inhibitor of apoptosis protein (XIAP), Embelin binds to the BIR3 domain shared by the IAP family, and can inhibit the functions of NAIP, cIAP1, cIAP2, and XIAP, thereby inducing apoptosis of various types of cancer cells, achieving the purpose of inhibiting the growth, proliferation, and migration of cancer cells. The inhibitor of apoptosis protein (IAP) family contains a group of evolutionarily conserved apoptosis inhibitors, and inhibiting apoptosis is one of the important mechanisms for tumor cells to resist the killing effects of chemotherapy drugs such as oxaliplatin. Embelin restores the apoptosis of tumor cells induced by oxaliplatin by inhibiting the function of IAP, achieving the effect of increasing the sensitivity of intestinal cancer cells to oxaliplatin. In chemotherapy-resistant colorectal cancer, there is an over-activated cIAP2 molecular signaling pathway. Embelin restores the apoptosis induced by oxaliplatin by inhibiting the anti-apoptotic function of cIAP2. Therefore, Embelin combined with oxaliplatin has a good effect in the treatment of chemotherapy-resistant colorectal cancer.

[0004] In in vivo studies, most small-molecule anti-tumor drugs, including embonic acid, have problems such as poor water solubility, short in vivo half-life, poor selectivity, and high toxicity and side effects on normal tissues, thus limiting their clinical applications. To solve this problem, scientists have used various nanoscale materials as carriers for anti-tumor drugs. These carriers have many advantages such as increasing the biocompatibility of small-molecule anti-tumor drugs, prolonging the circulation time of drugs in the blood, reducing drug toxicity and side effects, and increasing the accumulation amount of drugs at the tumor site, so as to achieve the purpose of improving drug efficacy. However, most drug carriers themselves have no therapeutic effect, and excessive intake can cause various unpredictable problems, such as toxicity to normal tissues and inflammation. In response to this problem, it has been found that nano-metal-organic frameworks (MOFs) are a class of highly ordered crystalline porous coordination polymers with advantages such as clear structure, high specific surface area and porosity, adjustable pore size, and easy chemical functionalization, which can achieve targeted drug delivery, increase cell uptake, and control drug release.

[0005] ZIF-8 nanoparticles are formed by the coordination of zinc ions with 2-methylimidazole. Zinc ions and 2-methylimidazole are dissolved in water or organic solvents and directly synthesized by heating the solution of metal ions and ligands. Due to its high porosity, large specific surface area, high drug loading rate, and pH responsiveness, ZIF-8 can remain stable under physiological conditions and release drugs in the weakly acidic microenvironment of tumors, making it an ideal carrier for loading drugs and achieving drug sustained release. Summary of the Invention

[0006] The purpose of the present invention is to provide a nano-system for loading anti-tumor drugs embonic acid and oxaliplatin and a preparation method thereof. This system uses ZIF-8 nano-metal-organic framework (MOF) as a carrier, and realizes the synergistic release and treatment of drugs in tumor cells by co-loading hydrophilic anti-cancer drug oxaliplatin and hydrophobic anti-cancer drug embonic acid.

[0007] To achieve the above purpose, the present invention provides the following technical solutions: A nano-system for loading anti-tumor drugs embonic acid and oxaliplatin is obtained by in-situ encapsulation or post-synthesis modification of embonic acid and oxaliplatin into highly ordered crystalline porous coordination polymers.

[0008] The preparation method of the nano-system for loading anti-tumor drugs embonic acid and oxaliplatin according to the present invention is to use a solution containing Zn(NO 3 ) 2 ·6H 2An aqueous solution of O and oxaliplatin was rapidly added to a methanol solution containing 2-methylimidazole and enbeic acid, and a homogeneous and stable nano-dispersion was obtained through stirring, centrifugation, and washing with pure water; then, surface modification was carried out using F127, and the nano-system was obtained through stirring, centrifugation, washing with pure water, and vacuum drying.

[0009] Specifically, it includes the following steps: (1) Synthesis of ZIF-8 nanoparticles loaded with enbeic acid-oxaliplatin: An aqueous solution containing Zn(NO 3 ) 2 ·6H 2 O and oxaliplatin was rapidly added to a methanol solution containing 2-methylimidazole and enbeic acid; the mixed solution was stirred at room temperature, then the product was centrifuged, washed with pure water, and then redispersed in pure water for further use; (2) Surface modification using F127: An aqueous solution of ZIF-8 nanoparticles loaded with enbeic acid-oxaliplatin was added to deionized water containing F127; the mixed solution was stirred at room temperature, then the product was centrifuged, washed with deionized water, and dried in vacuo to obtain a dry sample of F127-ZIF-8-oxaliplatin-enbeic acid nanoparticles, that is, a nano-system loaded with the anti-tumor drugs enbeic acid and oxaliplatin.

[0010] Among them, in the step (1), the mass ratio of oxaliplatin to enbeic acid is 1:1. The molar ratio of Zn(NO 3 ) 2 ·6H 2 O to 2-methylimidazole is 1:5 - 10.

[0011] Among them, in the step (1), the centrifugation rate is 12000 rpm and the centrifugation time is 5 minutes.

[0012] Among them, in the step (2), the volume ratio of the aqueous solution of ZIF-8 nanoparticles loaded with enbeic acid-oxaliplatin to deionized water containing F127 is 1:2. The content of F127 in the deionized water containing F127 is 0.005 g / mL.

[0013] Among them, in the step (2), the centrifugation rate is 12000 rpm and the centrifugation time is 5 minutes. The vacuum drying temperature is 50 °C and the time is 6 hours.

[0014] Among them, enbeic acid can significantly increase the sensitivity of colorectal cancer to oxaliplatin.

[0015] Compared with the prior art, the beneficial effects of the present invention are: The present invention first realizes the loading of enbeic acid - oxaliplatin into MOF through in - situ encapsulation or post - synthesis modification strategies to form a co - delivery system, and demonstrates its synergistic therapeutic effect on colorectal cancer in vitro, providing new ideas for the drug treatment of colorectal cancer. The principle of the nano - system of the present invention is based on the design of the nano - drug delivery system and its application in tumor treatment. The high - efficient loading, stable delivery and tumor microenvironment - responsive release of drugs are realized through the nano - metal - organic framework (ZIF - 8).

[0016] (1) Characteristics of the nano - carrier: The ZIF8 nano - material is composed of zinc ions and 2 - methylimidazole, with high porosity and a large specific surface area, enabling it to load a high amount of drugs. ZIF8 is stable under neutral and physiological conditions but degrades in the weakly acidic environment of tumors. This pH - responsive property enables it to selectively release drugs at the tumor site, reducing toxicity to normal tissues.

[0017] (2) Drug synergy: Oxaliplatin is a chemotherapy drug and has a significant effect on inducing apoptosis of tumor cells as a first - line treatment for colorectal cancer. Enbeic acid is a natural inhibitor of apoptosis proteins (IAP inhibitor), which enhances the sensitivity of tumor cells to chemotherapy drugs (such as oxaliplatin) by inhibiting the functions of IAP family members such as cIAP2 and XIAP. By co - loading the two drugs into ZIF8, the nano - system can degrade within tumor cells and simultaneously release enbeic acid and oxaliplatin. Enbeic acid restores oxaliplatin - induced apoptosis of tumor cells and enhances the anti - tumor effect of the drugs.

[0018] (3) Targeted delivery and release: The pH - responsive property of ZIF8 ensures the release of drugs in the tumor microenvironment. The acidic conditions at the tumor site cause ZIF8 to decompose, releasing the loaded oxaliplatin and enbeic acid, achieving local and efficient drug delivery and reducing systemic toxicity. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the preparation process of the nano - system loaded with the anti - tumor drugs enbeic acid and oxaliplatin.

[0020] Figure 2 It is the measurement result of crystallinity.

[0021] Figure 3 It is the measurement result of the particle size and distribution of the nanoparticles.

[0022] Figure 4 It is the measurement result of the ZETA potential.

[0023] Figure 5 It is the measurement result of oxaliplatin loading.

[0024] Figure 6Results of embelin loading determination.

[0025] Figure 7 Results of absorbance determination after loading.

[0026] Figure 8 To detect the cytotoxicity of embelin-oxaliplatin nano-MOF in vitro. Using colorectal cancer cell lines SW620 and HCT116, treated with embelin-oxaliplatin nano-MOF, a drug dose-response curve was plotted. The IC50 of SW620 was 0.036 μM, and the IC50 of HCT116 was 0.029 μM.

[0027] Figure 9 To verify the therapeutic effect of embelin-oxaliplatin nano-MOF on colorectal cancer in vivo. After in vitro expansion of the stably growing colorectal cancer cell line, it was injected into nude mice to construct a subcutaneous tumor-bearing model. Drugs were administered through the tail vein (Group 1: control; Group 2: oxaliplatin; Group 3: embelin; Group 4: embelin-oxaliplatin nano-MOF), and the growth of tumors in mice was observed. Embelin-oxaliplatin nano-MOF significantly inhibited the growth of colorectal cancer in mice. Detailed implementation mode

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Example 1

[0029] As Figure 1 shown, a preparation method of a nano-system loaded with anti-tumor drugs embelin and oxaliplatin, the specific steps are as follows: ① Synthesis of ZIF-8 nanoparticles loaded with embelin-oxaliplatin: A 11.3 mL aqueous solution containing 297.5 mg Zn(NO 3 ) 2 ·6H 2 O and 20 mg oxaliplatin was quickly added to a 11.3 mL methanol solution containing 660 mg 2-methylimidazole and 20 mg embelin. The mixed solution was further stirred at room temperature for 60 minutes, and then the product was centrifuged (12,000 rpm, 5 minutes), washed with pure water, and then redispersed in 10 mL of pure water for further use.

[0030] ②Surface modification using F127: 10 mL of an aqueous solution of ZIF-8 nanoparticles loaded with embelin-oxaliplatin was added to 20 mL of deionized water containing 100 mg of F127. The mixed solution was further stirred at room temperature for 12 hours. Subsequently, the product was centrifuged (12000 rpm, 5 minutes), washed with deionized water, and dried in vacuo at 50 °C for 6 hours to obtain 129 mg of dry sample of F127-ZIF-8-oxaliplatin-embelin nanoparticles, namely Embelin-oxaliplatin nano-MOF.

[0031] ①Determination of crystallinity: A small amount of the sample prepared in Example 1 was taken, and the sample was detected using a multifunctional X-ray diffractometer to obtain a PXRD pattern. It can be seen that the crystallinity of the sample is good, as Figure 2 shown.

[0032] ②Determination of the particle size and distribution of nanoparticles: The Embelin-oxaliplatin nano-MOF was measured on a nanoparticle size analyzer using dynamic light scattering. The average particle size of the nanoparticles was 137.5 nm, as Figure 3 shown.

[0033] ③Determination of ZETA potential: The sample was measured using a Zeta potential analyzer to determine the solid-liquid interface electric property of the particulate matter in the dispersion system. It can be seen that the surface of the nanoparticles is negatively charged, as Figure 4 shown.

[0034] ④Determination of oxaliplatin loading: 2.15 mg of the sample prepared in Example 1 was taken and the encapsulation rate of oxaliplatin in the nanoparticles was detected using an inductively coupled plasma mass spectrometer. The measured platinum content was 0.3267 μg. Therefore, the CCCP content was 0.6654 μg. The encapsulation rate of oxaliplatin in the F127-ZIF-8 nanoparticles was approximately 0.04177%. The embelin loading rate was approximately 0.035%. The particle concentration was approximately 4.3 mg / mL, as Figures 5 - 7 shown.

[0035] Experimental subjects: Human intestinal cancer cell lines.

[0036] Colorectal cancer cell lines that could grow stably were selected for in vitro chemotherapeutic drug susceptibility tests. The CCK8 method was used to detect cell viability. The cells were seeded in 96-well plates (10 3 cells, 100 μL per well). After drug treatment, at 37 °C, 5% CO 2Incubate for 3 days. Add 10 μL of CCK8 reagent to each well, and measure the absorbance at 450 nm using a spectrophotometer after incubating at 37 °C for 2 hours. Draw a drug dose-response curve with a drug dose of 0 μM as the baseline, and the data is expressed as the percentage of growth starting from 0 μM. The closer the curve is to the Y-axis, the more sensitive it is to the chemotherapeutic drug.

[0037] In vitro detection of the cytotoxicity of Embelin-oxaliplatin nano-MOF: Use common colorectal cancer cell lines SW620 and HCT116, treat them with Embelin-oxaliplatin nano-MOF, and draw a drug dose-response curve. The IC50 of SW620 is 0.036 μM, and the IC50 of HCT116 is 0.029 μM. As Figure 8 shown.

[0038] Experimental subjects: Human intestinal cancer cell lines, immunodeficient mice.

[0039] Prepare a serum-free suspension of the colorectal cancer cell line with stable growth. Take male nude mice at 4-6 weeks of age. For the subcutaneous tumor-bearing model, inject tumor cells into the right lower abdomen of the mice. Administer drugs via the tail vein injection according to the grouping (Group 1: solvent control; Group 2: oxaliplatin; Group 3: embelin; Group 4: oxaliplatin + embelin MOF), once every 3 days. Measure the tumor diameter with a vernier caliper every 7 days and continuously observe for 4 weeks. Sacrifice the mice after 4 weeks and examine the tumor growth. The experimental results confirm that embelin-oxaliplatin nano-MOF can significantly inhibit the growth of colorectal cancer in mice, as Figure 9 shown.

[0040] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nanosystem loaded with anti-tumor drugs enbemic acid and oxaliplatin, characterized in that: Enbemic acid and oxaliplatin were loaded into highly ordered crystalline porous coordination polymers via in situ encapsulation or post-synthesis modification.

2. The method for preparing the nanosystem loaded with anti-tumor drugs enbemic acid and oxaliplatin according to claim 1, characterized in that: An aqueous solution containing Zn(NO3)2·6H2O and oxaliplatin is quickly added to a methanol solution containing 2-methylimidazole and enbenic acid, and a uniform and stable nano-dispersion is obtained through stirring, centrifugation and pure water washing. F127 is then used for surface modification, and the nano-system is obtained through stirring, centrifugation, pure water washing and vacuum drying.

3. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 2, characterized in that , including the following steps: (1) Synthesis of ZIF-8 nanoparticles loaded with enbemic acid and oxaliplatin: An aqueous solution containing Zn(NO3)2·6H2O and oxaliplatin was quickly added to a methanol solution containing 2-methylimidazole and enbemic acid; the mixed solution was stirred at room temperature, and the product was then centrifuged, washed with pure water, and then dispersed in pure water for further use; (2) Surface modification using F127: an aqueous solution of ZIF-8 nanoparticles loaded with enbemic acid-oxaliplatin was added to deionized water containing F127; the mixed solution was stirred at room temperature, and the product was then centrifuged, washed with deionized water, and dried in air to obtain a dry sample of F127-ZIF-8-oxaliplatin-enbemic acid nanoparticles, i.e., a nanosystem loaded with the anti-tumor drugs enbemic acid and oxaliplatin.

4. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 3, characterized in that: In the step (1), the mass ratio of oxaliplatin to enbemic acid is 1:

1.

5. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 4, characterized in that: In the step (1), the molar ratio of Zn(NO3)2·6H2O to 2-methylimidazole is 1:5-10.

6. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 5, characterized in that: In the step (1), the centrifugal speed is 12000 rpm and the centrifugal time is 5 minutes.

7. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 3, characterized in that: In the step (2), the volume ratio of the aqueous solution of ZIF-8 nanoparticles loaded with enbemic acid-oxaliplatin to the deionized water containing F127 is 1:

2.

8. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 7, characterized in that: In the step (2), the content of F127 in the deionized water containing F127 is 0.005 g / mL.

9. The method for preparing the nanosystem loaded with anti-tumor drugs enbenic acid and oxaliplatin according to claim 8, characterized in that: In the step (2), the centrifugal speed is 12000 rpm and the centrifugal time is 5 minutes.

10. The method for preparing the nanosystem loaded with anti-tumor drugs enbemic acid and oxaliplatin according to claim 9, characterized in that: In the step (2), the vacuum drying temperature is 50° C. and the time is 6 hours.

Images