Tumor cell membrane bionic nano-drug entrapped with lonidamine as well as preparation and application of tumor cell membrane bionic nano-drug

Abstract

The invention belongs to the technical field of medicines, and discloses a lonidamine-entrapped tumor cell membrane bionic metal organic framework nanoparticle as well as preparation and application thereof. The preparation method comprises the following steps: firstly, preparing lonidamine-entrapped metal organic framework nanoparticles by adopting a one-pot method, then coating tumor cell membranes on the surfaces of the nanoparticles through repeated membrane extrusion or ultrasonic treatment, and finally preparing the lonidamine-entrapped tumor cell membrane bionic nano-drug. The preparation process is simple, the cost is low, the stability is good, and the repeatability is high. The bionic nano-drug disclosed by the invention is helpful for solving the problems of poor solubility, low bioavailability and the like of an anti-cancer drug lonidamine. And meanwhile, the tumor cell membrane camouflage technology can promote the nano-drug to realize relatively high tumor tissue accumulation by utilizing the dual effects of EPR passive targeting effect and homologous active targeting, so that the anti-tumor effect is improved, and the toxic and side effects of lonidamine are reduced.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a tumor cell membrane bionic nanomedicine encapsulating lonidamine, and its preparation and application. Background Art

[0002] Malignant tumors have a complex pathogenesis, poor treatment effects, and high recurrence and metastasis rates, so they have become the number one killer that threatens human health. Compared with normal cells, tumor metabolic reprogramming is an important biological feature of most tumors, which is mainly manifested in the high-efficiency aerobic glycolysis energy supply mode. Even under conditions of sufficient oxygen, tumor cells will still preferentially take up glucose and use the glycolysis process to produce a large amount of ATP and metabolites to provide sufficient energy and biomacromolecule raw materials for cell proliferation, growth, invasion and metastasis. Therefore, aerobic glycolysis plays an important role in the occurrence, development, invasion and metastasis of tumors. Lonidamine is a hexokinase inhibitor in the glycolysis process that can block aerobic glycolysis metabolism, destroy the energy supply of tumor cells, and then kill tumor cells. At present, lonidamine has been proven in preclinical studies to have effective therapeutic effects in a variety of tumors, including breast cancer, melanoma, colon cancer and lung cancer. However, lonidamine has poor water solubility and low bioavailability in the body. At the same time, lonidamine has poor targeting to tumor tissues and can be distributed to various tissues and organs throughout the body, resulting in serious toxic side effects, especially liver toxicity, which limits its clinical application. To solve this problem, lonidamine is encapsulated in a rationally designed intelligent tumor-targeted drug delivery carrier to form a nanodrug that can target tumor tissues, which has important guiding significance for promoting its clinical transformation and application in tumor treatment.

[0003] Metal-organic frameworks (MOFs) are promising nanomaterials for constructing drug delivery carriers. Zeolitic imidazolate frameworks 8 (ZIF8) is a tetrahedral MOF material formed by dimethylimidazole and metal Zn ions connected by coordination bonds. Compared with traditional nano drug delivery carriers such as liposomes and polymer micelles, ZIF8 nano drug delivery carriers have a porous structure and a large specific surface area, so they can improve the encapsulation efficiency and drug loading of drugs. At the same time, ZIF-8 nano drug delivery carriers can be degraded under the acidic conditions of lysosomes / endosomes to release drug molecules, thereby achieving controllable and precise drug release in tumor cells and exerting anti-tumor effects in a timely manner. In order to improve the targeting of nano drug delivery carriers to tumor tissues, natural tumor cell membrane bionic camouflage technology has been widely explored by researchers. Natural tumor cell membranes are coated on the surface of nano drug delivery carriers, which can disguise them as endogenous cells, thereby inheriting the homologous binding and immune escape capabilities similar to endogenous cells. Therefore, nanodrug carriers with biomimetic camouflage of tumor cell membranes can utilize the dual effects of EPR passive targeting effect and homologous active targeting to achieve higher tumor tissue accumulation and improve the anti-tumor effect. Summary of the invention

[0004] The technical problem solved by the present invention is to overcome the poor water solubility, low bioavailability, poor tumor targeting, strong toxic and side effects of the anti-tumor drug lonidamine, and provide a tumor cell membrane bionic nano drug encapsulating lonidamine, preparation and application thereof. The present invention first adopts a one-pot method to prepare metal organic framework nanoparticles encapsulating lonidamine, and then coats the tumor cell membrane on the surface of the nanoparticles by repeated membrane extrusion or ultrasound, and finally prepares a tumor cell membrane bionic nano drug encapsulating lonidamine. The nano drug can utilize the dual effects of EPR passive targeting effect and homologous active targeting to achieve higher tumor tissue accumulation, improve anti-tumor effect, and reduce toxic and side effects.

[0005] Specifically, the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a tumor cell membrane bionic nano drug encapsulating lonidamine for the first time, wherein the nanoparticles are formed by repeatedly extruding or ultrasonically coating tumor cell membranes on the surface of metal organic framework nanoparticles. The encapsulated drug is the anti-tumor drug lonidamine. The metal organic framework material is ZIF8. The tumor cell membrane is selected from one or more of breast cancer cell membranes, melanoma cell membranes, brain glioma cell membranes, lung cancer cell membranes and cervical cancer cell membranes.

[0007] The second aspect of the present invention provides for the first time a method for preparing a tumor cell membrane bionic nano drug encapsulating lonidamine, which comprises the steps of: dissolving lonidamine and zinc nitrate hexahydrate in a dimethyl sulfoxide solution, adding a methanol solution containing dimethylimidazole, reacting for a period of time, and obtaining metal organic framework nanoparticles encapsulating lonidamine by centrifugation, washing, and drying. The nanoparticles and tumor cell membranes are mixed in a certain proportion, and the tumor cell membrane bionic nano drug encapsulating lonidamine is obtained by membrane extrusion or ultrasonic treatment, and centrifugation and washing.

[0008] The method for preparing the bionic nano drug, wherein the mass ratio of lonidamine to zinc nitrate hexahydrate is 1:3-1:30, preferably 1:20; the molar ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:3 to 1:72, preferably 1:40; after adding the methanol solution containing dimethylimidazole, the reaction time is 5-120 minutes, preferably 30 minutes; the mass ratio of the nanoparticles to the tumor cell membrane is 1:1-10:1, preferably 1:1.

[0009] The preparation method of the bionic nano drug, wherein the preparation of the tumor cell membrane comprises the steps of: lysing the tumor cells, ultrasonically destroying them, taking the supernatant after gradient centrifugation to obtain tumor cell membrane fragments, and obtaining the tumor cell membrane after membrane extrusion filtration.

[0010] The preparation method of the bionic nano drug, the preferred preparation method is:

[0011] Dissolve 1 mg of lonidamine (Lon) and 20 mg of zinc nitrate hexahydrate in 1 ml of dimethyl sulfoxide and stir for 10 minutes. Add 2 ml of dimethylimidazole methanol solution containing 800 mg at room temperature and react for 30 minutes. Centrifuge at 14000 rpm / min for 15 minutes. Wash three times with DMSO to obtain Lon@ZIF8; disperse 1 mg of Lon@ZIF8 and 1 mg of tumor cell membrane in aqueous solution, squeeze through 200 nm membrane 12 times, centrifuge at 14000 rpm / min for 15 minutes, and wash with PBS to obtain tumor cell membrane bionic nanomedicine encapsulating lonidamine.

[0012] The present invention also provides an application of a tumor cell membrane bionic nano drug encapsulating lonidamine in anti-tumor. The method and steps are as follows:

[0013] The tumor cell membrane bionic nanodrugs loaded with lonidamine were diluted with serum-containing culture medium to a solution of a predetermined concentration. Five replicate wells were set for each concentration, and a control group and a zero adjustment group were set. An appropriate amount of breast cancer 4T1 cells in the logarithmic growth phase were inoculated in a 96-well plate. After overnight culture and adherence, tumor cell membrane bionic nanodrugs loaded with lonidamine at different concentrations were added and cultured for 24 hours. 200 μl of CCK-8 reagent at a concentration of 10% was added to each well. After incubation for 1 hour, the absorption value was measured at 450 nm. The cell viability was calculated using the following formula:

[0014] Cell viability (%) = [(OD experimental - OD zero adjustment) / (OD control - OD zero adjustment)] × 100

[0015] The beneficial effects of the present invention are as follows:

[0016] 1 The preparation process of the bionic nanomedicine encapsulating lonidamine described in the present invention is simple, low in cost, good in stability and high in repeatability.

[0017] 2 The bionic nanomedicine of the present invention helps to solve the problems of poor solubility and low bioavailability of the anticancer drug lonidamine. At the same time, the tumor cell membrane camouflage technology can enable nanomedicines to achieve higher tumor tissue accumulation by utilizing the dual effects of EPR passive targeting effect and homologous active targeting, thereby improving the anti-tumor effect and reducing the toxic side effects of lonidamine.

[0018] 3 Compared with free lonidamine, the bionic nanomedicine described in the present invention exhibits more effective anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Electron microscope image of biomimetic nanomedicine

[0020] Figure 2 :Particle size diagram of biomimetic nanomedicine

[0021] Figure 3 : Potential diagram of biomimetic nanomedicine

[0022] Figure 4 :In vitro anti-tumor performance

[0023] Figure 5 : Live and dead cell staining results DETAILED DESCRIPTION

[0024] The following examples are intended to illustrate the present invention rather than to further limit the present invention. The present invention is further described in detail below with reference to the examples, but the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art can carry out equivalent replacement, combination, improvement or modification to the present invention based on the description of the present invention, but these will all be included in the scope of the present invention.

[0025] Example 1: Preparation of tumor cell membrane biomimetic nanomedicine containing lonidamine

[0026] Dissolve 1 mg of lonidamine (Lon) and 20 mg of zinc nitrate hexahydrate in 1 ml of dimethyl sulfoxide and stir for 10 minutes. Add 2 ml of dimethylimidazole methanol solution containing 800 mg at room temperature and react for 30 minutes. Centrifuge at 14000 rpm / min for 15 minutes. Wash three times with DMSO to obtain Lon@ZIF8; disperse 1 mg of Lon@ZIF8 and 1 mg of tumor cell membrane in aqueous solution, squeeze through 200 nm membrane 12 times, centrifuge at 14000 rpm / min for 15 minutes, and wash with PBS to obtain tumor cell membrane bionic nanomedicine (Lon@CM-ZIF8) loaded with lonidamine.

[0027] The physical properties of the bionic nanomedicine: Figure 1 As shown in the figure, the transmission electron microscopy results show that the bionic nanomedicine (Lon@CM-ZIF8) has a spherical structure, a smooth surface, good dispersion, and a layer of cell membrane can be seen on the surface. Figure 3 and 4 As shown, the particle size of the bionic nanodrug (Lon@CM-ZIF8) measured by dynamic light scattering was 142.9 nm, the PDI was 0.041, and the Zeta potential was -15.5 V; the drug loading of the nanodrug Ce6@CM-ZIF8 was determined to be 16.03% by high performance liquid chromatography.

[0028] Example 2: In vitro anti-tumor studies

[0029] The tumor cell membrane bionic nanodrug encapsulating lonidamine was diluted with serum-containing culture medium to a solution of predetermined concentration. Six replicate wells were set for each concentration, and a control group and a zero adjustment group were set. An appropriate amount of breast cancer 4T1 cells in the logarithmic growth phase were inoculated in a 96-well plate. After overnight culture and adherence, different concentrations of tumor cell membrane bionic nanodrugs encapsulating lonidamine (Lon@CM-ZIF8) were added and cultured for 24 hours. 200 μl of CCK-8 reagent at a concentration of 10% was added to each well. After incubation for 1 hour, the absorption value was measured at 450 nm. The cell viability was calculated using the following formula:

[0030] Cell viability (%) = [(OD 实验 -OD 调零 ) / (OD 对照 -OD 调零 )]×100

[0031] like Figure 4The results showed that the cytotoxicity of biomimetic nanomedicine (Lon@CM-ZIF8) to breast cancer cells was concentration-dependent, and the higher the concentration, the stronger the anti-tumor effect. Due to the homologous targeting effect of the tumor membrane, the in vitro anti-tumor effect of biomimetic nanomedicine (Lon@CM-ZIF8) was significantly higher than that of free lonidamine and nanomedicine Lon@ZIF8 without cell membrane coating.

[0032] Example 3: Live and dead cell staining

[0033] An appropriate amount of breast cancer 4T1 cells in the logarithmic growth phase were inoculated into a 96-well plate, cultured overnight to adhere to the wall, and then free lonidamine and Lon@CM-ZIF8 were added for incubation and continued to be cultured for 24 hours. Finally, the cells in the culture plate were digested and collected, and the cell uptake of biomimetic nanoparticles was observed using a live-dead cell staining kit.

[0034] The results are as follows Figure 5 As shown, the biomimetic nanodrug (Lon@CM-ZIF8) induced higher tumor cell death compared with free lonidamine.

Claims

1. A tumor cell membrane bionic nanomedicine encapsulating lonidamine, characterized in that: First, a one-pot method is used to prepare metal organic framework nanoparticles encapsulating lonidamine. Then, the tumor cell membrane is coated on the surface of the nanoparticles by repeated membrane extrusion or ultrasound, and finally a tumor cell membrane bionic nanodrug encapsulating lonidamine is prepared.

2. The tumor cell membrane bionic nanomedicine containing lonidamine according to claim 1, characterized in that: The metal organic framework material is ZIF8.

3. The tumor cell membrane bionic nanomedicine containing lonidamine according to claim 1, characterized in that: The tumor cell membrane is selected from one or more of breast cancer cell membrane, melanoma cell membrane, glioma cell membrane, lung cancer cell membrane and cervical cancer cell membrane.

4. The method for preparing a tumor cell membrane bionic nanomedicine containing lonidamine according to claim 1, characterized in that: The preparation method comprises the following steps: dissolving lonidamine and zinc nitrate hexahydrate in a dimethyl sulfoxide solution, adding a methanol solution containing dimethyl imidazole, reacting for a period of time, and obtaining metal organic framework nanoparticles encapsulating lonidamine by centrifugation, washing, and drying. The nanoparticles and tumor cell membranes are mixed in a certain proportion, and the tumor cell membrane bionic nanomedicine encapsulating lonidamine is obtained by membrane extrusion or ultrasonic treatment, and centrifugation and washing.

5. The preparation method according to claim 4, characterized in that: The mass ratio of lonidamine to zinc nitrate hexahydrate is 1:3-1:30, preferably 1:20; the molar ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:3 to 1:72, preferably 1:40; after adding the methanol solution containing dimethylimidazole, the reaction time is 5-120 minutes, preferably 30 minutes; the mass ratio of nanoparticles to tumor cell membranes is 1:1-10:1, preferably 1:

1.

6. The preparation method according to claim 4, characterized in that: The preparation of the tumor cell membrane comprises the steps of: lysing the tumor cells, ultrasonically destroying them, taking the supernatant after gradient centrifugation to obtain tumor cell membrane fragments, and obtaining the tumor cell membrane after membrane extrusion filtration.

7. Use of the tumor cell membrane bionic nanomedicine encapsulating lonidamine according to claim 1 in the preparation of tumor therapeutic drugs.

8. Use according to claim 9, characterized in that The tumor types include one or more of breast cancer cells, melanoma, glioma, lung cancer and cervical cancer.

Images