Homologous cancer cell membrane coated ZIF-8 bionic nanoparticles and preparation method thereof

Abstract

The invention discloses homologous cancer cell membrane coated ZIF-8 bionic nanoparticles and a preparation method thereof, and the homologous cancer cell membrane coated ZIF-8 bionic nanoparticles enhance the breast cancer treatment effect by combining a chemotherapy and a gene therapy in a bionic cell membrane nano system. According to the nano system, a unique formula is utilized, and a metal organic framework (ZIF-8s) and DNAzymes are combined together, so that the limitation of traditional chemotherapy is solved. The nanoparticles are coated with a membrane produced by homologous breast cancer cells. According to the bionic coating, the nanoparticles can better escape from the recognition of an immune system, and the homologous targeting ability of the nanoparticles is enhanced. The nano-particles have the advantages that the nano-particles can simulate cancer cells, improve targeting and biocompatibility, degrade and release DOX, DNAzymes and Zn < 2 + > ions in an endosome acid environment, activate the DNAzymes, cut a specific mRNA sequence, cause down-regulation of survivin expression, enable the cancer cells to be sensitive to chemotherapy and enhance the cytotoxic effect of the DOX. The use of the nanoparticles is expected to improve the curative effect of chemotherapy and reduce the side effect of chemotherapy.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to homologous cancer cell membrane-coated ZIF-8 bionic nanoparticles and a preparation method thereof. Background Art

[0002] Today, cancer is still one of the leading causes of death worldwide. Among women, breast cancer is one of the most common types of cancer and one of the leading causes of cancer death among women in many countries and regions. According to the latest data released by the International Agency for Research on Cancer (IARC), breast cancer is the second most common cancer after lung cancer, and is the leading cause of cancer morbidity and mortality in women, accounting for 23.8% and 15.4% respectively, seriously threatening women's lives and health.

[0003] Doxorubicin (DOX) is a broad-spectrum anti-tumor drug that inhibits nucleic acid synthesis by embedding into DNA and has a killing effect on tumor cells in various growth cycles. However, the non-specificity caused by systemic administration of chemotherapy drugs leads to significant toxic side effects and drug resistance in normal tissues or cells, which greatly limits the efficiency of cancer treatment. Deoxyribozyme (DNAzyme) is a single-stranded DNA sequence with catalytic activity that can catalyze a variety of different chemical reactions. The structure of DNAzyme includes a catalytic core sequence and two substrate binding arm sequences located on both sides. After the binding arm sequence recognizes and targets the substrate mRNA molecule, the catalytic core cuts the mRNA molecule at a specific phosphodiester bond. By changing the binding arm sequence, DNAzyme can be designed to target different RNA substrates. However, DNAzyme still has major challenges to be solved, and an efficient DNAzyme delivery platform with sufficient auxiliary factor supply has not yet been developed.

[0004] In the field of drug delivery, ZIF-8 nanoparticles have attracted much attention due to their unique physicochemical properties. ZIF-8 nanoparticles have high specific surface area, good chemical stability, and controllable drug release under different conditions, which make them potential drug carriers. However, ZIF-8 nanoparticles also have some limitations, such as insufficient biocompatibility, low nonspecific targeting, and easy clearance by the immune system in the body. Summary of the invention

[0005] In view of the existing problems, the object of the present invention is to provide a homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticle and a preparation method thereof.

[0006] The specific technical solutions are as follows: A method for preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles comprises the following steps: 1) Preparation of ZIF-8 biomimetic nanoparticles for drug loading: zinc ion solution and DOX solution were mixed, then added dropwise to 2-methylimidazole solution, stirred continuously, centrifuged and washed, and mixed with nucleic acid drug solution; 2) Extraction of homologous cancer cell membranes: Collect cells, wash with pre-cooled PBS solution, and extract and purify cell membranes; 3) Preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles: mixing the nanoparticles of step 1) and the cell membrane of step 2) to obtain homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles.

[0007] Furthermore, the mass ratio of ZIF-8 biomimetic nanoparticles and homologous cancer cell membranes was 3:1.

[0008] Furthermore, the zinc ion is zinc nitrate hexahydrate, the mass ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:10, and the nucleic acid drug is one of siRNA, DNAzyme, and ASO.

[0009] Furthermore, homologous cancer cell membranes were extracted using the Bio-Time Cell Membrane Protein Extraction Kit P0033.

[0010] Furthermore, the mixing process in step 3) is one of room temperature incubation, mechanical extrusion and ultrasonic treatment.

[0011] A homologous cancer cell membrane-coated ZIF-8 prepared by the above preparation method.

[0012] Advantages and beneficial effects of the present invention The method provided by the present invention cleverly constructs a homologous cancer cell membrane bionic nano drug delivery system, which carries drugs through a simple "one-pot method" and electrostatic interaction. The presence of the bionic membrane not only improves the biocompatibility and immune escape ability of the nanoparticles, but also improves the synergistic anti-cancer effect through cell membrane coating technology, and further enhances the synergistic anti-cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The particle size and potential distribution diagram of ZIF-8, NPs / DOX / DNAzyme and CM@NPs / DOX / DNAzyme in Example 1.

[0014] Figure 2 The cleavage activity of the DNAzymes of Example 2 under different concentrations of auxiliary factors in vitro.

[0015] Figure 3 In Example 3, flow cytometry analysis was performed to determine whether homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles were taken up by MCF-7 cells.

[0016] Figure 4This is Example 4, flow cytometry analysis of the immune escape characteristics of homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles.

[0017] Figure 5 Viability analysis and IC50 value of cells uptake of homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles as in Example 5. DETAILED DESCRIPTION

[0018] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention.

[0019] Example 1

[0020] Preparation of ZIF-8 biomimetic nanoparticles coated with homologous cancer cell membrane (1) Homologous cancer cell membrane extraction MCF-7 cells were cultured in a culture dish. When the cell density grew to 90%, the cells were digested with a trypsin-free Versene solution, collected by centrifugation at 500g for five minutes, washed once with pre-cooled PBS, and counted. Membrane protein extraction reagent A containing 1mM PMSF was added to the cells, the cells were fully suspended, and placed on ice for 15 minutes. The samples were then placed in liquid nitrogen-room temperature for 3 cycles, the samples were centrifuged at 800g for 10 minutes (4°C), the precipitate was discarded, and then the cell membrane precipitate was collected by centrifugation at 14000g for 30 minutes (4°C) for use.

[0021] Use the Bio-Tech Cell Membrane Protein and Cytoplasm Protein Extraction Kit (P0033) (2) Preparation of ZIF-8 biomimetic nanoparticles loaded with drugs Dissolve 5 mg of doxorubicin (dobiprofen), 100 mg of zinc nitrate hexahydrate and 1000 mg of 2-methylimidazole in 4 mL, 0.4 mL and 4 mL of deionized water, respectively, and slowly drop the zinc nitrate hexahydrate solution into the doxorubicin solution and stir at 1000 rpm for 5 min. Slowly drop the mixed solution of doxorubicin and zinc ions into the 2-methylimidazole solution and stir at 1200 rpm for 10 min. Centrifuge the above mixture solution at 8000 g for 10 min, discard the supernatant, and collect the precipitate. Wash the precipitate 3 times with an aqueous solution containing 2% ethanol. That is, the preparation of ZIF-8 biomimetic nanoparticles carrying drugs is obtained, which is recorded as NPs / DOX.

[0022] (3) Preparation of homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles The washed nanoparticles were mixed with DNAzyme solution at a mass ratio of 40:1 at room temperature for 30 minutes, which was recorded as NPs / DOX / DNAzyme. The extracted homologous cancer cell membrane was mixed with nanoparticles carrying doxorubicin and DNAzyme at a mass ratio of 3:1, sonicated with an ultrasonic probe for 8 minutes, and recovered at room temperature for 30 minutes to obtain homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles, which were recorded as CM@NPs / DOX / DNAzyme.

[0023] like Figure 1 A dynamic light scattering (DLS) in the figure shows that the average particle size of ZIF-8 after hydration dynamics is 213.48nm, the average particle size after loading with DOX and DNAzyme drugs is 212.94nm, and the average particle size after coating with homologous cancer cell membrane is 279.90nm. Zeta potential results Figure 1 B in the figure shows that the potential of ZIF-8 was 43.86 mV. After loading with DOX and DNAzyme drugs, the potential was reversed to -6.04 mV. After being coated with the homologous cell membrane, the charge was -32.82 mV.

[0024] Example 2

[0025] DNAzyme in vitro cleavage activity assay To test the self-activation of DNAzyme to examine the cleavage activity, DNAzyme (1 μM) and substrate strand (1 μM) were mixed in Hepes buffer and various concentrations of Zn 2+ or Mg 2+ Incubate for 30 min. Lysates were separated by 20% non-denaturing PAGE, and the cleavage rate was analyzed using ImageJ software.

[0026] like Figure 2 As shown in A, with Zn 2+ With the increase of Zn concentration, after 30 min of incubation, the gel band of the cleaved substrate gradually increased, indicating that there was obvious 2+ activity-dependent. In contrast, Mg 2+ Even at 1 mM, the cleavage activity of DNAzyme was quite weak. The cleavage rate was then quantified to obtain Figure 2 The result of B.

[0027] Example 3

[0028] Cellular uptake experiments MCF-7 cells were seeded in 24-well plates at a cell density of 1×10 5 / well, placed in a 5% CO2, 37℃ incubator. After 24 hours, free FAM-DNAzyme (fluorescently labeled DNAzyme), free DOX, NPs / DOX / DNAzyme and CM@NPs / DOX / DNAzyme were given. Eight hours after administration, the medium was removed, washed once with PBS, digested with trypsin, and washed again by centrifugation three times. The samples were placed on flow cytometry for analysis. FAM-DNAzyme labeling was detected using the FITC fluorescence channel, and DOX was detected using the PE fluorescence channel.

[0029] like Figure 3 As shown in the figure, compared with nanoparticles not coated by homologous cell membranes, MCF-7 cells have a higher uptake of homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles. Since DOX drug itself has a positive charge, it interacts with the negatively charged cell membrane and is easier to enter the cell. On the contrary, negatively charged DNAzyme is more difficult to actively enter the cell.

[0030] Example 4

[0031] Immune escape assay Raw264.7 cells were seeded on a 24-well plate. When the cell density grew to 70%, an equal amount of CM@NPs / DOX / DNAzyme and NPs / DOX / DNAzyme were added to each well. After incubation for 8 h, the cells were washed once with PBS, digested with trypsin, and centrifuged and washed again three times. The cells were placed on a flow cytometer for analysis. FAM-labeling was detected using the FITC fluorescence channel, and DOX was detected using the PE fluorescence channel.

[0032] like Figure 4 As shown, the cell uptake of CM@NPs / DOX / DNAzyme was significantly reduced, which was beneficial to reduce nonspecific endocytosis and had immune escape characteristics.

[0033] Example 5

[0034] Cytotoxicity experiment of ZIF-8 biomimetic nanoparticles coated with homologous cancer cell membrane (1) MCF-7 cells were seeded in 96-well plates at a cell density of 5×10 3, after 24 hours of culture, drugs were administered, including DOX, DNAzyme, nanoparticles loaded with DOX (NPs / DOX), nanoparticles loaded with DOX and DNAzyme (NPs / DOX / DNAzyme), and unloaded homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles (CM@NPs), and drug-loaded homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles (CM@NPs / DOX / DNAzyme). After 48 hours of administration, the culture medium was removed and a culture medium containing 10% CCK-8 solution was added to each well. After culturing in a 5% CO2, 37℃ incubator for 30 minutes, the OD450 value of each well was detected by a multifunctional microplate reader. Cell survival rate (%) = (OD value of the experimental group - OD value of the blank control group) / (OD value of the control group - OD value of the blank control group) × 100%.

[0035] like Figure 5 As shown in A in the figure, free DNAzyme is difficult to actively enter cells due to its negative charge and does not show cytotoxicity. Figure 5 As shown in B, the biomimetic nanoparticles without drug loading have excellent biocompatibility (e.g. Figure 5 B). Under incubation conditions with different DOX concentrations, the cell viability decreased in the order of NPs / DOX, NPs / DOX / DNAzyme, and CM@NPs / DOX / DNAzyme (e.g. Figure 5 C in the figure), among which CM@NPs / DOX / DNAzyme showed stronger cancer cell killing ability than NPs / DOX / DNAzyme (e.g. Figure 5 D in.

Claims

1. A method for preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles, characterized in that: The steps include: 1) Preparation of ZIF-8 biomimetic nanoparticles for drug loading: zinc ion solution and DOX solution were mixed, then added dropwise to 2-methylimidazole solution, stirred continuously, centrifuged and washed, and mixed with nucleic acid drug solution; 2) Extraction of homologous cancer cell membranes: Collect cells, wash with pre-cooled PBS solution, and extract and purify cell membranes; 3) Preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles: mixing the nanoparticles of step 1) and the cell membrane of step 2) to obtain homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles.

2. The method for preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles according to claim 1, characterized in that: The mass ratio of ZIF-8 nanoparticles and homologous cancer cell membranes was 3:

1.

3. The method for preparing a homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticle according to claim 1, characterized in that: The zinc ion is zinc nitrate hexahydrate, the mass ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:10, and the nucleic acid drug is one of siRNA, DNAzyme, and ASO.

4. The method for preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles according to claim 1, characterized in that: Homologous cancer cell membranes were extracted using the Bio-Time Cell Membrane Protein Extraction Kit P0033.

5. The method for preparing homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles according to claim 1, characterized in that: The mixing process in step 3) is one of room temperature incubation, mechanical extrusion and ultrasonic treatment.

6. Homologous cancer cell membrane-coated ZIF-8 biomimetic nanoparticles prepared by the preparation method according to claims 1-5.

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