Preparation and application of catechin nano-lipid particles of cisplatin combined ChR2 gene targeting cervical cancer cells
By preparing catechin nanolipid particles loaded with cisplatin and ChR2 genes, the delivery problem of ChR2 gene in cervical cancer cells and the lack of targeting cisplatin is solved, and efficient and safe targeted cervical cancer cell therapy is achieved.
Patent Information
- Application Number
- CN202411879078.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to achieve efficient and safe delivery of the ChR2 gene in cervical cancer cells, and the chemotherapy drug cisplatin lacks targeting, resulting in damage to normal cells.
Porous catechin nanoparticles and cisplatin were bound by hydrogen bonding, and the plasmid carrying the ChR2 gene was bound to the nanoparticles by electrostatic adsorption, and the liposome membrane modified with EGF was outsourced to prepare catechin nanolipid particles loaded with cisplatin and ChR2 genes ELCC@AR.
Targeted delivery of cisplatin and ChR2 genes was achieved, increasing drug delivery rate and cell transfection rate, synergistically promoting apoptosis of cervical cancer cells, and inhibiting tumor growth.
Smart Images
Figure CN119656335A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of biomedicine and nanotechnology, and in particular to the preparation and application of cisplatin-combined ChR2 gene-targeted catechin nanolipid particles for cervical cancer cells. Background Art
[0002] Cervical cancer is a common malignant tumor that threatens women's life and health. Infection with high-risk human papillomavirus (HPV) is the main cause of cervical cancer. At present, common treatments for cervical cancer include surgery, chemotherapy, and immunotherapy. Although the above treatments can alleviate the disease progression of cervical cancer patients to a certain extent, they also have certain toxic side effects. For example, the chemotherapy drug cisplatin lacks targeting and is easy to cause damage to normal cells. It also requires long-term use and a long treatment cycle. Therefore, a single treatment method cannot meet the current status of cervical cancer treatment, and new treatment methods are urgently needed to solve the current problem.
[0003] Channelrhodopsin-2 (ChR2 for short) is an important light-sensitive channel protein. ChR2 was first discovered in Chlamydomonas reinhardtii in 1991 and has attracted much attention for its unique electrophysiological properties. The protein is a non-selective cation channel protein with 7 transmembrane structures, and its characteristics enable it to be controlled by light pulses. Studies have shown that ChR2 can cause apoptosis of cervical cancer cells, thereby inhibiting tumor proliferation. However, the problem of how to accurately deliver the ChR2 gene to cervical cancer cells remains to be solved.
[0004] In recent years, a variety of nanocarriers have been developed for gene delivery, including liposomes, polymer nanoparticles, inorganic nanoparticles, and viral vectors. However, although nanocarrier delivery technology has shown great potential in ChR2 gene delivery, it still faces a series of technical problems to be solved, such as potential immunogenicity and safety, as well as the need to optimize efficient and specific light-controlled apoptosis effects. Therefore, selecting suitable nanocarriers and optimizing them to achieve efficient and safe delivery of ChR2 genes in cervical cancer cells is an issue that needs to be solved urgently. Summary of the invention
[0005] The present invention combines the porous catechin nanoparticles (AR) and cisplatin through hydrogen bonds, and then uses electrostatic adsorption to combine the plasmid pc DNA 3.1 (+) -ChR2-GFP carrying the ChR2 gene with AR, followed by external wrapping of the liposome membrane modified by EGF, and finally obtains the catechin nanolipid particles ELCC@AR loaded with cisplatin and ChR2 genes. The nanoparticles have the function of targeting cervical cancer cells, and the liposome encapsulation can make cisplatin and the plasmid carrying the ChR2 gene safely reach the cervical cancer cells, increasing the drug delivery rate and cell transfection rate. In addition, the combined treatment of catechins, chemotherapy drugs cisplatin and ChR2 genes, the two synergistically promote the apoptosis of cervical cancer cells, thereby inhibiting tumor growth.
[0006] The present invention also provides a method for preparing catechin nanolipid particles ELCC@AR that are combined with cisplatin and ChR2 gene targeting cervical cancer cells, which specifically comprises the following steps:
[0007] (1) Preparation of AR nanoparticles: 150 mg of catechins, 100 mg of F127 and 1000 mg of NaCl were dispersed and dissolved in 10 mL of 40% ethanol solution; 1 mL of toluene and 150 μL of formaldehyde were added, and the mixture was stirred at 500 rpm for 30 min. Then, 400 μL of NH 3 ·H 2 O was added dropwise for reaction. After reacting at room temperature for 4 h, the mixture was centrifuged at 12000 rpm for 5 min, and then washed three times with a 1:1 mixture of water and ethanol, and the precipitate was dried in a vacuum drying oven at 80 °C for 12 h.
[0008] (2) Preparation of C@AR: AR and cisplatin were mixed in deionized water and incubated at 37°C for 2 days. The mixed solution was transferred to a centrifugal filter with a molecular weight of 50,000 Da and centrifuged at 2000 × g and 26°C for 15 min to obtain C@AR. The C@AR was then dispersed in deionized water and purified by centrifugation three times according to the above conditions.
[0009] (3) Preparation of pcDNA3.1(+)-ChR2-GFP plasmid: The insert ChR2-GFP was extracted from the plasmid FCK-ChR2-GFP, digested with BamHI and EcoRI, and subcloned using the plasmid vector pcDNA3.1(+) as a vector to obtain the luminescent plasmid pc DNA 3.1(+)-ChR2-GFP carrying the ChR2 gene.
[0010] (4) Preparation of CC@AR nanoparticles: C@AR and plasmid pc DNA 3.1(+)-ChR2-GFP were mixed in deionized water and then shaken for 24 h to allow the two to adsorb and bind under electrostatic action to obtain CC@AR nanoparticles.
[0011] (5) ELCC@AR: Using the membrane dispersion method, 0.4 g soybean phospholipid A, 0.1 g cholesterol, and 0.03 g octadecylamine were weighed and dissolved in 10 mL of anhydrous ethanol and transferred to a 250 mL eggplant-shaped bottle. CC@AR nanoparticles were then added to the above solution according to the ratio to the total liposome mass. The organic solvent was removed by rotary evaporation in a constant temperature water bath at 45 °C to form a uniform lipid film. 10 mL of 0.02 mol / L citric acid solution was added, and the solution was hydrated at 50 °C. The solution was ultrasonicated in an ice bath for 5 min, and the solution was extruded through a 0.22 μm microporous filter membrane. 1.0 mL of 0.5 mg / mL EGF stock solution was added. The pH value was adjusted to 6.8 with saturated disodium hydrogen phosphate solution. The solution was incubated at a constant temperature for 25 min to obtain ELCC@AR nanoparticles.
[0012] Preferably, in step (2), the mass ratio of AR to cisplatin is 10:1.
[0013] Preferably, in step (4), the mass ratio of C@AR to plasmid pc DNA 3.1(+)-ChR2-GFP is 5:1.
[0014] Preferably, the mass ratio of the total liposomes to the CC@AR nanoparticles in step (5) is 5:1.
[0015] Application of the ELCC@AR particles prepared by the method of the present invention in the preparation of anti-cervical cancer tumor drugs.
[0016] Preferably, the anti-tumor drug is a drug that carries and specifically expresses ChR2 protein, catechins and cisplatin.
[0017] Preferably, the anti-tumor drug is an anti-tumor drug used for chemotherapy and gene combination therapy; the anti-tumor drug inhibits the growth of cervical cancer cells, promotes calcium ion influx, and promotes apoptosis of cervical cancer cells.
[0018] Preferably, the anti-tumor drug is an anti-tumor drug for intravenous injection combined with laser irradiation; the intravenous injection, the injection dose is 5 mg / kg; the laser irradiation conditions are 460nm blue light irradiation for 3-6min, the light intensity is 0.4W cm -2 .
[0019] The advantages of the present invention are:
[0020] 1. The present invention is the first to simultaneously load cisplatin and pc DNA 3.1(+)-ChR2-GFP onto a nanocarrier, constructing a nanoparticle system for the combined and targeted treatment of cervical cancer. The ELCC@AR nanoparticles of the present invention have the ability to synergistically treat cervical cancer through chemotherapy and gene therapy, and combined with catechin nanoparticles, it has been proven that they can inhibit the growth of cervical cancer cells and have a good anti-tumor effect.
[0021] 2. The ELCC@AR nanoparticles of the present invention carry pc DNA 3.1(+)-ChR2-GFP for the first time and are successfully combined for the treatment of cervical cancer. The modification based on EGF and liposome coating further enhances the targeting of the nanoparticles, allowing the ELCC@AR nanoparticles to accurately reach the tumor site to express ChR2 protein. After irradiation with 460nm blue light, calcium ions flow in, synergizing with cisplatin and catechins to promote apoptosis of cervical cancer cells, and are expected to be widely used in clinical applications of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Transmission electron microscope image of ELCC@AR nanoparticles prepared in the present invention: Figure 1 (a) is CC@AR, Figure 1 (b) is ELCC@AR, Figure 1 (c) Agarose gel electrophoresis of ELCC@AR.
[0023] Figure 2 For in vitro cytotoxicity data: Figure 2 (a) Cell activity diagram of ELCC@AR nanoparticles prepared by the present invention on normal human cervical epithelial cells (HCerEpiC), Figure 2 (b) Cell activity of ELCC@AR nanoparticles on cervical cancer cells (HeLa), ** indicates P < 0.01, **** indicates P < 0.0001.
[0024] Figure 3 This is the expression of ChR2 in HeLa cells: Figure 3 (a) is the expression of ChR2 protein in HeLa cells. Figure 3 (bc) is intracellular Ca 2+ Concentration map, **** P<0.0001.
[0025] Figure 4 Dot plot (a) of HeLa cell apoptosis detected by flow cytometry and statistical graph of apoptotic cell ratio (b), * indicates P < 0.05, *** indicates P < 0.001.
[0026] Figure 5 In vitro anti-tumor performance data: A comparative diagram of the inhibition of HeLa activity by AR, C@AR, and ELCC@AR nanoparticles prepared by the present invention, *** indicates P<0.001, **** indicates P<0.0001.
[0027] Figure 6 For in vivo anti-tumor efficacy and safety data: Figure 6 (ac) are the tumor sizes of mice in each group and their statistical graphs; Figure 6(d) H&E staining results of tissue sections of major organs of mice, * indicates P < 0.05.
[0028] Figure 7 To promote apoptosis of tumor tissue in vivo: Figure 7 (ab) are immunofluorescence images and statistical analysis of apoptotic cells in tumor tissues. Figure 7 (c) is the expression diagram of ChR2 and apoptosis-related proteins in tumor tissues.
[0029] Figure 8 For in vivo targeting performance data: Figure 8 (a) In vivo fluorescence images of tumor-bearing mice at different times after intravenous injection of ELCC@AR. Figure 8 (b) In vitro fluorescence images of major organs and tumor tissues 24 hours after injection. DETAILED DESCRIPTION
[0030] The technical solutions described in the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described in this specification are only part of the feasible technical solutions of the present invention. Other implementation methods obtained by ordinary technicians in this field based on the embodiments of the present invention without any creative work should be deemed to belong to the scope of protection of the present invention.
[0031] Example 1: Preparation of ELCC@AR nanoparticles
[0032] (1) Preparation of AR nanoparticles:
[0033] Disperse 150 mg of catechin, 100 mg of F127 and 1000 mg of NaCl in 10 mL of 40% ethanol; add 1 mL of toluene and 150 μL of formaldehyde, stir at 500 rpm for 30 min, then add 400 μL of NH3·H2O to react, react at room temperature for 4 h, centrifuge at 12000 rpm for 5 min, then wash three times with a 1:1 mixture of water and ethanol, and dry the precipitate in a vacuum drying oven at 80°C for 12 h.
[0034] (2) Preparation of cisplatin-loaded AR nanoparticles (C@AR):
[0035] AR was prepared into deionized water at a concentration of 5 mg / mL and cisplatin at a concentration of 0.5 mg / mL, stirred to form a mixture, and then incubated at 37°C for 2 days (until cisplatin was fully loaded onto AR nanoparticles through hydrogen bonding). The mixed solution was transferred to a centrifugal filter (molecular weight: 50,000 Da), and centrifuged at 2000×g and 26°C for 15 min to obtain C@AR, which was then repeatedly dispersed in deionized water and purified by centrifugation three times under the above conditions.
[0036] (3) Preparation of pcDNA3.1(+)-ChR2-GFP plasmid
[0037] The insert ChR2-GFP was extracted from the plasmid FCK-ChR2-GFP, digested with BamHI and EcoRI, and subcloned using the plasmid vector pcDNA3.1(+) as a vector to obtain the plasmid pc DNA 3.1(+)-ChR2-GFP that can emit light and carries the ChR2 gene.
[0038] (4) Preparation of CC@AR nanoparticles
[0039] C@AR and plasmid pc DNA 3.1(+)-ChR2-GFP were mixed in deionized water at a mass ratio of 5:1, and then shaken for 24 hours to allow the two to adsorb and bind under electrostatic action. The mixture was centrifuged at 10,000×g and 26°C for 15 minutes, and the supernatant (containing the unbound plasmid) was removed to obtain CC@AR nanoparticles.
[0040] (5) EGF-modified liposome membrane-coated CC@AR nanoparticles
[0041] Using the membrane dispersion method, 0.4g soybean phospholipid A, 0.1g cholesterol, and 0.03g octadecylamine were weighed, dissolved in 10mL anhydrous ethanol and transferred to a 250mL eggplant-shaped bottle, and then 1 / 5CC@AR nanoparticles of the total liposome mass were added to the above solution. The organic solvent was removed by rotary evaporation in a constant temperature water bath at 45°C to form a uniform lipid film, and 10mL 0.02mol / L citric acid solution was added. The mixture was hydrated at 50°C, ultrasonicated in an ice bath for 5min, and sized with a 0.22μm microporous filter membrane. 1.0mL 0.5mg / mL EGF stock solution was added and stirred to mix evenly. The pH value was adjusted to 6.8 with saturated disodium hydrogen phosphate solution, and incubated at a constant temperature for 40min to obtain ELCC@AR nanoparticles.
[0042] Example 2: Characterization of the ELCC@AR nanoparticles prepared in Example 1
[0043] (1) Transmission electron microscopy can see structures smaller than 0.2 μm. 5 μL of ELCC@AR sample was carefully dropped on the copper grid, and the excess liquid was absorbed with filter paper. Then, 5 μL of 3% phosphotungstic acid was dropped on the copper grid and stained for 1 min. The excess dye was then absorbed with filter paper. The copper grid was dried at room temperature and then placed in a transmission electron microscope for observation.
[0044] Transmission electron microscopy observed Figure 1 As shown, Figure 1(a) The prepared CC@AR nanoparticles have a uniform spherical nanostructure with a diameter of about 120 nm. The black dots on the surface are cisplatin loading, which is then linked to the plasmid pc DNA 3.1(+)-ChR2-GFP and then coated with EGF-modified liposomes. The electron microscopy image of the obtained ELCC@AR is shown in Figure 2. Figure 1 As shown in (b), its shape is approximately spherical, with uniform size and a diameter of about 200 nm. Membrane-like substances can be seen on its surface and black particles inside, indicating that the liposomes have successfully encapsulated CC@AR.
[0045] (2) Agarose gel electrophoresis experiment: In order to explore whether the EGF-modified liposomes successfully encapsulate the pc DNA 3.1(+)-ChR2-GFP plasmid, the present invention conducted an agarose gel electrophoresis experiment. After the electrophoresis gel is prepared, 20 μL of pcDNA 3.1(+)-ChR2-GFP plasmid solution, CC@AR solution, and ELCC@AR solution are mixed evenly in 4 μL of 6×loading buffer blue indicator in proportion, and then added to the gel card slot, and 5 μL of DNA Marker is added to both sides of the gel. After the preparation work is completed, install the electrophoresis tank cover, and connect the positive and negative poles of the electrophoresis tank to the positive and negative poles of the power supply through wires. Adjust the power supply voltage to a constant voltage of 120V for 35 minutes. After the experiment, observe the DNA bands of the sample using a gel imager.
[0046] like Figure 1 As shown in (c), both the pc DNA plasmid solution and CC@AR solution had corresponding DNA bands, while ELCC@AR did not show corresponding DNA bands, which indicated that ELCC@AR successfully encapsulated the pc DNA 3.1(+)-ChR2-GFP plasmid.
[0047] Example 3: Cytotoxicity evaluation of the ELCC@AR nanoparticles prepared in Example 1
[0048] Normal human cervical epithelial cells (HCerEpiC) and cervical cancer cells (HeLa) were inoculated into 96-well plates, 5000 cells per well, and cultured overnight at 37°C and 5% CO2 until the cells adhered to the wall. ELCC@AR was prepared into solutions of different concentrations by gradient dilution method, and 100 μL of solution was added to each well of the 96-well plate to make the final concentrations in the well plate 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL; after the material and cells were co-cultured for 6 hours, the cells were taken out and incubated at 0.4 W cm -2Excite with 460nm light for 3min, and return to the cell culture incubator for 24 hours after the end of illumination; after the incubation, discard the original culture medium in the 96-well plate, add 5mg / mL sterile MTT in the dark, 10μL per well, and continue to culture in the dark in the incubator at 37C for 4 hours. After 4 hours, discard MTT, add 150μL DMSO to fully dissolve the crystal formazan in the well plate, and use a microplate reader to measure different absorbances at 490nm to analyze cell viability.
[0049] Figure 2 The results of MTT experiments of HCerEpiC and HeLa cells treated with different concentrations of ELCC@AR. Figure 2 (a) It can be seen that the nanoparticles ELCC@AR have no obvious cytotoxic effect on HCerEpiC cells at the detected concentrations, and only have a slight inhibitory effect at a concentration of 16 μg / mL; Figure 2 (b) It can be seen that compared with the blank group, the nanoparticles ELCC@AR can inhibit the growth of HeLa cells with increasing concentration, and the HeLa growth begins to be significantly inhibited at a concentration of 8 μg / mL. At the same time, there is no obvious toxic effect on HCerEpiC at this concentration. The subsequent anti-tumor performance experiment selected 8 μg / mL for experimental research.
[0050] Example 4: In vitro transfection of Hela cells with pc DNA 3.1(+)-ChR2-GFP plasmid
[0051] (1) ChR2 protein expression in Hela cells
[0052] Hela cells were seeded in 6-well plates at a density of 2 × 10 5 The cells were grown in a humidified incubator (5% CO2) at 37°C overnight. The ELCC@AR prepared in Example 1 was added to the well plate to a final concentration of 8 μg / mL. The control group was added with PBS solution. After 6 h of co-culture, the cells were illuminated with a 460 nm blue light laser (0.4 W cm -2 ) for 3 min and continued incubation for 12 h. After that, total cell protein was extracted and the expression of ChR2 protein was observed by Western Blot experiment.
[0053] The results of band analysis are as follows Figure 3 As shown in (a), ChR2 protein is expressed in Hela cells after blue light irradiation, indicating that ELCC@AR nanoparticles have a good gene delivery effect.
[0054] (2) Intracellular calcium ion status of Hela cells
[0055] Intracellular Ca2+ Use Rhod-2 AM to measure levels. Rhod-2 can stay inside the cell and bind to calcium ions, emitting red fluorescence. By measuring the intensity of the red fluorescence inside the cell, the intracellular Ca 2 + The concentration can be evaluated. The cells were inoculated in 6 confocal culture dishes, and the ELCC@AR prepared in Example 1 was added to the well plate to a final concentration of 8 μg / mL. The control group was added with PBS solution. After 6 hours of co-culture, the blue light 460nm laser (0.4W cm -2 ) for 3 min and continued to incubate for 24 h. After the incubation, the cells were washed with PBS and incubated with 5 μM Rhod-2 AM at 37 ° C for 30 min in the dark. The level was observed using a confocal microscope, then measured by flow cytometry, and statistical analysis was performed using GraphPad Prism 8.0.2 software.
[0056] Nanoparticles ELCC@AR carry the ChR2 gene into Hela cells to express the protein ChR2, which is a cell membrane channel protein. After irradiation with 460nm blue light, the channel opens, allowing Ca 2+ Ions flow into the cytoplasm. Figure 3 (b), the red fluorescence of the ELCC@AR group was significantly stronger than that of the Control group. The results of statistical analysis Figure 3 (c) It can be seen that the Ca in the cytoplasm of the ELCC@AR group 2+ The ion concentration was significantly higher than that of the Control group, indicating that the plasmid was carried into the cells by the nanoparticles and expressed the ChR2 protein, which opened the calcium ion channel under blue light stimulation.
[0057] Example 6: In vitro apoptosis-promoting experiment of ELCC@AR nanoparticles
[0058] Hela cells were seeded in 6-well plates at a density of 2 × 10 5 The cells were cultured in a humidified incubator (5% CO2) at 37°C overnight. AR, C@AR, and ELCC@AR prepared in Example 1 were added to the corresponding wells to make the final concentrations of 8 μg / mL. The control group was added with an equal amount of PBS solution. After 6 hours of co-culture, the ELCC@AR group was illuminated with a 460nm blue light laser (0.4W cm -2 ) for 3 min, and then continue to culture for 24 h, and then take out the cells for flow cytometry detection.
[0059] Figure 4 HeLa cells were treated with AR, C@AR, and ELCC@AR nanoparticles and then double-stained with PI / annexinV-FITC, and cell apoptosis was observed by flow cytometry. Figure 4 (a) It can be observed that compared with the Control group, the number of apoptotic cells in the AR, C@AR, and ELCC@AR groups increased. According to the statistical results Figure 4 (b) It can be seen that compared with the AR group, the number of apoptotic cells in the C@AR group increased, which may be due to the enhanced killing effect of cisplatin on HeLa cells; when cisplatin combined with ChR2 protein was irradiated with blue light, ELCC@AR nanoparticles showed a more significant performance in promoting HeLa cell apoptosis compared with C@AR, which indicates that after blue light irradiation, protein ChR2 opens the cell's Ca 2+ Channels may promote the pro-apoptotic effect of cisplatin, which shows that cisplatin combined with catechins and protein ChR2 has a stronger ability to promote cancer cell apoptosis.
[0060] Example 6: In vitro anti-tumor performance experiment of ELCC@AR nanoparticles
[0061] Hela cells were seeded into 96-well plates at a density of 5000 cells per well and cultured in a cell culture incubator for 24 h. AR, C@AR, and ELCC@AR prepared in Example 1 were added to the corresponding wells to make their final concentrations all 8 μg / mL. The control group was added with an equal amount of PBS solution. After 6 h of co-culture, the ELCC@AR group was illuminated with a blue light 460 nm laser (0.4 W cm -2 ) for 3 min and then cultured for 24 h. After incubation, the CCK8 method was used to determine the cell survival rates of different groups.
[0062] According to the experimental results Figure 5 It can be seen that catechins did not significantly inhibit HeLa proliferation. When cisplatin was added, the effect of inhibiting tumor growth was more significant. In addition, after the ChR2 protein gene was transferred, the nanoparticles ELCC@AR treated with light showed better anti-tumor effects than the nanoparticles C@AR without the ChR2 gene. This shows that the ChR2 gene was successfully transferred into Hela cells and expressed ChR2 protein. After blue light irradiation, calcium ions flowed into the cytoplasm, which reduced the mitochondrial membrane potential and promoted the production of ROS, and synergized cisplatin and catechins to promote cell apoptosis. Nanoparticles ELCC@AR promoted the apoptosis of Hela cells through the synergistic effect of chemotherapy and gene therapy, which reflected the good anti-tumor performance of ELCC@AR.
[0063] Example 7: In vivo anti-tumor performance and safety experiments of ELCC@AR nanoparticles
[0064] In order to evaluate the therapeutic effect of ELCC@AR nanoparticles in cervical cancer tumor-bearing mice, we selected 16 nude mice with cervical tumors to evaluate the efficacy and safety of in vivo tumor treatment. The mice were randomly divided into four groups, and the average initial tumor volume of all groups was similar, ranging from 40 to 60 mm. 3 Then, mice were injected with PBS, AR, C@AR, and ELCC@AR at a dose of 5 mg / kg through the tail vein four times (6, 8, 11, and 13 days after tumor inoculation), respectively. Twelve hours after each injection, the blue light 460 nm laser (0.4 W cm -2 ) irradiated the tumor site for 6 min. The body weight and tumor volume of each mouse were measured every day. On the 18th day, the mice were killed by cervical dislocation, and the tumors were collected, photographed and weighed. The main organs of the mice, heart, liver, spleen, lung, kidney, and thymus were collected and used for H&E staining in tissue fixative.
[0065] The experimental results are as follows Figure 6 As shown in (ab), compared with the Control group, the tumor growth rate in the AR group was slightly slowed, and the tumor growth in the C@AR group and the ELCC@AR group was effectively inhibited. C@AR and AR showed antitumor activity after the 3rd and 4th treatments, respectively (12th and 14th days after tumor inoculation, P<0.05). However, compared with the AR group, ELCC@AR significantly induced tumor reduction after the second injection (P<0.05). After the last treatment on the 14th day after tumor inoculation, ELCC@AR showed a significant reduction in tumor volume at the end of the follow-up period compared with C@AR. This result is consistent with Figure 6 (c) is consistent with the growth curve shown. After an 18-day treatment period, the average tumor weight of the ELCC@AR group was the lowest among all the drug-dosing groups. These results indicate that the ELCC@AR group has stronger in vivo antitumor activity than the C@AR group. In addition, the body weight of the mice was monitored daily as an indicator of systemic toxicity of drug administration. The arrow indicates the drug injection time. After 4 injections at a dose of 5 mg / kg, the body weight of the ELCC@AR group was significantly decreased compared with the other groups (P<0.05). Finally, a systematic evaluation of the biosafety of the ELCC@AR group was carried out, and the results are shown in Figure 6 As shown in (d), compared with the Control group, the tissue morphology of the organs in the ELCC@AR group did not change significantly after staining, indicating that the ELCC@AR nanoparticles had no toxic side effects on the main organs of mice and had no adverse effects on liver and kidney functions, and had good biosafety.
[0066] Example 8: Study on the promotion of tumor tissue apoptosis by ELCC@AR nanoparticles in vivo
[0067] Mice were treated as described in Example 7. At the end of the anti-tumor experiment, the tumor tissues of the mice were collected and frozen in liquid nitrogen. A portion of the mouse tumor tissue was taken for WB experiment to observe the expression of ChR2 and C-caspase3 proteins in the tissue; another portion of the tumor tissue was taken for frozen section (5 μm), and the sections were stained with TUNEL kit according to the instructions, and the presence of TUNEL signal in the cell nucleus was determined by co-localization of DAPI staining. Fluorescence images were taken using an inverted fluorescence microscope and analyzed using Image J software. The quantification of TUNEL staining was calculated based on the ratio of the average green (TUNEL) to blue (DAPI) fluorescence area in each section.
[0068] In order to observe the effect of AR, C@AR and ELCC@AR in promoting tumor cell apoptosis in vivo, the present invention observed the expression of apoptotic proteins by WB experiment and observed the tumor tissue sections of mice by immunofluorescence staining. The experimental results are as follows Figure 7 As shown in (ab), the ELCC@AR group showed the highest number of TUNEL-positive tumor cells compared with the other groups, indicating that a large amount of apoptosis occurred in the tumor tissue. Figure 7 (c) As shown in the WB band, the expression level of ChR2 protein in tumor tissue of the ELCC@AR group was higher than that in other groups, indicating that the ELCC@AR nanoparticles carrying the ChR2 gene were successfully transfected into cancer cells and expressed the ChR2 protein after blue light irradiation; while the expression level of C-caspase3 protein in the ELCC@AR group was higher than that in the C@AR group, indicating that the ChR2 protein opened the calcium ion channel and promoted the cell apoptosis activity caused by cisplatin. This result showed that ELCC@AR in vivo can induce more tumor cell death, reflecting the efficient anti-tumor effect of the combination of chemotherapy and gene therapy.
[0069] Example 9: In vivo targeting study of FL-Ru@Pt-C nanoparticles
[0070] In order to further evaluate the ability of ELCC@AR to target tumor tissue in vivo, the present invention used Hela tumor-bearing mouse model to study the tumor accumulation and biological distribution of ELCC@AR. A group of healthy mice with a tumor volume of about 900mm 2 Nude mice were injected with ELCC@AR (5 mg / kg) through the tail vein, and the fluorescence imaging ability of ELCC@AR in nude mice was detected using a small animal in vivo imager. Fluorescence imaging was performed at different times (0h, 2h, 4h, 6h, 12h and 24h). After imaging, the mice were euthanized and their tissues and organs (heart, liver, spleen, lung, kidney, tumor) were dissected and fluorescence imaging was performed under the same excitation wavelength.
[0071] The experimental results are as follows Figure 8As shown in (a), the fluorescence spread throughout the body within 2 hours of injection. As time went on, the fluorescence at the tumor site became stronger and stronger, and the fluorescence at the tumor site was strongest around 10-12 hours. In addition, 24 hours after ELCC@AR injection, although the fluorescence at the tumor site partially decreased, the fluorescence signal could still be clearly observed. This shows that ELCC@AR can successfully target the tumor site. Subsequently, the nude mice were euthanized, and the heart, liver, spleen, lung, kidney and tumor were taken for imaging to observe the distribution of ELCC@AR in organs and tissues. Figure 8 (b) It can be seen that after the tail vein injection of ELCC@AR, due to the targeting recognition performance of EGF and the passive EPR effect, ELCC@AR can be targeted to the cervical cancer tumor cell site for fluorescence imaging. The above results show that EGF specifically recognizes and binds to the EGF receptor on the membrane of human cervical cancer Hela cells, and is transported into the cell by endocytosis ELCC@AR, and is enriched in the tumor site in large quantities, achieving the purpose of targeting anticancer drugs to tumor cells.
[0072] In summary, the ELCC@AR nanoparticles of the present invention can target the tumor site, open calcium ion channels through blue light irradiation, and promote apoptosis of cervical cancer cells in combination with catechins and cisplatin. Experiments have verified that the nanoparticles combined with chemotherapy and gene therapy have good cervical cancer treatment effects in vivo and in vitro, and have no obvious toxic side effects on organisms, providing a new nano-drug delivery system for the clinical treatment of cervical cancer.
[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.
Claims
1. A cisplatin combined with ChR2 gene targeting catechin nanolipid particles for cervical cancer cells, characterized in that: The nanoparticles are prepared from plasmid pc DNA 3.1(+)-ChR2-GFP, AR, cisplatin, and EFG-modified liposome membranes. The nanoparticles are ELCC@AR, which combine AR and cisplatin through hydrogen bonds, and then combine plasmid pc DNA 3.1(+)-ChR2-GFP and AR through electrostatic adsorption, and then are externally wrapped with liposome membranes modified by EGF to finally obtain nanoparticles ELCC@AR.
2. A method for preparing the cisplatin combined with ChR2 gene targeted catechin nanolipid particles for cervical cancer cells according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Preparation of AR nanoparticles: 150 mg of catechin, 100 mg of F127 and 1000 mg of NaCl were dispersed and dissolved in 10 mL of 40% ethanol solution; 1 mL of toluene and 150 μL of formaldehyde were added, and the mixture was stirred at 500 rpm for 30 min, and then 400 μL of NH3·H2O was added dropwise for reaction. After reacting at room temperature for 4 h, the mixture was centrifuged at 12000 rpm for 5 min, and then washed three times with a mixture of 1:1 water and ethanol, and the precipitate was dried in a vacuum oven at 80 °C for 12 h; (2) Preparation of C@AR: AR and cisplatin were mixed in deionized water and incubated at 37°C for 2 days. The mixed solution was transferred to a centrifugal filter with a molecular weight of 50,000 Da and centrifuged at 2000 × g and 26°C for 15 min to obtain C@AR. The C@AR was then dispersed in deionized water and purified by centrifugation three times according to the above conditions. (3) Preparation of pcDNA3.1(+)-ChR2-GFP plasmid: The insert ChR2-GFP was extracted from the plasmid FCK-ChR2-GFP, digested with BamHI and EcoRI, and subcloned using the plasmid vector pcDNA3.1(+) as a vector to obtain the luminescent plasmid pc DNA 3.1(+)-ChR2-GFP carrying the ChR2 gene; (4) Preparation of CC@AR nanoparticles: C@AR and plasmid pc DNA 3.1(+)-ChR2-GFP were mixed in deionized water and then shaken for 24 h to allow the two to adsorb and bind under electrostatic action to obtain CC@AR nanoparticles; (5) ELCC@AR: Using the membrane dispersion method, 0.4 g soybean phospholipid A, 0.1 g cholesterol, and 0.03 g octadecylamine were weighed, dissolved in anhydrous ethanol, and transferred to a 250 mL eggplant-shaped bottle. CC@AR nanoparticles were then added to the above solution according to the ratio to the total liposome mass. The organic solvent was removed by rotary evaporation in a constant temperature water bath at 45 °C to form a uniform lipid film. 10 mL of 0.02 mol / L citric acid solution was added, and the mixture was hydrated at 50 °C. Ultrasonication was performed on an ice bath for 5 min, and the mixture was extruded through a 0.22 μm microporous filter membrane. 1.0 mL of 0.5 mg / mL EGF stock solution was added, and the pH value was adjusted to 6.8 with saturated disodium hydrogen phosphate solution. The mixture was incubated at a constant temperature for 25 min to obtain ELCC@AR nanoparticles.
3. The method for preparing a cisplatin combined with ChR2 gene targeted catechin nanolipid particles for cervical cancer cells according to claim 2, characterized in that: The mass ratio of AR to cisplatin in step (2) is 10:
1.
4. The method for preparing a cisplatin combined with ChR2 gene targeted catechin nanolipid particles for cervical cancer cells according to claim 2, characterized in that: Step (4) The mass ratio of C@AR and plasmid pc DNA 3.1(+)-ChR2-GFP is 5:
1.
5. The method for preparing a cisplatin combined with ChR2 gene targeted catechin nanolipid particles for cervical cancer cells according to claim 2, characterized in that: In step (5), the mass ratio of total liposomes to CC@AR nanoparticles was 5:
1.
6. Use of the cisplatin combined with ChR2 gene-targeted catechin nanolipid particles for cervical cancer cells as claimed in claim 1 in anti-cervical cancer drugs.
7. The use according to claim 6, characterized in that The anti-tumor drug is an anti-tumor drug used for chemotherapy and gene combined therapy.
8. The use according to claim 6, characterized in that The anti-tumor drug can inhibit the growth of cervical cancer cells, promote the influx of calcium ions, and promote the apoptosis of cervical cancer cells.
9. The use according to claim 6, characterized in that: The anti-tumor drug is an anti-tumor drug for intravenous injection combined with laser irradiation administration; The intravenous injection dose is 5 mg / kg; The laser irradiation conditions are 460nm blue light irradiation for 3-6min, and the light intensity is 0.4W cm -2 .