Application of hypericin nanoparticles subjected to photodynamic treatment in preparation of drugs for inhibiting liver cancer cells

Abstract

The invention provides application of hypericin nanoparticles subjected to photodynamic treatment in preparation of a medicine for inhibiting liver cancer cells. The hypericin nanoparticles (HC-NPs) are formed by wrapping a traditional Chinese medicine monomer hypericin (HC) with distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000). The hypericin nano-particles provided by the invention have an obvious inhibition effect on liver cancer cells under a specific illumination condition, so that the expression of the gene SERPINE1 is up-regulated in a time-dependent and dose-dependent manner. The gene participates in regulation of biological behaviors of liver cancer, and provides a new view angle for treatment of liver cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of drugs for treating liver cancer, and relates to the application of hypericin nanoparticles treated with photodynamic therapy in the preparation of drugs for inhibiting liver cancer cells. Background Art

[0002] Hepatocellular carcinoma (hereinafter referred to as liver cancer) is the fifth most common malignant tumor in the world. Since most liver cancer patients are in the middle and late stages when they seek medical treatment, their overall prognosis is poor, and it has become the third most lethal cancer in the world. Liver cancer brings a serious burden to human health and social development, and is a health problem that needs to be solved urgently. With the development of medical technology, the current clinical treatment methods for liver cancer are mainly surgical resection, ablation, and comprehensive treatment. The treatment effect of liver cancer is limited by many factors, and drug resistance may occur during the treatment process. Although there are still other treatment options, such as broad-spectrum tyrosine kinase inhibitors (TKI)-sorafenib and lenvatinib, as well as immunotherapy drugs atezolizumab and anti-angiogenic drugs bevacizumab, these treatments can only prolong patient survival to a certain extent.

[0003] Photodynamic therapy (PDT) is a method of diagnosing and treating diseases based on three elements: photosensitizer, light, and reactive oxygen species. Its basic principle is that photosensitizer (PS) absorbs the energy of excitation light of a specific wavelength, and then undergoes a photochemical reaction with oxygen around the tissue to generate cytotoxic reactive oxygen species (ROS), thereby killing tumor cells. Therefore, photodynamic therapy is a new and promising treatment strategy for liver cancer.

[0004] Hypericin (HC), as an effective photosensitizer, has also shown great application potential in the field of photodynamic therapy. However, judging from the pharmacokinetic test results, the water solubility and bioavailability of hypericin are not ideal. Nanoencapsulation technology can effectively enhance the solubility and bioavailability of drugs, enhance the therapeutic effect, and improve the physical and chemical properties of drugs. It can also make drugs targeted and reduce toxic side effects.

[0005] In summary, hypericin, as an excellent photosensitizer, is expected to show outstanding potential in the treatment of liver cancer with the support of nanoencapsulation technology. Summary of the invention

[0006] In view of the problem that hypericin is a highly efficient photodynamic therapy photosensitizer in the prior art and is expected to improve the therapeutic effect of tumors, but the effect and mechanism of photodynamic therapy in liver cancer are still unclear, the present invention provides the use of hypericin nanoparticles after photodynamic treatment in the preparation of drugs for inhibiting liver cancer cells. Based on the hypericin nanoparticles after photodynamic treatment, the present invention proposes an in-depth study on the inhibitory effect and mechanism of action on liver cancer. The effect of hypericin nanoparticles after photodynamic treatment on the biological behavior of liver cancer cells and the possible mechanism of action are evaluated through in vitro experiments, which will provide more ideas for the preparation of clinical liver cancer drugs.

[0007] The technical route of the present invention is as follows:

[0008] 1) CCK-8 assay, growth curve, Transwell assay, etc. were performed in two liver cancer cell lines (Bel-7402 and Huh7) to detect the effects and differences of hypericin nanoparticles and photodynamic therapy on liver cancer cells;

[0009] 2) Use bioinformatics database and drug simulation molecular docking system to analyze and obtain hypericin-related targets, and use protein immunoblotting (WB) and real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods to detect the protein and mRNA levels of related targets in cell samples;

[0010] Application of photodynamically treated hypericin nanoparticles in photodynamic anti-hepatocancer drugs.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The method for preparing hypericin nanoparticles after photodynamic treatment comprises the following steps:

[0013] 1) Preparation of Hypericin Nanoparticles:

[0014] Hypericin nanoparticles (HC-NPs) were prepared by nano-coprecipitation method. Hypericin (2 mg) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) (20 mg) were mixed in 2 ml of tetrahydrofuran (THF) to fully dissolve them. Ultrapure water was slowly added dropwise at room temperature and under ultrasonic conditions every 30 min. The ratio of hypericin, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and ultrapure water was 2 mg:20 mg:4 mL.

[0015] Under ventilation conditions, stir with a magnetic stirrer overnight to remove tetrahydrofuran;

[0016] If solid precipitates are still found the next day, tetrahydrofuran is added continuously and the above operation is repeated until no more precipitates appear. Ultrasonic oscillation is continued for 72 hours and the volume is fixed to 4 ml to finally obtain the working mother solution of hypericin nanoparticles, which is stored at 4°C in the dark.

[0017] The particle diameter was then measured using a transmission electron microscope (JEOL JEM-2100);

[0018] 2) Add the hypericin nanoparticles obtained in the previous step to the cell culture dish and irradiate with light for 10 min using a xenon lamp light source with a light intensity of 100 mW / cm 2 , and obtain hypericin nanoparticles after photodynamic treatment.

[0019] Furthermore, the molecular formula of the hypericin is 9,11,13,16,18,20-hexahydroxy-5,24-dimethyloctacyclo[13.11.1.12,10.03,8.04,25.019,27.021,26.014,28]octane-1(26),2,4(25),5,8,10,12,14(28),15(27),16,18,20,23-tridecane-7,22-dione, which is prepared by Desit Biotechnology Co., Ltd. with a purity of ≥98.00%.

[0020] Furthermore, the xenon lamp light source described in step 2) was purchased from Beijing Zhongjiao Jinyuan Technology Co., Ltd., model: CEL-S500.

[0021] The distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) described in step 1) is used as a nano-encapsulation agent and is purchased from Shanghai Pengshuo Biotechnology Co., Ltd. Many current research reports have confirmed the amphiphilicity of DSPE-PEG2000, so it is often considered to be a good packaging material for nano-formulations. DSPE-PEG2000 has excellent biocompatibility and biodegradability and has been approved by the U.S. FDA for intravenous injection.

[0022] Furthermore, the photodynamically treated hypericin nanoparticles are used in the preparation of drugs for inhibiting liver cancer cells, specifically, the photodynamically treated hypericin nanoparticles act by promoting the expression of SERPINE1 in liver cancer cells.

[0023] Furthermore, the photodynamically treated hypericin nanoparticles are used in the preparation of a drug for inhibiting liver cancer cells. The drug is a pharmaceutical composition, which uses the photodynamically treated hypericin nanoparticles as the main active ingredient, and is added with pharmaceutically acceptable excipients or auxiliary ingredients to prepare a clinically acceptable pharmaceutical preparation. The content of the photodynamically treated hypericin nanoparticles in the pharmaceutical composition is generally 0.01-95.0% (w / w).

[0024] Generally speaking, as a drug, it is used clinically after being prepared into a preparation. The drug of the present invention, as a pharmaceutical composition, can be prepared according to methods known in the art, and can be prepared into any dosage form suitable for human or animal use by combining the drug of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0025] Furthermore, the photodynamically treated hypericin nanoparticles are used in the preparation of drugs for inhibiting liver cancer cells, and the drug preparations include two dosage forms: oral preparations and injection preparations.

[0026] Furthermore, the hypericin nanoparticles after photodynamic treatment are used in the preparation of drugs for inhibiting liver cancer cells, the oral preparation is an oral capsule, and the injection preparation is an intravenous injection.

[0027] The drug or pharmaceutical composition of the present invention can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0028] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w types, w / o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0029] The drug of the present invention can be made into a common preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation and various microparticle delivery systems. In order to make the drug of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants and glidants. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropanol, etc.; the adhesive may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0030] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0031] In order to prepare the dosing unit into a capsule, the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient of the present invention can also be first made into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard capsule or a soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.

[0032] To prepare the drug of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent and an appropriate amount of a solubilizer, a cosolvent, a pH adjuster, and an osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a support agent.

[0033] Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents or other additives may be added to the pharmaceutical preparations.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. The use of the photodynamically treated hypericin nanoparticles of the present invention in the preparation of drugs for inhibiting liver cancer cells. The inhibitory effect of the photodynamically treated hypericin nanoparticles combined with photodynamic therapy on the cell viability, migration and invasion ability of liver cancer cells is significantly better than that of the single application of hypericin nanoparticles or photodynamic therapy (p < 0.0001).

[0036] 2. The use of the photodynamically treated hypericin nanoparticles of the present invention in the preparation of drugs for inhibiting liver cancer cells, through the analysis results of the biological information database and drug molecule docking, found 4 hypericin-related targets, among which SERPINE1 was experimentally proved to be the key target of hypericin.

[0037] 3. The cell experiments of the present invention show that the hyperforin nanoparticles treated with photodynamic therapy can inhibit the cell viability, migration ability and invasion ability of liver cancer cells in vitro, and induce apoptosis of liver cancer cells; and the hyperforin nanoparticles treated with photodynamic therapy can promote the expression of SERPINE1. The research of the present invention shows that the hyperforin nanoparticles treated with photodynamic therapy can provide a new perspective for liver cancer treatment drugs by regulating the biological behavior of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a diagram of the molecular structure of hypericin in the experimental example of the present invention and an electron microscope image of hypericin nanoparticles;

[0039] Figure 2 It is a comparison diagram of the conditions of hypericin combined with photodynamic therapy and the effects before and after nano-encapsulation in the experimental example of the present invention;

[0040] Figure 3 This is a graph of the reactive oxygen production capacity of hypericin nanoparticles combined with photodynamic therapy detected by DCFH-DA in the experimental example of the present invention;

[0041] Figure 4 This is a diagram of the Transwell experiment in the experimental example of the present invention to detect the effect of hypericin nanoparticles combined with photodynamic therapy on the migration and invasion ability of liver cancer;

[0042] Figure 5 is a diagram of hypericin-related targets obtained by using a bioinformatics database and molecular docking simulation in an experimental example of the present invention;

[0043] Figure 6 is a graph of protein immunoblotting results in an experimental example of the present invention;

[0044] Figure 7 This is a diagram of immunofluorescence analysis of hypericin nanoparticles combined with photodynamic therapy to enhance SERPINE1 expression in an experimental example of the present invention;

[0045] Figure 8This is a graph showing the changes in mRNA and protein in Huh7 48 hours after photodynamic therapy in the experimental example of the present invention. DETAILED DESCRIPTION

[0046] The specific implementation modes of the present invention are further described below in conjunction with examples.

[0047] Embodiment 1:

[0048] Application of hypericin nanoparticles after photodynamic treatment in the preparation of drugs for inhibiting liver cancer cells:

[0049] According to the existing process requirements, hard capsules are prepared.

[0050] Embodiment 2:

[0051] Application of hypericin nanoparticles after photodynamic treatment in the preparation of drugs for inhibiting liver cancer cells:

[0052] According to the existing process requirements, it is prepared into an injection.

[0053] Experimental example:

[0054] 1 Materials and methods

[0055] 1.1 Experimental Materials

[0056] 1.1.1 Origin of liver cancer cells

[0057] There are two liver cancer cell lines used in this study, namely Bel-7402 and Huh7 human liver cancer cells, which were donated by Professor Chen Hongsong (Institute of Liver Diseases, Peking University).

[0058] 1.1.2 Preparation of hypericin nanoparticles

[0059] Hypericin (structural formula see Figure 1 A) Hypericin nanoparticles were obtained. Transmission electron microscopy (TEM) showed that the hypericin was coated with a thin layer of membrane, with good dispersion and a particle size of <200nm ( Figure 1 B), reaching the nanoparticle standard, proving that the hypericin nanoparticles were successfully encapsulated.

[0060] Figure 1 The molecular structure of hypericin and the electron micrograph of hypericin nanoparticles (scale bar: 100 nm);

[0061] 1.2 Experimental Reagents

[0062] 1.2.1 Antibodies used in immunoblotting

[0063] SERPINE1 antibody (13801-1-AP, rabbit monoclonal antibody, 45 KDa, 1:1000 dilution, Proteintech); β-Actin antibody (66009-1-AP, mouse monoclonal antibody, 53 KDa, 1:1000 dilution, Proteintech).

[0064] 1.2.2 Primer sequences

[0065] Table 2 Primer sequences

[0066]

[0067] 2 Results

[0068] 2.1 Design of conditions for hypericin nanoparticles combined with photodynamic therapy

[0069] In order to study the effects of hypericin and photodynamic therapy on human liver cancer cell lines (Bel-7402 and Huh7), different concentrations of hypericin nanoparticles and different illumination times were used for cell viability detection. The different treatment groups were named as follows: HC only (hypericin water solvent alone), HC with irradiation (hypericin water solvent combined with photodynamic therapy), HC-NPs only (hypericin nanoparticles alone), HC-NPs with irradiation (HC-NPs-PDT, hypericin nanoparticles combined with photodynamic therapy), Control group (blank control group), Irradiation only (photodynamic therapy alone).

[0070] Steps: Select Bel-7402 and Huh7 cells that are in good growth state and in the logarithmic growth phase and culture them at 8×10 3 The cells were inoculated at a density of 100 / well in a 96-well plate (4 replicates per group). The cells were placed in a 37°C, 5% CO2 incubator for 24 hours. After the cells were stably attached to the wall, the old culture medium was removed and new culture medium containing hypericin nanoparticles at concentrations of 0 ng / ml, 40 ng / ml, 80 ng / ml, 120 ng / ml, 160 ng / ml, and 200 ng / ml was added. After 24 hours, the cells were illuminated (power: 100 mW / cm 2), the illumination time was 0min, 5min, 10min, and 15min respectively. Among them, 0min was the dark light group (only adding hypericin nanoparticle solutions of various concentration gradients, without illumination treatment), which served as the control group. After the illumination was completed, the DMEM complete medium without hypericin nanoparticles was replaced and placed in a 37°C, 5% CO2 incubator for further culturing for 24h. Next, in a light-proof environment, the complete medium containing CCK-8 solution was added and incubated for 2h, and the absorbance at 450nm was recorded by a continuous wavelength microplate reader (Molecular Devices, USA) to calculate the cell viability.

[0071] The results showed that both liver cancer cells were affected by hypericin nanoparticles and photodynamic therapy ( Figure 2 A and 2C), the dose- and time-dependent relationships were significant, p < 0.0001. The optimal IC50 concentration was 120 ng / mL for Bel-7402 cell line and 80 ng / mL for Huh7 cell line, and the illumination time was 10 min.

[0072] Under non-photodynamic conditions, there was no significant difference in the inhibitory effect of hyperforin nanoparticles and ordinary hyperforin aqueous solvent on cell viability. Under photodynamic conditions, the inhibitory effect of hyperforin nanoparticles on cell viability was more significant, p<0.0001. When the drug concentration reached 1000ng / mL, hyperforin nanoparticles or aqueous solvents combined with photodynamics could significantly inhibit the viability of the two liver cancer cells ( Figure 2 B and 2D). However, the concentration of hypericin aqueous solvent required to achieve the same inhibitory effect was much higher than that of hypericin nanoparticles.

[0073] To further evaluate the effect of hypericin nanoparticles combined with photodynamic therapy on the long-term viability of the two liver cancer cell lines, the inventors designed a series of growth curve experiments at different time points (6h, 24h, 48h, 72h, 96h, and 120h).

[0074] The results showed that the long-term inhibitory effect of hyperforin nanoparticles or photodynamic therapy alone on cell viability was poor. The viability of Huh7 cells was enhanced by hyperforin nanoparticles alone ( Figure 2 F), which may be due to the low concentration that failed to produce sufficient inhibitory effect. The long-term cell viability inhibition effect of hyperforin nanoparticles combined with photodynamic therapy was more significant, p<0.0001. Therefore, hyperforin nanoparticles combined with photodynamic therapy had a significant inhibitory effect on liver cancer cells.

[0075] Figure 2Comparison of the conditions of hypericin combined with photodynamic therapy and the effects before and after nanoencapsulation ((A) and (C) represent the effects of hypericin nanoparticles of different concentrations under different illumination times; (B) and (D) represent the effects of hypericin of different concentrations with or without nanoencapsulation under the same illumination time; (E) and (F) represent the 120 h growth curves of Bel-7402 and Huh7 under fixed photodynamic conditions, respectively).

[0076] 2.2 Detection of reactive oxygen species generation during photodynamic therapy

[0077] Since the key mechanism of photodynamic therapy includes the generation of reactive oxygen species (ROS), this application further explores the efficiency of hypericin nanoparticles combined with photodynamic therapy to generate reactive oxygen species, such as Figure 3 As shown in A and 3B, the generation of intracellular reactive oxygen species in different treatment groups was assessed by DCFH-DA staining.

[0078] The results showed that compared with the other experimental groups, only the hypericin nanoparticles combined with photodynamic therapy group significantly enhanced the fluorescence signal of DCFH-DA, indicating that this treatment group can efficiently produce a large amount of reactive oxygen species. This further confirms the advantage of hypericin nanoparticles combined with photodynamic therapy in generating reactive oxygen species.

[0079] Figure 3 DCFH-DA was used to detect the reactive oxygen production capacity of hypericin nanoparticles combined with photodynamic therapy ((A) Bel-7402 reactive oxygen species-related fluorescence detection and (C) statistics; (B) Huh7 reactive oxygen species-related fluorescence detection and (D) statistics; scale bar: 200 μm).

[0080] 2.3 Hypericin nanoparticles combined with photodynamic therapy can inhibit the migration and invasion of liver cancer cells

[0081] Based on the working conditions obtained in the previous study, the inventors further tested the effects of hypericin nanoparticles on liver cancer cells in vitro. The results of Transwell (without matrix gel) are shown in Figure 4 A (rows 1 and 3) shows that hyperforin nanoparticles combined with photodynamic therapy have a significant inhibitory effect on the migration ability of liver cancer cells (p<0.0001). Figure 4 A (rows 2 and 4) showed that this group also had a significant inhibitory effect on the invasion ability of liver cancer cells (p<0.0001).

[0082] Figure 4Transwell assay to detect the effect of hypericin nanoparticles combined with photodynamic therapy on the migration and invasion of liver cancer ((A) Transwell assay to detect the effect of hypericin nanoparticles combined with photodynamic therapy on the migration and invasion of liver cancer (B, C, D, E) and the statistics of each group; Scale bar: 200μm).

[0083] 2.4 PCR results analysis

[0084] Preliminary screening was performed based on the NCBI database, String protein interaction network, and Cytoscape algorithm, and then the molecule-protein binding simulation was performed using the drug molecular docking simulation software PYMOL ( Figure 5 A) Screening candidate targets for hypericin. Based on the bioinformatics analysis, the inventors obtained four candidate gene targets for hypericin, and extracted mRNA from the control group, single photodynamic therapy group, single hypericin nanoparticle group, and hypericin nanoparticle combined with photodynamic therapy group of the two cell lines.

[0085] The results showed that, by qRT-PCR analysis, compared with the control group, the changes in the mRNA level of gene SERPINE1 in Bel-7402 and Huh7 cells were the most obvious ( Figure 5 B and 5C), and the expression levels of the other three genes changed less, and the relevant results are not presented. Therefore, it is preliminarily believed that SERPINE1 is an important target of hypericin.

[0086] Figure 5 The bioinformatics database and molecular docking simulation were used to obtain hypericin-related targets ((A) hypericin-related molecular docking results; (B) mRNA expression levels of Bel-7402 and (C) Huh7 target SERPINE1).

[0087] 2.5 Analysis of Western Blot Results

[0088] Proteins of liver cancer cells in the blank control group, light-only group, hyperforin nanoparticles group, and hyperforin nanoparticles combined with photodynamic therapy group were extracted and collected. Western blotting results showed that in Bel-7402 and Huh7 cell lines, the expression of SERPINE1 protein in the combined group was significantly increased ( Figure 6 ), which was consistent with the qRT-PCR results. There was no such phenomenon in the light-only group and the hypericin nanoparticles-only group. The SERPINE1 protein expression in the light-only group was slightly reduced but not statistically significant. In the combined group, while the SERPINE1 protein expression increased, the P53 / p-P53 and AKT / p-AKT protein expressions decreased ( Figure 6), suggesting that the decrease of P53 / p-P53 and AKT / p-AKT may be related to the increase of SERPINE1. AKT and its phosphorylated form (p-AKT) are important in regulating the biological behavior of liver cancer cells. In summary, hypericin nanoparticles combined with photodynamic therapy can significantly increase the protein expression level of SERPINE1 in liver cancer cells.

[0089] Figure 6 Western blotting results ((A) shows the Western blotting bands of different groups of Huh7 and Bel-7402; (B) shows the statistical graph related to the Western blotting of Bel-7402; (C) shows the statistical graph related to the Western blotting of Huh7.).

[0090] 2.6 Immunofluorescence analysis

[0091] To further understand the expression of SERPINE1, the inventors used immunofluorescence staining in in vitro experiments. The results showed that in Huh7 cells, the SERPINE1 fluorescence signal of the hyperforin nanoparticles combined with photodynamic therapy was significantly enhanced, while no significant changes were observed in the blank control group, photodynamic therapy group, and hyperforin nanoparticles group ( Figure 7 ). This finding is consistent with previous results.

[0092] Figure 7 Immunofluorescence analysis of hypericin nanoparticles combined with photoactivation to enhance SERPINE1 expression (Scale bar: 100 μm).

[0093] 2.7 Analysis of results 48 hours after treatment

[0094] When observing the in vitro results of hypericin nanoparticles combined with photodynamic therapy, the inventor unexpectedly discovered a time-dependent change. The inventor set up different groups of Huh7 liver cancer cell lines and collected samples at 8h, 24h, and 48h. Then, through qRT-PCR and Western blot analysis, it can be seen that ( Figure 8 ), with the development of time, the mRNA and protein expression of SERPINE1 gradually increased, while the protein expression of P53 and p-P53 gradually decreased over time within 24 hours. At 48 hours, the expression of P53 protein still decreased over time, but p-P53 showed a similar recovery phenomenon.

[0095] Therefore, the inventors found that hypericin nanoparticles combined with photodynamic therapy triggered a P53 protein-dependent depletion of SERPINE1 enhancement.

[0096] Figure 8Changes of mRNA and protein in Huh7 cells 48 hours after photodynamic therapy. (A) Changes of SERPINE1 mRNA expression at 48 hours; (B) Changes of SERPINE1, P53, p-P53, and β-Actin protein expression at 48 hours.

[0097] 3 Conclusion

[0098] This application verifies the effectiveness of hyperforin nanoparticles combined with photodynamic therapy in the fight against liver cancer, reveals the potential mechanism of action of SERPINE1 as a key target, and reveals that hyperforin nanoparticles treated with photodynamic therapy can be used to prepare P53 protein-dependent, SERPINE1-enhanced drugs. This discovery provides new ideas and methods for the application of hyperforin nanoparticles in photodynamic anti-liver cancer drugs, and lays the foundation for further clinical research.

[0099] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Use of photodynamically treated hypericin nanoparticles in the preparation of drugs for inhibiting liver cancer cells, characterized in that: Specifically, the hypericin nanoparticles after photodynamic treatment work by promoting the expression of SERPINE1 in liver cancer cells; The method for preparing the hypericin nanoparticles after photodynamic treatment comprises the following steps: 1) Preparation of Hypericin Nanoparticles: Hypericin nanoparticles were prepared by nano-coprecipitation method. 2 mg of hypericin and 20 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 were mixed in 2 ml of tetrahydrofuran to fully dissolve them. Ultrapure water was slowly added dropwise at room temperature under ultrasonic conditions every 30 min. The ratio of hypericin, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and ultrapure water was 2 mg:20 mg:4 mL. Under ventilation conditions, stir with a magnetic stirrer overnight to remove tetrahydrofuran; If solid precipitates are still found the next day, tetrahydrofuran is added continuously and the above operation is repeated until no more precipitates appear. Ultrasonic oscillation is continued for 72 hours and the volume is fixed to 4 ml to finally obtain the working mother solution of hypericin nanoparticles, which is stored at 4°C in the dark. Then, the particle diameter was measured using a transmission electron microscope JEOL JEM-2100; 2) Add the hypericin nanoparticles obtained in the previous step to the cell culture dish and irradiate with light for 10 min using a xenon lamp light source with a light intensity of 100 mW / cm 2 , and obtain hypericin nanoparticles after photodynamic treatment.

2. The use of the photodynamically treated hypericin nanoparticles according to claim 1 in the preparation of a drug for inhibiting liver cancer cells, characterized in that: The hypericin is prepared by Desite Biotechnology Co., Ltd., with a purity of ≥98.00%.

3. Use of the hypericin nanoparticles after photodynamic treatment according to claim 1 in the preparation of a drug for inhibiting liver cancer cells, characterized in that: The xenon lamp light source described in step 2) was purchased from Beijing Zhongjiao Jinyuan Technology Co., Ltd., model: CEL-S500.

4. The use of the hypericin nanoparticles after photodynamic treatment according to claim 1 in the preparation of a drug for inhibiting liver cancer cells, characterized in that: The photodynamically treated hyperforin nanoparticles are used in the preparation of a drug for inhibiting liver cancer cells. The drug is a pharmaceutical composition, which uses the photodynamically treated hyperforin nanoparticles as the main active ingredient and is added with pharmaceutically acceptable excipients or auxiliary ingredients to prepare a clinically acceptable pharmaceutical preparation. The content of the photodynamically treated hyperforin nanoparticles in the pharmaceutical composition is usually 0.01-95.0% w / w.

5. Use of the hypericin nanoparticles after photodynamic treatment according to claim 1 in the preparation of a drug for inhibiting liver cancer cells, characterized in that: The hypericin nanoparticles after photodynamic treatment are used in the preparation of drugs for inhibiting liver cancer cells. The drug preparation includes two dosage forms: oral preparation and injection preparation.

6. Use of the hypericin nanoparticles after photodynamic treatment according to claim 5 in the preparation of a drug for inhibiting liver cancer cells, characterized in that: The hypericin nanoparticles after photodynamic treatment are used in the preparation of drugs for inhibiting liver cancer cells. The oral preparation is an oral capsule, and the injection preparation is an intravenous injection.

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