HIC-M nano particle and preparation method, application and medicine thereof

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to HICM nanoparticles, a preparation method and application thereof and a drug. The HIC (at) M nanoparticles take hydrazide hyaluronic acid as a carrier, load cis-platinum and indocyanine green, and coat a chorion cancer cell membrane. The HIC (at) M nano particle can treat chorionic carcinoma subcutaneous tumor through caudal vein injection and sonodynamic combined action, can act on uterus in-situ tumor through an innovative uterine cavity perfusion administration mode, is combined with ultrasonic treatment, has a remarkable anti-tumor effect, and has good biological safety. The invention provides a new method for efficiently loading cis-platinum and indocyanine green onto the nano-carrier, and also shows a huge prospect of treating choriocarcinoma by combining an intrauterine perfusion administration mode with sonodynamic chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of anticancer drugs, and specifically relates to HIC@M nanoparticles and a preparation method, application and drug thereof. Background Art

[0002] Choriocarcinoma is a malignant gestational trophoblastic tumor caused by abnormal proliferation of placental trophoblasts. It has a high potential for proliferation and invasion, which seriously endangers women's reproductive safety and life health. Choriocarcinoma can be secondary to any form of pregnancy, such as hydatidiform mole, miscarriage and normal pregnancy. Its typical clinical manifestations are vaginal bleeding and metastatic lesions in the lungs, reproductive system, liver, brain, etc. At present, the clinical treatment of choriocarcinoma is highly dependent on chemotherapy. Some patients may undergo hysterectomy according to the disease condition, which seriously affects the patient's quality of life and fertility needs. The extensive killing effect of traditional chemotherapy can easily lead to serious toxic and side effects, such as liver damage, gastrointestinal reactions, mucosal damage, bone marrow suppression, etc., as well as varying degrees of drug resistance, resulting in poor prognosis or even death in patients. Therefore, the development of more efficient drug administration and treatment strategies with fewer side effects is of great significance to solving the clinical treatment problems of choriocarcinoma.

[0003] Local drug delivery is the delivery of therapeutic drugs to the diseased area. Compared with systemic drug delivery, the required drug dose is low, the targeting is strong, and the drug degradation is less. These factors are conducive to increasing the drug utilization in the tumor area and reducing systemic side effects. The vagina and uterus are both structures rich in blood vessels, which are very suitable for local drug delivery for female reproductive system diseases. Existing vaginal and intrauterine drug delivery are generally based on traditional dosage forms such as tablets, suppositories, and solutions. They have the advantages of easy application and removal, bypassing the first metabolism, and increasing the permeability of small molecule drugs. However, problems such as poor retention and inability to deliver specifically still limit its application in the treatment of female reproductive system tumors. Therefore, intrauterine perfusion drugs that can be delivered specifically and have a long retention time, high comfort and good efficacy will be of great significance for female reproductive system tumors. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a HIC@M nanoparticle and a preparation method, application and medicine thereof.

[0005] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a HIC@M nanoparticle, wherein the HIC@M nanoparticle uses hydrazide hyaluronic acid as a carrier, is loaded with cisplatin and indocyanine green, and is coated with a choriocarcinoma cell membrane.

[0006] The HIC@M nanoparticles of the present invention can be homologously recognized by choriocarcinoma cells, increase the cellular uptake of drugs, and the rich disulfide bonds can decompose and consume GSH at the tumor site, promoting the accumulation of ROS. This process can not only amplify the anti-tumor effect of SDT, but also avoid the detoxification effect of CDDP, further induce cell apoptosis and ferroptosis, and ultimately achieve "killing three birds with one stone". Secondly, the HIC@M nanoparticles are dispersed in clinical hyaluronic acid to form a gel-like preparation, which is retained in the uterine cavity through perfusion and directly acts on the lesion site. The ultrasound probe is then positioned in the lower abdomen to act on the uterine site to enhance the synergistic effect of SDT and achieve a balance between the best efficacy and the minimum toxic side effects.

[0007] In a second aspect, the present invention provides a method for preparing the HIC@M nanoparticles, comprising the following steps: Dissolve cisplatin, indocyanine green and acylhydrazide hyaluronic acid in water and mix to obtain a mixed solution A, react in the dark at 36-37°C for 22-24 hours, centrifuge and wash the product until the supernatant is colorless, and the obtained precipitate is HIC nanoparticles; the mass ratio of cisplatin, indocyanine green and acylhydrazide hyaluronic acid is 1:1:2-3; Under ice bath conditions, the HIC nanoparticle solution and the choriocarcinoma cell membrane are mixed to obtain a mixed solution B, the mixed solution B is subjected to ultrasonic treatment for 0.5 to 0.6 h, and then extruded to obtain the HIC@M nanoparticles.

[0008] Furthermore, the hydrazide hyaluronic acid is prepared by the following steps: Adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to a hyaluronic acid solution, reacting at 29-31° C. for 0.5-0.6 h, then adding 3,3'-dithiodipropionyl hydrazide solution, reacting for 36-40 h, dialyzing the resulting solution for 48-50 h, and freeze-drying to obtain the hydrazide hyaluronic acid; The mass volume ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid solution and 3,3'-dithiodipropionyl hydrazide solution is 0.48 g: 0.288 g: 10-12 mL: 35-40 mL; The concentration of the hyaluronic acid solution is 0.08-0.1 g / mL, and the concentration of the 3,3'-dithiodipropionyl hydrazide solution is 0.05-0.6 g / mL.

[0009] In a third aspect, the present invention provides the use of the HIC@M nanoparticles in the preparation of anti-tumor drugs.

[0010] Furthermore, the tumor includes choriocarcinoma.

[0011] Furthermore, the drug is a drug for sonodynamic therapy.

[0012] Furthermore, the sonodynamic therapy includes locally applying sound waves to the human body.

[0013] Furthermore, the sound wave is ultrasonic wave.

[0014] In a fourth aspect, the present invention provides a drug for sonodynamic therapy against choriocarcinoma, wherein the drug contains the HIC@M nanoparticles as an active ingredient.

[0015] Furthermore, the drug is in the form of intrauterine infusion or intravenous injection.

[0016] The present invention has the following beneficial effects: (1) The cancer cell membrane-coated nanoparticles in the HIC@M nanoparticles of the present invention can be recognized by cell homology, increasing the cellular uptake of the drug and showing better targeting when combined with the intrauterine perfusion strategy.

[0017] (2) The abundant disulfide bonds in HIC@M nanoparticles can consume endogenous GSH enriched in the tumor site, avoid detoxification, and improve the therapeutic efficiency of CDDP. A large amount of ROS accumulates in the tumor site, which can amplify the effect of SDT and enhance the synergistic therapeutic effect of ultrasound chemotherapy.

[0018] (3) ROS accumulation and GSH depletion can further induce apoptosis and ferroptosis, which together promote the clearance of tumor cells. HIC@M nanoparticles showed good biosafety in both tail vein injection and intrauterine perfusion mouse models, and this method may become a new paradigm for the treatment of choriocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Characterization of HHA and HIC, where A is the image of HIC analyzed by TEM, B is the particle size distribution of HHA and HIC analyzed by DLS, C is the Zeta potential of HHA and HIC, and D is the infrared spectrum of HHA, ICG, CDDP and HIC.

[0020] Figure 2 The results of the ROS generation and GSH consumption capacity test of HIC, where A is the DPBF test 1 Quantitative analysis of O2, B is the average fluorescence intensity of DCFH-DA under different treatments, C is the ability of DTNB probe to detect the consumption of GSH by HIC at different concentrations, and D is the ability of DTNB probe to detect the consumption of GSH by different groups.

[0021] Figure 3Characterization comparison of HHA and HIC, where A is the TEM analysis image of HHA and HIC, B is the Coomassie blue staining of JEG-3 cell lysate, JEG-3 cell membrane, HIC@M and HIC, M is the protein marker, and I~IV are JEG-3 cell lysate, JEG-3 cell membrane, HIC@M and HIC, respectively.

[0022] Figure 4 Results of the assay of specific homologous targeting of HIC@M to cells.

[0023] Figure 5 The results of in vitro anti-tumor ability test of HIC@M+US, where A is the relative cell viability of different groups, B is the hCG level of different groups, and C is the quantitative analysis of single cell cloning experiment. Among them, a~f are Control group, US group, ICG+US group, CDDP+US group, HIC group and HIC+US group respectively.

[0024] Figure 6 Figure 2 shows the tumor progression curves of mice in different treatment groups. a~e are Control group, ICG+US group, CDDP+US group, HIC@M group and HIC@M+US group, respectively.

[0025] Figure 7 is the tumor weight of mice in different treatment groups, a~e are Control group, ICG+US group, CDDP+US group, HIC@M group and HIC@M+US group, respectively.

[0026] Figure 8 H&E, TUNEL, Ki67 and GPX4 staining of tumor tissues of mice in different treatment groups.

[0027] Fig. 9 These are the in vivo imaging pictures of mice in different treatment groups at different time points.

[0028] Fig.10 Masson staining and H&E staining images of the uterine body and uterine horns of mice in different treatment groups, A is the Masson staining image, and B is the H&E staining image.

[0029] Fig.11 In vivo bioluminescence images of mice at different time points for monitoring tumor growth, wherein the second column on the seventh day and the second and third columns on the tenth day indicate the death of mice.

[0030] Fig.12 These are ex vivo images of potential metastatic organs, lung, liver, uterus, and intestine, isolated from JEG-3 orthotopic tumor-bearing mice on day 10.

[0031] Fig.13 Shown are the survival curves of mice in different treatment groups.

[0032] Fig.14 H&E and TUNEL staining of tumor tissues of mice in different treatment groups.

[0033] Fig.15 H&E staining of intestinal tissues of mice in different treatment groups.

[0034] Fig.16 These are the results of blood routine and biochemical examinations of mice in the MTX intravenous injection and HIC@M+US intrauterine perfusion groups. A is the hemoglobin content, B is the red blood cell content, and C is the ALT content. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0036] The key terms and abbreviations involved in the following embodiments are: HA: hyaluronic acid; EDC·HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS: N-hydroxysuccinimide; DTP: 3,3'-dithiodipropionylhydrazide; DCFH-DA: 2,7-dichlorodihydrofluorescein diacetate; ROS: reactive oxygen species; DPBF: 1,3-diphenylisobenzofuran; 1 O2: singlet oxygen; GSH: glutathione; DTNB: 5,5'-dithiobis(2-nitrobenzoic acid); US: ultrasound; MTT: CRD: cell proliferation mortality; CC: choriocarcinoma; CDDP: cisplatin; EDX: energy dispersive x-ray spectroscopy; EPR: hyperpermeability and retention effect; ESR: electron paramagnetic resonance spectrometry; FIGO: International Federation of Gynecology and Obstetrics; GTN: gestational trophoblastic neoplasm; H&E: hematoxylin and eosin; HHA: hydrazide hyaluronic acid; ICG: indocyanine green; MMC: mitochondrial membrane potential; MTX: methotrexate; SDT: sonodynamic therapy; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; RBC: red blood cell; ALT: alanine aminotransferase.

[0037] Example 1: Preparation of HIC@M nanoparticles and their ability to generate ROS and consume GSH.

[0038] 1. Test method.

[0039] (1) Preparation of hydrazide hyaluronic acid.

[0040] First, prepare 10 mL of 0.1 g / mL HA aqueous solution, and add 0.48 g of EDC·HCl and 0.288 g of NHS in sequence. Stir the mixed solution in a 30°C water bath for 30 minutes to activate the carboxyl groups on HA. Dissolve 2.38 g of DTP in 40 mL of ultrapure water, add it to the above solution, stir for 36 hours, and continue to dialyze the resulting solution for 48 hours. Freeze-dry to obtain the final product, hydrazide hyaluronic acid, referred to as HHA.

[0041] (2) Preparation of HIC nanoparticles.

[0042] 1 mL of 1 mg / mL CDDP solution and 1 mL of 1 mg / mL ICG solution were mixed with 2 mL of 1 mg / mL HHA solution and stirred in the dark at 37°C for 24 h. Centrifugation and washing were repeated until the supernatant was colorless to obtain HHA nanoparticles loaded with CDDP and ICG, referred to as HIC.

[0043] (3) Preparation of HIC@M nanoparticles.

[0044] An appropriate amount of JEG-3 cells were collected, and the cell membrane of JEG-3 cells was extracted using the Bio-Tech membrane protein extraction kit according to the instructions. The protein amount of the cell membrane was determined by the BCA method. The protein concentration was calculated by mixing equal volumes of 1 mg / mL JEG-3 cell membrane solution and 1 mg / mL HIC solution. The mixture was ultrasonically treated at 40KHZ and 100W for 0.5h under ice bath conditions. The mixture was then repeatedly extruded 20 times using an Avanti min extruder equipped with a 400nm filter membrane to obtain HIC@M nanoparticles, referred to as HIC@M.

[0045] (4) ROS detection.

[0046] DCFH-DA fluorescent probe detects total ROS. 2 μL of 10 mM DCFH-DA solution was mixed with 80 μL of 10 mM NaOH solution and fixed to 1 mL with PBS. 25 μL of the above buffer was mixed with different groups of drugs, and then ultrasonically treated or not ultrasonically according to the group, and then the fluorescence emission spectrum in the range of 500-600 nm was detected.

[0047] 20 μL of 10 mM DPBF was used to detect singlet oxygen, and the absorbance of DPBF was recorded every 2 min by UV-visible spectrophotometer. Remaining DPBF (%) = absorbance after treatment / initial absorbance of solution × 100%. Three independent replicates were performed.

[0048] (5) Glutathione consumption capacity detection.

[0049] The glutathione consumption capacity was detected by the Ellman method. First, the glutathione consumption of HIC@M at different concentrations was determined. HIC solutions with concentrations of 0μg / mL, 12.5μg / mL, 25μg / mL, 50μg / mL, and 100μg / mL were prepared and incubated with 5mM GSH at room temperature for 2h. Then 5μL of DTNB DMSO solution with a concentration of 4mg / mL was added as a glutathione indicator. After standing for 1min, the sample was scanned by 415nm UV-visible absorption spectrum to detect glutathione loss. Then the glutathione consumption of different treatment groups was determined using basically the same method. Remaining DTNB (%) = absorption value after treatment / initial absorption value of solution × 100%.

[0050] 2. Test results.

[0051] In order to improve the efficiency of anti-tumor combined therapy and reduce toxic side effects, HIC@M was designed and constructed in this example. The raw materials used were all FDA-approved clinical drugs, and safety was guaranteed. First, DTP was grafted onto HA by the carbodiimide method to prepare HHA. The results of Fourier transform infrared spectroscopy analysis showed that HHA had a peak at 1558 cm −1 The absorption peak belonging to the amide II band in the spectrum is significantly stronger than that of HA. 1 This is also confirmed by the H NMR spectrum. Compared with HA, 1 A new chemical shift was observed at 2.91 ppm in the HNMR spectrum. The above results all indicate that DTP was successfully grafted onto HA, proving the successful synthesis of HHA.

[0052] The collected HIC was characterized by TEM, and it can be seen that the prepared HIC has a uniform morphology and is in the shape of a hollow sphere with a particle size of about 150nm to 210nm. Figure 1 As shown in A. Using HAADF-STEM, EDX element mapping of HIC revealed that C, N, O, and Pt elements were uniformly distributed throughout the structure. The hydrated particle size of HIC was measured, and the results were similar to those of TEM, and the particle size distribution showed a narrow single peak distribution, as shown in Figure 1 As shown in B, this indicates that these nanoparticles have good monodispersity. Compared with HHA, the particle size of HIC is slightly reduced. This may be because the cross-linking density of CDDP in the HHA network increases after drug loading, which promotes the transformation of the particle structure from loose to dense. The size of nanocarriers plays an important role in the targeted delivery of therapeutic drugs to tumors. Generally speaking, nanoparticles smaller than 200nm can be preferentially accumulated in tumors by enhancing the EPR effect after systemic administration. The negative Zeta potential is beneficial to improve the stability of HIC@M nanoprodrugs, prolong their circulation time in the blood, and prevent them from being phagocytosed and cleared by the reticuloendothelial system. Figure 1The infrared absorption spectrum of HIC shows that the characteristic absorption peaks of HHA, ICG and CDDP are all present in HIC, indicating that HIC is successfully formed. Figure 1 D. The fluorescence spectrum showed that compared with free ICG, the absorption peak of HIC blue-shifted from 831 nm to 828 nm, again indicating that ICG was successfully loaded on HIC.

[0053] After confirming that HIC was successfully synthesized, we further verified its ability to generate ROS and consume GSH under the combined action of sonodynamics. ICG, as a sonosensitizer, generates a large amount of ROS under the action of ultrasound, directly killing tumor cells. The abundant disulfide bonds in HIC can consume the abundant GSH in the tumor site, thereby reducing the detoxification effect of GSH on cisplatin, improving the chemotherapy efficiency of cisplatin, and avoiding the consumption of generated ROS. Use DPBF as a singlet oxygen probe to monitor US-triggered 1 O2 generation, HIC generation was analyzed by incubating the mixture of nanoparticles and DPBF under US irradiation. 1 With the increase of US irradiation time, the characteristic absorption peak of DPBF at 416nm decreased significantly, indicating that 1 O2. Figure 2 As shown in A, compared with the Control group, US group, ICG+US group and HIC group, the HIC+US group had the highest oxidation rate of DPBF. At 10 minutes, the consumption rate of DPBF in each group was counted. The HIC+US group had the highest consumption rate, with the remaining DPBF at 65.33%, which was statistically significant compared with 95.35% in the Control group. This shows that HIC can generate a large amount of ROS under US conditions to kill tumor cells. The DCFH-DA generation experiment also proved the ROS generation ability of HIC. The fluorescence peak increment of the HIC+US treatment group was 9.57 times, and the increment of the Control group was 3.56 times, indicating that HIC nanoparticles can generate a large amount of ROS under ultrasonic treatment. The results are consistent with the results of the DPBF consumption experiment, as shown in Figure 2 As shown in B.

[0054] As an abundant endogenous antioxidant, GSH can maintain the intracellular redox balance and accumulate in large quantities at the tumor site to prevent ROS-induced tumor cell damage. It can also degrade cisplatin and reduce the chemotherapy efficiency of cisplatin. Most oxidative anticancer strategies are limited by the high level of intracellular GSH. Therefore, consuming intracellular GSH can significantly improve the SDT effect. Therefore, this example further explores the ability of HIC to consume GSH. HIC is rich in disulfide bonds and can consume glutathione by cleaving disulfide bonds. The Ellman reagent DTNB probe was used to detect GSH consumption. The probe reacted with GSH to generate a yellow product with a maximum ultraviolet absorption wavelength of 412nm. The glutathione solution was incubated with nanomicelles of different concentrations for 2 hours, and glutathione was detected with DTNB. The results are as follows: Figure 2 As shown in Figure 3C, the absorbance peak at 412 nm decreased in a dose-dependent manner, indicating that the consumption of GSH was dose-dependent. The higher the HIC concentration, the stronger its ability to consume GSH. In addition, in order to compare the GSH consumption capacity of different treatment groups, DTNB was used to detect the GSH content of the Control group, US group, HIC group, and HIC+US group before and after treatment. The results are shown in Figure 3C. Figure 2 As shown in Figure D, the UV absorption peak at 412 nm in the HIC+US group decreased most significantly, indicating that HIC nanoparticles have obvious GSH consumption ability under US radiation conditions, which is beneficial to maintain high ROS levels, further enhance SDT performance, and improve the efficiency of cisplatin chemotherapy, laying a solid foundation for subsequent cell and animal anti-tumor experiments.

[0055] Example 2: Living cell uptake and anti-tumor effect of HIC@M.

[0056] 1. Test method.

[0057] MTT colorimetric method was used to detect cytotoxicity. JEG-3 cells were seeded in 12-well plates and cultured overnight. The cells were incubated with different concentrations of HIC@M and the intensity was 0.5 W / cm 2 , 1.0W / cm 2 and 1.5W / cm 2 The cells were irradiated with US to screen the optimal drug administration conditions. Then the cells were incubated with ICG, CDDP, and HIC@M for 4 h, 1.0 W / cm 2 Ultrasound for 3 min or no ultrasound. After 20 h, wash the cells with PBS and incubate with 5 mg / mL MTT solution for 4 h. Remove the MTT solution, add DMSO to each well to dissolve methoxylamide, and measure the absorbance at 490 nm with an enzyme-labeled instrument.

[0058] 2. Test results.

[0059] The rich disulfide bonds of HIC enable it to be enriched and respond to tumor sites with high GSH expression. Coating the choriocarcinoma cell membrane with HIC can further enhance its homologous targeting to the tumor site, enabling it to better exert a specific killing effect at the tumor site, thereby maximizing the efficacy and reducing drug side effects. Figure 3 The TEM electron microscope image shown in A preliminarily shows the successful coating of JEG-3 cell membrane. The Coomassie brilliant blue gel staining experiment further confirms the complete preservation of the surface proteins of the cancer cell membrane. Figure 3 As shown in Figure B, the JEG-3 cell lysate has a richer protein spectrum than the extracted JEG-3 cell membrane and HIC@M, which may be because the isolated cancer cell membrane lacks nuclear, organelle and cytoplasmic proteins, etc. The HIC@M protein band is consistent with the JEG-3 cell membrane protein band, while the uncoated material group HIC shows no protein, which means that the cell membrane protein is retained after coating.

[0060] We then examined the time-dependent cellular uptake of HIC@M by JEG-3 cells, and the results showed that the uptake was highest after 4 hours of incubation. Next, we explored the specific homologous targeting of HIC@M to cells in various placenta-derived cell lines. Figure 4 As shown in the figure, after co-incubation with HIC@M, JEG-3 cells showed stronger ICG fluorescence than HTR-8 / SVneo and JAR cells, which proved that the JEG-3 cell membrane enhanced the specific self-recognition of HIC@M. In summary, HIC@M coated with JEG-3 cell membrane showed excellent homology and could perform specific homologous targeting to cancer cells, providing a wide range of potential applications for the treatment of choriocarcinoma.

[0061] Next, the in vitro antitumor ability of HIC@M+US was evaluated. 2 The cell survival rate of the 1.5W / cm 2 Ultrasound of low intensity will cause most of the adherent cells to float, and the survival rate is only 8.22±3.15%, which will cause a large error in subsequent cell experiments. Finally, 1.0W / cm 2 The intensity was used as the ultrasound condition for subsequent experiments. Under this condition, the survival rate of JEG-3 cells was 77.18±12.66%. In order to compare the synergistic anti-tumor effect of HIC@M, six groups were further set up: Control group, US group, ICG+US group, CDDP+US group, HIC@M group, and HIC@M+US group. Figure 5As shown in A, the HIC@M+US group had the strongest cell killing ability, with a cell survival rate of 42.19%±8.37%, which was much lower than 100.00%±19.90% in the Control group, 81.37%±11.95% in the US group, 63.25%±21.30% in the ICG+US group, 60.55%±18.07% in the CDDP+US group, and 63.41%±6.73% in the HIC@M group. Single-cell cloning experiments are usually used to estimate CRD and evaluate the ability of single cells to develop into colonies. Compared with other groups, the HIC@M+US group had the least cell colony formation, as shown in the statistics. Figure 5 As shown in C, HIC@M+US significantly inhibited the colony-forming ability of JEG-3 cells. The anti-proliferative effect of HIC@M+US on JEG-3 cells was evaluated by EdU experiment. The control group showed strong green fluorescence, representing cell proliferation, while the cells treated with HIC@M+US produced slight green fluorescence, indicating an inhibitory effect on cell proliferation. hCG is a significant marker for early monitoring of trophoblast invasion and prognosis during treatment in choriocarcinoma. The ELISA experiment was used to detect the hCG content in the supernatant of JEG-3 cells in different intervention groups to evaluate the effect of HIC@M+US on endocrine activity. The results are shown in Figure 5 As shown in B, the hCG level of untreated JEG-3 cells was 168.20±12.19 mIU / mL, while the hCG level of HIC@M+US-treated cells was significantly reduced to 106.35±8.10 mIU / mL.

[0062] Example 3: Efficacy of intravenous injection in treating subcutaneous tumors.

[0063] 1. Test method.

[0064] Subcutaneous transplantation 70~100cm 3 The tumor-bearing mice were randomly divided into five groups, with five mice in each group: (1) Control group, intravenous injection of normal saline; (2) ICG+US group, intravenous injection of 0.5 mg / kg ICG+1.5 W / cm 2 (3) CDDP+US group, intravenous injection of 0.5 mg / kg CDDP+1.5 W / cm 2 US treatment for 3 minutes; (4) HIC@M group, intravenous injection of HIC@M, the amount of CDDP in HIC@M was the same as that in CDDP+US group; (5) HIC@M+US group, based on the HIC@M group, 1.5W / cm 2The mice were treated with US for 3 minutes and 0.5 hours after intravenous injection, which was repeated every 3 days. After 10 days of monitoring, the mice were euthanized, and their tumor tissues and other important organs were collected for immunohistochemical staining, TUNEL staining, Ki67 staining, and GPX4 staining.

[0065] 2. Test results The results of in vitro experiments showed that HIC@M+US had a good inhibitory effect on JEG-3 cells by generating a large amount of ROS and inducing cell ferroptosis, which prompted further exploration of the in vivo anti-tumor effect of HIC@M+US. The in vivo anti-cancer properties of HIC@M+US were verified in the JEG-3 subcutaneous tumor-bearing mouse model. First, a choriocarcinoma xenograft nude mouse model was constructed by subcutaneous injection of JEG-3 cells. Tumor-bearing mice were injected with 1% paraformaldehyde and 1% paraformaldehyde to achieve a tumor volume of 70-100 mm. 3 The mice were then randomly divided into five groups, namely, the Control group, ICG+US group, CDDP+US group, HIC@M group and HIC@M+US group. The Control group was intravenously injected with normal saline, the HIC@M+US group was ultrasonically treated 0.5h after the tail vein injection of the drug, and the other groups were given corresponding treatments according to the situation, once every 3 days, for a total of 3 times. During the treatment period, the changes in the weight and tumor volume of mice in each group were recorded on time. It was observed that there was no significant difference in the weight gain trend of mice in each group, indicating that the adverse reactions of HIC@M+US and each group were small. The tumors of mice with choriocarcinoma subcutaneous tumors grew rapidly, but the tumor growth rate of the HIC@M+US group was significantly slowed down due to its obvious anti-tumor effect. The relative tumor volume of each group of mice was calculated based on the measured data to determine whether the treatment was effective. The results showed that the final relative tumor volume of mice treated with HIC@M+US was 2.32 times, which was much lower than 10.58 times of the Control group, 9.48 times of the ICG+US group, 6.50 times of the CDDP+US group and 9.61 times of the HIC@M group. Figure 6 All mice were euthanized after treatment, and the subcutaneous tumors of each mouse were dissected for photographing, weighing, and sampling. Figure 7 The tumor weight of each group of mice was displayed. The above results show that the tumor growth of the ICG+US group and the CDDP+US group was slightly inhibited, indicating that the efficacy of sonodynamic therapy or chemotherapy alone on tumors is limited, while HIC@M+US can significantly inhibit tumor growth and achieve good tumor treatment effects in the in vivo JEG-3 subcutaneous tumor model, further indicating that the combined sonodynamic / chemotherapy based on HIC@M has a more ideal anti-tumor effect than a single mode.

[0066] Tumor tissues of mice in different treatment groups were further collected for H&E, TUNEL, Ki67 and GPX4 staining. Figure 8As shown in the figure, H&E staining photos of tumor tissues of mice in the HIC@M+US group showed severe tissue destruction, with a large number of vacuoles in the loose tissue, indicating that HIC@M+US has a strong tumor destruction potential. Tumor TUNEL staining showed that the apoptosis rate of green fluorescent labeled cells in the HIC@M+US group was much higher than that in other groups. Ki-67, a biomarker of cell proliferation, was significantly lower in the HIC@M+US group than in other groups, showing a significant inhibition of tumor cell proliferation. Immunohistochemistry detected the expression of GPX4 in xenograft tumors, and found that HIC@M+US treatment significantly reduced the expression of GPX4 in tumor tissues. The above experimental results all indicate that HIC@M+US exhibits an inhibitory effect on tumor growth in the treatment of choriocarcinoma, and this effect is closely related to the promotion of reactive oxygen species generation, apoptosis and ferroptosis in tumor cells.

[0067] Example 5: Uterine intraperfusion retention and biosafety of HIC@M nanoparticles.

[0068] 1. Test method.

[0069] The needle of a 5mL medical syringe was ground and bent to an angle suitable for the uterine perfusion position, and then replaced with the syringe of a 1mL syringe, thus making a simple intrauterine perfusion device. The top and rear ends of the 200mL gun head were removed, and the middle part was about 1.5cm long, and then both ends were ground flat, thus making a simple mouse dilator. First, use the simple dilator to find the mouse's cervical opening, then place the perfusion device in a position parallel to the mouse's vaginal opening, gently and slowly insert it until resistance is encountered, and then gently push the syringe to inject the drug into the uterus.

[0070] The mice in the intrauterine perfusion group were divided into two groups, with six mice in each group: (1) Control group, intrauterine perfusion of normal saline; (2) HIC@M in HA group, intrauterine perfusion of HIC@M dissolved in HA calculated according to the dosage of 0.5 mg / kg CDDP; (3) HIC@M in H2O group, intrauterine perfusion of HIC@M dissolved in ddH2O calculated according to the dosage of 0.5 mg / kg CDDP. Treatment was once every 3 days for a total of 3 times. 15 days after treatment, blood and major organs were collected by the same method for routine blood tests, biochemical tests, H&E staining and Masson staining.

[0071] 2. Test results In order to explore the feasibility of intrauterine perfusion administration of HIC@M, biosafety experiments and drug retention time measurements of intrauterine perfusion were conducted. HIC@M was dissolved in ddH2O and HA, respectively. After standing for a certain period of time, it can be seen that the HIC@M in H2O group solution is still in a fluid state. It has a certain fluidity and injectability, and can be injected into the uterine cavity through a catheter. Next, the mice were randomly divided into two groups, and HIC@M dissolved in ddH2O and HIC@M dissolved in HA were intrauterinely perfused, respectively. In vivo imaging was performed at different time points to observe its distribution and metabolism in the body. Fig. 9 As shown in the figure, HIC@M dissolved in ddH2O diffused rapidly to other parts such as the peritoneum and intestines, while HIC@M dissolved in HA was basically confined to the uterus and did not diffuse to surrounding tissues, which had a better local aggregation effect. The fluorescence intensity of the nanoparticles was the strongest from 6 to 12 hours after perfusion, and the nanoparticles in the uterus of the mice in the HIC@M in H2O group were basically metabolized after 48 hours, while the nanoparticles in the uterus of the mice in the HIC@M in HA group were still significantly enriched. The ex vivo uteri of the two groups of mice were taken for fluorescence imaging 12 hours and 48 hours after perfusion, respectively. The results showed that the HIC@M in the uterus of the mice in the HIC@M in H2O group was absorbed into the blood 12 hours after intrauterine perfusion, and was absorbed and metabolized by organs such as the liver and kidney, while the HIC@M in HA group did not have this phenomenon; after 48 hours of intrauterine perfusion, the fluorescence intensity of the uterus of the mice in the HIC@M in HA group was significantly higher than that of the HIC@M in H2O group. This indicates that after HIC@M is dissolved in HA, it can remain in the mouse uterus for a long time without diffusing into surrounding tissues and being absorbed by organs such as the liver and kidneys, laying a strong foundation for its subsequent therapeutic effects.

[0072] Uterine cavity biosafety: Uterine tissues of mice in the HIC@M in HA group and the Control group were dissected for in vitro photography, Masson staining and H&E staining. Two sites of the uterine body and uterine horn were taken from each mouse to observe whether fibrosis and other histological damage occurred. Fig.10 As shown in the figure, the uterine Masson staining and H&E staining of the treatment group and the control group showed similar tissue structures, and the Masson staining statistical graph showed no statistical difference between the two groups. During the experiment, no significant difference was observed in the weight gain trend of mice in each group, indicating that the adverse reactions of HIC@M+US and each group were relatively small. The above experimental results all prove that the administration method of HIC@M intrauterine perfusion is safe.

[0073] Example 6: Efficacy in treating in situ tumors.

[0074] 1. Test method.

[0075] Female BALB / c nude mice aged 4-6 weeks were anesthetized using a small animal anesthesia machine. The lower abdomen of each mouse was strictly disinfected with medical iodine, and then a 15 mm long longitudinal incision was made along the midline of the abdomen. After opening the peritoneum, the intestine was arranged, the tissue around the uterus was properly separated, and the uterine body and uterine horns were fully exposed. The bifurcation of the uterine horns was exposed, and JEG-3 cancer cells stably transfected with luciferase were injected into the myometrium. The surgical incision was then sutured with 5-0 absorbable sutures. The mice were placed in a warm environment after surgery, and their physical signs were monitored until they fully recovered.

[0076] In vivo synergistic therapeutic effect of orthotopic xenograft tumor model: The orthotopic xenograft tumor model was divided into three groups, with four mice in each group: (1) Control group, intrauterine perfusion of normal saline; (2) MTX group, intravenous injection of 5 mg / kg methotrexate; (3) HIC@M+US group, intrauterine perfusion of HIC@M calculated according to the dosage of 0.5 mg / kg CDDP, and 1.5 W / cm 2 The mice were treated with US for 3 min. Six hours after intrauterine perfusion, the mice were treated with US, which was repeated every 3 days. 150 mg / kg of D-luciferin substrate was injected intraperitoneally, and the tumor growth trend was analyzed every 2 days based on the in vivo bioluminescence using an in vivo imaging system. After 10 days of monitoring, the mice were euthanized and their tumor tissues were collected for further analysis.

[0077] 2. Test results.

[0078] After verifying the retention and safety of HIC@M in the uterine cavity, we further performed intrauterine perfusion therapy on an orthotopic tumor model. In situ tumor in vivo imaging showed that the tumor volume of mice in different groups increased over time, and the increase in the Control group was the largest, while the increase in the HIC@M+US group was the smallest, and the tumor growth rate was the slowest. Fig.11 As shown. After the treatment, the mice in each group were euthanized, and the organs to which the in situ tumors were prone to metastasis were dissected and imaged again. The most common metastatic lesion in the clinical treatment of choriocarcinoma is the lung. In this experimental animal model, because the uterine in situ transplanted tumor was too large, it broke through the uterus and infiltrated the surrounding digestive tract tissues to varying degrees. Therefore, the uterus, lungs, liver, and intestines were imaged to observe the degree of metastasis in mice in different treatment groups. After HIC@M intrauterine perfusion combined with sonodynamic therapy, the volume of the uterine in situ tumor was significantly smaller than that of the Control group and the MTX group, and there was almost no intestinal metastasis. In the Control group, the volume of the uterine in situ tumor increased significantly, there were multiple metastatic tumor lesions in the intestine, and fluorescent imaging of metastatic tumors was also visible in the liver, as shown Fig.12As shown. It is worth noting that the clinical treatment of choriocarcinoma relies on serum hCG detection. Most patients can be diagnosed with the disease during routine monitoring after pregnancy. This may be the reason why intestinal metastasis and damage are less common in clinical patients. The tumors of mice with choriocarcinoma in situ tumors grew very rapidly. During the treatment period, 3 mice in the Control group and 1 mouse in the MTX group died of tumor progression, while there were no unplanned deaths in the HIC@M+US group. This also preliminarily indicates the anti-tumor ability of HIC@M+US treatment. Fig.13 shown. Fig.14 The uterine morphology of mice in each treatment group is shown, which more intuitively shows the inhibitory effect of the HIC@M+US group on tumors.

[0079] H&E staining photos of mouse tumor tissues showed that mice in the HIC@M+US group had severe tissue damage, and TUNEL staining showed that the apoptosis rate of green fluorescent labeled cells was much higher than that of other groups, indicating that HIC@M+US treatment has a strong tumor destruction potential. Fig.14 As shown. The above experimental results all indicate that HIC@M+US treatment has an inhibitory effect on tumor growth in the treatment of choriocarcinoma. The toxic and side effects of the HIC@M+US group and the MTX treatment group were further compared. The blood and intestinal tissues of mice were collected for routine blood tests, blood biochemical tests and H&E staining of intestinal tissues. Fig.15 As shown in the figure, the intestinal tissue of the HIC@M+US group showed no obvious damage, while the MTX treatment group and the Control group showed different degrees of damage due to direct infiltration and metastasis of tumor tissue. The results of blood routine and blood biochemical tests showed that the MTX group showed different degrees of anemia and liver function damage, such as Fig.16 As shown in the figure, none of the above manifestations were found in the HIC@M+US group. Compared with intravenous injection, local treatment theoretically has lower toxicity and side effects, better targeting, and the rich blood vessels in the uterus are conducive to drug absorption. HIC@M, with its high biosafety, good retention and sonodynamic responsiveness, perfectly combines the advantages of local treatment with the advantages of combined tumor treatment, providing a new treatment idea for choriocarcinoma and other uterine solid tumors.

[0080] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A HIC@M nanoparticle, characterized in that: The HIC@M nanoparticles use hydrazide hyaluronic acid as a carrier, are loaded with cisplatin and indocyanine green, and are coated with choriocarcinoma cell membranes.

2. The method for preparing HIC@M nanoparticles according to claim 1, characterized in that: The following steps are involved: Dissolve cisplatin, indocyanine green and hydrazide hyaluronic acid in water and mix to obtain a mixed solution A, react in the dark at 36-37°C for 22-24 hours, centrifuge and wash the product until the supernatant is colorless, and the obtained precipitate is HIC nanoparticles; the mass ratio of cisplatin, indocyanine green and hydrazide hyaluronic acid is 1:1:2-5; Under ice bath conditions, the HIC nanoparticle solution and the choriocarcinoma cell membrane are mixed to obtain a mixed solution B, the mixed solution B is subjected to ultrasonic treatment for 0.5 to 0.6 h, and then extruded to obtain the HIC@M nanoparticles.

3. The method for preparing HIC@M nanoparticles according to claim 2, characterized in that: The hydrazide hyaluronic acid is prepared by the following steps: Adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to a hyaluronic acid solution, reacting at 29-31° C. for 0.5-0.6 h, then adding 3,3'-dithiodipropionyl hydrazide solution, reacting for 36-40 h, dialyzing the resulting solution for 48-50 h, and freeze-drying to obtain the hydrazide hyaluronic acid; The mass volume ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid solution and 3,3'-dithiodipropionyl hydrazide solution is 0.48 g: 0.288 g: 10-12 mL: 35-40 mL; The concentration of the hyaluronic acid solution is 0.08-0.1 g / mL, and the concentration of the 3,3'-dithiodipropionyl hydrazide solution is 0.05-0.6 g / mL.

4. Use of the HIC@M nanoparticles according to claim 1 in the preparation of anti-tumor drugs.

5. The use of HIC@M nanoparticles in the preparation of anti-tumor drugs according to claim 4, characterized in that: The tumors include choriocarcinoma.

6. The use of HIC@M nanoparticles in the preparation of anti-tumor drugs according to claim 5, characterized in that: The drug is a drug for sonodynamic therapy.

7. The use of HIC@M nanoparticles in the preparation of anti-tumor drugs according to claim 6, characterized in that: The sonodynamic therapy involves locally applying sound waves to the human body.

8. The use of HIC@M nanoparticles in the preparation of anti-tumor drugs according to claim 7, characterized in that: The sound wave is ultrasonic wave.

9. A drug for sonodynamic therapy against choriocarcinoma, characterized in that: The drug uses the HIC@M nanoparticles described in claim 1 as an active ingredient.

10. The drug according to claim 9, characterized in that: The medicine is in the form of intrauterine infusion or intravenous injection.

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