A kind of HIC@M nanoparticles, their preparation method, application and medicine

Abstract

The present invention belongs to the technical field of anti-cancer drugs, and particularly relates to a HIC@M nanoparticle, a preparation method, an application and a drug thereof. The HIC@M nanoparticle uses hydrazide-modified hyaluronic acid as a carrier, loads cisplatin and indocyanine green, and is coated with choriocarcinoma cell membrane. The HIC@M nanoparticle of the present invention can not only treat subcutaneous choriocarcinoma tumors through the combined action of tail vein injection and sonodynamic therapy, but also act on uterine in-situ tumors through an innovative intrauterine perfusion administration method, combined with ultrasound treatment, to achieve a significant anti-tumor effect, and both have good biosafety. The present invention provides a new method for efficiently loading cisplatin and indocyanine green onto a nanocarrier, and also demonstrates the great prospect of treating choriocarcinoma by the combined intrauterine perfusion administration method and sonodynamic chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-cancer drugs, and particularly relates to a HIC@M nanoparticle, a preparation method, an application thereof, and a drug. Background Art

[0002] Choriocarcinoma is a malignant gestational trophoblastic tumor caused by abnormal proliferation of placental trophoblasts, with high potential for proliferation and invasion, seriously endangering the reproductive safety and life health of women. Choriocarcinoma can occur secondary to any form of pregnancy, such as hydatidiform mole, miscarriage, and normal pregnancy, etc. Its typical clinical manifestations are vaginal bleeding, and manifestations of metastatic foci in the lungs, reproductive system, liver, brain, etc. At present, the clinical treatment of choriocarcinoma highly relies on chemotherapy, and some patients can be combined with hysterectomy according to the disease situation, which seriously affects the quality of life and fertility needs of patients. The broad-spectrum killing effect of traditional chemotherapy easily leads to serious toxic and side effects, such as liver injury, gastrointestinal reactions, mucosal injury, myelosuppression, etc., as well as varying degrees of drug resistance, resulting in poor prognosis or even death of patients. Therefore, developing a more efficient and less side-effect drug delivery and treatment strategy is of great significance for solving the clinical treatment problems of choriocarcinoma.

[0003] Local drug delivery, that is, delivering therapeutic drugs to the diseased site, requires a lower drug dose, has stronger targeting, and less drug degradation compared with systemic drug delivery. These factors are beneficial to increasing the drug utilization rate in the tumor area and reducing systemic side effects. Both the vagina and the uterus are structures rich in blood vessels, and are very suitable for local drug delivery of female reproductive system diseases. The existing vaginal and intrauterine drug deliveries generally mainly use traditional dosage forms such as tablets, suppositories, solutions, etc., which have advantages such as easy application and removal, can bypass the first metabolism, and increase the permeability of small molecule drugs, but problems such as poor retention and inability to specifically deliver still limit their application in the treatment of female reproductive system tumors. Therefore, an intrauterine perfusion drug that can specifically deliver, has a long retention time, high comfort, and good curative effect will be of great significance for female reproductive system tumors. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a HIC@M nanoparticle, a preparation method, an application thereof, and a drug.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a HIC@M nanoparticle, wherein the HIC@M nanoparticle uses hydrazide-modified hyaluronic acid as a carrier, loads cisplatin and indocyanine green, and is coated with choriocarcinoma cell membrane.

[0007] The HIC@M nanoparticles of the present invention can be homologously recognized by choriocarcinoma cells, increasing the cellular uptake of drugs. The abundant 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 inducing apoptosis and ferroptosis, ultimately achieving "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 by perfusion and directly acts on the lesion site. Then, the ultrasonic probe is positioned on the lower abdomen to act on the uterine site to enhance the synergistic effect of SDT, achieving a balance between the best therapeutic effect and the minimum side effects.

[0008] In a second aspect, the present invention provides a method for preparing the HIC@M nanoparticles, comprising the following steps:

[0009] Dissolve cisplatin, indocyanine green and hydrazide-modified hyaluronic acid in water and mix them to obtain a mixed solution A. React in the dark at 36 - 37 °C for 22 - 24 h. The product is centrifuged and washed until the supernatant is colorless. The obtained precipitate is the HIC nanoparticles; the mass ratio of cisplatin, indocyanine green to hydrazide-modified hyaluronic acid is 1:1:2 - 3;

[0010] Under ice bath conditions, mix the HIC nanoparticle solution and choriocarcinoma cell membrane to obtain a mixed solution B. Ultrasonically treat the mixed solution B for 0.5 - 0.6 h, and then extrude to obtain the HIC@M nanoparticles.

[0011] Further, the hydrazide-modified hyaluronic acid is prepared by the following steps:

[0012] Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the hyaluronic acid solution, react at 29 - 31 °C for 0.5 - 0.6 h, then add 3,3'-dithiodipropionyl hydrazide solution, react for 36 - 40 h, and continue to dialyze the obtained solution for 48 - 50 h, and freeze-dry to obtain the hydrazide-modified hyaluronic acid;

[0013] The mass-to-volume ratio of 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;

[0014] 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.

[0015] In a third aspect, the present invention provides the application of the HIC@M nanoparticles in the preparation of anti-tumor drugs.

[0016] Furthermore, the tumor includes choriocarcinoma.

[0017] Furthermore, the drug is a drug for sonodynamic therapy.

[0018] Furthermore, the sonodynamic therapy includes locally applying sound waves to the human body.

[0019] Furthermore, the sound wave is an ultrasonic wave.

[0020] In a fourth aspect, the present invention provides a drug for sonodynamic therapy against choriocarcinoma, and the drug uses the HIC@M nanoparticles as an active ingredient.

[0021] Furthermore, the drug is in the form of intrauterine perfusion or intravenous injection.

[0022] The present invention has the following beneficial effects:

[0023] (1) The nanoparticles coated with cancer cell membranes in the HIC@M nanoparticles of the present invention can be recognized by cell homology, increasing the cellular uptake of the drug. Combined with the intrauterine perfusion strategy, it shows better targeting.

[0024] (2) The abundant disulfide bonds in the HIC@M nanoparticles can consume the endogenous GSH enriched in the tumor site, avoid detoxification, and improve the treatment efficiency of CDDP. A large amount of ROS accumulates in the tumor site, which can amplify the effect of SDT and enhance the synergistic treatment effect of sonochemotherapy.

[0025] (3) The accumulation of ROS and the depletion of GSH can further trigger apoptosis and ferroptosis, jointly promoting the clearance of tumor cells. The HIC@M nanoparticles show good biosafety in both the tail vein injection and intrauterine perfusion mouse models, and this method may become a new paradigm for the treatment of choriocarcinoma. Description of the Drawings

[0026] Figure 1 Characterizations of HHA and HIC, where A is the TEM image of HIC analysis, 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 spectra of HHA, ICG, CDDP, and HIC.

[0027] Figure 2 Detection results of the ROS generation and GSH consumption ability of HIC, where A is the quantitative analysis of 1 O2 by DPBF detection, B is the average fluorescence intensity of DCFH-DA under different treatments, C is the ability of DTNB probe to detect the GSH consumption of HIC at different concentrations, and D is the ability of DTNB probe to detect the GSH consumption of different groups.

[0028] Figure 3 For the characterization comparison of HHA and HIC, where A is the TEM analysis images 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 in sequence.

[0029] Figure 4 For the results of specific homologous targeting determination of HIC@M on cells.

[0030] Figure 5 For the results of in vitro anti-tumor ability detection 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 in sequence.

[0031] Figure 6 For 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 in sequence.

[0032] Figure 7 For the tumor weights 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 in sequence.

[0033] Figure 8 For the H&E, TUNEL, Ki67 and GPX4 staining of tumor tissues of mice in different treatment groups.

[0034] Figure 9 For the in vivo imaging pictures of mice at different time points for monitoring tumor growth.

[0035] Figure 10 For the Masson staining and H&E staining pictures of the uterine body and uterine horn of mice in different treatment groups, A is the Masson staining picture, and B is the H&E staining picture.

[0036] Figure 11 For the in vivo bioluminescence images of mice at different time points used for monitoring tumor growth, where the second column on the seventh day and the second and third columns on the tenth day indicate mouse death.

[0037] Figure 12 For the ex vivo images of potential metastatic organs lung, liver, uterus and intestine isolated from JEG-3 orthotopic tumor-bearing mice on the 10th day.

[0038] Figure 13 Survival curves of mice in different treatment groups.

[0039] Figure 14 H&E and TUNEL staining of tumor tissues of mice in different treatment groups.

[0040] Figure 15 H&E staining of intestinal tissues of mice in different treatment groups.

[0041] Figure 16 Results of blood routine and biochemical examinations of mice in the MTX intravenous injection and HIC@M + US intrauterine perfusion group. A is the hemoglobin content, B is the red blood cell content, and C is the ALT content. Detailed implementation mode

[0042] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0043] Key terms and abbreviations involved in the following embodiments:

[0044] HA: Hyaluronic acid; EDC·HCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS: N-Hydroxysuccinimide; DTP: 3,3'-Dithiobispropionylhydrazide; 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 reproductive mortality; CC: Choriocarcinoma; CDDP: Cisplatin; EDX: Energy dispersive x-ray spectroscopy; EPR: Enhanced permeability and retention effect; ESR: Electron paramagnetic resonance spectrometer; FIGO: International Federation of Gynecology and Obstetrics; GTN: Gestational trophoblastic neoplasia; H&E: Hematoxylin and eosin; HHA: Hydrazide-modified hyaluronic acid; ICG: Indocyanine green; MMC: Mitochondrial membrane potential; MTX: Methotrexate; SDT: Sonodynamic therapy; TUNEL: Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling; RBC is red blood cell; ALT: Alanine aminotransferase.

[0045] Example 1: Preparation of HIC@M nanoparticles and their ability to generate ROS and consume GSH.

[0046] 1. Test method.

[0047] (1) Preparation of hydrazide-modified hyaluronic acid.

[0048] First, prepare 10 mL of an HA aqueous solution with a concentration of 0.1 g / mL, and sequentially add 0.48 g of EDC·HCl and 0.288 g of NHS. 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 dialysis of the resulting solution for 48 hours, followed by freeze-drying to obtain the final product hydrazide-modified hyaluronic acid, abbreviated as HHA.

[0049] (2)Preparation of HIC nanoparticles.

[0050] Mix 1 mL of a CDDP solution with a concentration of 1 mg / mL, 1 mL of an ICG solution with a concentration of 1 mg / mL, and 2 mL of an HHA solution with a concentration of 1 mg / mL, and stir in the dark at 37 °C for 24 h. Centrifuge and wash repeatedly until the supernatant is colorless to obtain HHA nanoparticles loaded with CDDP and ICG, abbreviated as HIC.

[0051] (3)Preparation of HIC@M nanoparticles.

[0052] Collect an appropriate amount of JEG-3 cells, extract the cell membranes of JEG-3 cells using the Beyotime Membrane Protein Extraction Kit according to the instructions, and determine the protein content of the cell membranes by the BCA method. Calculate based on the protein concentration, mix an equal volume of a 1 mg / mL JEG-3 cell membrane solution and a 1 mg / mL HIC solution, and perform ultrasonic treatment at 40 KHZ and 100 W for 0.5 h under ice bath conditions. Then, repeat the extrusion of the mixture 20 times using an Avanti min extruder equipped with a 400 nm filter membrane to obtain HIC@M nanoparticles, abbreviated as HIC@M.

[0053] (4)ROS detection.

[0054] The DCFH-DA fluorescent probe is used to detect total ROS. Mix 2 μL of a DCFH-DA solution with a concentration of 10 mM and 80 μL of a NaOH solution with a concentration of 10 mM, and fix it to 1 mL with PBS. Mix 25 μL of the above buffer with different groups of drugs, and perform ultrasonic treatment or not according to the group, and then detect the fluorescence emission spectrum in the range of 500 - 600 nm.

[0055] 20 μL of a DPBF solution with a concentration of 10 mM is used to detect singlet oxygen, and the UV-visible spectrophotometer records the absorbance of DPBF every 2 minutes. The remaining DPBF (%) = absorbance value after treatment / initial absorbance value of the solution × 100%. Conduct 3 independent replicates.

[0056] (5)Detection of glutathione depletion ability.

[0057] 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%.

[0058] 2. Test results.

[0059] 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.

[0060] 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 1as shown in C of []. The infrared absorption spectrum of HIC shows that the characteristic absorption peaks of HHA, ICG, and CDDP are all present in HIC, indicating the successful formation of HIC, as Figure 1 shown in D of []. The fluorescence spectrum shows that compared with free ICG, the absorption peak of HIC blue-shifts from 831 nm to 828 nm, further indicating that ICG is successfully loaded onto HIC.

[0061] After confirming the successful synthesis of HIC, its ability to generate ROS and consume GSH under the combined action of sonodynamic was further verified. As a sonosensitizer, ICG generates a large amount of ROS under the action of ultrasound to directly kill tumor cells, and 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 at the same time avoiding the consumption of the generated ROS. DPBF was used as a singlet oxygen probe to monitor the 1 generation of ¹O₂ triggered by US. By co-incubating the mixture of nanoparticles and DPBF, the ability of HIC to generate 1 ¹O₂ was analyzed under US irradiation conditions. As the US irradiation time increased, the characteristic absorption peak of DPBF at 416 nm decreased significantly, indicating the generation of 1 ¹O₂. As Figure 2 shown in A of []. Compared with the Control group, US group, ICG+US group, and HIC group, the oxidation rate of DPBF in the HIC+US group was the highest. The consumption rate of DPBF in each group was statistically analyzed at 10 min. The consumption rate in the HIC+US group was the highest, and the remaining DPBF was 65.33%, which was significantly different from 95.35% in the Control group, indicating that HIC can generate a large amount of ROS under US conditions to kill tumor cells. The DCFH-DA generation experiment also demonstrated the ROS generation ability of HIC. The fluorescence peak increment in the HIC+US treatment group was 9.57 times, and the increment in the Control group was 3.56 times, indicating that HIC nanoparticles can generate a large amount of ROS under ultrasound treatment, and the results were consistent with the results of the DPBF consumption experiment, as Figure 2 shown in B of [].

[0062] As a rich endogenous antioxidant, GSH can maintain the intracellular redox balance, accumulate in large amounts at the tumor site, prevent ROS-induced damage to tumor cells, and can also degrade cisplatin, reducing the chemotherapy efficiency of cisplatin. Most oxidative anticancer strategies are limited by the high level of GSH in cells. Therefore, depleting intracellular GSH can significantly improve the SDT effect. Thus, in this example, the ability of HIC to deplete GSH was further explored. HIC is rich in disulfide bonds and can consume glutathione through disulfide bond cleavage. The Ellman reagent DTNB probe was used to detect GSH consumption. This probe reacts with GSH to produce a yellow product with a maximum ultraviolet absorption wavelength of 412 nm. The glutathione solution was incubated with nanomicelles at different concentrations for 2 h, and glutathione was detected with DTNB. The results are as Figure 2 shown in C of Figure 2 . The absorbance peak at 412 nm decreased in a dose-dependent manner, indicating that the consumption of GSH was dose-dependent, and the greater the concentration of HIC, the stronger its ability to consume GSH. In addition, to compare the GSH consumption ability of different treatment groups, the GSH content before and after treatment of the Control group, US group, HIC group, and HIC+US group was detected with DTNB. The results are as Figure 2 shown in D of Figure 2 . The ultraviolet absorption peak at 412 nm in the HIC+US group decreased most significantly, indicating that HIC nanoparticles have obvious GSH consumption ability under the condition of US irradiation, which is beneficial to maintaining a high ROS level, further enhancing the SDT performance, and at the same time improving the efficiency of cisplatin chemotherapy, laying a solid foundation for subsequent cell and animal anti-tumor experiments.

[0063] Example 2: Uptake of HIC@M by living cells and anti-tumor effect.

[0064] 1. Test method.

[0065] Cell cytotoxicity was detected by the MTT colorimetric method. JEG-3 cells were seeded in 12-well plates and cultured overnight. The cells were incubated with HIC@M at different concentrations and irradiated with US at intensities of 0.5 W / cm 2 , 1.0 W / cm 2 and 1.5 W / cm 2 to screen the optimal drug administration conditions. Then, the cells were incubated with ICG, CDDP, and HIC@M for 4 h, and sonicated with 1.0 W / cm 2 ultrasound for 3 min or without ultrasound. After 20 h, the cells were washed with PBS, incubated with 5 mg / mL MTT solution for 4 h. The MTT solution was removed, and DMSO was added to each well to dissolve formazan, and the absorbance value at 490 nm was detected with an enzyme-linked immunosorbent assay instrument.

[0066] 2. Test results.

[0067] The abundant disulfide bonds in HIC enable it to accumulate and respond at tumor sites with high GSH expression. Coating HIC with choriocarcinoma cell membranes can further enhance its homologous targeting at tumor sites, enabling it to better exert specific killing effects at tumor sites, maximizing the therapeutic effect and reducing drug side effects. As Figure 3 The TEM electron micrograph shown in A of Figure 3 preliminarily demonstrated the successful coating of JEG-3 cell membranes. Coomassie Brilliant Blue gel staining experiments further confirmed the intact preservation of cell membrane surface proteins. As Figure 3 shown in B of Figure 3 , the JEG-3 cell lysate had a richer protein profile than the extracted JEG-3 cell membranes and HIC@M. This may be because the isolated cancer cell membranes lack nuclear, organelle, and cytoplasmic proteins, etc. The protein bands of HIC@M were consistent with those of JEG-3 cell membranes, while the uncoated material group HIC did not show proteins, indicating that cell membrane proteins were retained after coating.

[0068] The time-dependent cellular uptake of HIC@M by JEG-3 cells was continuously detected, and the results showed that the maximum uptake occurred after 4 hours of incubation. Next, the specific homologous targeting of HIC@M to cells was explored in various placenta-derived cell lines. As Figure 4 shown, after co-incubation with HIC@M, JEG-3 cells showed stronger ICG fluorescence than HTR-8 / SVneo and JAR cells, which demonstrated that the JEG-3 cell membrane enhanced the specific self-recognition of HIC@M. In summary, HIC@M coated with JEG-3 cell membranes exhibited excellent homology and could perform specific homologous targeting of cancer cells, providing broad potential applications for choriocarcinoma treatment.

[0069] Next, the in vitro anti-tumor ability of HIC@M+US was evaluated. It was found that the cell survival rate at an ultrasonic intensity of 0.5 W / cm 2 was 99.58±3.85%, showing no significant difference from the survival rate of 100±3.32% in the Control group cells; while the ultrasonic intensity of 1.5 W / cm 2 caused most adherent cells to float, and the survival rate was only 8.22±3.15%, resulting in a large error in subsequent cell experiments. Finally, an ultrasonic intensity of 1.0 W / cm 2 was selected as the ultrasonic condition for subsequent experiments, and the survival rate of JEG-3 cells under this condition was 77.18±12.66%. To compare the synergistic anti-tumor effect of HIC@M, 6 more groups were further set up: Control group, US group, ICG+US group, CDDP+US group, HIC@M group, HIC@M+US group. As Figure 5As shown in A of , the HIC@M+US group had the strongest cell-killing ability, with a cell survival rate of 42.19%±8.37%, far lower than 100.00%±19.90% of the Control group, 81.37%±11.95% of the US group, 63.25%±21.30% of the ICG+US group, 60.55%±18.07% of the CDDP+US group, and 63.41%±6.73% of the HIC@M group. Single-cell cloning experiments are usually used to estimate CRD and evaluate the ability of a single cell to develop into a colony. Compared with other groups, the HIC@M+US group formed the fewest cell colonies, and the statistics are shown in Figure 5 As shown in C of , it indicated that HIC@M+US significantly inhibited the colony-forming ability of JEG-3 cells. The anti-proliferation effect of HIC@M+US on JEG-3 cells was evaluated by EdU assay. The Control group showed strong green fluorescence, representing cell proliferation, while the cells treated with HIC@M+US produced weak 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 content of hCG in the supernatant of JEG-3 cells in different intervention groups was detected by ELISA assay to evaluate the effect of HIC@M+US on endocrine activity, and the results are as Figure 5 As shown in B of , the hCG level of untreated JEG-3 cells was 168.20±12.19 mIU / mL, while the hCG level of the cells treated with HIC@M+US decreased significantly, to 106.35±8.10 mIU / mL.

[0070] Example 3: Efficacy of intravenous injection in treating subcutaneous tumors.

[0071] 1. Test method.

[0072] The tumor-bearing mice transplanted subcutaneously with tumors of 70 - 100 cm 3 were randomly divided into five groups of five each. The groups were: (1) Control group, intravenous injection of normal saline; (2) ICG+US group, intravenous injection of 0.5 mg / kg of ICG + 1.5 W / cm 2 of US treatment for 3 minutes; (3) CDDP+US group, intravenous injection of 0.5 mg / kg of CDDP + 1.5 W / cm 2 of US treatment for 3 minutes; (4) HIC@M group, intravenous injection of HIC@M, with the same CDDP dosage in HIC@M as in the CDDP+US group; (5) HIC@M+US group, on the basis of the HIC@M group, 1.5 W / cm 2The US treatment was carried out for 3 minutes, and the US treatment was performed 0.5 hours after intravenous injection, and 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, GPX4 staining, etc.

[0073] 2. Test Results

[0074] In vitro experimental results showed that HIC@M+US had a good inhibitory effect on JEG-3 cells by generating a large amount of ROS and inducing ferroptosis, which prompted further exploration of the in vivo antitumor effect of HIC@M+US. The in vivo anticancer characteristics of HIC@M+US were verified in a JEG-3 subcutaneous tumor-bearing mouse model. First, a choriocarcinoma xenograft nude mouse model was constructed by subcutaneous injection of JEG-3 cells. When the tumor volume of the tumor-bearing mice reached 70-100 mm 3 ³, they were randomly divided into five groups: Control group, ICG+US group, CDDP+US group, HIC@M group, and HIC@M+US group. The Control group was injected with normal saline intravenously. The HIC@M+US group was treated with ultrasound 0.5 h after intravenous injection of the drug. The other groups were given corresponding treatments according to the situation, once every 3 days, for a total of 3 times. During the treatment, the body weight and tumor volume changes of the mice in each group were recorded regularly. It was observed that there was no significant difference in the increasing trend of the body weight of the mice in each group, indicating that the adverse reactions of HIC@M+US and each group of treatments were relatively small. The tumors of the choriocarcinoma subcutaneous tumor mice grew rapidly, but the tumor growth rate of the HIC@M+US group was significantly slowed down due to its obvious antitumor effect. The relative tumor volumes of the mice in each group were calculated based on the measured data to determine whether the treatment was effective. The results showed that the final relative tumor volume of the mice treated with HIC@M+US was 2.32 times, 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, as Figure 6 shown. All the mice were euthanized after the treatment, and the subcutaneous tumors of each mouse were dissected for photographing, weighing, and sampling, Figure 7 showing the tumor weights of the mice in each group. The above results showed that the tumor growth of the ICG+US group and the CDDP+US group was slightly inhibited, indicating that the efficacy of simple sonodynamic therapy or chemotherapy on tumors was limited, while HIC@M+US could significantly inhibit tumor growth and achieved good tumor treatment effects in the in vivo JEG-3 subcutaneous tumor model, further indicating that the sonodynamic / chemotherapy combination therapy based on HIC@M had a more ideal antitumor effect than the single mode.

[0075] Furthermore, the tumor tissues of the mice in different treatment groups were taken for H&E, TUNEL, Ki67, and GPX4 staining. As Figure 8As shown, the H&E staining photographs of the tumor tissues of the mice in the HIC@M+US group showed severe tissue damage with a large number of vacuoles in the loose tissue, indicating that HIC@M+US has strong tumor-destroying potential. Tumor TUNEL staining showed that the apoptosis rate of the cells labeled with green fluorescence in the HIC@M+US group was much higher than that in other groups. The biomarker of cell proliferation, Ki-67, was significantly lower in the HIC@M+US group than in other groups, showing an obvious inhibition of tumor cell proliferation. Immunohistochemical detection of the expression of GPX4 in xenograft tumors 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 shows an effect of inhibiting tumor growth in the treatment of choriocarcinoma, and this effect is closely related to promoting the generation of reactive oxygen species, apoptosis, and ferroptosis of tumor cells.

[0076] Example 5: Intrauterine perfusion retention and biosafety of HIC@M nanoparticles.

[0077] 1. Test method.

[0078] The needle of a 5 mL medical syringe was ground flat and bent to an angle suitable for uterine perfusion, and then the barrel of a 1 mL syringe was replaced, thus making a simple intrauterine perfusion device. The top and the rear end of a 200 mL pipette tip were removed, and the middle part with a length of about 1.5 cm was taken and the two ends were ground flat, thus making a simple mouse speculum. First, the mouse's cervical orifice was found with the simple speculum, and then the perfusion device was placed parallel to the mouse's vaginal orifice and gently and slowly inserted until resistance was encountered, and then the syringe was gently pushed to inject the drug into the uterus.

[0079] The mice in the intrauterine perfusion group were divided into two groups with six mice in each group, and the grouping was as follows: (1) Control group, intrauterine perfusion of normal saline; (2) HIC@M in HA group, intrauterine perfusion of HIC@M dissolved in HA according to the dosage of 0.5 mg / kg of CDDP; (3) HIC@M in H2O group, intrauterine perfusion of HIC@M dissolved in ddH2O according to the dosage of 0.5 mg / kg of CDDP. Treatment was carried out once every 3 days for a total of 3 times. At 15 days after treatment, blood and major organs were collected in the same way for routine blood tests, biochemical examinations, H&E staining, and Masson staining.

[0080] 2. Test results

[0081] To explore the feasibility of the intrauterine perfusion administration method of HIC@M, a biosafety experiment of the drug intrauterine perfusion administration method and the measurement of drug retention time were carried out. HIC@M was dissolved in ddH2O and HA respectively. After standing for a certain period of time, it was found that the solution in the HIC@M in H2O group was still in a flowing state. It has certain fluidity and injectability and can be injected into the uterine cavity through a catheter. Next, the mice were randomly divided into 2 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 their distribution and metabolism in the body. As Figure 9 shown, HIC@M dissolved in ddH2O rapidly diffused to other parts such as the peritoneum and intestines, while HIC@M dissolved in HA was basically confined to the uterine position and did not diffuse to the surrounding tissues, showing a better local aggregation effect. The fluorescence intensity of the nanoparticles was the strongest from 6 h to 12 h after perfusion. After 48 h, the nanoparticles in the uterus of the mice in the HIC@M in H2O group were basically metabolized, while the nanoparticles in the uterus of the mice in the HIC@M in HA group were still significantly enriched. Then, the excised uteri of the two groups of mice were taken for fluorescence imaging at 12 h and 48 h after perfusion respectively. The results showed that at 12 h after intrauterine perfusion, the HIC@M in the uterus of the mice in the HIC@M in H2O group was absorbed into the blood and absorbed and metabolized by organs such as the liver and kidneys, while this phenomenon was not observed in the mice in the HIC@M in HA group; at 48 h after intrauterine perfusion, the fluorescence intensity of the uterus of the mice in the HIC@M in HA group was significantly higher than that in the HIC@M in H2O group. It shows that HIC@M can stay in the uterus of mice for a long time after being dissolved in HA, does not diffuse to the surrounding tissues, and is not absorbed by organs such as the liver and kidneys, laying a strong foundation for its subsequent therapeutic effect.

[0082] Intrauterine biosafety: The uterine tissues of the mice in the HIC@M in HA group and the Control group were dissected and photographed in vitro, stained with Masson and H&E. Two parts, the uterine body and the uterine horn, were taken from each mouse to observe whether fibrosis and other histological injuries occurred. As Figure 10 shown, the Masson staining and H&E staining of the uteri in the treatment group and the Control group showed similar tissue structures, and the statistical chart of Masson staining showed no statistical difference between the two groups. During the experiment, it was observed that there was no significant difference in the weight gain trend of the mice in each group, indicating that the adverse reactions of HIC@M+US and each treatment were relatively small. The above experimental results all prove that the intrauterine perfusion administration method of HIC@M is safe.

[0083] Example 6: Efficacy in treating in-situ tumors.

[0084] 1. Test method.

[0085] Anesthetize female BALB / c nude mice aged 4 - 6 weeks using a small animal anesthesia machine. Sterilize the lower abdomen of each mouse strictly with medical iodine tincture, and then make a 15 - mm long longitudinal incision along the mid - ventral line. After opening the peritoneum, arrange the intestinal tract, appropriately separate the tissues around the uterus, and fully expose the uterine body and uterine horns. Expose the bifurcation of the uterine horns, and inject JEG - 3 cancer cells stably transfected with luciferase into the uterine muscle layer. Subsequently, suture the surgical incision with 5 - 0 absorbable thread. After the operation, place the mice in a warm environment and monitor their physical signs until they fully recover.

[0086] In - situ xenograft tumor model's in - vivo synergistic treatment effect: The in - situ xenograft tumor model was divided into three groups, with four mice in each group. The grouping was as follows: (1) Control group, intra - uterine perfusion of normal saline; (2) MTX group, intravenous injection of 5 mg / kg methotrexate; (3) HIC@M + US group, according to the dosage of 0.5 mg / kg CDDP, intra - uterine perfusion of HIC@M, and treated with US at 1.5 W / cm 2 for 3 min. Six hours after intra - uterine perfusion, the mice received US treatment, which was repeated every 3 days. Inject 150 mg / kg D - luciferin substrate intraperitoneally, and analyze the tumor growth trend according to the in - vivo bioluminescence situation through the in - vivo imaging system every 2 days. After 10 days of monitoring, euthanize the mice and collect their tumor tissues for further analysis.

[0087] 2. Test results.

[0088] After verifying the retention and safety of HIC@M in the uterine cavity, further in - situ tumor model intra - uterine perfusion treatment was carried out. In - situ tumor in - vivo imaging showed that the tumor volumes of mice in different groups increased with time, and the increase amplitude of the Control group was the largest, while the amplitude of the HIC@M + US group was the smallest, and the tumor growth rate was the slowest, as Figure 11 shown. After the treatment ended, euthanize the mice in each group, and dissect and image the organs where the in - situ tumors are prone to metastasize again. The most common metastatic site in the clinical treatment of choriocarcinoma is the lung. In this experimental animal model, due to the excessive volume of the in - situ uterine transplant tumor, it broke through the uterus and invaded the surrounding digestive tract tissues to varying degrees. Therefore, the uterus, lung, liver, and intestine were all sampled for imaging to observe the metastatic degree of mice in different treatment groups. After intra - uterine perfusion of HIC@M combined with sonodynamic therapy, the volume of the in - situ uterine 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 in - situ uterine tumor increased significantly, there were multiple metastatic tumor lesions in the intestine, and fluorescence imaging of metastatic tumors was also visible in the liver, as Figure 12As shown. It is worth noting that the clinical treatment of choriocarcinoma relies on serum hCG detection. Most patients can detect the disease during routine monitoring after pregnancy, which may be the reason for the relatively rare occurrence of intestinal metastasis and injury in clinical patients. The tumor growth of the in-situ tumor mice of choriocarcinoma is very rapid. During the treatment period, 3 and 1 mice in the Control group and the MTX group died respectively due to tumor progression, and there were no unplanned deaths in the HIC@M+US group of mice, which also preliminarily demonstrated the anti-tumor ability of HIC@M+US treatment, such as Figure 13 shown. Figure 14 The uterine morphology of the mice in each treatment group is shown, which more intuitively shows the inhibitory effect of the HIC@M+US group on tumors.

[0089] The H&E staining photos of the tumor tissues of the mice show that severe tissue damage occurred in the mice in the HIC@M+US group. TUNEL staining shows that the apoptosis rate of the cells labeled with green fluorescence is much higher than that of other groups, indicating that HIC@M+US treatment has strong tumor destruction potential, such as Figure 14 shown. The above experimental results all show that HIC@M+US treatment shows an effect of inhibiting 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 the mice were collected for routine blood tests, blood biochemical tests and H&E staining of intestinal tissues. As Figure 15 shown, there was no obvious damage to the intestinal tissues in the HIC@M+US group, while the MTX treatment group and the Control group showed varying degrees of damage due to the direct infiltration and metastasis of tumor tissues. The results of routine blood tests and blood biochemical tests show that the MTX group showed varying degrees of anemia and liver function damage, as Figure 16 shown, and none of the above manifestations were found in the HIC@M+US group. Compared with intravenous injection treatment, local treatment theoretically has lower toxic and side effects and better targeting, and the rich blood vessels in the uterus are conducive to drug absorption. Due to its high biological safety, good retention and sonodynamic responsiveness, HIC@M combines the advantages of local treatment and tumor combined treatment extremely well, providing new treatment ideas for choriocarcinoma and other uterine solid tumors.

[0090] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

Claims

1. A HIC@M nanoparticle, characterized in that, The HIC@M nanoparticles use hydrazide-modified hyaluronic acid as a carrier, load cisplatin and indocyanine green, and are coated with choriocarcinoma cell membranes; the preparation method of the HIC@M nanoparticles includes the following steps: Dissolve cisplatin, indocyanine green and hydrazide-modified hyaluronic acid in water and mix them to obtain a mixed solution A. React in the dark at 36-37 °C for 22-24 h. The product is centrifuged and washed until the supernatant is colorless. The obtained precipitate is the HIC nanoparticles; the mass ratio of cisplatin, indocyanine green and hydrazide-modified hyaluronic acid is 1:1:2-5; Under ice bath conditions, mix the HIC nanoparticle solution and choriocarcinoma cell membranes to obtain a mixed solution B. Ultrasonically treat the mixed solution B for 0.5-0.6 h, and then extrude to obtain the HIC@M nanoparticles; The hydrazide-modified hyaluronic acid is prepared by the following steps: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the hyaluronic acid solution, react at 29-31 °C for 0.5-0.6 h, then add 3,3'-dithiobispropionylhydrazide solution, and react for 36-40 h. The obtained solution is continuously dialyzed for 48-50 h and freeze-dried to obtain the hydrazide-modified hyaluronic acid; The mass-to-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid solution and 3,3'-dithiobispropionylhydrazide 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'-dithiobispropionylhydrazide solution is 0.05-0.6 g / mL.

2. Use of the HIC@M nanoparticles according to claim 1 in the preparation of an anti-tumor drug.

3. Use of the HIC@M nanoparticles according to claim 2 in the preparation of anti-tumor drugs, characterized in that, The tumor includes choriocarcinoma.

4. Use of the HIC@M nanoparticles according to claim 3 in the preparation of anti-tumor drugs, characterized in that, The drug is a drug for sonodynamic therapy.

5. Use of the HIC@M nanoparticles according to claim 4 in the preparation of anti-tumor drugs, characterized in that, The sonodynamic therapy includes locally acting on the human body with sound waves.

6. Use of the HIC@M nanoparticles according to claim 5 in the preparation of an anti-tumor drug, characterized in that, The sound wave is an ultrasonic wave.

7. 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.

8. The medicament according to claim 7, wherein, The drug is in the form of intrauterine perfusion or intravenous injection.

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