Medicament combining Complete with iron agent as well as preparation method and application of medicament

Abstract

The invention discloses a medicine of a combination of Complete and an iron agent and a preparation method and application thereof, and belongs to the technical field of medicines.The medicine of the combination of Complete and the iron agent comprises the composition of Complete and the iron agent, the iron agent is a Fe < 3 + > donor, and the Fe < 3 + > donor can be selected from ferric salt or iron oxide of ferric iron or nano-particles of ferric salt or iron oxide. The pharmaceutical composition also comprises a pharmaceutically acceptable carrier and an additive, the inhibition effect of the pharmaceutical composition on the growth of tumor cells is obviously superior to the effect of independently using the Complete, the condition of drug resistance of the tumor cells to the Complete is improved, the pharmaceutical composition can be applied to preparation of the medicine for treating squamous cell carcinoma of lung, and the use dosage of the Complete can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Kanglaite combined iron preparation and a preparation method and application thereof. Background Art

[0002] Lung cancer is a malignant tumor with high morbidity and mortality worldwide, of which non-small cell lung cancer (NSCLC) accounts for the majority, and lung squamous cell carcinoma (LUSC) is one of its main subtypes. Although targeted therapy and immunotherapy have made certain progress in the treatment of NSCLC, patients with LUSC still face problems such as recurrence and drug resistance, and new and effective treatments are urgently needed. Ferroptosis is a programmed cell death method that depends on iron ions and is particularly suitable for dealing with cancer cells that are resistant to traditional treatments.

[0003] Kanglaite is an anticancer active ingredient extracted from the Chinese medicine Coix seed. The drug is an anticancer Chinese medicine injection preparation, mainly used in the clinical treatment of lung adenocarcinoma. When used in combination with chemotherapy, radiotherapy or targeted therapy, Kanglaite can improve the patient's objective response rate, disease control rate and overall quality of life. However, in actual application, it was found that the treatment effect of Kanglaite preparation alone or Kanglaite preparation alone combined with chemotherapy, radiotherapy or targeted agents was not good, the dosage was large, and the probability of adverse reactions during treatment was also high. Summary of the invention

[0004] The purpose of the present invention is to improve the above-mentioned problems and provide a drug of Kanglaite combined with iron agent and its preparation method and application. The obtained drug can be used in the preparation of drugs for treating lung squamous cell carcinoma. The inhibitory effect of the drug on tumor cell growth is significantly better than the effect of Kanglaite alone.

[0005] The present invention is achieved through the following technical solutions: A drug for combining Kanglaite with an iron agent, comprising a combination of Kanglaite and an iron agent, wherein the iron agent is Fe 3+ Donor.

[0006] Furthermore, Fe 3+ The donor includes ferric iron salts or iron oxides or nanoparticles thereof.

[0007] Furthermore, it also includes a pharmaceutically acceptable carrier.

[0008] Furthermore, the carrier comprises dendritic mesoporous silica.

[0009] Furthermore, it also includes at least one pharmaceutically acceptable additive.

[0010] The preparation method of the Kanglaite combined iron agent drug as mentioned above comprises the steps of: selecting dendritic mesoporous silica nanoparticles as a carrier, loading Fe 3+ After the donor is successfully loaded, Kanglaite is added to form DMSN / Fe 3+ -Kanglaite nanomedicine.

[0011] Furthermore, Fe 3+ The donor is Fe3O4 nanoparticles with a diameter of 4 nm.

[0012] A use of any of the above-mentioned Kanglaite combined with an iron agent in the preparation of a drug for treating lung squamous cell carcinoma.

[0013] Furthermore, the Kanglaite combined with iron agent is used in the preparation of drugs for inhibiting the proliferation and migration of cancer cells.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention proposes a drug combining Kanglaite with an iron agent. The combined use of Kanglaite and an iron agent can enhance the effect of Kanglaite in inhibiting tumor cell growth. The iron agent is preferably Fe 3+ Donors include trivalent iron salts or iron oxides or their nanoparticles, etc. The Kanglaite combined iron drug can significantly enhance the level of reactive oxygen species (ROS) in tumor cells, thereby effectively inducing ferroptosis of tumor cells.

[0015] 2. In the present invention, the proposed Kanglaite combined with iron agent drug improves the drug utilization rate of Kanglaite by introducing Fe3O4 particles compared to the use of Kanglaite alone, and significantly inhibits the tumor growth of lung squamous cell carcinoma in a mouse xenograft model. It can be seen that the Kanglaite combined with iron agent drug can be used in the preparation of drugs for the treatment of lung squamous cell carcinoma.

[0016] 3. The present invention also proposes a preferred Kanglaite combined with iron agent drug, which, in addition to Kanglaite and iron agent, also includes a pharmaceutically acceptable carrier, preferably a dendritic mesoporous silica carrier. The introduction of the carrier can achieve the controlled release of Kanglaite and iron agent, reduce the dosage of Kanglaite drug, and reduce possible adverse reactions during treatment. The carrier is preferably a nanocarrier, which can improve the bioavailability of the drug and shorten the therapeutic effect time of the drug.

[0017] 4. In the present invention, the iron preparation of the Kanglaite combined iron preparation drug is preferably Fe 3+ Donor nanoparticles, such as Fe3O4 nanoparticles, have a better effect.

[0018] 5. In the present invention, the proposed drug of Kanglaite combined with iron is a combination of Kanglaite and iron, which is significantly better than Kanglaite alone in inhibiting the growth of lung squamous cell carcinoma cells. 3+ -Kanglaite nanomedicine is a new treatment strategy that provides a new approach for the treatment of squamous cell lung carcinoma and also expands the application scope of Kanglaite in the treatment of other tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the cell survival rate of NCL-H1703 cells in Example 1 after being treated with Kanglaite alone for 24, 48 or 72 hours.

[0020] Figure 2 The figure is the flow cytometry analysis result of Annexin V / PI staining of NCL-H1703 cells at different time points.

[0021] Figure 3 The figure is the result of cell survival rate of NCL-H1703 cells after being treated with different concentrations of FeCl3 and / or Kanglaite for 72 hours.

[0022] Figure 4 This is the result of Annexin V / PI flow cytometry analysis of NCL-H1703 cells treated with 100μM FeCl3, 2mg / mL Kanglaite, and 2mg / mL Kanglaite + 100μM FeCl3 for 72h.

[0023] Figure 5 The figure shows the cell survival rate of NCL-H1703 cells after being treated with FeCl3, Fe3O4 and Fe3O4 nanoparticles alone and in combination with Kanglaite reagent.

[0024] Figure 6 It is Kanglait and Fe 3+ The results of the combined use on ROS in NCL-H1703 cells.

[0025] Figure 7 It is Kanglait and Fe 3+ The results of the combined use on the GPX4 protein level in NCL-H1703 cells.

[0026] Figure 8 It is Kanglait and Fe 3+ The results of the combined effect on lipid peroxidation in NCL-H1703 cells.

[0027] Fig. 9 The addition of ferroptosis inhibitor Fer-1 has an effect on the 3+ Graph showing the impact of combined use.

[0028] Fig.10 It is a flow chart of the synthesis of DMSN / Fe3O4-Kanglaite (D / FK) nanomedicine.

[0029] Fig.11 This is an electron microscope image of dendritic mesoporous silica nanoparticles loaded with Fe3O4 particles.

[0030] Fig.12 This is the element mapping analysis result of DMSN / Fe3O4 structure.

[0031] Fig.13 It is the Kanglaite standard curve.

[0032] Fig.14 This is the result of the test on the loading amount of Kanglaite in DMSN / Fe3O4-Kanglaite (D / FK) nanomedicine.

[0033] Fig.15 This is the release trend of Kanglaite from DMSN / Fe3O4(D / F) nanomedicine in the presence of 10% serum.

[0034] Fig.16 Blank group, 2mg / mL Kanglaite + 100μM Fe 3+ The results of the ability of the D / FK nanoparticles (Kanglaite content 13.25μg / mL) group and the D / FK nanoparticles (Kanglaite content 13.25μg / mL) group to induce cell ferroptosis.

[0035] Fig.17 This is a graphic representation of the tumor sites in the experimental mice 51 days after being injected with the control group, Kanglaite and DMSN / Fe3O4-Kanglaite nanomedicines respectively.

[0036] Fig.18 This is a statistical chart of tumor volume in each group of experimental mice 51 days after injection of the control group, Kanglaite and DMSN / Fe3O4-Kanglaite nanodrugs.

[0037] Fig.19 This is a statistical chart of tumor weight in each group of experimental mice 51 days after injection of control group, Kanglaite and DMSN / Fe3O4-Kanglaite nanodrugs.

[0038] Fig. 20 This is a statistical chart of tumor growth in each group of experimental mice 51 days after injection of the control group, Kanglaite and DMSN / Fe3O4-Kanglaite nanomedicines. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0040] Example 1 1. Materials and reagents involved in the experiment 1.1 Cells Human lung squamous cell carcinoma cells NCL-H1703 were purchased from the Cell Resource Bank of the Chinese Academy of Sciences (Shanghai). NCL-H1703 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / L penicillin. The cells were subcultured in a 37°C, 5% CO2 cell culture incubator.

[0041] 1.2 Mouse Model Female BALB / c (nu / nu) mice (18–20 g) aged 5–6 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd. (Jiangsu, China). All animals were raised in the Experimental Animal Center of the School of Medicine of the University of Electronic Science and Technology of China in a sterile environment with free access to food and water. All experiments involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Sichuan Provincial People's Hospital in accordance with animal ethics requirements.

[0042] 1.3 Reagents Kanglaite (China Food and Drug Administration approval number: Z10970091), Ferrostatin-1 (HY-100579, MedChemExpress), PBS (C0221A, Beyotime), dendritic mesoporous silica (Ruixi Biotechnology, Xi'an), Fe3O4 nanoparticles (Ruixi Biotechnology, Xi'an), FeCl3 (Cologne Chemical, Chengdu), Fe3O4 (Cologne Chemical, Chengdu), BODIPY™581 / 591C11 (HY-D1301, MedChemExpress).

[0043] 2. Experimental Methods 2.1 Cell culture 2.1.1 Cell recovery (1) Take out the cells frozen in the liquid nitrogen tank, quickly thaw them in a 37°C water bath for about 1 minute, then add them to a 15 mL centrifuge tube containing 5-6 mL of complete culture medium and centrifuge at 1500 rpm for 3 minutes; (2) Discard the supernatant and add 1 mL of fresh culture medium to resuspend the cell pellet; (3) Add the cells to a new 10 cm culture dish, add 7-8 mL of culture medium, shake gently to evenly disperse the cells, and place in a cell culture incubator for culture.

[0044] 2.1.2 Cell passaging (1) Take out the cells to be passaged, discard the supernatant, add 2 mL of preheated trypsin, and return to the cell culture incubator for 3-5 minutes; (2) Observe the digestion of cells. When the cells become round and shrink, add 1 ml of culture medium to terminate the digestion reaction. Collect the cells into a new 15 ml centrifuge tube and centrifuge at 1500 rpm for 3 minutes. (3) Discard the supernatant and add 1 mL of fresh culture medium to resuspend the cells; (4) Add the cells to a new 10 cm culture dish, add 7-8 mL of culture medium, gently shake to evenly disperse the cells, and return to the cell culture incubator for culture.

[0045] 2.1.3 Cell cryopreservation (1) Prepare the freezing solution at a ratio of DMSO: culture medium = 1:9; (2) Take out the cells to be frozen, discard the supernatant, add 2 mL of preheated trypsin, and place it back in the incubator for 3-5 minutes; (5) Observe the digestion of cells. When the cells become round and shrink, add 1 ml of culture medium to terminate the digestion reaction. Collect the cells into a new 15 ml centrifuge tube and centrifuge at 1500 rpm for 3 minutes. (3) Discard the supernatant, resuspend the cells with freezing solution, divide them into cryopreservation tubes, and place them in a gradient cooling box; (4) Place the gradient cooling box in a -80°C refrigerator. After 3-4 days, place the cells in a liquid nitrogen tank for long-term storage.

[0046] 2.2 CCK-8 assay for cell viability CCK-8, or Cell Counting Kit-8, is a commonly used reagent for detecting cell viability. WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow formazan, and the depth of the color is proportional to the number of living cells. The experimental steps are as follows: (1) Inoculate an appropriate amount of cells into a 96-well plate and treat with drugs according to experimental requirements; (2) Aspirate the culture medium, wash three times with serum-free culture medium, then add 100 μL of detection solution (10 μL CCK-8 and 90 μL culture medium) to each well and react in a 37°C incubator for 1-2 h; (3) Detect the light absorbance at 450nm and 630nm, and the difference is the absorbance value of each well; (4) Calculate the cell survival rate.

[0047] 2.3 Annexin V-FITC / PI detection of cell apoptosis (1) Prepare cells according to experimental requirements; (2) Aspirate the cell culture medium into a 15 mL centrifuge tube, wash the cells once with PBS, and add an appropriate amount of trypsin to digest the cells. After digestion, add complete culture medium to terminate digestion and collect the cells into a 15 mL centrifuge tube.

[0048] (3) Centrifuge at 1500 rpm for 5 minutes to collect the cell pellet. Then add 200 μL of staining buffer to resuspend the cells; (4) Add 5 μL of Annexin V-FITC and 10 μL of PI to each sample and mix well; (6) Incubate at room temperature in the dark for 20 min, inverting the cells several times during the incubation process to ensure uniform staining. (7) After incubation, place the sample in an ice bath for a short period of time; (8) Detection was performed using flow cytometry.

[0049] 2.4 Intracellular ROS detection (1) Prepare DCFH-DA staining solution with HBSS. The final concentration of DCFH-DA is 10 μM. Keep in a 37°C water bath before use. (2) After the cells are treated, the culture medium is removed, the cells are washed twice with HBSS, and DCFH-DA staining solution is added at a ratio of 1.5 mL of staining solution per well of a 6-well plate, and the cells are incubated in a 37°C incubator for 30 minutes. (3) After incubation, remove the DCFH-DA staining solution, wash twice with HBSS, and then add 1 mL of HBSS to cover the cell surface; (4) Use a fluorescence microscope or flow cytometer for detection with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0050] 2.5 Lipid peroxidation detection (1) Prepare BODIPY™ 581 / 591C11 staining solution with HBSS to a final concentration of 5 μM; (2) After the cells are treated, remove the culture medium, wash twice with HBSS, add BODIPY™ 581 / 591C11 staining solution at a ratio of 1.5 mL per well of a 6-well plate, and incubate in a 37°C incubator for 30 minutes. (3) After incubation, remove the BODIPY™ 581 / 591C11 staining solution, wash twice with HBSS, and then add 1 mL of HBSS to cover the cell surface; (4) Detection was performed using an inverted fluorescence microscope.

[0051] 2.6 Western blot 2.6.1 Extraction of total cell protein (1) After cell treatment, remove the supernatant and wash the cells twice with pre-cooled PBS; (2) Add 1× loading buffer containing protease inhibitors, collect the lysate into a 1.5 mL centrifuge tube using a cell scraper, and heat at 98°C for 10 minutes; (3) Centrifuge at 10,000 rpm for 2 minutes and transfer the supernatant to a new centrifuge tube to obtain the extracted total cellular protein.

[0052] 2.6.2 SDS-polyacrylamide gel electrophoresis (1) Prepare the separation gel and stacking gel according to the molecular weight of the target protein; (2) Boil the protein samples at 95°C for 5 minutes and then add them to the gel wells in order; (3) Set the voltage to 60 V at the beginning of electrophoresis, and adjust the voltage to 150 V after the sample enters the separation gel until the end.

[0053] 2.6.3 Western blot (1) After electrophoresis, place the gel and PVDF membrane in the electrophoresis clip in order, taking care to remove bubbles, pour in the electrophoresis solution, and transfer at a constant current of 300 mA for 2 h, keeping the ice bath during the process; (2) After electroporation, place the PVDF membrane in 5% skim milk and block for 1 hour; (3) Discard skim milk, rinse with TTBS for 5-10 minutes, add corresponding primary antibody, and incubate overnight at 4°C; (4) Recover the primary antibody and rinse the PVDF membrane with TTBS buffer three times, 10 minutes each time. Then add the secondary antibody and incubate for 1 hour; (5) Place the PVDF membrane on the stage of the developer, absorb excess water with filter paper, add an appropriate amount of developer, and detect protein bands.

[0054] 2.7 Synthesis of dendritic mesoporous silica / Fe3O4-KLT nanoparticles First, dendritic mesoporous silica was synthesized by dissolving 0.96 g of CTATos and 0.17 g of TEA in 50 mL of deionized water and reacting at 80 ° C for 1 h. 1.95 mL of TEOS was quickly added to the reaction mixture and the reaction was continued at 80 ° C for 2 h. After the reaction, the precipitate was collected by centrifugation and washed three times with deionized water and ethanol. The collected material was dispersed in 100 mL of ethanol solution containing 15 mL of HCl and reacted at 70 ° C for 24 h. After the reaction, the precipitate was collected by centrifugation, washed three times with ethanol, and dried at 60 ° C to obtain dendritic mesoporous silica (DMSN).

[0055] Next, dendritic mesoporous Fe3O4 nanoparticles were synthesized. The iron-oleate complex was synthesized and then pyrolyzed to produce uniform ultrasmall iron oxide nanoparticles (Fe3O4NPs) with an average diameter of 4nm. The synthesis process started with the dispersion of FeCl3•6H2O (5.4g) and sodium oleate (18.266g) in a solvent mixture consisting of ethanol (40mL), deionized water (30mL) and n-hexane (70mL). The mixture was heated to 70℃ and maintained for 4h. Subsequently, the upper layer of the solution was partially removed, washed and evaporated to obtain a waxy solid iron-oleate complex. Next, 4.5g of the iron-oleate complex and 8.05g of oleyl alcohol were dissolved in 25g of diphenyl ether. Under an inert atmosphere, the solution was heated to 200℃ at a rate of 10℃ / min. After maintaining the temperature for 30min, it was quickly cooled to 25℃ and then 50mL of acetone was added. Fe3O4 nanoparticles were obtained by centrifugation. Finally, DMSN / Fe3O4 nanoparticles were synthesized by stirring the Fe3O4 nanoparticle mixture for 12 h and subsequently centrifuged.

[0056] DMSN / Fe3O4 nanoparticles were mixed with Kanglaite and stirred overnight. After centrifugation to remove the supernatant, the precipitate was redispersed to obtain DMSN / Fe3O4-Kanglaite nanoparticles (abbreviated as D / FK NPs).

[0057] 2.8 Calculation of the loading amount of Kanglaite in DMSN / Fe3O4-Kanglaite nanocomposites Kanglaite has a maximum absorbance at 200nm. Accurately prepare Kanglaite solutions with concentrations of 0, 10, 14, 17, 20, and 25mg / mL, measure their absorbance at 200nm using a UV spectrophotometer, draw a standard curve, and refer to Fig.13 A known amount of D / FK NPs was then taken and the particle structure was destroyed to completely release Kanglaite. After centrifugation to remove the remaining nanoparticle fragments, the absorbance of the solution was measured at 200 nm and the concentration of Kanglaite was determined using the standard curve.

[0058] 2.9 Release kinetics of KLT from DMSN / Fe3O4-KLT nanocomposites To simulate the in vivo environment, D / FK NPs were dispersed in a release medium containing 10% serum and incubated at 37°C with gentle shaking. Every 2 h, the nanoparticles were separated from the supernatant by high-speed centrifugation. The amount of Kanglaite released in the supernatant was measured at 200 nm using a UV-visible spectrophotometer and quantified according to the previously established standard curve. The concentration of Kanglaite in the supernatant was compared with the initial total drug loading in the nanoparticles to calculate the percentage of drug released. This process was repeated at multiple time points to generate a release kinetic curve for Kanglaite.

[0059] 2.10 Nude mouse xenograft tumor model Five-week-old BALB / c nude mice were acclimated for one week before the experiment. 6 NCI-H1703 cells suspended in 200 μL PBS containing 50% Matrigel (C0387, Beyotime) were injected subcutaneously into the left abdomen of mice. The tumor grew to approximately 70 mm 3 When the size (volume V = length × width × width / 2) was measured, the mice were randomly divided into three groups: control group, Kanglaite group and Kanglaite / Fe3O4 nanoparticles group, with 8 mice in each group. Each mouse was injected with solvent control, 10mg / kg Kanglaite or Kanglaite / Fe3O4 nanoparticles through the tail vein once every three days for a total of 51 days. Tumor growth was monitored every three days. At the end of the experiment, the mice were euthanized, the tumors were removed, weighed and photographed for record.

[0060] 3. Experimental Results and Analysis 3.1 Iron ions significantly enhance the killing effect of Kanglaite on NCL-H1703 cells NCL-H1703 cells were treated with Kanglaite (2 mg / mL) for 24, 48 or 72 h, and the cell survival rate was detected using CCK-8. The results are shown in Table 1. Figure 1 .Depend on Figure 1 It can be seen that Kanglaite slightly induced the death of NCL-H1703 cells, with a time-dependent effect.

[0061] The effect of Kanglaite on NCL-H1703 cells was further confirmed by flow cytometric analysis of Annexin V / PI staining at multiple time points. Figure 2 .

[0062] Ferroptosis is an iron-dependent programmed cell death, driven by the accumulation of lipid peroxides and oxidative stress. Polyunsaturated fatty acids (PUFAs) are key substrates for lipid peroxidation, and Kanglaite is rich in PUFAs, suggesting that it may be involved in the iron-mediated cell death process. Therefore, this protocol considers adding iron to enhance the effect of Kanglaite.

[0063] In this embodiment, FeCl3 is used as Fe 3+ Donor, treated NCL-H1703 cells with different concentrations of FeCl3 and / or Kanglaite (2 mg / mL) for 72 h, and detected the cell survival rate by CCK-8. The results were referenced to Figure 3 .

[0064] Depend on Figure 3 It can be seen that due to Fe 3+The addition of FeCl3 significantly enhanced the cytotoxicity of Kanglaite, and the higher the concentration of FeCl3, the more obvious the enhancement effect on Kanglaite. The combination of 100μM FeCl3 and Kanglaite was further tested by Annexin V / PI flow cytometry analysis. The results are shown in Table 2. Figure 4 .Depend on Figure 4 It can be seen that the combined effect of Kanglaite and iron preparations is far better than using Kanglaite alone.

[0065] Further, in this embodiment, Fe 3+ The effects of using donor FeCl3, Fe3O4 and Fe3O4 nanoparticles in combination with Kanglaite were tested. The results are shown in Table 2. Figure 5 .

[0066] Depend on Figure 5 It can be seen that FeCl3, Fe3O4 and Fe3O4 nanoparticles all have the effect of significantly enhancing Kanglaite, which shows that both inorganic and organic iron agents can be used as combined reagents for Kanglaite.

[0067] 3.2 Iron supplementation plus Kanglaite significantly induced ferroptosis in NCL-H1703 cells To further evaluate the 3+ The combined effect on the ferroptosis pathway of NCL-H1703 cells. In this example, some classic key markers of ferroptosis were detected. The core events of ferroptosis include the generation of reactive oxygen species (ROS), the decrease in GPX4 expression, and the increase in lipid peroxidation. Figure 6-9 ,in: Figure 6 It is Kanglait and Fe 3+ The results of the combined use on ROS in NCL-H1703 cells; Figure 7 This is the result diagram of the effect on GPX4 protein level in NCL-H1703 cells; Figure 8 This is the result diagram of the effect on lipid peroxidation of NCL-H1703 cells; Fig. 9 The addition of ferroptosis inhibitor Fer-1 has an effect on the 3+ Graph showing the impact of combined use.

[0068] Depend on Figure 6 It can be seen that Kanglaite and Fe 3+ The combination resulted in an increase in ROS production in NCL-H1703 cells. Figure 7 It can be seen that the addition of Kanglaite and Fe 3+ After the combination of reagents, the GPX4 protein level in NCL-H1703 cells decreased significantly. Figure 8 It can be seen that Kanglaite and Fe 3+ The combination of agents resulted in increased lipid peroxidation in NCL-H1703 cells. Fig. 9It can be seen that the ferroptosis inhibitor Fer-1 effectively reversed the effects of Kanglaite and Fe 3+ The above investigation results show that Kanglaite and Fe 3+ Significantly induced ferroptosis in NCL-H1703 cells.

[0069] 3.3 Synthesis and characterization of dendritic mesoporous silica / Fe3O4-Kanglaite nanomedicine The present invention proposes a nano drug delivery system that can more effectively utilize the synergistic effect of Kanglaite and iron ions, providing a basis for the sustained release and controlled release of Kanglaite combined with iron agents.

[0070] The specific operation method is: in Kanglaite and iron (Fe 3+ In this embodiment, dendritic mesoporous silica is used as an example to examine the drug release performance.

[0071] refer to Fig.10 According to this process, dendritic mesoporous silica was prepared, and Fe3O4 nanoparticles with a diameter of 4 nm were selected as Fe 3+ First, dendritic mesoporous silica nanoparticles (DMSNs) were used as nanocarriers to load Fe3O4 nanoparticles to form DMSN / Fe3O4 (D / F) nanomedicines. Transmission electron microscopy (TEM) images showed the successful loading of Fe3O4 particles in DMSN / Fe3O4 nanoparticles. Fig.11 Elemental mapping analysis shows that Si, O and Fe elements are evenly distributed in the DMSN / Fe3O4 structure. Fig.12 Next, Kanglaite was loaded onto DMSN / Fe3O4 nanoparticles to form DMSN / Fe3O4-Kanglaite (D / FK) nanoparticles. The Kanglaite loading and encapsulation efficiency of D / FK nanoparticles were calculated by referring to the calculation method of Kanglaite loading in DMSN / Fe3O4-Kanglaite nanocomposites described in Item 2.8 above. Fig.14 .according to Fig.14 It can be seen that at this time, the Kanglaite drug loading and encapsulation efficiency of D / FK nanoparticles are 5.3% and 53%, respectively.

[0072] Subsequently, the release kinetics of Kanglaite was measured under simulated conditions containing 10% serum in this example. Fig.15 .Depend on Fig.15 It can be seen that the cumulative release of Kanglaite gradually increases with time, reaching 80% after 20 hours and tending to be stable. This shows that DMSN / Fe3O4-Kanglaite nanoparticles have a significant sustained release effect, and the drug can be released continuously and stably without being quickly exhausted in a short time.

[0073] 3.4 Dendritic mesoporous silica / Fe3O4-Kanglaite nanomedicine significantly improves its efficacy In this protocol, in order to further evaluate the ability of D / FK nanomedicine to induce ferroptosis, NCL-H1703 cells were divided into three groups: control group, 2 mg / mL Kanglaite + 100 μM Fe 3+ , D / FK nanoparticles (Kanglaite content 13.25μg / mL) were treated for 16h to investigate the ability of each group of reagents to induce cell ferroptosis. Fig.16 shown.

[0074] according to Fig.16 It can be seen that the effect of low-dose D / FK nanoparticles is significantly better than that of high-dose Kanglaite+Fe 3+ , indicating that nanoparticles have a higher efficiency in inducing ferroptosis.

[0075] 3.5 Dendritic mesoporous silica / Fe3O4-Kanglaite nanomedicine inhibits tumor growth in mice 7-week-old BALB / c nude mice were subcutaneously inoculated with 1×10 6 NCL-H1703 cells, wait until the tumor volume reaches about 70mm 3 Afterwards, they were randomly divided into three groups: empty control group, Kanglaite group and DMSN / Fe3O4-Kanglaite nanodrug group. The corresponding drugs were injected through the tail vein every 3 days for 51 days, and the tumor growth was monitored. The experimental mice in each group were injected with control group, Kanglaite and DMSN / Fe3O4-Kanglaite nanodrugs respectively for 51 days. Tumor location, tumor volume and tumor weight are shown in Figure 17-19 , tumor growth curve is shown in Fig. 20 .

[0076] according to Fig. 20 The results show that compared with the control group, Kanglaite has a certain inhibitory effect on lung squamous cell carcinoma. The DMSN / Fe3O4-Kanglaite nanomedicine group performed more significantly in tumor inhibition, showing a superior anti-tumor effect than other groups. Figure 17-19 The experimental results also show that the DMSN / Fe3O4-Kanglaite nanomedicine group is significantly better than the group using Kanglaite alone in inhibiting mouse tumor growth. This further proves that the nano drug delivery system can significantly enhance the anti-tumor activity of Kanglaite and show stronger therapeutic potential.

[0077] In summary, the drug of Kanglaite combined with iron can be used in the preparation of drugs for treating lung squamous cell carcinoma, and iron can enhance the effect of Kanglaite. Preferably, the drug of Kanglaite combined with iron can be added with pharmaceutically acceptable carriers and additives. Taking the carrier as an example, the drug of Kanglaite combined with iron also includes a dendritic mesoporous silica carrier, which can achieve sustained release and control of Kanglaite, expanding the application range of Kanglaite in tumor treatment.

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Kanglaite combined with iron preparation, characterized in that: A composition comprising Kanglaite and an iron agent, wherein the iron agent is Fe 3+ Donor.

2. The Kanglaite combined iron preparation according to claim 1, characterized in that: Fe 3+ The donor includes ferric iron salts or iron oxides or nanoparticles thereof.

3. The Kanglaite combined with iron preparation drug according to claim 1, characterized in that: Also included are pharmaceutically acceptable carriers.

4. The Kanglaite combined with iron preparation drug according to claim 3, characterized in that: The support includes dendritic mesoporous silica.

5. The Kanglaite combined with iron preparation drug according to claim 1, characterized in that: At least one pharmaceutically acceptable additive is also included.

6. The method for preparing the Kanglaite combined iron preparation drug according to claim 5, characterized in that: The steps include: selecting dendritic mesoporous silica nanoparticles as carriers, loading Fe 3+ After the donor is successfully loaded, Kanglaite is added to form DMSN / Fe 3+ -Kanglaite nanomedicine.

7. The method for preparing the Kanglaite combined with iron preparation according to claim 6, characterized in that: Fe 3+ The donor is Fe3O4 nanoparticles with a diameter of 4 nm.

8. Use of the Kanglaite combined with iron agent as claimed in any one of claims 1 to 7 in the preparation of a drug for treating lung squamous cell carcinoma.

9. The use according to claim 8, characterized in that: The Kanglaite combined with iron agent is used in the preparation of a drug for inhibiting the proliferation and migration of cancer cells.

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