Synergistic nano chemotherapy preparation for treating pancreatic cancer and preparation method thereof

Abstract

The invention relates to the technical field of medicines, and provides a synergetic nano chemotherapy preparation for treating pancreatic cancer and a preparation method of the synergetic nano chemotherapy preparation. According to the synergistic nano chemotherapy preparation, an RGD peptide modified lipid-mesoporous silica nano delivery system is used for simultaneously encapsulating a ferroptosis inducer Erastatin and a chemotherapy drug gemcitabine hydrochloride hcGEM. The nano synergistic treatment system not only can actively deliver the drug to the pancreatic tumor tissue with high expression of the integrin receptor in a targeted manner through the surface-modified RGD peptide, but also can realize drug synergistic treatment, and improves the killing effect of the drug on pancreatic cancer cells while reducing the toxic and side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to drugs for treating pancreatic cancer, and specifically to a synergistic nano-chemotherapeutic preparation for treating pancreatic cancer and a preparation method thereof. Background Art

[0002] Pancreatic cancer is a malignant tumor with a very high mortality rate. Due to its difficult early diagnosis, strong invasiveness, and distant metastasis, the 5-year survival rate of patients is less than 10%. Currently, the clinical efficacy of single-drug or combined chemotherapy regimens based on gemcitabine hydrochloride is poor; new cancer therapies, such as immune checkpoint inhibitor therapy and gene therapy, are basically ineffective for pancreatic cancer. Therefore, there is an urgent need to explore new strategies and methods for the treatment of pancreatic cancer.

[0003] Ferroptosis is a form of cell death that is different from apoptosis, pyroptosis, and necrosis, and can significantly inhibit pancreatic cancer cells with KRAS mutations. Ferroptosis can reverse the resistance of pancreatic cancer to traditional apoptotic chemotherapy drugs, and its combination with traditional chemotherapy can produce a significant synergistic inhibitory effect. Therefore, the combined treatment strategy based on ferroptosis-chemotherapy provides a new treatment idea for pancreatic cancer that is not well treated by traditional therapies.

[0004] Lipid-coated mesoporous silica nanoparticles (Lip-MSN) are nanoparticles formed by coating lipid membranes on the surface of mesoporous silica. As a combined drug delivery carrier, Lip-MSN not only has the advantages of mesoporous silica and liposomes, but also overcomes the disadvantages of both: the inner core mesoporous silica can serve as an internal loading area to accommodate drug molecules and provide a supporting skeleton for the overall structure to stabilize the outer lipid membrane; the outer lipid membrane can serve as a loading area for fat-soluble drugs and achieve controlled release of the internally loaded drugs by coating on the surface of mesoporous silica, which not only improves the biocompatibility and colloidal stability of mesoporous silica, but is also more suitable for functional modification of targeted molecules or stimuli-responsive materials.

[0005] RGD peptide can specifically bind to integrin αvβ3 and αvβ5 receptors, and the surface of pancreatic tumor cells (such as PANC-1 cells, etc.) and the overexpressed fibrinogen, plasminogen, laminin, etc. in tumor stroma all contain integrin αvβ3 and αvβ5 receptors. Therefore, RGD peptide can be used as an important ligand for targeting pancreatic cancer. Summary of the invention

[0006] Based on the ferroptosis combined with chemotherapy treatment strategy for pancreatic cancer, the present invention constructs a pancreatic cancer targeted nano-delivery system integrating ferroptosis inducers and chemotherapeutic drugs, and prepares an RGD peptide-modified lipid-mesoporous silica nano-delivery system to simultaneously encapsulate Erastin (Era), a ferroptosis inducer with synergistic efficacy, and gemcitabine hydrochloride (hcGEM), a chemotherapeutic drug, while enhancing the synergistic anti-tumor effect, reducing the drug dosage and reducing the systemic toxicity of the drug. The nano-synergistic treatment system can not only actively deliver drugs to pancreatic tumor tissues with high expression of integrin receptors through surface-modified RGD peptides, but also realize drug synergistic treatment, while reducing toxic side effects and improving the killing effect of drugs on pancreatic cancer cells.

[0007] The research process of the present invention is summarized as follows: first, Erastin, a ferroptosis inducer with the best synergistic anticancer effect with gemcitabine hydrochloride, was screened out through cell experiments, the synergistic ratio of the two drugs was established, and pH-sensitive lipid-mesoporous silica nanoparticles (hcGEM / Era RGD-Lip-MSN) co-loaded with hcGEM and Era were prepared; then the formulation process of hcGEM / Era RGD-Lip-MSN was optimized, and then the anti-tumor effect of hcGEM / Era RGD-Lip-MSN was evaluated in vitro and in vivo. The details are as follows:

[0008] In the early stage, the present invention investigated the synergistic inhibitory effect of different ferroptosis inducers (Erastin, SASP (sulfasalazine), ART (artesunate)) combined with hcGEM on human pancreatic cancer cell line PANC-1 cells and gemcitabine-resistant PANC-1 cells at the cellular level, and found that the hcGEM-Era4:1 group could synergistically inhibit PANC-1 cells and PANC-1 / hcGEM cells at all concentrations. Therefore, the subsequent prescription was used to construct hcGEM / Era-RGD-lip-MSN with the goal of achieving a drug loading of hcGEM and Era in a molar ratio of 4:1.

[0009] Secondly, a series of MSNs and hcGEM / EraRGDLip-MSNs were prepared by sol-gel method and thin film hydration method, respectively. The optimal synthesis conditions for preparing MSNs with a minimum average particle size of about 116 nm and the preparation formula with the best synergistic inhibition molar ratio of hcGEM and Era of 4:1 were screened out through formula optimization. The average particle size of hcGEM / Era RGD-Lip-MSN synthesized by the optimal formula was 140.57±2.93nm, PDI was 0.22±0.13, and Zeta potential was -58.80±0.65mV; the encapsulation efficiency and drug loading of hcGEM were 37.45% and 11.05% respectively; the encapsulation efficiency and drug loading of Era were 78.96% and 4.89% respectively, and the molar ratio of hcGEM to Era was 4.13:1. hcGEM / Era RGD-Lip-MSN releases slowly under neutral release conditions, but shows a "burst release" effect at the initial stage of release under acidic release conditions. At 48 hours, the cumulative release rates of hcGEM and Era were 86.46% and 79.24%, respectively. The overall release characteristics showed pH-responsive release characteristics, and can be basically released according to the molar ratio of hcGEM to Era of 4:1. The prepared hcGEM / Era RGD-Lip-MSN meets the expected requirements and lays a good foundation for subsequent in vitro and in vivo experimental studies.

[0010] Third, the killing effect of hcGEM / Era RGD-Lip-MSN on tumor cells was investigated. The results showed that blank RGD-Lip-MSN had almost no toxicity to PANC-1 cells; the cell inhibition rate of the hcGEM / Era RGD-Lip-MSN treatment group was similar to that of the hcGEM+Era treatment group (P>0.05). Compared with the hcGEM / Era Lip-MSN treatment group, hcGEM / Era RGD-Lip-MSN can increase the intracellular ROS content, reduce the intracellular GSH content, and enhance the ferroptosis effect. The results of in vitro pharmacodynamic studies suggest that hcGEM / Era RGD-Lip-MSN can effectively deliver hcGEM and Era into tumor cells, enhance the proliferation inhibition of tumor cells by the two drugs, reduce the intracellular GSH content, increase the intracellular ROS content, and induce cell ferroptosis.

[0011] Fourth, in vivo pharmacodynamic studies were conducted. A nude mouse model of subcutaneous pancreatic cancer transplanted tumors was established by tumor tissue block transplantation, and the in vivo antitumor efficacy and systemic toxicity of different drug preparations were evaluated by analyzing the tumor volume, weight, and body weight changes of nude mice. The results showed that the combined use of hcGEM and Era can significantly improve the tumor inhibition effect. The co-delivery of hcGEM and Era by hcGEM / Era RGD-Lip-MSN can improve the tumor inhibition effect of the two drugs and reduce the toxic side effects of the drugs. In vivo pharmacodynamic studies suggest that the co-delivery of hcGEM and Era by hcGEM / Era RGD-LipMSN can reduce the toxic side effects of hcGEM while improving the tumor inhibition effect of the drug.

[0012] Based on the above research, the technical solution to be protected by the present invention is as follows:

[0013] In the first aspect of the present invention, a synergistic nano-chemotherapeutic preparation for the treatment of pancreatic cancer is provided, wherein the ferroptosis inducer Erastin and the chemotherapeutic drug gemcitabine hydrochloride hcGEM are simultaneously encapsulated by an RGD peptide-modified lipid-mesoporous silica nano-delivery system, wherein the mass ratio between hcGEM and the ferroptosis inducer Erastin is 4:1.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned synergistic nano-chemotherapeutic preparation for treating pancreatic cancer, comprising the following steps:

[0015] A. Preparation of Mesoporous Silica MSN

[0016] The template agent CTAB is added to a reaction container filled with deionized water at a molar volume ratio of 1:160, and then a strong base is added in excess at a molar ratio of 3:7, and water bath ultrasound is performed until the solution is clarified, and the reaction container is heated in an oil bath at 80°C under magnetic stirring; a silicon source with a molar amount 5 times that of CTAB is slowly added to the reaction container at a certain speed, and after constant temperature stirring for a certain period of time, centrifuged at 4000g under room temperature, the precipitate is washed with ethanol to remove impurities, and then the precipitate is resuspended and dispersed with a small amount of deionized water, frozen with liquid nitrogen and freeze-dried into a fine powder, and then the freeze-dried powder is added to a 550°C muffle furnace for calcination to remove CTAB, so as to obtain mesoporous silica powder.

[0017] Preferably, the strong base is selected from sodium hydroxide or potassium hydroxide;

[0018] During the oil bath heating process, the magnetic stirring speed was 600 rpm;

[0019] The silicon source is selected from TEOS, which is slowly added to the reaction container at 1 mL / min, stirred at constant temperature for 2 hours, centrifuged at 4000g for 30 minutes at room temperature, the supernatant is discarded, the precipitate is washed with ethanol and centrifuged three times; then the precipitate is resuspended and dispersed with a small amount of deionized water, frozen with liquid nitrogen and freeze-dried into fine powder. The freeze-dried powder is then added to a muffle furnace at 550°C and calcined for 6 hours to remove CTAB, and finally mesoporous silica powder is obtained.

[0020] B. Preparation of hcGEM-MSNs loaded with mesoporous silica

[0021] After hcGEM aqueous solution was vortex dispersed, MSN of the same mass as hcGEM was added, and after repeated water bath sonication for several times, it was magnetically stirred at room temperature for at least 20 h and then packaged for the preparation of drug-loaded lipid mesoporous silica on the same day.

[0022] Preferably, in the hcGEM aqueous solution, the mass volume ratio of hcGEM to deionized water is 20:1; water bath sonication is repeated 3 times, each time for 30 minutes, and then magnetic stirring is performed at room temperature for 24 hours.

[0023] C. Preparation of RGD-targeted pH-sensitive lipid mesoporous silica loaded with hcGEM and Erastin

[0024] The lipid materials Lip and Erastin composed of specific materials are mixed with dichloromethane and then ultrasonically dispersed; then the hcGEM-MSN suspension obtained in step B is centrifuged and the precipitate is added and ultrasonically dispersed again; after the mixed solution is fully reduced-pressure rotary evaporation, a ddH2O solution preheated to 45°C is added, and hydrated at normal pressure at the same temperature, and then internally ultrasonicated, and centrifuged at 10000rpm after ultrasonication. After the precipitate is washed and centrifuged with ddH2O for multiple times, the supernatant of each centrifugation is collected and mixed to obtain the product hcGEM / Era RGD-Lip-MSN, which is stored in a refrigerator at 4°C.

[0025] Among them, the feeding mass ratio of MSN to lipid material Lip is 10:30; the feeding ratio of Erastin to hcGEM in hcGEM-MSN suspension is 1:4.

[0026] Preferably, the lipid material consists of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), cholesterol (CHOL), cholesterol succinate (CHEMS), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG) and RGD-modified distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEGRGD) in a molar ratio of 16:8:8:2:1:0.1.

[0027] Among them, DOPE has the characteristic of differential release in the environment of pH 6.0-7.4. CHEMS is a good pH-responsive material. The four-membered ring steroid skeleton in its structure is prone to severe deformation when the pH decreases, so it has pH-sensitive properties.

[0028] Preferably, the ultrasonic dispersion time is 5 min for both times;

[0029] The mass volume ratio of lipid material Lip to dichloromethane is 20 mg:1 ml;

[0030] The centrifugation conditions for the hcGEM-MSN suspension were 10000 rpm for 10 min;

[0031] The conditions for fully vacuum rotary evaporating the mixed solution are as follows: the reaction container is vacuum rotary evaporated on a rotary evaporator at 45°C and 60 rpm to obtain a relatively uniform film and then continue vacuum rotary evaporation to completely remove the solvent;

[0032] The hydration time at normal pressure was 60 min; the internal ultrasound conditions were 15 / 15 s, 32.5 W, 20 min.

[0033] Functions and Effects of the Invention

[0034] The present invention successfully prepared RGD peptide-modified pH-sensitive lipid-mesoporous silica nanoparticles co-loaded with hcGEM and Era at an optimal synergistic inhibition molar ratio of 4:1. In vivo and in vitro pharmacodynamic experiments confirmed that hcGEM / Era RGD-Lip-MSN can effectively deliver hcGEM and Era into tumor cells, thereby improving the synergistic killing effect of the two drugs on tumor cells. While enhancing the synergistic anti-tumor effect, the drug dosage is reduced and the systemic toxicity of the drug is reduced. The nano-synergistic treatment system can not only actively deliver drugs to pancreatic tumor tissues with high expression of integrin receptors through surface-modified RGD peptides, but also achieve drug synergistic treatment, thereby reducing toxic side effects while improving the killing effect of drugs on pancreatic cancer cells. Therefore, the present invention provides a new basis for the treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The particle size and distribution of nanoparticles are shown, where A, MSN; B, hcGEM / Era RGD-Lip-MSN.

[0036] Figure 2The effects of different drug treatment groups on PANC-1 cell survival are shown; A, the effect of blank RGD-Lip-MSN on cell survival; B, the effects of four preparations, hcGEM, hcGEM+Era, hcGEM RGD-Lip-MSN, and hcGEM / Era RGD-Lip-MSN, on cell survival; *P<0.05.

[0037] Figure 3 The intracellular ROS levels of PANC-1 cells in different drug treatment groups are shown: A, flow cytometry of intracellular ROS levels in different drug treatment groups; B, bar chart of intracellular ROS levels in different drug treatment groups; ns P>0.05, *P<0.05, **P<0.01.

[0038] Figure 4 The intracellular GSH levels of PANC-1 cells in different drug treatment groups are shown; ns P>0.05,*P<0.05,**P<0.01.

[0039] Figure 5 Flow cytometry and bar graphs showing cell survival in different drug treatment groups; *P<0.05, **P<0.01.

[0040] Figure 6 Shows the changes in tumor volume in nude mice after treatment with different preparations ns P>0.05, *P<0.05.

[0041] Figure 7 Shows the changes in tumors in different drug groups Among them, A. tumor images of different drug administration groups; B. tumor weights of different drug administration groups; ns P>0.05,*P<0.05,**P<0.01.

[0042] Figure 8 Shows the weight changes of tumor-bearing nude mice in different drug administration groups *P<0.05. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0044] In the early stage of this invention, the synergistic inhibitory effect of three ferroptosis inducers Erastin, SASP (sulfasalazine), ART (artesunate) combined with gemcitabine hydrochloride on human pancreatic cancer cell line PANC-1 cells and gemcitabine-resistant PANC-1 cells was investigated at the cellular level, and the ferroptosis inducer Erastin with synergistic inhibitory effect with gemcitabine hydrochloride was screened out by calculating the synergistic index and the synergistic ratio was: hcGEM-Era 4:1. On this basis, we used lip-MSN as a carrier to construct a pancreatic cancer-targeted, pH-sensitive synergistic nanotherapy system hcGEM / Erastin-RGD-lip-MSN modified with RGD peptide and co-loaded with hcGEM and Erastin.

[0045] 1. Preparation of hcGEM / Erastin-RGD-lip-MSN

[0046] 1. Preparation of Mesoporous Silica (MSN)

[0047] Weigh 3mmol of CTAB (template) and add it to a round-bottom flask containing 480mL of deionized water, add 7mmol of NaOH, and ultrasonicate in a water bath until the solution is clear. After the silicone oil in the collector constant temperature magnetic stirrer is heated to 80°C, the round-bottom flask is heated in an oil bath and magnetically stirred at 600rpm. 15mmol of TEOS (silicon source) is added dropwise to the flask at 1mL / min. After constant temperature stirring for 2h, centrifuge at 4000g for 30min at room temperature. Discard the supernatant, wash the precipitate with ethanol and centrifuge 3 times. Resuspend the precipitate with a small amount of deionized water, freeze it with liquid nitrogen and freeze it into a fine powder. Then add the freeze-dried powder to a 550°C muffle furnace and calcine it for 6h to remove CTAB, and finally obtain mesoporous silica powder.

[0048] 2. Preparation of hcGEM-loaded Mesoporous Silica (hcGEM-MSN)

[0049] The hcGEM-loaded mesoporous silica (hcGEM-MSN) was prepared by solution impregnation method. Weigh 100 mg hcGEM in a centrifuge tube, add 5 mL ddH2O and vortex to disperse. Add 100 mg MSN and repeat water bath sonication 3 times, 30 min each time. After magnetic stirring at room temperature for 24 h, evenly divide into 10 EP tubes, each with 0.5 mL suspension (containing 10 mg hcGEM and 10 mg MSN), which is used to prepare drug-loaded lipid mesoporous silica on the same day.

[0050] 3. Preparation of RGD-targeted pH-sensitive lipid mesoporous silica loaded with hcGEM and Erastin

[0051] (hcGEM / Erastin-RGD-lip-MSN)

[0052] (1) Lipid materials

[0053] Dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), cholesterol (CHOL), cholesterol succinate monoester (CHEMS), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG) and RGD-modified distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEGRGD) were selected as lipid membrane materials. Among them, DOPE has the characteristics of differential release in the environment of pH 6.0-7.4. CHEMS is a good pH-responsive material. The four-membered ring steroid skeleton in its structure is prone to severe deformation when the pH is reduced, so it has pH-sensitive properties.

[0054] (2) Preparation method

[0055] Keep the mass ratio of MSN to Lip at 10:30, and the amount of Era is 2.1 mg. Weigh 12.51 mg DOPE, 6.63 mg DSPC, 3.24 mg CHOL, 4.08 mg CHEMS, 3.24 mg DSPE-MPEG, 0.3 mg DSPE-PEG-RGD (a total of 30 mg lipid material), and 2.1 mg Era in a 25 mL eggplant-shaped bottle. Add 1.5 mL of dichloromethane and disperse by ultrasonication for 5 min. Centrifuge 0.5 mL of the suspension hcGEM-MSN (containing 10 mg hcGEM and 10 mg MSN) prepared in step 2 at 10000 rpm for 10 min. Collect the supernatant, add the precipitate to the above-mentioned eggplant-shaped bottle, and disperse by ultrasonication for 5 min. Place the eggplant-shaped bottle on a rotary evaporator at 45 ° C, 60 rpm, and evaporate under reduced pressure for 30 min to obtain a relatively uniform film and continue to evaporate under reduced pressure for 30 min to completely remove the solvent. Add 2 mL of ddH2O solution preheated to 45°C and hydrate at 45°C for 60 min. After hydration, perform internal ultrasound for 20 min (15 / 15s, 32.5W). After ultrasound, centrifuge at 10000 rpm for 10 min, wash and centrifuge 3 times with 0.2 mL of ddH2O, collect and mix the supernatant of each centrifugation, and store the product hcGEM / Era RGD-Lip-MSN in a 4°C refrigerator.

[0056] Similarly, hcGEM / Era Lip-MSNs co-loaded with hcGEM and Era (hcGEM / Era Lip-MSNs), hcGEM-loaded RGDLip-MSNs (hcGEM RGD-Lip-MSNs), and blank RGD-Lip-MSNs were prepared. When preparing hcGEM / Era Lip-MSNs, 0.3 mg DSPE-PEG-RGD was replaced with 0.3 mg DSPE-MPEG, and the other conditions remained unchanged; when preparing hcGEM RGDLip-MSNs, Era was not added; when preparing blank RGD-Lip-MSNs, blank MSNs were used instead of hcGEM MSNs and Era was not added.

[0057] 2. Characterization of HcGEM / Erastin-RGD-lip-MSN

[0058] 1. Particle size: The particle size and PDI of each hcGEM / Era RGD-Lip-MSN product were measured using a Malvern laser particle size analyzer. The results are shown in Figure 1 The average particle sizes of MSN and hcGEM / Era RGD-Lip-MSN were 116.47±0.91nm and 140.57±2.93nm, respectively.

[0059] 2. Encapsulation efficiency and drug loading: Centrifuge the sample, take 1 mL of the supernatant, add 1 mL of methanol and sonicate for 20 min, dilute 25 times with ddH2O, filter through a 0.22 μm membrane, and determine the hcGEM content in the supernatant by HPLC; take another 1 mL of the supernatant, dilute 5 times with 50% methanol aqueous solution, sonicate for 20 min, filter through a 0.22 μm membrane, and determine the Era content in the supernatant by HPLC. The encapsulation efficiency (EE) and drug loading (DL) of hcGEM and Era were calculated by the indirect method.

[0060] EE(%)=(M0-M1) / M0×100%DL(%)=(M0-M1) / M2

[0061] Where M0 is the total mass of hcGEM (or Era) added when preparing hcGEM / Era RGD-Lip-MSN; M1 is the mass of free hcGEM (or Era) in the mixed supernatant; M2 is the total mass of the prepared hcGEM / Era RGD-Lip-MSN after reduced pressure drying. Under this prescription, the encapsulation efficiency and drug loading of hcGEM were 37.45% and 11.05%, respectively; the results showed that the encapsulation efficiency and drug loading of Era were 78.96% and 4.89%, respectively, and the molar ratio of hcGEM to Era was 4.13:1.

[0062] 3. In vitro drug release: 1 mL of freshly prepared hcGEM / Era RGD-Lip-MSN was measured and placed in a dialysis bag (molecular weight cutoff 14000 Da, pre-boiled in PBS and stored at 4°C). The dialysis bags were placed in 45 mL of PBS (pH 7.4) or PBS (pH 6.5) containing 1% SDS. Constant temperature oscillation was performed at 37°C and 100 rpm for 48 h. 1.5 mL of release medium was taken out at each time point (0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 h), and the same amount of fresh release medium was added. The sample was centrifuged at 10000 g for 5 min, all the supernatant was carefully removed, and 2 mL of new release medium was added at the same time. The content of hcGEM and Erastin in the release medium was detected by high performance liquid chromatography.

[0063] 3. Results of the synergistic efficacy study at the cell level

[0064] (1) Cytotoxicity assay

[0065] PANC-1 cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), and the cell culture dishes were placed in a constant temperature and humidity incubator at 37°C and 5% CO2. The CCK-8 method was used to detect the effects of five preparations (including blank RGD-Lip-MSN, hcGEM, hcGEM+Era, hcGEM-RGD-Lip-MSN and hcGEM / Era-RGD-LipMSN) on the proliferation of PANC-1 cells. PANC-1 cells in the logarithmic growth phase were inoculated in a 96-well plate with a seeding density of 5000 cells per well. After the cells adhered, the supernatant was discarded, and 100 μL of complete culture medium containing blank RGD-Lip-MSN, hcGEM, hcGEM+Era, hcGEM RGD-Lip-MSN and hcGEM / Era RGDLip-MSN was added to each of the three replicate wells, and each replicate well of the blank control group and the untreated group was set up. Among them, the concentration gradient of blank RGDLip-MSN was 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 mg / mL; the concentration gradient of hcGEM was 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 μmol / L, and the combined Era concentration was 1 / 4 of the hcGEM concentration. After the drug and cells were co-cultured for 48 hours, 100 μL of DMEM culture medium containing 10% CCK-8 was added to each well. After culturing at constant temperature for 2 h, the OD value of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument.

[0066] from Figure 2It can be seen that blank RGD-Lip-MSN has almost no toxicity to PANC-1 cells, and the survival rate of PANC-1 cells after administration of 16 mg / mL RGDLip-MSN is still >85%. Based on the hcGEM concentration, the IC50 of the hcGEM / Era RGD-Lip-MSN treatment group, hcGEM RGD-Lip-MSN treatment group, hcGEM+Era treatment group and hcGEM treatment group were 0.4302, 1.635, 0.3159 and 1.327 μmol / L, respectively. The cell inhibition rate of the hcGEM / Era RGD-Lip-MSN treatment group was significantly higher than that of the hcGEM treatment group and the hcGEM RGD-LipMSN treatment group (P<0.05), while compared with the hcGEM+Era treatment group, the cell inhibition rates of the two were close. These results showed that hcGEM+Erastin had a synergistic inhibitory effect on PANC-1 cells, and the inhibitory effect was maintained when RGD-Lip-MSNs were co-loaded.

[0067] (2) ROS levels

[0068] The level of ROS in tumor cells can reflect the intracellular ferroptosis to a certain extent. The intracellular ROS content was determined using the oxidation-sensitive fluorescent probe DCFH-DA (Shanghai Bio-Tian Biotechnology Co., Ltd., China) according to the manufacturer's instructions. PANC-1 cells were plated at 1×10 6 The cells were inoculated into two 6-well plates at a density of 100 μg / mL and cultured in a cell culture incubator at 37°C for 24 hours. After aspirating the culture medium, 2 mL of DMEM complete culture medium containing 0.02% DMSO, hcGEM, Era, hcGEM+Era, hcGEM / Era-Lip-MSN, hcGEM / Era-RGD-Lip-MSN, and hcGEM / Era-RGD-Lip-MSN+Ferrostatin-1 (Fer-1) were added. The cells were cultured in the drug-containing culture medium for 48 hours, and then 0.1% Rosup solution was added to the positive control well for 30 minutes. After digestion and centrifugation, 0.1% DCFH-DA was added to each EP tube and placed in a cell culture incubator for 25 minutes. After centrifugation and washing 3 times, the cells were taken for flow cytometry to detect ROS, and the Rosup group was used as the control to calculate the relative percentage of ROS in each group.

[0069] The DCFH-DA probe was used to detect the ROS level of cells in the drug-treated group. The non-fluorescent DCFH-DA probe can easily penetrate the cell membrane and enter the cell, and then be decomposed by esterases in the cytoplasm into DCFH that cannot penetrate the cell membrane. The reactive oxygen species in the cell can oxidize DCFH into DCF, causing the probe to change from a non-fluorescent state to a fluorescent state. The fluorescence intensity of intracellular DCF detected by flow cytometry can reflect the intracellular ROS level.

[0070] from Figure 3 As can be seen, both hcGEM and Erastin as single agents can increase the level of reactive oxygen species (ROS) in PANC-1 cells. When the two drugs were used in combination, the level of ROS in PANC-1 cells was significantly increased to a greater extent than when either drug was used alone, indicating that the two drugs have a synergistic effect in inducing ROS production in PANC-1 cells. When the two drugs were co-loaded into the nanocarrier, they still maintained the ability to induce ROS generation. In addition, RGD-modified hcGEM / Erastin-RGD-lip-MSN was also able to induce higher levels of ROS in PANC-1 cells. This may be related to the fact that RGD modification enhanced the uptake of nanocarriers by PANC-1 cells. The group of PANC-1 cells treated with hcGEM / Erastin-RGD-lip-MSN in combination with the ferroptosis inhibitor Fer-1 showed a significant reduction in intracellular reactive oxygen species (ROS). This finding suggests that the increased ROS levels observed in PANC-1 cells after hcGEM / Erastin-RGD-lip-MSN treatment are related to the ferroptosis mechanism.

[0071] (3) GSH level

[0072] Glutathione (GSH) plays a key role in cellular defense against oxidative stress and toxic compounds. In cancer cells, GSH levels are essential for DNA synthesis, growth, and resistance to a variety of drugs and radiation. Therefore, GSH is considered an important target for cancer therapy, and various therapeutic approaches based on GSH depletion in cancer cells have been proposed.

[0073] PANC-1 cells were plated at 1×10 6The cells were seeded at a density of 100 μg / mL into two 6-well plates and cultured in a 37°C cell culture incubator for 24 h, and then added to complete medium containing different treatments (0.02% DMSO, hcGEM, Era, hcGEM+Era, hcGEM / Era Lip-MSN, hcGEM / Era RGD-Lip-MSN, hcGEM / Era RGD-LipMSN+Ferrostatin-1). After 48 h of culture, PANC-1 cells were collected and the relative GSH and lipid peroxide concentrations were assessed using a lipid peroxide detection kit and a glutathione detection kit according to the manufacturer's instructions.

[0074] The results are as follows Figure 4 As shown in the figure, the increase in intracellular GSH content in the PANC-1 cells in the hcGEM treatment group is due to the fact that the ROS produced by PANC-1 cells induced by hcGEM activated the intracellular antioxidant mechanism, causing the negative feedback increase of intracellular GSH. Erastin is an inducer of ferroptosis and can significantly reduce the level of intracellular GSH. When hcGEM and Erastin were used in combination, the GSH content decreased significantly, which was lower than the level of Erastin and hcGEM single-drug groups, confirming that hcGEM and Erastin can synergistically inhibit GSH in PANC- cells. In the hcGEM / Eras-RGD-lip-MSN nanocarrier group, the intracellular GSH content was significantly lower than that of the hcGEM / Eras-lip-MSN nanocarrier group without RGD targeted modification, and was equivalent to the level of the hcGEM+Erastin group, indicating that the synergistic therapeutic effect of the two drugs can still be exerted by simultaneously encapsulating hcGEM and Erastin in the nanocarrier, and RGD targeted modification can significantly increase the absorption of nanocarriers by PANC-1 cells, making the level of GSH decrease more significantly. After adding the ferroptosis inhibitor Fer-1, the GSH content increased significantly, proving that the decrease in intracellular GSH content is closely related to the ferroptosis mechanism.

[0075] (4) Flow cytometry cell viability analysis

[0076] PANC-1 cells (1×10 6cells / well) were inoculated into 6-well plates and cultured for 24 hours. Subsequently, the cells were cultured with different treatment groups (DMSO as negative control, hcGEM, Era, hcGEM / Era-Lip-MSN, hcGEM / Era RGD-Lip-MSN, hcGEM / Era RGD-Lip-MSN+Fer-1) for 48 hours (the equivalent dose of hcGEM was 1μg / mL and Era was 0.25μg / mL). The treated cells were washed twice, trypsinized and centrifuged, and then resuspended in AnnexinV binding buffer. The cells were stained with Annexin V / FITC fluorescent dye for 5 minutes and detected by flow cytometry. Figure 5 As shown, the survival rate of the hcGEM / Era-RGD-Lip-MSN group was significantly lower than that of the hcGEM / Era-Lip-MSN group and the free single drug group (P<0.05 and P<0.01, respectively). This indicates that the RGD targeted modification synergistic nanodelivery system hcGEM / Era-RGD-Lip-MSN can enhance the inhibitory effect on the survival rate of PANC-1 cells. In addition, when hcGEM / Era-RGD-Lip-MSN was combined with the ferroptosis inhibitor Fer-1, the survival rate of cells was significantly improved (P<0.01), indicating that the inhibitory effect of hcGEM / EraRGD-Lip-MSN on the survival rate of PANC-1 cells is mediated by the ferroptosis mechanism.

[0077] IV. Investigation of synergistic efficacy in animal models

[0078] 1. Experimental methods:

[0079] To evaluate the antitumor effect of hcGEM / Era-RGD-Lip-MSN in vivo, nude mice were subcutaneously injected with Panc-1 cells (1×10 7 ) prepared a subcutaneous Panc-1 tumor model. When the tumor volume reached about 150mm 3 Afterwards, the mice were randomly divided into 7 groups (n=4): (1) saline group; (2) blank RGD-Lip-MSN group; (3) hcGEM group; (4) Era group; (5) hcGEM+Era group; (6) hcGEM / Era-Lip-MSN group; (7) hcGEM / Era-RGD-Lip-MSN group. Mice were treated once every 3 days with intravenous injection of 60 mg / kg hcGEM, 27.4 mg / kg Era, and equal amounts of hcGEM / Era-Lip-MSN and hcGEM / Era-RGD-Lip-MSN. The body weight and tumor volume of each animal were recorded every three days. The longest length (L) and width (S) of the tumor were measured with a vernier caliper according to the formula V=0.5×L×S 2The tumor volume was calculated. After 2 weeks of administration, the nude mice were killed by cervical dislocation, the experiment was terminated, and the tumor weight was measured.

[0080] 2. Experimental results:

[0081] (1) Subcutaneous tumor volume change curve

[0082] from Figure 6 It can be seen that the tumor volumes of different drug administration groups all showed a trend of continuous increase. Among them, the tumor volumes of the saline group and the blank vector RGD-Lip-MSN administration group increased most significantly over time, while the tumor volumes of the hcGEM / Era-RGD-Lip-MSN and hcGEM / Era-Lip-MSN administration groups did not increase much over time. The tumor volumes of the hcGEM / Era-RGD-Lip-MSN administration group and the hcGEM / Era-Lip-MSN administration group were significantly smaller than those of the hcGEM+Era administration group (P<0.05), indicating that hcGEM / Era-RGD-Lip-MSN and hcGEM / Era LipMSN had stronger antitumor activity than the free hcGEM and Era combination group. The tumor volume of the hcGEM+Era administration group was significantly smaller than that of the hcGEM administration group or the Era administration group (P<0.05), indicating that the combined use of hcGEM and Era can produce a stronger tumor inhibitory effect than single drugs. The tumor volume of the hcGEM / Era-RGD-Lip-MSN administration group was always slightly smaller than that of the hcGEM / Era-Lip-MSN administration group, indicating that the tumor inhibition effect of the hcGEM / Era RGD-Lip-MSN administration group was slightly better than that of the hcGEM / Era-Lip-MSN administration group.

[0083] (2) Tumor weight after administration

[0084] from Figure 7 It can be seen that the tumor volume and weight of the hcGEM+Era administration group were significantly smaller than those of the hcGEM administration group or the Era administration group (P<0.05), indicating that the combination of the two drugs can significantly improve the tumor inhibition effect; the tumor volume and weight of the hcGEM / Era-RGDLip-MSN administration group were significantly lower than those of the other groups except the hcGEM / Era-Lip-MSN administration group (P<0.05), indicating that the co-delivery of hcGEM and Era by hcGEM / Era-RGD-Lip-MSN can significantly improve the tumor inhibition effect of the two drugs. The tumor volume and weight of the hcGEM / Era-Lip-MSN administration group were significantly smaller than those of the hcGEM+Era administration group (P<0.05), indicating that hcGEM / Era-Lip-MSN can improve the tumor inhibition effect of hcGEM and Era.

[0085] (3) Weight changes of nude mice

[0086] Depend on Figure 8 It can be seen that during the 12-day treatment period, the body weight of nude mice in the hcGEM administration group and the hcGEM+Era administration group showed a trend of first increasing and then decreasing significantly, indicating that the toxic and side effects of hcGEM intravenous injection were relatively large. The body weight of nude mice in the hcGEM / Era Lip-MSN administration group and the hcGEM / Era RGD-Lip-MSN administration group showed a relatively slow upward trend, indicating that hcGEM / Era-RGD-Lip-MSN or hcGEM / Era Lip-MSN can effectively deliver hcGEM and Era, reduce the toxic and side effects of the two drugs, and increase the dosage safety range of the two drugs. The body weight of nude mice in the Era administration group showed a relatively slow upward trend, indicating that the systemic toxicity of Era intravenous injection was relatively small.

[0087] In summary, the present invention successfully prepared RGD peptide-modified pH-sensitive lipid-mesoporous silica nanoparticles co-loaded with hcGEM and Era with an optimal synergistic inhibition mass ratio of 4:1. In vivo and in vitro pharmacodynamic experiments confirmed that hcGEM / Era RGD-Lip-MSN can effectively deliver hcGEM and Era into tumor cells, thereby enhancing the synergistic killing effect of the two drugs on tumor cells.

[0088] The basic principles, main features and significant advantages of the present invention have been described in detail above. The purpose of the description in the embodiments and the specification is only to further explain the operating principle of the present invention. Without violating the core idea of ​​the present invention and the statutory protection scope, the present invention allows and covers all kinds of reasonable changes and improvements. These changes and improvements will be deemed to be within the scope of protection requested by the present invention. Specifically, the scope of protection of the present invention will be clearly and strictly defined by the attached claims and their equivalents with equal legal effect.

Claims

1. A synergistic nano-chemotherapeutic preparation for the treatment of pancreatic cancer, characterized in that: An RGD peptide-modified lipid-mesoporous silica nanodelivery system was used to simultaneously encapsulate the ferroptosis inducer Erastin and the chemotherapeutic drug gemcitabine hydrochloride hcGEM.

2. The synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 1, characterized in that: The mass ratio between hcGEM and ferroptosis inducer Erastin was 4:

1.

3. The method for preparing the synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 1 or 2, characterized in that: The steps include: A. Preparation of Mesoporous Silica MSN The template agent CTAB is added to a reaction container containing deionized water at a molar volume ratio of 1:160, and then a strong base is added in excess at a molar ratio of 3:7, and a water bath is ultrasonicated until the solution is clarified, and the reaction container is heated in an oil bath at 80°C under magnetic stirring; a silicon source with a molar amount 5 times that of CTAB is slowly added to the reaction container at a certain speed, and after constant temperature stirring for a certain period of time, centrifuged at 4000g under room temperature, the precipitate is washed with ethanol to remove impurities, and then the precipitate is resuspended and dispersed with a small amount of deionized water, frozen with liquid nitrogen, and then freeze-dried into a fine powder, and then the freeze-dried powder is added to a muffle furnace at 550°C for calcination to remove CTAB, thereby obtaining mesoporous silica powder; B. Preparation of hcGEM-MSNs loaded with mesoporous silica After hcGEM aqueous solution was vortexed and dispersed, MSN of the same mass as hcGEM was added, and after repeated water bath sonication, it was magnetically stirred at room temperature for at least 20 h, and then packaged and used to prepare drug-loaded lipid mesoporous silica on the same day; C. Preparation of RGD-targeted pH-sensitive lipid mesoporous silica loaded with hcGEM and Erastin The lipid materials Lip and Erastin composed of specific materials are mixed with dichloromethane and then ultrasonically dispersed; then the hcGEM-MSN suspension obtained in step B is centrifuged, the precipitate is taken out and added and ultrasonically dispersed again; After the mixed solution was fully evaporated under reduced pressure, ddH2O solution preheated to 45°C was added, and hydrated at normal pressure at the same temperature, followed by internal ultrasound. After ultrasound, centrifugation was performed at 10,000 rpm. The precipitate was washed with ddH2O and centrifuged several times. The supernatant of each centrifugation was collected and mixed to obtain the product hcGEM / EraRGD-Lip-MSN. Among them, the feeding mass ratio of MSN to lipid material Lip is 10:30; the feeding ratio of Erastin to hcGEM in hcGEM-MSN suspension is 1:

4.

4. The method for preparing the synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 3, characterized in that: In step A, the strong base is selected from sodium hydroxide or potassium hydroxide; During the oil bath heating process, the magnetic stirring speed was 600 rpm; The silicon source is selected from TEOS, which is slowly added to the reaction container at 1 mL / min, stirred at constant temperature for 2 hours, centrifuged at 4000g for 30 minutes at room temperature, the supernatant is discarded, the precipitate is washed with ethanol and centrifuged three times; then the precipitate is resuspended and dispersed with a small amount of deionized water, frozen with liquid nitrogen and freeze-dried into fine powder. The freeze-dried powder is then added to a muffle furnace at 550°C and calcined for 6 hours to remove CTAB, and finally mesoporous silica powder is obtained.

5. The method for preparing the synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 3, characterized in that: In step B, the mass volume ratio of hcGEM to deionized water in the hcGEM aqueous solution is 20:1; Repeat water bath sonication three times, 30 min each time, and then magnetically stir at room temperature for 24 h.

6. The method for preparing the synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 3, characterized in that: In step C, the lipid material is composed of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), cholesterol (CHOL), cholesterol succinate (CHEMS), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG) and RGD-modified distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEGRGD) in a molar ratio of 16:8:8:2:1:0.

1.

7. The method for preparing the synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 3, characterized in that: In step C, the ultrasonic dispersion time is 5 min for both times; The mass volume ratio of lipid material Lip to dichloromethane is 20 mg:1 ml; The centrifugation conditions for the hcGEM-MSN suspension were 10000 rpm for 10 min; The conditions for fully vacuum rotary evaporating the mixed solution are as follows: the reaction container is vacuum rotary evaporated on a rotary evaporator at 45°C and 60 rpm to obtain a relatively uniform film and then continue vacuum rotary evaporation to completely remove the solvent; The hydration time at normal pressure was 60 min; the internal ultrasound conditions were 15 / 15 s, 32.5 W, 20 min.

8. The method for preparing the synergistic nano-chemotherapeutic preparation for treating pancreatic cancer according to claim 3, characterized in that: In step C, the product hcGEM / Era RGD-Lip-MSN was stored in a refrigerator at 4°C.

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