Construction Of A Targeted Lipid-coated Mesoporous Silica Nano-delivery System For Pancreatic Cancer Based On Ferroptosis-chemotherapy Synergistic Therapy And Its Anti-tumor Activity In Vitro And In Vivo

Pancreatic cancer is a malignant tumor with a high mortality rate.Due to late diagnosis,strong invasiveness,and distant metastasis,the 5-year survival rate of patients is less than 10%.Currently,single-drug or combined chemotherapy regimens based on gemcitabine hydrochloride are not effective clinically,and new cancer therapies such as immune checkpoint inhibitor therapy and gene therapy are basically ineffective against pancreatic cancer.Therefore,there is an urgent need to explore new strategies and methods for the treatment of pancreatic cancer.Ferroptosis is a form of cell death different from apoptosis,pyroptosis,and necrosis,and it can have a significant suppressive effect on KRAS-mutated pancreatic cancer cells.Ferroptosis can reverse the resistance of pancreatic cancer to traditional apoptotic chemotherapeutics,and combined with traditional chemotherapy,it can produce a significant synergistic inhibitory effect.Therefore,the combined treatment strategy based on ferroptosis-chemotherapy can provide a new treatment strategy for pancreatic cancer which is poorly treated by traditional therapies.Lipid-coated mesoporous silica nanoparticle（Lip-MSN）is a kind of nanoparticle formed by wrapping lipid film on the surface of mesoporous silica.As a drug nano-delivery carrier,Lip-MSN not only has the advantages of mesoporous silica and liposome,but also overcomes the shortcomings of both.Its inner core mesoporous silica can be used as an internal drug loading area to accommodate drug molecules,and provide a supporting framework for the overall structure to stabilize the outer lipid film;the outer lipid film can act as a loading area for fat-soluble drugs,and through its coating on mesoporous silica,it can achieve controlled release of internally loaded drugs,as well as improve the biocompatibility and colloidal stability of mesoporous silica.Also it is easier to be modified by targeting molecules or stimuli-responsive materials.RGD peptide can specifically bind to integrinαvβ3 andαvβ5 receptors,while pancreatic tumor cells and the overexpressed fibrinogen,plasminogen,laminin in pancreatic tumor stroma all contain integrinαvβ3 andαvβ5 receptors.Thus,RGD peptide may serve as an important ligand for targeting pancreatic cancer.This subject designed a p H-responsive lipid-coated mesoporous silica nano-delivery system modified by RGD peptide to simultaneously entrap and deliver the ferroptosis inducer Erastin（Era）and the chemotherapeutic drug gemcitabine hydrochloride（hcGEM）,in order to enhance the synergistic anti-tumor effect and reduce the dosage and the systemic toxicity of the drug.This delivery system can not only actively target and deliver drugs to pancreatic tumor tissue with high expression of integrin receptors through the surface-modified RGD peptide,but also release synergistic therapeutic drugs p H-sensitively in acidic tumor environment,reducing toxic and side effects while improving the killing effect of drugs on pancreatic cancer cells.Firstly,the synergistic inhibitory effect of different ferroptosis inducers combined with hcGEM on pancreatic cancer cells was investigated by CCK8 method,and it was found that all three ferroptosis inducers could synergistically inhibit PANC-1 cells and PANC-1/hcGEM cells to varying degrees.Among them,only the hcGEM-Era 4:1 group can synergistically inhibit PANC-1 cells and PANC-1/hcGEM cells at all concentrations,so the follow-up prescription research was carried out with hcGEM and Era as the main drugs.Secondly,the determination method of hcGEM and Era was established by high performance liquid chromatography.The results showed that within 2～250μg/m L,the concentration peak area of hcGEM was linearly correlated with the concentration（R²=1）,and the linear regression equation was A=0.4572 C+0.3902;within 2～250μg/m L,the concentration peak area of Era was linearly correlated with the concentration（R²=0.9999）,and the linear regression equation was A=0.3352 C+0.0969.After investigation and verification,the specificity,standard curve,precision and extraction recovery of the two drug content determination methods all met the determination requirements,and could be used for the content determination and in vitro release content determination of hcGEM and Era.The research has laid a good experimental foundation.Then,mesoporous silica nanoparticles（MSN）were prepared by the sol-gel method,and the synthesis process was optimized by single factor analysis with the particle size of MSN as an index,and the optimal reaction conditions for the preparation were determined,that was,the molar ratio of CTAB to TEOS was 1:5,the stirring speed was600 rpm,the reaction temperature was 80°C,the reaction time was 2 h and the titration rate was 1 m L/min.The average particle size of MSN synthesized under the optimal reaction conditions was 116.47±0.91 nm,and the PDI was 0.24±0.01.Scanning electron microscopy and transmission electron microscopy showed that the particle size of a single MSN was 50 nm,with its morphology spherical,dispersion good and mesoporous channels orderly.The results of nitrogen adsorption and desorption experiments indicated that the specific surface area of the MSN was 745.799 m²/g,the pore volume was 0.978 cm³/g,and the average pore diameter was 5.687 nm.On this basis,RGD-modified lipid mesoporous silica loaded with hcGEM and Era（hcGEM/Era RGD-Lip-MSN）was prepared by two thin-film hydration methods,and by changing the mass ratio of MSN and Lipid as well as the input amount of Era screened out the prescription of drug loading with the best synergistic inhibitory molar ratio of 4:1between hcGEM and Era.Under this formulation,the average particle size of hcGEM/Era RGD-Lip-MSN was 140.57±2.93 nm,the PDI was 0.22±0.13,and the Zeta potential was-58.80±0.65 m V;the encapsulation efficiency and drug loading capacity of hcGEM were 37.45%and 11.05%,respectively;the encapsulation efficiency and drug loading capacity of Era were 78.96%and 4.89%,respectively,and the molar ratio of hcGEM to Era was 4.13:1.Transmission electron microscopy showed that the particle size of RGD-Lip-MSN was about 70 nm,and the surface was covered with a lipid film.hcGEM/Era RGD-Lip-MSN could slowly release hcGEM and Era under neutral release conditions,and the cumulative release rates of hcGEM and Era were 30.23%and 44.71%at 48 h,respectively;the initial stage of release in an acidic release environment showed a"burst release"effect,with its cumulative release rates of hcGEM and Era were 86.46%and 79.24%at 48 h,respectively.The overall release characteristics showed p H response characteristics,which could basically be released according to the molar ratio of hcGEM and Era 4:1.The prepared hcGEM/Era RGD-Lip-MSN could carry the two drugs with the optimal tumor cell synergistic inhibitory molar ratio,and the in vitro release characteristics met the expected requirements,laying the foundation for subsequent experimental research in vivo and in vitro.In vitro experiment,the cytotoxicity experiment was first carried out by the CCK8method.The IC₅₀of 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 1.327μmol/L,respectively.The cell inhibition rate of hcGEM/Era RGD-Lip-MSN treatment group was significantly higher than that of hcGEM treatment group or hcGEM RGD-Lip-MSN treatment group（P<0.05）,and compared with hcGEM+Era treatment group,the cell inhibition rates of two groups were similar.The results of the cell scratch experiment showed that the hcGEM/Era RGD-Lip-MSN treatment group had a significantly stronger inhibitory effect on the migration of PANC-1 cells than the hcGEM treatment group or Era treatment group（P<0.05）,but compared with hcGEM+Era treatment group,the cell inhibition rates of the two groups were similar.By detecting the contents of ROS,MDA and GSH in cells treated with different drugs,it was found that hcGEM/Era RGD-Lip-MSN could significantly increase the contents of ROS and MDA in cells,and reduce the contents of GSH in cells（P<0.05）,compared with the hcGEM/Era Lip-MSN treatment group.Besides,compared with DMSO treatment group,hcGEM could significantly increase the content of intracellular ROS and MDA,and reduce the content of intracellular GSH（P<0.05）.The results of Western blot showed that the intracellular GPX4 and xCT protein levels in the hcGEM/Era RGD-Lip-MSN treatment group were significantly reduced（P<0.05）,compared with the hcGEM/Era Lip-MSN treatment group.The uptake of fluorescent substances by cells in different drug treatment groups was detected by confocal microscopy,and it was found that the intracellular red fluorescence intensity in the RGD-modified lipid-coated mesoporous silica nanoparticle co-loading the water-soluble fluorescent dye rhodamine B and the fat-soluble fluorescent dye coumarin6（Rh B/C6 RGD-Lip-MSN）treatment group was the strongest,while the green fluorescence intensity was comparable to that of the free group,indicating that the Rh B/C6 RGD-Lip-MSN could effectively deliver two fluorescent substances into the cell.Finally,the cell survival of different drug treatment groups was detected by flow cytometry.The results showed that the cell survival rate of the hcGEM/Era RGD-Lip-MSN treatment group was significantly lower than that of the hcGEM/Era RGD-Lip-MSN+Ferrostatin-1（P<0.05）,indicating that the inhibitory effect of hcGEM/Era RGD-Lip-MSN on the proliferation of PANC-1 cells was partly derived from ferroptosis,which could be inhibited by the Ferrostatin-1（Fer-1）.The cell survival rate of the hcGEM/Era RGD-Lip-MSN treatment group was significantly lower than that of the hcGEM/Era Lip-MSN treatment group（P<0.05）,which indicated that hcGEM/Era RGD-Lip-MSN could deliver hcGEM and Era more effectively into the cells to exert a stronger proliferation inhibitory effect.In vivo pharmacodynamics study,a nude mouse model of subcutaneous tumor transplantation of pancreatic cancer was established by transplanting tumor tissue blocks,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 after intravenous injection of different drug preparations.The results showed that the tumor volume of the hcGEM+Era administration group was significantly smaller than that of the hcGEM administration group or Era administration group（P<0.05）,indicating that the application of hcGEM combined with Era could significantly improve the tumor suppression effect.The tumor volume of the hcGEM/Era RGD-Lip-MSN administration group was significantly smaller than that of the hcGEM+Era administration group（P<0.05）,and the body weight of the nude mice in the hcGEM/Era RGD-Lip-MSN administration group gradually increased while that of the hcGEM+Era administration group decreased significantly（P<0.05）,indicating that the co-delivery of hcGEM and Era by hcGEM/Era RGD-Lip-MSN could improve the tumor suppression effect of the two drugs and reduce the side effects of the 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 suppression effect of the hcGEM/Era RGD-Lip-MSN administration group was slightly better than that of hcGEM/Era Lip-MSN administration group.Therefore,the co-delivery of hcGEM and Era by hcGEM/Era RGD-Lip-MSN could improve the tumor suppressive effect of the two drugs while reducing the side effects of the drugs.All the above research results show that the hcGEM/Era RGD-Lip-MSN designed in this project can simultaneously deliver the ferroptosis inducer Era and the chemotherapeutic drug hcGEM,and by combining different cell death mechanisms,RGD targeted delivery,and p H-sensitive release,it can reduce the toxic and side effects of the two drugs while improving the tumor inhibitory effect of the drugs,which can provide a data reference for the nano-delivery system based on the ferroptosis-chemotherapy combined treatment strategy.