Construction Of Nanoprecursor Drug Delivery System Modified By Cell Transmembrane Peptide And Its Application In Lung Cancer Treatment

Lung cancer is a malignant tumor disease that seriously endangers human life and health.Chemotherapy,as the main treatment method for lung cancer,is widely used,but can cause many side effects to the body.In particular,the application of lung cancer treatment chemotherapy drugs is greatly limited by the method of administration,such as the poor compliance of patients with systemic toxic and side effects of intravenous administration,and the low bioavailability of oral administration and large gastric irritation.Pulmonary drug delivery has attracted extensive attention because it can directly penetrate drugs into lung tissue through trachea,significantly reduce the dose of chemotherapy drugs and increase the effective drug concentration in lung,thus greatly reducing systemic toxic and side effects and improving drug efficacy.In addition,in order to overcome the disadvantages of poor solubility and high toxicity of chemotherapy drugs,this study constructed an inhalable nano-prodrug drug delivery system modified by cell-penetrating peptide for the treatment of lung cancer model mice.In this study,insoluble 7-ethyl-10-hydroxycamptothecin（SN38）and curcumin were used as model drugs.The amphiphilic prodrug TAT-PEG-SN38 was synthesized by using polyethylene glycol（PEG）as the linker of SN38 and cell transmembrane peptide TAT.Subsequently,TAT-PEG-SN38 was used as a part of the liposome membrane,and a liposome containing SN38 prodrug and curcumin was prepared by microfluidic method.The solubility problem of SN38 and curcumin was solved by the co-delivery system at the same time,and the uptake efficiency of the drug was greatly improved due to the introduction of membrane penetrating peptide,too.Pulmonary drug delivery can significantly reduce the dose of chemotherapy drugs,improve the concentration of drugs in the lung and avoid the toxic and side effects of drugs on other tissues and organs as much as possible.The main contents of this study are as follows:1.Preparation and characterization of SN38 prodrug and curcumin co-loaded liposomeTBDPS·Cl was used to selectively protect the C₁₀ position of SN38,then glycine was used as the linker to connect the C₂₀ position of SN38 to the carboxyl terminal of Mal-PEG-COOH,and TBAF was used to remove the protection group,and then Mal-PEG-SN38 was linked to the amino group of TAT.The amphiphilic prodrug TAT-PEG-SN38 was synthesized.According to the results of ~1H-NMR,the characteristic peaks of SN38C₂₀ hydroxyl group disappeared,while the characteristic peaks of C₁₀hydroxyl group and PEG and TAT still existed,which proved the successful synthesis of SN38 prodrug.Liposomes containing SN38 prodrug and curcumin were prepared by microfluidic method,and their particle size,zeta potential encapsulation rate and drug loading rate were determined.The advantages of microfluidic method were proved by using ethanol injection method as control.The particle size of co-loaded liposome prepared by microfluidic method was 171.21±1.10 nm,the polydispersion coefficient was0.124±0.03,and the encapsulation efficiency of curcumin was 88.37±2.94%.The co-loaded liposome had good storage stability,and its spherical shape with obvious core-shell structure was observed by transmission electron microscope.2.In vitro antitumor activity of SN38 prodrug and curcumin co-loaded liposomeAfter characterization of co-liposome,its anti-proliferation effect on A549 cell line was studied.Firstly,the cellular internalization of co-loaded and single-loaded curcumin liposomes by A549 cells was analyzed.The results showed that the uptake efficiency of co-loaded liposomes was significantly higher in A549 cells than that of curcumin liposomes alone,which verified the effect of transmembrane peptide on improving cell uptake efficiency.Cytotoxicity experiments showed that the improvement of dosage form and drug co-loading could enhance the antitumor effect of the drug.Cell apoptosis and cell cycle arrest experiments showed that the preparation had good ability to induce cell apoptosis and induce S phase cell cycle arrest.3.In vivo antitumor activity of SN38 prodrug and curcumin co-loaded liposomeUsing coumarin as an indicator,the body distribution of co-loaded liposome by pulmonary delivery was studied.The results showed that the drug accumulated in lung preferently after administration,and there was still a certain concentration of drug in lung24 h later.Subsequently,BALB/c lung cancer model mice were established,and the in vivo antitumor effect of the preparation was studied by pulmonary administration.The results showed that Lip-TAT-PEG-SN38/CUR group had the lowest number of lung tumor nodules,and the appearance of lung was similar to the healthy lung,which proved that the co-liposome group had the best anti-tumor effect.During the treatment period,the body weight of mice in each group did not change significantly,which proved the safety of the preparation.The results of the tissue section experiment showed that the lung sections of the mice in the Lip-TAT-PEG-SN38/CUR group did not show compactness.Finally,the expression level of Ki-67 in the lungs of each group was analyzed by immunohistochemical assay,and the results showed that the expression level of LIP-TAT-PEG-SN38/CUR group was the lowest.