Transferrin-conjugated Liposomes Loaded With Novel Dihydroquinoline Derivatives As Potential Anticancer Agents

Cancer is a complex disease that occurs and develops through cell signal transduction pathways.Malignant tumor cells have uncontrolled biological characteristics of proliferation,metastasis,and apoptosis.The threat of malignant tumors to human health is increasing day by day with the continuous improvement of living standard,the life expectancy of human beings is also prolonged year by year.Traditional anti-tumor agents have the disadvantages of large side effects,narrow therapeutic window,and long-term use and are liable to produce drug resistance.Therefore,it is imperative to develop a safe,effective and selective new anti-tumor drug.1,2-dihydroquinoline?EEDQ?and its derivatives are widely found in nature,have a variety of biological activities,and their anti-tumor activity is remarkable.A series of EEDQ derivatives were synthesized and screened for their antitumor activity.EEDQ2had the best killing effect on HeLa and HepG2 cells.However,the EEDQ2 compound is hardly soluble in water and has no selectivity for tumor cells.The direct administration has obvious side effects.This article designed targeted liposomes for the delivery of EEDQ2 to increase the tumor cell selectivity of EEDQ2,increase cellular uptake,enhance cytotoxicity,and reduce side effects.The idea of this article:1.Screening of anti-tumor activity of EEDQ derivatives and detection of physicochemical propertiesEight kinds of EEDQ derivatives were synthesized and the antitumor activities were selected using HeLa and HepG2 cell lines.The HEK-293T cell line was used to evaluate its toxicity for normal cells.The experimental results showed that the half maximal inhibitory concentration(IC₅₀)of EEDQ2 on HeLa and HepG2 cell lines were 18.55?g/ml and 14.53?g/ml,respectively.The cytotoxicity of EEDQ2 was similar to the positive control drug of paclitaxel,but it had relatively weakly cytotoxicity for normal cells.EEDQ2 was an anti-tumor compound with promising application.EEDQ2 is insoluble in water,soluble in ethanol,and has a strong affinity for lipids,so the establishment of a liposome-based delivery system to deliver EEDQ2improves the water solubility of the drug and increases cellular uptake.2.Establishment transferrin liposomes delivery system of EEDQ2Liposomes is a vesicle with a phospholipid bilayer structure,which encapsulates EEDQ2 within the liposome bilayer,utilizes the biocompatibility of the artificial membrane with cell membrane to deliver agent.Transferrin receptor?TfR?on the surface of malignant tumor cells is highly expressed compared with normal cells,and transferrin-modified liposomes can be used to target tumor cells.In this study,liposomes were composed using HSPC,Chol,and DSPE-PEG.The mass ratio of HSPC:Chol:DSPE-PEG₂₀₀₀000 was 36:10:1.EEDQ2 liposomes with 10%drug loading efficiency?LP-EEDQ2?was prepared and characterized.The particle size of LP-EEDQ2 was 92 nm,zeta potential of LP-EEDQ2 was-3.12 mV.After incubation with transferrin?Tf?,the particle size of liposomes significantly increased to 103 nm with,zeta potential had no significant change.The surface was coated with PEG so that the liposomes had a long blood circulation effect.The appropriate particle size could be accumulated at the tumor site by passive targeting.3.In vitro evaluation of Tf-LP-EEDQ2In vitro evaluation of Tf-LP-EEDQ2 delivery systems was performed in this paper.In the results of cytotoxicity experiments,Tf-LP-EEDQ2 showed higher growth inhibition of HeLa and HepG2 cells.Tf-LP-EEDQ2 also showed the greatest cytotoxicity compared with EEDQ2 and LP-EEDQ.In the active oxygen production experiment,the ROS level of the cells was significantly increased when HeLa and HepG2 cells were treated with Tf-LP-EEDQ2,the high level of reactive oxygen could lead to mitochondrial dysfunction.In addition,Tf-LP-EEDQ2 also could induces mitochondrial membrane potential reversal.Tf-LP-EEDQ2 may induce apoptosis of tumor cells by changing mitochondrial membrane potential.By fluorescence quantitative analysis,Tf-LP-EEDQ2 increased the cellular uptake of EEDQ2 about3.7 times than LP-EEDQ.Subsequent laser confocal and flow cytometry experiments preliminarily demonstrated that Tf-LP-EEDQ2 could enhance cellular uptake of EEDQ.In conclusion,these data suggest that Tf-LP-EEDQ2 is a promising strategy for anti-tumor.Tf-LP-EEDQ2 solved the limitions of poor water solubility of EEDQ2,toxic of direct administration,and low bioavailability to reach the aim of targeting tumor cells and improve anti-tumor effect,which laid the foundation for further experiments in vivo.