| Aims:Raw264.7 stable transfected cell line overexpressing tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)that targeting death receptor 5(DR5)highly expressed on the malignant melanoma A375 cells was established.Therefrom,their secreted exosomes expressing TRAIL(TRAIL-Exo)can be obtained.TPL was then encapsulated into TRAIL-Exo to prepare TRAIL-Exo/TPL delivery system.The synergistic function of TRAIL-Exo/TPL against malignant melanoma was evaluated both in vivo and in vitro,and its mechanism was also elucidated.Methods:1.TRAIL overexpressing Raw264.7 cells were constructed using lentiviral expression plasmid containing TRAIL gene sequence,lentivirus packaging,transfection,and resistance screening.TRAIL expression was verified by RT-q PCR,Western Blot and flow cytometry.2.The TRAIL-Exo was isolated and purified by gradient ultracentrifugation and characterized.The tumor targeting of TRAIL-Exo in vitro was evaluated by laser confocal microscopy.The efficacy and potential mechanism of TRAIL-Exo in inducing A375 cell apoptosis in vitro were verified using flow cytometry.3.TRAIL-Exo/TPL was prepared by sonication and ultracentrifugation and subjected to physical and chemical characterization.The encapsulation efficiency and drug loading were determined by HPLC.The cumulative release of TRAIL-Exo/TPL under different p H conditions was also calculated by HPLC.The stability of TRAIL-Exo/TPL was evaluated by particle size analysis.4.The uptake efficiency of TRAIL-Exo/TPL by A375 cells was investigated by flow cytometry.The intracellular drug content was determined by HPLC-MS/MS.The uptake pathways of TRAIL-Exo/TPL by A375 cells were explored by flow cytometry.The intracellular distribution and localization of TRAIL-Exo/TPL were observed through laser confocal microscopy.5.The in vitro synergistic anti-tumor effect of TRAIL-Exo/TPL was evaluated through cytotoxicity,apoptosis,cycle,invasion,and wound healing experiments.The expression of extrinsic and intrinsic apoptotic pathway-related proteins was detected by Western Blot.6.The dynamic distribution,tumor targeting of TRAIL-Exo/TPL in tumor-bearing nude mice and local distribution in isolated organs were observed through in vivo imaging system.The TPL concentration of TRAIL-Exo/TPL in plasma and different tissues was determined by HPLC-MS/MS.7.The in vivo synergistic antineoplastic activity of TRAIL-Exo/TPL was evaluated by tumor volume,tumor weight,tumor inhibition rate,survival time,tumor HE section,TUNEL positive rate,and Ki67 positive rate.The biosafety of TRAIL-Exo/TPL was evaluated by body weight,pathological HE sections of main organs,blood routine,and biochemical indexes.Results:1.Raw264.7 cells transfected with lentiviral overexpression plasmid containing TRAIL gene sequence could overexpress TRAIL m RNA and TRAIL protein.2.TRAIL-Exo possessed the typical structural characteristics of exosomes,with an average particle size of about 100 nm,and expressed exosome-specific proteins CD9,CD63,and TSG101.TRAIL was also expressed on TRAIL-Exo and displayed a high positive binding rate with TRAIL antibody.TRAIL-Exo exhibited more remarkable tumor targeting and greater uptake efficiency compared with Exo and could induce cell apoptosis by specifically binding with DR5.3.The highest encapsulation rate was 53.61±3.14%of TRAIL-Exo/TPL and the corresponding drug loading was 5.09±0.28%when the mass ratio of TRAIL-Exo to TPL was 10:1.TRAIL-Exo/TPL still maintained the structural and morphological characteristics of TRAIL-Exo and had a sustained-release behavior under normal physiological conditions,but can promote drug release under acidic conditions,and showed good stability.4.TRAIL-Exo/TPL showed higher cellular internalization and enhanced drug delivery efficiency in A375 cells compared with Exo/TPL and TPL and significantly increased the intracellular drug content.TRAIL-Exo/TPL was internalized by A375 cells mainly through energy-dependent endocytosis mediated by caveolin and DR5.After being internalized by A375 cells,TRAIL-Exo/TPL can co-localize with lysosomes,endoplasmic reticulum,and mitochondria.The order of preferential positioning was lysosome>endoplasmic reticulum>mitochondria.5.The cell viability of the TRAIL-Exo/TPL group was the lowest among all groups(29.27%),and its cytotoxicity was concentration-dependent.Similarly,the TRAIL-Exo/TPL group also demonstrated the highest apoptotic rate among all groups(74.47%)and significantly increased the early and late apoptosis and necrosis rates.TRAIL-Exo/TPL simultaneously arrested the G0/G1 phase(59.14%)and S phase(39.32%)of A375 cells.TRAIL-Exo/TPL group displayed the least number of transmembrane cells and the lowest invasion rate(27.35%)among all groups.In TRAIL-Exo/TPL group,the scratch distance after 48 h changed the least from the initial scratch distance,and the migration rate was the lowest(10.46%)among all groups.The expression level of caspase-3,caspase-8,caspase-9,Bax,Bid,and cytochrome c of A375 cells treated with TRAIL-Exo or TPL was upregulated and was further upregulated after TRAIL-Exo/TPL treatment.The expression level of Bcl-2,NF-κB,VEGF,and survivin in A375 cells treated with TRAIL-Exo or TPL was downregulated and was further downregulated after treatment with TRAIL-Exo/TPL,which indicating that TRAIL-Exo/TPL may exert a synergistic anti-tumor effect by activating extrinsic and intrinsic apoptosis signaling pathways and inhibiting the expression of NF-κB,VEGF and survivin.6.The fluorescence intensity of TRAIL-Exo/TPL of tumor sites in tumor-bearing nude mice after intravenous injection gradually increased with time,reaching a maximum about 6 h,and then slowly decreased.Even after 24 h,there was an apparent fluorescence signal at the tumor.Furthermore,the TPL content of TRAIL-Exo/TPL in plasma,heart,liver,spleen,lung,and kidney gradually decreased over time.On the contrary,in the tumor site,it presented a trend of first increasing and then decreasing.The drug content in tumor peaked(402.33 ng/g)at 4 h.After 24 h,there was still a certain amount of TPL accumulation.7.TRAIL-Exo/TPL group had the smallest tumor volume(139.26 mm~3),the lightest tumor weight(0.11 g),the highest inhibition rate(91.20%),the most obvious pathological damage of the tumor,the highest TUNEL positive rate(78.44%)and the lowest Ki67 positive proliferation rate(5.47%)among all groups,indicating the most remarkable effect of inhibiting tumor growth.TRAIL-Exo/TPL also significantly prolonged the survival time of tumor-bearing nude mice.No obvious influence on the body weight,main organs,and biochemical function,blood routine of nude mice treated with TRAIL-Exo/TPL were observed,proving its good biosafety and biocompatibility.Conclusion:In this study,TRAIL-Exo/TPL delivery system was successfully constructed.The results both in vitro and in vivo proved that TRAIL-Exo/TPL has outstanding tumor targeting and synergistic anti-neoplastic effects,which can be used as a novel,safe and effective delivery nanoplatform.This study is expected to open up a new way for pharmaceutical research of TPL and provide new sights and references for the targeted therapy of malignant melanoma. |
PDF Full Text Request