Study Of The Anti-tumor Effect And Cellular Uptake Mechanisms Of GE11Peptide Modified Targeting Liposomes

Objective: In this study, we constructed peptide GE11decorated DOX-loadedliposomes in order to actively target to EGFR over-expressed A549cells. The in vitrocell experiments were used to evaluate the targeting efficiency, cellular cytotoxicity,cellular uptake. We also evalued the tumor accumulation of GE11modified liposomesin A549tumor-bearing nude mice. At last, the cellular uptake mechanism of GE11decorated DOX-loaded liposomes was studied. All of these experiments aimed toprovide evidences for this active targeting drug delivery system against NSCLC.Methods: The DSPE-PEG2000-GE11were synthesized by addition reactionbetween DSPE-PEG2000-Mal and Cys-GE11, then the structure of the product wascharacterized by1H-NMR and FTIR and the conjugation efficiency of GE11toDSPE-PEG2000-Mal was quantitatively determined by HPLC. The GE11-LP/DOXwas prepared by combination of the thin film hydration and post insertion method.Then the particle size, zeta potential, morphology and encapsulation efficiency ofliposomes were evaluated by DLS, TEM and HPLC. The MTT assay was used tochoose the optimal GE11targeting density and evaluate the IC₅₀for free DOX,PEG-LP/DOX and GE11-LP/DOX. Qualitative and quantitative analysis of liposomescellular uptake by A549cells was detected by fluorescence microscope and flowcytometry. The real-time distribution of liposomes in A549tumor-bearing nude micewas determined by a near-infrared （NIR） fluorescence imaging system. At last, thecellular uptake mechanism and the subcellular localization of the liposomes wereexamined by flow cytometry and confocal microscopy.Results: The results of1H-NMR and FTIR verified the synthesis of theDSPE-PEG2000-GE11and the calculated conjugated efficiency of GE11toDSPE-PEG2000-Mal was above90%detected by HPLC. The particle size of the different GE11density modified liposomes were about124nm, the zeta potential wasabout10mv. The morphology of GE11-LP/DOX was regularly spherical in shapeand the encapsulation efficiency of doxorubicin was about92%. It demonstrated thatthe density of GE11was of great importance to the targeting efficiency andDOX-loaded liposomes with10%GE11density showed the optimal tumor selectivityto A549cells. The calculated IC₅₀for10%GE11-LP/DOX was1.29μg/mL, whichwas2.6times lower than PEG-LP/DOX （3.35μg/mL）. It was clear that A549cellstreated with GE11-LP/DOX took up more liposomes in comparison withPEG-LP/DOX. In vivo imaging study demonstrated that both the liposomes graduallyaccumulated in tumor via EPR effect. The fluorescence intensity of Cy7-labeledGE11-LP in tumor was much higher but lower in the body, compared with thePEG-LP group. Low temperature and ATP-deprived reduced the cellular uptake ofPEG-LP/DOX and GE11-LP/DOX. Compared with the control, the cellular uptake ofPEG-LP/DOX or GE11-LP/DOX decreased in the presence of chlorpromazine andcolchicine. After liposomes were internalized by A549cells, GE11-LP/DOX wasdelivered to lysosomes.Conclusion: In this study, DSPE-PEG2000-GE11was synthetized andGE11-LP/DOX was prepared through the post insertion method. The in vitro cellexperiments demonstrated that the novel GE11could specially recognize and bind toEGFR, then the cellular internalization efficiency was promoted and the cellularcytotoxicity was increased. The vivo imaging study indicated that GE11-LP wasdelivered to tumor tissue more effectively. The internalization of the PEG-LP/DOXand GE11-LP/DOX by A549cells was an active energy-dependent way.GE11-LP/DOX was internalization into cells through clathrin-mediated endocytosisand macropinocytosis pathway. Then the liposomes were transferred into endosome.The GE11modified liposome delivery device may provide a novel strategy for targettreatment of non-small cell lung cancer.