Preparation Of PAMAM Dendrimer-based System For Targeted And Co-delivery Of SiRNA And PTX

Objective: In this paper, G4PAMAM dendrimers were used for the delivery ofsiRNA targeting VEGF mRNA, with HA as coating material for active targeting, to controlthe tumor growth by anti-angiogenesis. However, anti-angiogenic gene therapy can noteliminate tumor quickly and completely. Besides, PAMAM/HA complexes can not avoidphagocytose by RES in vivo. So, to further solve these prolems, PEGylated HA wassynthesized to coating PAMAM dendrimers for the co-delivery of siRNA and antitumorchemotherapeutic drug-PTX, with dual functions, targeting and long-circulating, in orderto reduce toxity and achieve a higher anti-tumor efficacy.Methods: siRNA/PAMAM/HA（SPH）complexes with a different amount of HA wereprepared, siRNA retardation assay was studied by agarose gel electrophoresis, particle sizeand potential were measured by laser particle size analyzer. MTT assay was used toevaluate cytotoxicity, the cellular uptake was studied by fluorescence microscopy and flowcytometry, selecting the best HA coating amount. HA-PEG was successfully synthesizedby amidation reactions, and the structure was verified by1H-NMR analysis afterpurification. The siRNA/PTX/PAMAM/HA-PEG（SPPG）complexes were prepared forco-delivery of siRNA and PTX, by electrostatic adsorption and physical entrapment. ThesiRNA retardation assay, stability experiment, particle size, potential, morphology andencapsulation efficiency were investigated. Cytotoxicity was studied by MTT assay. Flowcytometry and HPLC were preformed to determined the cellular uptake quantitatively. Theeffect of RNAi was estimated by RT-PCR. Moreover, the cellular uptake mechanism andsubcellular localization were elucidated by flow cytometry and confocal microscopy. Thedistribution of complexes in vivo was also observed by fluorescence imaging system.Results: The results measured by laser particle size analyzer showed that the particlesize of SPH increased and the zeta potential decreased, as the ratio of HA increasing. Theresults of MTT assay showed that the shielding with HA can reduce the toxicity. Obviously increased uptake and active targeting ability of the complexes shielding with HA could beobserved in the uptake test, and the highest cellular uptake was observed at25%coatingratio of HA(charge ratio). The results of1H-NMR verified the formation of HA-PEG. Theparticle size of siRNA/PTX/PAMAM (SPP)， siRNA/PTX/PAMAM/HA (SPPH) andsiRNA/PTX/PAMAM/HA-PEG（SPPG）increased gradually, and the potential decreased.The strongest cellular uptake and RNAi effect could be ovserved in the complexesshielding with HA in vivo. Compared with that without shielding, complexes shieldingwith HA-PEG showed relatively less uptake, but there were no significant differencesbetween the two complexes in the effect of RNAi. The uptake mechanism results indicatedthat the transit of the three complexes were energy-dependent. SP employed theclathrin-mediated pathway to enter cells, while SPH and SPG employed various pathwaysincluding the clathrin-and caveolin-mediated pathways. The results in vivo showed thatboth SPPH and SPPG could accumulate in tumor, while SPPG displayed a longer timeaccumulation.Conclusions: In this paper, SPPG, a co-delivery system for siRNA and PTX (SPPG)was designed and constructed, with active targeting and long circulation properties, basedon G4PAMAM dedrimers and PEGylated HA. The system combined gene therapy withconventional chemotherapy, which was expected to achieve higher antitumor effect invivo.