LHRH-TAT-chitosan / SiRNA Complex And Macrophage And The Effect Of TPP-LHRH-MPG ^{â–³ NLS / AFP-siRNA Complex On Tumor Growth}

RNA interference (RNAi), a post-transcriptional reculatory mechanism, is widespread in eukaryote and could degrade mRNA with specific sequence.RNAi shows a good application in treating various tumor diseases, including hepatic carcinoma. However, the easily degradation and hardly cell member penetration of RNA molecule makes RNAi apply to clinical application difficultly. Novel gene vector could improve the efficient of RNA delivery, but the biocompatibility, the efficiency of RNA releasing and RNA interference should be deeply evaluaed.The characteristics of nanoparticles are observed by agarose gel retardation assay and light scattering, while analyse the variation of cytokines (IL-1β, IL-6, IL-12, TNF-α, IL-10) concentration after coincubating nanoparticles with RAW264.7 through ELISA. Flow cytometry and experiment of uptaking the chicken red blood cell by mouse peritoneal macrophage are utilized to detect the macrophage phagocytic activity influenced by polyplex in vitro and in vivo. It was demonstrated that TLC had stronger siRNA condensing power than unmodified chitosan(N/P ratios arel-2,1:1, 2:1,5:1,10:1, respectively), and the size of TLC/siRNA polyplexes (N/P ratios are 10:1 and 20:1) ranged from around 93 to 150 nm with zeta potential ranging from+2.7 to+19.3 mV. TLC/siRNA polyplexes include 200 nmol/L siRNA cannot induce the release of cytokines above. It was revealed that TLC exerted no significant impact on the function of macrophages in terms of phagocytic activity and cytokine production, even though the macrophages internalized more TLC/siRNA polyplexes than unmodified chitosan/siRNA polyplexes. It is demonstrated that TLC is a safe and valuable siRNA vector.According to the previous research that modified chitosan, PEG vector with TPP (sodium tripolyphosphate) to improve the efficiency of drug delivery. In this article, we covalently linked TPP into amino terminal of LHTH-MPGΔNLS to product new type of TPP-LHRH-MPGΔNLS carrier and make it pack AFP-siRNA. Compare with the size and zeta potential of new vector TAT-LHRH-MPGΔNLS/siRNA polyplexes and LHRH-MPGaΔLS/siRNA polyplexes, the zeta potential became larger while the size have no obvious change. In the experiment of suppressing of tumor cell growth, we choose two hepatic carcinoma cell (HepG2 and Be17402) and human liver cell L02 to transfect AFP-siRNA by the two type of vector. The survival rate of hepatoma cell were lower than L02 after 48 h, and the RT-PCR test for the three type of cells after 24h AFP-siRNA transfecting determine that TPP-LHRH-MPGΔNLS/APF-siRNA nanoparticle has little function to HepG2 but could suppress the growth of Be17402, and the linked TPP would influence the targeting of vector.