| Objective: In this paper, NSC-LA was used as a carrier, PTX as a model drug. SiRNA/LDL complex was prepared with the cholesterol-modified small interference RNA(siRNA) and LDL via incubation. NSC-LA was prepared siRNA/LDL coupled micelles loaded with PTX(PTX-siRNA/LDL-NSC-LA); LDL as a gene carrier, as well as a targeting ligand. LDL coupled micelles loaded with PTX have active targeting and passive targeting dual role, to increase the susceptibility of PTX. And this tumor targeting polymeric physical can mitigate the multidrug resistance.Methods:(1) LDL was separated from plasma of human beings by density gradient ultracentrifugation adjusted with KBr. The properties and tumor-targeted characters were verified. NSC-LA was synthesized and characterized by Fourier Transform Infrared Spectromete(FT-IR) and 1H-NMR. Chol-siRNA/LDL was mixed with cholesterol-conjugated siRNA and LDL via incubation. The structure of micelles was verified by FT-IR. The drug release was investigated in differient medium. The particle size, morphology and surface potential of the PTX-siRNA/LDL-NSC-LA were characterized.(2) The security of micelles was evaluated by hemolytic test in vitro and MTT assay.(3) PTX-siRNA/LDL-NSC-LA was prepared and the cytotoxicities, celluar uptake mechanism, transfection as well as effect of reversing MDR were studied.(4) The subcutaneous tumor model was built to study the distribution of DiR-labeled nanoparticles by near-infrared fluorescence imaging.Result:(1) The result of FT-IR and transmission showed that LDL was successfully coupled to micelles and its uptake was mediated by LDL receptors. NSC-LA was synthesized and characterized. 30:1 was the best ratio of siRNA/LDL and siRNA could be protected from RNase and serum stability in LDL. The mean size of the optimized micelles was(171.6±6.42) nm and Zeta potential was(-22.52±0.47) mV. The entrapment efficiency and loading capacity were(93.92±1.06)% and(14.45±0.78)%.respectively. The results of transmission electron microscope, FT-IR and electrophoresis showed that LDL was successfully coupled to micelles rather than simple mixture. The polymeric micelle has obvious reduction sensitivity and a certain pH sensitivity, and quickly release drugs in acidic environment.(2) The cotransporter micelles almost incompatible with the blood and the blank vector LDL-NSC-LA less toxic to tumor cells compared with the PTX.(3) Compared to Taxol? and PTX/NSC-LA, LDL-coupled micelles had a stonger cytotoxicity against MCF-7 and MCF-7/Taxol cells but not against L929 cells. The micelles might be taken up by clathrin- and caveolae-mediated endocytosis and scavenger receptor-B family might be involved in the process. The lipid raft also had an effect on the uptake. The transfection and RT-PCR results demonstrated that the co-delivering micelles could be internalized by the cells effectively and inhibited the expression of mdr1 mRNA and protein.(4) The NIR fluorescence imaging results showed that the micelles had a good targeting effect on subcutaneous tumors.Conclusions: We successfully prepared and characterized LDL-NSC-LA micelles co-delivering MDR1 siRNA and antitumor drug. These binary polymers were sensitive to pH and reduction. The ―binary polymer‖ consisted of PTX-loaded micelles and siRNA-loaded nanoparticle. The two different parts of the ―binary polymer‖ were connected through amide bond. NSC-LA was surrounded by LDL coupled with siRNA which was essential for the micelles to be recognized by cancer cells. SiRNA protected by LDL, which is an endogenous protein, survived when it encountered RNase and macrophages as well as assisted the antitumor activity of PTX. Moreover, powerful gene silencing activities and tumor-targeting effects were the highlights of the micelle mechanism in the proposed cancer therapy. Thus, micelles modified by endogenous protein and loaded with siRNA or antitumor drugs may provide an innovative and high-efficiency method for co-delivery of genes and chemotherapy agents. |
PDF Full Text Request