Polyethylenimine-based Non-viral Oligonucleotide Delivery System Mediated By NGR Peptides

The suppression of hTERT in tumor cells by ASON that lead to telomerase inhibition, destabilization and consequently cell growth inhibition and/or cell death represents a promising anticancer strategy. The condensation of antisense oligonucleotide into well-defined particles is an integral part of several approaches to non-viral cellular delivery system. The cationic polymer PEI that was found to have the ability to condense ASON into nanoparticles is a very important non-viral vector. PEI can combine strongly with ASON to generate stable and positive nano-condensates that can adhere to the negative charged cellular membrane and penetrate the cell membrane predominantly via fluid-phase endocytosis. Recently, PEI/ASON condensates with the characteristics of being simple to prepare and easy to modify have become a promising vector for ASON transfection in vivo.In the study, the optimal preparation conditions of B-PEI/ASON condensates were optimized by single factor and uniform design experiment. The physicochemical properties, anti-enzyme activities of condensates and the anti-aggregation effects of hydrophilic polymer were investigated. The results were as the following: the incorporation efficiency of B-PEI/ASON condensates was nearly 100% at the N/P ratio higher than 3. 5; The NaCl strongly influenced the stability of the condensates and the size of condensates became lager significantly at the concentration of NaCl higher than 50 mmol/L; The size of the condensates rose dramatically at the pH higher than 8. 0 while the protonation rate of amino nitrogen of B-PEI declined rapidly; for the ASON, its concentration don' t affect on the stability and size of condensates in designed scale. The discrete and uniform spheroidal particles with the average size of 120.6 nm and zeta potential 33. 84 mv were obtained at the optimal conditions of pH 7. 0 N/P =4. 0 and C_ASON=100μg/mLThe influence of N/P on the incorporation efficiency of L-PEI/ASON condensates and the protection of Dextran-70 from aggregation of condensates was investigated. The condensates with the average size of 156. 7 nm and zeta potential of 36. 28 mv were obtained under the conditions of pH 7.0 N/P =10.0 and C_ASON=100μg/mLFor the preparation of condensates targeting to tumor cells, L-PEI/ASON condensates at N/P =6.5 were modified by NGR peptides through electrostatic reaction. The discrete and uniform particles with the average size of 128.1 nm and zeta potential of 32. 14 mv were generated under the conditions of pH 7.0 N/P_PEI=6.5 N/P_NGR=0. 3 and C_ASON=100μg/ml, ASON were loaded completely and protected perfectly from DNase I by the condensates.The efficacy to promote transfection in vitro, tumor-targeting and anti-tumor activity of PEI were investigated. The delivery of ASON into EC 9706 cells were dramatically promoted by NGR/L-PEI/ASON condensates compared with the free ASON, the fluorescence intensity in the nuclei was even higher than the cytoplasm. Condensates decorated with NGR peptides could be transported into tumor tissue after administering intravenously or subcutaneously and the quantity of the ASON in tumor tissue increased with time increment. However the condensates unmodified couldn' t enter into tumor tissue.The volume of esophageal carcinoma tumor xenografts on the BALB/C nude mice of the therapeutic groups (183.6 mm³ and 172.5 mm³, respectively) being treated by administering condensates intravenously or subcutaneously was smaller than on the BALB/C nude mice of the negative control groups (469.1 mm³ and 393.8mm³, respectively) being treated by administered physiological saline or NGR/L-PEI/SON condensates intravenously, apoptosis in esophageal carcinoma cells appeared in the therapeutic groups. The result from transmission electron microscopy showed that the tumor cells displayed cytoplasmic and nuclear shrinkage, chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation and no typical apoptosis body formed in the control groups.The well-defined and stable condensates with the ability of targeting to tumor cells were generated by use of PEI to condense ASON into nanoparticles as a core and then by use of NGR peptides to modify the surface of the particles through electrostatic reaction and also its anti-tumor activity was investigated in the study.