| DNAzymes are synthetic, single-stranded, catalytic nucleic acids that bind and cleave target mRNA in a sequence-specific manner, which have been explored for geno-therapeutics. Bottleneck restricting their application is the lack of an efficient delivery system. As a new inorganic nanomaterial with wide application prospects, hydroxyapatite nanoparticles (HAP) have attracted increasing attention as new candidates of non-viral vectors.Objective:Establish the high-efficient HAP/DNAzyme delivery system; Clarify the HAP/DNAzyme delivery system of cell membrane transport mechanism, its bioavailability and biological safety; Improve the DNAzyme technology platform, overcome the bottleneck of DNAzyme in clinical application, establish a new delivery system using nano material as the representative, and implement the clinical transformation of the nucleic acid drugs.Methods:In this study, we were the first to establish an HAP-based delivery system, explored its cellular uptake mechanisms, intracellular localization and biological effects using transient transfection technique, fluorescence microscopy, laser scanning confocal fluorescence microscopy and Western blot technology. Results:The encapsulation of Arg-nHAP and DZ1(LMP1targeted) reached almost100%efficiency under in vitro conditions. Using specific inhibitors, the cellular uptake of Arg-nHAP/DZlcomplex was shown to be through the energy-dependent endocytosis pathway. Furthermore, effective intracellular delivery and nuclear localization of the complex was evidenced through the confocal microscopy. Biologically, the complex successfully down regulated the expression of the LMP1in nasopharyngeal carcinoma cells. In a tumor xenograft mouse model, the complex was shown to be efficiently delivered into the tumor tissue, down-regulated the LMP1expression and suppressed tumor growth.Conclusion:Arg-nHAP may be an efficient vector for nucleic acid-based drugs with potential clinical applications. |
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