199. Plasmid DNA Delivery by Nanoparticule Loaded Coronary Stent

Abstract

Top of pageAbstract Introduction: Cardiovascular gene therapy has been hampered by a lack of effective gene delivery system. Although stent-based percutaneous gene delivery showed great promising, insufficient gene loading on stents remains a big problem. In this paper, we reported a new approach that gene loaded dodecylated chitosan nanoparticles (DCDNPs) were tethered on coronary stent and demonstrated efficient and highly localized plasmid DNA delivery and gene expression. Materials and Methods: Dodecylated chitosan (50kDa) was a gift from KD Yao. Plasmid EGFP-C1 was obtained from Clontech (Palo Alto, CA). Coronary stents (13mm) were kindly provided by the Microport Medical Co Ltd. (Shanghai, China). The NPs were prepared as Mao's description. The DCDNPs were attached on stents by spraying coating. The morphology of DCDNP-loaded stent (NP-stent) was observed by Scanning Electron Microscopy (SEM). The NP-stents loaded with EGFP-C1 plasmid were evaluated for gene delivery and expression in rat aortic smooth muscle cell (A10 cell) culture and in rabbit common carotid artery (CCA) studies. Results: When mixed with plasmid DNA in aqueous solution, dodecylated chitosan formed positive-charged nanospheres with mean diameter of around 90nm and zeta potential of +28|[plusmn]|3mV. Under physiologic condition, the DCDNPs did not show significant change in size and zeta potential for one week of incubation. TEM microphotographs showed spherical shape and homogeneous particle size distribution of the nanosparticles. EMA studies demonstrated that the DCDNPs could effectively protect plasmid DNA from degradation by DNase I digesting. A thin layer of dense DCDNPs was obverted on the metal struts of both unexpanded and expanded stents. SEM confirmed that the DCDNPs were successfully loaded on the endovascular stents. In cell culture, A10 cells grown on the surface and along the adjacent area of the EGFP- C NP-stents showed high level of GFP expression. Chitosan-coated or naked stents (without plasmid DNA) showed no detectable GFP expression. The NP-stents had no harmful effects on A10 cell growth as indicated by cell culture. Rabbit CCA studies with the pEGFPC1-loaded NP-stent demonstrated efficient gene transduction, with about12.3% of neointimal cells expressing GFP positivity, which is much higher than directly injecting the same quantity of EGFP-C1 into the rabbit common carotid artery. Furthermore, the GFP positive cells were only detected in the neointimal layer closely contacted to the NP-stent. Immunohistochemical analyses using anti-GFP antibody confirmed positive expression in the rabbit common carotid artery implanted with NP-stent. Conclusions: This result demonstrated a great progress in delivering plasmid DNA into intravascular tissue with high efficiency and good hemo- and bio- compatibility and without adverse immune response. The same technique could be used for a wide array of single or multiple therapeutic plasmid DNAs.