| It has been demonstrated that the clinical risks of using viral vectors for gene therapy are obstacles for their practical use even though virus-mediated gene delivery offers high transfection efficiency. As a result, many trials of nonviral gene delivery have been focused on the design of synthetic cationic vectors for nucleic acid condensation and delivery to overcome the clinical obstacles of virus-mediated gene delivery. In spite of the accomplishments of clinically useful nonviral vectors, most nonviral gene carriers provide less efficient gene transfer, especially in escape from endosomal vesicles. In this study, Ca-alginate nanoparticles were used as the gene carriers due to their biocompatible and biodegradable.; Once internalized via phagocysosis/endocytosis pathway, they can undergo quick erosion and elicit osmotic swelling, hence facilitating phagosomal/endosomal escape of gene to the cytoplasm. For the preparation of alginate-based nanoprticles, reverse emulsion technique was employed for phagocytosis-mediate gene delivery in J774A.1 macrophage (with an average nanoparticle size of 780 nm in diameter). And, w/o reverse microemulsion template was also employed with (2ethylhexyl) sulfosuccinate (AOT) as surfactant for endocytosis-mediated gene delivery in NIH 3T3 (with an average nanoparticle size of 80 mn in diameter). After gelation of alginate microemulsion, Ca-alginate nanoparticles were collected by centrifugation. For the intracellular tracking of Ca-alginate nanoparticles, those were labeled with Lucifer-yellow under 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDAC). To examine the efficacy of Ca-alginate nanoparticles in facilitating endosomal escape of DNA, NIH 3T3 cells were incubated with nanoparticles obtained from the microemulsion template for 24, 48 and 72 h, respectively. Transfection rates were also quantitatively determined by measuring the intensity of eGFP expression in cells using a fluorimetric microplate reader. The results showed that Ca-alginate nanoparticles with an average size around 80 nm in diameter led to high gene expression which is comparable to the efficacy using polyethyleneimine (PEI)/DNA complexes. |
