Resumen de Diseño y desarrollo de nuevas formulaciones para la vehiculización de genes terapéuticos con aplicación al cáncer de hígado y colon
Gemma Navarro Díez, María Concepción Tros de Ilarduya Apaolaza
The success of gene therapy is largely dependent on the development of vectors (new pharmaceutical dosage forms) capable of effectively transfering therapeutic genes into cells. One mayor approach in non-viral gene therapy is based on cationic polymers, like PAMAM (polyamidoamine) dendrimers. In the latest years, the growing interest in the use of dendrimers as gene carries has lead into numerous in vitro and less in vivo studies. The combination of DNA with dendrimers forms complexes that reduce the size and the charge of DNA, protects the genetic material against degradation by serum nucleases and enhances the uptake into cells by endocytosis. However, the mechanisms of internalization and the influence of biophysical features in transfection efficacy are not fully elucidated. Moreover, the safe limits in terms of toxicity of dendrimers when condensed with DNA are still not clear. More systematic studies are needed to gain a better understanding of the rules that govern dendrimer-based gene delivery. In this study, we have developed, characterized and optimized nanoparticles composed of PAMAM dendrimers (14.000 Da and 28.000 Da) and plasmid DNA, which can deliver genetic material into tumour cells. The quantities of both components were calculated in order to prepare complexes at charge ratio (+/-) from 1/1 to 10/1 (dendrimer/DNA). The size of the complexes ranged from 150 nm to 170 nm for those prepared with the 14.000 Da dendrimer at charge ratio 2/1 to 10/1. At the same charge ratios, the complexes prepared with the highest molecular weight polymer showed lower values (around 100 nm). The zeta potential was positive in all cases, except for the ratio 1:1 (neutrality point). Complexes prevented nuclease digestion. The protection was observed in both polymer generations at all the charge ratios prepared. In vitro gene expression by complexes was performed in HepG2 cells (human hepatocellular carcinoma) and CT26 cells (murine colon adenocarcinoma). Transfection levels increased with the charge ratio and with the molecular weight with maximum levels at charge ratio 10/1 in both generations. Finally, in vitro toxicity assays showed a viability higher than 80% in all the complexes prepared.
