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GEMMA NAVARRO DÍEZ Y C. TROS DE ILARDUYA AN. R. ACAD. NAC. FARM.

mero de 14.000 Da presentaron tamaños de partícula de entre 150 y 170 nm,
mientras que a las mismas relaciones de carga, los complejos preparados con el
polímero de mayor peso molecular presentaron valores menores, de alrededor de
100 nm. El potencial de superficie fue positivo en todos los casos excepto cuando
se utilizó la misma cantidad de polímero y de DNA (punto de electroneutralidad).
Además, en este estudio se ha demostrado la capacidad de estas formulaciones
para proteger al DNA frente a la degradación por DNAasas independientemente del
peso molecular y cantidades utilizadas. Los resultados in vitro en células HepG2
(hepatocarcinoma humano) y CT26 (cáncer de colon murino) sugieren que a mayor
peso molecular y a mayor cantidad de dendrímero se obtienen mayores niveles de
expresión génica. Por último, señalar que los estudios de toxicidad determinaron
una viabilidad celular superior al 80% en todos los casos.

Palabras clave: Terapia génica.—Dendrímeros.—Polímeros.—Galénica.—PA-
MAM (poliamidoamina).

SUMMARY

Design and development of new pharmaceutical dosage forms
for therapeutic gene delivery applied to liver and colon cancer

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

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