Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment

Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very lo w. The objective of this study is to develop nontoxic biodegradable polymer -based cytokine gene delivery systems, which should enhance mIL-12 expressi on, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-L-glycolic acid] (PACA), a biodegradable nontoxi c polymer, by melting condensation. Plasmids used in this study encoded luc iferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid comp lexes were prepared at different (+/-) charge ratios and characterized in t erms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well a s pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PACA condensed and protected plasmids from nuclease degradation. The mean p article size and zeta potential of the polyplexes prepared in 5% (w/v) gluc ose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respecti vely. The surface characterization of polyplexes as determined by atomic fo rce microscopy showed complete condensation of DNA with an ellipsoidal stru cture in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of na ked pmIL-12. This is in good agreement with RT-PCR data, which showed signi ficant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-{4,5-dimethylthiaz ol-2-yl}-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumo r activity in vivo compared to naked pmIL-12. PACA/pmIL-12 complexes are no ntoxic and significantly enhance mIL-12 expression at mRNA and protein leve ls both in vitro and in vivo.