Ornithine-conjugated Polyamidoamine (PAMAM) Dendrimers for Gene Delivery: Preparation and Mechanism of their Uptake
A. Kumar, V.K. Yellepeddi, S. Palakurthi
Texas A&M HSC College of Pharmacy, US
Keywords: ornithine-conjugated PAMAM dendrimers, dendriplexes, polyamine transport system (PAT), transfection efficiency, endocytosis
Abstract:Aim of the study was to prepare ornithine-conjugated PAMAMG4 dendrimers and investigate their potential as carriers in gene delivery. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. 1H NMR and MALDI-TOF spectra showed that 60 molecules of ornithine (PAMAMG4-ORN60) were coupled to PAMAMG4 dendrimer. Dendriplexes were prepared using the pmaxGFP as the reporter gene and their stability was assessed by 1% agarose gel electrophoresis. Transfection efficiency increased with increase in generation number of PAMAM and degree of ornithine conjugation. Transfection efficiency of PAMAMG4-ORN60 in NCI H157G (non-small cell lung carcinoma) cells was significantly higher as compared to PAMAMG4. To assess reason for increased uptake, transfection experiments were carried out in HEK 293T (Human embryonic Kidney) cells in presence of excess of ornithine, polyamine transport (PAT) inhibitor, paraquat, in separate experiments. A comparative transfection study was also performed in PAT efficient and deficient cells (H147G and H157R, respectively). Transfection efficiency of PAMAMG4-ORN60 decreased in presence of ornithine, paraquat, and PAT deficient cells, while there was no effect on the PAMAMG4 dendrimers. It may be concluded that PAT is involved in the cellular internalization of ornithine-conjugated PAMAMG4-ORN60, suggesting that ornithine-conjugated dendrimers possess the potential to be novel gene carrier for cancer therapy.