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Polysaccharides (especially chitosan) have recently attracted much attention as gene therapy delivery vehicles for their unique properties such as biocompatibility, biodegradability, low toxicity, and controlled release. Nanoparticles have strong potential as a carrier of plasmid short hairpin RNA (p-shRNA). This study aimed to find the optimum conditions for obtaining Chitosan/triphosphate (TPP)/p-shRNA nanoparticles by the ionic gelation method, and investigating the cellular uptake of the optimized nanoparticles. After applying the central composite design of response surface methodology (RSM), the optimum conditions for preparation of nanoparticles with small size and high loading efficiency were: chitosan/TPP ratio = 10, pH = 5.5 and N/P ratio = 11. The resulting nanoparticles had an average size of 172.8 ± 7 nm and loading efficiency of 71.5 ± 5%. SEM images showed spherical and smooth nanoparticles. The nanoparticles complexed with p-shRNA and may protect it against nuclease digestion. Cytotoxicity studies with HeLa and PC3 human cancer cells demonstrated that chitosan/TPP nanoparticles had low toxicity. Cellular uptake studies using HeLa cells showed that the nanoparticles entered the cells (cellular uptake) and delivered DNA, probably due to their favorable Zeta potential (approximately +28 mV) and small size.

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To understand more about the lower generations of poly(amido amine) dendrimer (PAMAM) as a nonviral vector for antisense (ANS) therapy, a 21-mer epidermal growth factor receptor (EGFR) ANS was delivered by generation five of PAMAM in T47D breast carcinoma cells in culture. The semi-quantitative polymerase chain reaction (PCR) and western blot analysis were used to quantify the expression of EGFR mRNA and protein, respectively. The results showed that PAMAM G5/ANS nanoparticles were able to decrease the level of EGFR mRNA more than 40% even at the lower dendrimer primary amine to the antisense phosphate groups (N/P) ratio of 0.5. But, only the data of western blot analysis at the higher N/P ratios of 10 and 20 showed a decrease of the protein expression level similar to the mRNA expression level. Moreover, PAMAM dendrimer had a positive effect on the EGFR ANS action to inhibit the EGFR mRNA and protein expression. Further studies revealed that PAMAM G5 dendrimer as such inhibits the expression of EGFR in a concentration-dependent manner. Since PAMAM as such was able to inhibit the mRNA expression of p53 gene, we speculated that the effect of PAMAM G5 on the EGFR is a kind of its non-selective effect on the transcription and/or translation machinery of the cell.