Construction of PEG-PAMAM dendrimer-based TAG72-targeting nanocarrier for t-Bid gene delivery to colorectal tumor cells

The objective of this study is to develop and assess targeted PAMAM-PEG nanocarrier with anti-TAG72 nanobody for t-Bid gene coding construct delivery into the human colonic adenocarcinoma cells. Nanobody (Nb) coding sequence was subcloned into pSJ expression vector for large-scale production and then Nb was purified by Ni++ affinity chromatography. SDS-PAGE and western blot analysis were performed to verify purifiction. PAMAM was reacted with PEG at the ratio 1:2 (mol/mol) and anti-TAG72 Nb at the ratio 1:1 (mol/mol). Surface charge and size of resulting nanoparticles were evaluated by Malvern zeta sizer and Nanosight. Efficiency of constructed gene carrier for t-Bid, a killer gene, delivery into colonic adenocarcinoma cells in in vitro was assessed using real time PCR and cell counting assays. Production of nanoparticles with the average size of 162±92 nm and +4.57±0.52 zeta potential was confirmed by nanosight and Malvern zeta sizer in order. Gel retardation assay result verified efficiency of carrier for pDNA comlexation. Real time PCR results confirmed the target gene overexpression in the cancerous cell lines. The results of this research confirms the efficiency of PAMAM dendrimers for gene transferring, positive effect of PEGylation and targeting of nanoparticles by anti-TAG72 nanobody.