With the exception of Landi et al. [17] and Faham et al. [22], findings from Table 1 confirm that non-viral DC gene expression is dependent on DNA dosage and the size of polyplex used. Although one study [23] employed pDNA doses of up to 10 μg gene expression was only 0.005%. This may be due to the size of such complexes which ranged between 7 and 11.6 μm (Table 1).
Another analysis [24] employed pDNA doses of >5 μg and reported <0.05% gene expression. In the present study a dose of 20 μg led to up to 14% gene expression. A smaller dose of 10 μg was also used; however this led to extremely low gene expression (data not shown). This may be due to the prevalence of nucleases within DCs [16] that selleck chemical degrade nucleic acids as previous gene expression studies using 10 μg in CHO cells reported www.selleckchem.com/products/gsk1120212-jtp-74057.html higher gene expression profiles than complexes transfected into DCs [9]. This implies that at least three factors play a role in uptake and gene expression, these being; size, dosage and DNA topology. It is clear from this study that DNA topology is an important parameter to consider for non-viral gene delivery
to DCs for vaccination strategies. For polyplex gene expression this study recommends the use of SC-pDNA when complexed with PLL. DCs express various cell surface markers which contribute towards antigen presentation [2]. Fig. 4 shows flow cytometry scatter plots displaying the population of DCs and the level of expression of 9 surface markers following transfection of DNA polyplexes. SC-pDNA polyplexes were analysed, as these gave clear distinguishable population of cells positive for β-galactosidase that can be detected by flow cytometry (Fig. 4a). A comparison of the bulk transfected and nontransfected populations showed no evidence of increased expression of any of the markers (Fig. 4b). β-galactosidase expressing cells were gated, and the expression of the cell surface marker on gated and non-gated cells was compared directly (Fig. 4c). Markers such as DC-SIGN,
which mediates T-cell activation [25] did not change with polyplex gene expression (Fig. 4c). This could be due to below the low DNA dosage employed whereby 20 μg may not be enough to pass a certain threshold to elicit phenotypic changes. Table 1 summaries how previous studies employing similar DNA doses for non-viral DC gene delivery, failed to induce phenotypic changes, with the exception of one study which employed up to 0.2 mg DNA [22]. This suggests greater DNA dosage may be required for DC activation. PEI/DNA complexes were also reported to fail in inducing DC phenotypic changes [21]. Measuring such changes is important for clinical applications. Vaccines targeting DCs incorporate adjuvants that are designed to elicit phenotypic changes that activate DCs [21]. Therefore the findings from Fig. 4 reveal how PLL/DNA complexes could incorporate components (adjuvants) to induce DC activation.