Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations

Abstract

Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human β-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides

“…Luciferase-based assays are frequently used, and considered one of valuable tools to test efficacy of splice-switching ON delivery [ 38 , 39 ]. We have previously utilized such an assay to establish a robust and reliable approach to test the efficacy of a large number of transfection reagents to deliver splice-switching ON [ 32 , 40 ]. The procedure of complex formation was performed according to Saher et al with some modifications [ 32 ].…”

“…It is also a good candidate as drug delivery system for small molecules and macromolecular gene therapeutics [ 46 , 47 , 48 ]. Previously, we demonstrated that transfection efficiency of peptide dendrimers/lipids can be further improved by adding different types of saccharides or polyol compounds [ 40 ]. Among those screened polymeric excipients, PVA18 was one of the best excipients to enhance transfection efficiency of peptide dendrimer/lipid hybrids [ 40 ].…”

“…Previously, we demonstrated that transfection efficiency of peptide dendrimers/lipids can be further improved by adding different types of saccharides or polyol compounds [ 40 ]. Among those screened polymeric excipients, PVA18 was one of the best excipients to enhance transfection efficiency of peptide dendrimer/lipid hybrids [ 40 ]. In our current study, we observed that the addition of PVA18 to the lipophilic peptide dendrimers improved transfection efficiency significantly.…”

“…Interestingly, it seems that the enhancing effect of the excipient is linked to the lipid component. We have earlier reported an enhancing effect of PVA18 using lipofectin, [ 40 ]. Hence, we tested the addition of PVA18 to lipofectamine and we observed a 2-fold increase in transfection efficiency in OptiMEM ® .…”

“…Interestingly, for the gold standard L2000, there was no toxicity observed for the concentration tested ( Figure 8 C). As hypothesized, the relatively loose adsorption of PVA onto the surface of nanoparticles at pH 7 is sufficient to improve cellular uptake [ 40 ]. However, adsorption is expected to become much stronger during the endosomal stage due to pH reduction to around 5.5 and would likely enhance the protective properties of PVA and facilitate endosomal escape.…”