Fluorescent Peptide Dendrimers for siRNA Transfection: Tracking pH Responsive Aggregation, siRNA Binding, and Cell Penetration

Abstract

Transfecting nucleic acids into various cells is a key procedure in biological research also envisioned for therapeutic applications. In our effort to obtain simple reagents that would be readily accessible from commercial building blocks, we recently reported peptide dendrimers as single component siRNA transfection reagents accessible in pure form by solid-phase peptide synthesis. Here, we extend our studies of these dendrimers by identifying analogs bearing a coumarin or BODIPY fluorescent label in their core and displaying comparable siRNA transfection efficiencies, pH dependent aggregation, siRNA binding, and secondary structures. Fluorescence resonance energy transfer (FRET) studies show that the dendrimers are tightly associated with siRNA within the formed nanoparticles at pH 7.4 but are released into solution at pH 5.0 and can participate in endosome escape by destabilizing the membrane at this pH value. Colocalization studies furthermore suggest that peptide dendrimers and siRNA remain tightly associated throughout the transfection process.

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides

“…The activity of G 2,3 KL-(Leu) 4 in L configuration almost disappeared, meanwhile for G 2,3 KL-diPalmitamide (D) the activity was only slightly reduced in L configuration (Supplementary Figure S1). Our data follow the same trend and findings as previously published studies in which the amino acid chirality did affect the siRNA and DNA delivery [34][35][36].…”

“…Additionally, other 3rd generation controls were tested and highlighted the importance of having the lipid component in the dendrimer core (Supplementary Figure S1). Previous work confirms that lipids play an integral role regarding the transfection efficiency of peptide dendrimers, possibly because of their ability to facilitate cellular uptake as well as endosomal escape via membrane fusion [30,32,36].…”

“…Studies with fluorophore labeled dendrimers and oligonucleotides showed that endosome acidification induces protonation of the low pK a amino termini of the lipidated dendrimers, which releases part of the dendrimer from the nanoparticle. The latter can then disrupt the endosomal membrane and enable endosome escape [36]. In this study, we synthesized 18 different peptide dendrimers chemically conjugated tolipophilic moieties (fatty acids or hydrophobic amino acids) (Figure 1).…”

Transfecting tissue models with CRISPR/Cas9 plasmid DNA using peptide dendrimers

“…These experiments suggest that Z22 and its analogues mediate pDNA transfection through the same mechanism as dendrimers DMH13 and DMH18 for siRNA transfection, 21,22 despite the very different sizes of their oligonucleotide cargo. This mechanism involves the aggregation and complexation of the oligonucleotide at pH 7.4 to form nanoparticles, which enter cells by endocytosis.…”

“…12,13 We aimed for a peptide dendrimer 14 as a non-viral vector because dendrimers [15][16][17] and peptide-type oligomers [18][19][20] are both known to be favorable for this task. Specifically, we set out to obtain a peptide dendrimer for pDNA transfection by combining a hydrophobic core similar to the core of peptide dendrimers DMH13 and DMH18, which we recently reported as single component transfection reagents for siRNA, 21,22 with the branches of peptide dendrimer G123KL acting as a co-transfection reagent with lipofectin for DNA, 23 siRNA 24,25 and oligonucleotides. 26 We synthesized peptide dendrimers by standard Fmoc solidphase peptide synthesis (SPPS, Fig.…”