Multilayered Nanoparticles for Gene Delivery Used to Reprogram Human Foreskin Fibroblasts to Neurospheres

Abstract

Polycationic nanocomplexes are a robust means for achieving nucleic acid condensation and efficient intracellular gene deliveries. To enhance delivery, a multilayered nanoparticle consisting of a core of electrostatically bound elements was used. These included a histone-mimetic peptides, poly-l-arginine and poly-d-glutamic acid was coated with silicate before surface functionalization with poly-l-arginine. Transfection efficiencies and duration of expression were similar when using green fluorescent protein (GFP) plasmid DNA (pDNA) or GFP mRNA. These nanoparticles demonstrated significantly higher ( > 100%) and significantly longer (15 vs. 4 days) transfection efficiencies in comparison to a commercial transfection agent (Lipofectamine 2000). Reprogramming of human foreskin fibroblasts using mRNA to the Sox2 transcription factor resulted in three-fold higher neurosphere formation in comparison to the commercial reagent. These results demonstrate the potential of these nanoparticles as ideal vectors for gene delivery.

Polymeric Amines and Ampholytes Derived from Poly(acryloyl chloride): Synthesis, Influence on Silicic Acid Condensation and Interaction with Nucleic Acid

“…Study of Si(OH) 4 condensation in the presence of new polymers (Figure 7) revealed an acceleration of the condensation process with polymers containing a considerable amount of amine units similar to our results with poly(vinyl amine) [34]. The effect was visible at the very early stages of condensation (k third values) and the polymers decreased the equilibrium concentration of free silicic acid in the later stages.…”

“…Condensation of silicic acid at pH 7 proceeded mainly as an S N 2 reaction between the -Si-OH and -Si-O − moieties [33]. Silanol anions arose from the primary PSA particles as Si(OH) 4 is a very weak acid (pK = 9-10) [21,32] in comparison to PSA (pK 0 = 6-7). Interaction between PSA (including primary oligomers) and polyamine chain increased the amount of silanol anions (-SiO − + HN-) and an acceleration in condensation was observed.…”

“…Reactions in the presence of polymers proceeded through bimodal distribution of the particle size and after 1-2 days, uniformly distributed particles were observed. The size of the resulting particles increased with an increase of Si(OH) 4 concentration and with the introduction of carboxylic groups in the polymeric chain. Freeze-dried solutions of the composite nanoparticles were studied with transmission electron microscopy (TEM, Figure 10).…”

“…The structure and charge of the oligonucleotide-containing complexes are important factors that influence the transfection efficiency of the oligonucleotide. Various molecules have been investigated in this area and one approach is to use siliceous composites [4,5]. We studied the ability of composite siliceous nanoparticles based on new polymeric amines to interact with the oligonucleotide (Table 4, Figures S6 and S7 in the Supplementary Materials) and found the following:…”

“…The addition of HCl was done in <1 min using data from preliminary experiments. This allowed us to prevent Si(OH) 4 condensation at a pH higher than the desired value. Fifty mM HEPES buffer (pH = 7.4) was presented in the solutions prepared for study interactions with oligonucleotide.…”