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.

FIG. 1.

(A) Confocal at 30 h after transfection of mCherry DNA into MC3T3-E1 osteoblasts with the noted complexes and PLR tagged with FITC at a 1:100 FITC:PLRmer stoichiometric ratio.(+Silicate): No extracellular aggregation. DNA-PDGA-PLR: Efficient gene expression. Note the increased number of clustered particles. (+Silica): No extracellular aggregation. Significantly greater intracellular clustering of nanoparticles. DNA-HTP-PLR: Efficient gene expression. (+Silicate): No extracellular aggregation. Nuclear localization of nanoparticles is observed. DNA-HTP-PDGA-PLR: Efficient gene expression. (+Silicate): No extracellular aggregation. Nuclear localization and significant cytoplasmic accumulation is observed. (B) Transfection of GFP pDNA-loaded particles into murine bone marrow-derived cells to analyze retention of these particles in the primary cells for 3 weeks. FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HTP, histone tail peptide; PDGA, poly-d-glutamic acid; pDNA, plasmid DNA. Color images available online at www.liebertpub.com/tec

FIG. 2.

(A) Transmission electron microscope of a desiccated and lyophilized nanoparticle. (B) FTIR of silica-stabilized nanoparticles and demonstration of further layering with PLR. (C) Dynamic light scattering of nanoparticles during core formation, silica stabilization, and further layering (*p<0.05). FTIR, Fourier transform infrared. Color images available online at www.liebertpub.com/tec

FIG. 3.

(A) Fluorescent microscopy of primary bone marrow stromal cells transfected with pDNA encoding GFP using 800, 1600, and 2500 ng of DNA introduced via a single transfection with Lipofectamine (left) and three transfections with nanoparticles on days 2, 3, and 4 (right). (B) Flow cytometry of identical transfection conditions. (C) Quantitative representation of transfection efficiency via flow cytometry (p<0.5). ***indicates that groups are statistically different from one other (p<0.5). Color images available online at www.liebertpub.com/tec

FIG. 4.

(A) Fluorescent microscopy of human mesenchymal stem cells transfected with pDNA encoding GFP using 800, 1600, and 2500 ng of DNA introduced via a single transfection with Lipofectamine 2000 (left) and three transfections with nanoparticles on days 2, 3, and 4 (right). (B) Flow cytometry of identical transfection conditions. (C) Quantitative representation of transfection efficiency via flow cytometry. Color images available online at www.liebertpub.com/tec

FIG. 5.

(Upper Panel) Fluorescent imaging of human foreskin fibroblasts (HFF) transfected with GFP pDNA—expression is assessed for more than 21 days (Lower Panel). Fluorescent imaging of HFFs transfected with GFP mRNA synthesized using in-vitro mRNA synthesis kit—expression is assessed for more than 21 days. Color images available online at www.liebertpub.com/tec

FIG. 6.

(A, B) Gene expression analysis of HFFs transfected with Sox2 transcription factor using nanoparticles and Lipofectamine2000. The fold increase in gene expression was determined using ΔΔCt formula normalized against gene expression of cells treated with Lipofectamine2000. (C) Transfection of HFFs with GFP mRNA and Sox2 mRNA using nanoparticles and Lipofectamine. (D) Number of neurospheres generated by transfections HFFs with Sox2 using nanoparticles and Lipofectamine2000. * and *** indicates that groups are statistically different from one other (p<0.05). Color images available online at www.liebertpub.com/tec