A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

Abstract

Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1. This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo.

Keywords: DNA; anionic liposome; concentration; nanoparticles; siRNA; targeted gene delivery.

Figure 1

Effect of different concentrations of dextran on concentrating cationic LYD nanoparticles and on size and zeta potential. Notes: (A) Kinetics of the concentration of LYD nanoparticles over time when varying the concentration of dextran (100–300 g/L). (B) Size and charge measurements of LYD nanoparticles by dynamic light scattering before and after concentration with different amounts of dextran (100–300 g/L). Abbreviation: LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA.

Figure 2

Electron microscopy of nanocomplexes. Notes: Negative staining transmission electron microscopy was used to visualize (A) LYD nanoparticles before and after concentration, (B) PDL nanoparticles before and after concentration, and (C) PRL nanoparticles before and after concentration. Scale bar =500 nm for all nanoparticles. 300 g/L dextran was used to concentrate all three different nanoparticle formulations. Abbreviations: LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA; PDL, peptide Y, DNA, liposome L^AP1; PRL, peptide Y or RVG-9R, siRNA, liposome L^AP2.

Figure 3

In vitro transfections with concentrated nanocomplexes retain transfection efficiency with lack of cytotoxicity. Notes: (A) A total of 1.5 mL of LYD nanocomplexes were concentrated using different amounts of dextran (100–300 g/L) and were used in luciferase transfections in Neuro-2A cells. (B) LYD nanoparticles before and after concentration (concentrated for 3 hours and 5 hours, 15 minutes) were used in luciferase transfections in HBE cells. (C) siRNA silencing from anionic PRL nanocomplexes (with peptide Y) before or after concentration made at a 4:3:1 molar charge ratio using siRNA targeting luciferase in Neuro-2A-Luc cells at 50 nM. 24 hours later, luciferase assays were performed. L2K/siRNA nanocomplexes were used as a positive control in all in vitro silencing experiments. (D) Viability of Neuro-2A cells following transfection for 24 hours with cationic LYD and anionic PDL and PRL nanocomplexes. Cationic nanocomplexes were made at a weight ratio of 1:4:1 (liposome:peptide:DNA) and the anionic nanocomplexes at a molar charge ratio of 4:3:1 (liposome:peptide:siRNA). The viability values were normalized to the untransfected control cells. The dextran concentration in the counter-dialyzing solution was kept constant (300 g/L) in Figure 3B–D. All transfections were performed in groups of six and mean values were calculated. Asterisks indicate comparisons of specific formulations with statistical significance (**P<0.01; ***P<0.001). Abbreviations: RLU, relative light units; LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA; HBE, human bronchial epithelial cells 16HBE14o–; siRNA, small interfering RNA; siRNA IRR, irrelevant control small interfering RNA; L2K, Lipofectamine^® 2000; PRL, peptide Y or RVG-9R, siRNA, liposome L^AP2; conc, concentrated; PDL, peptide Y, DNA, liposome L^AP1; P, peptides; R, siRNA; D, DNA; h, hours.

Figure 4

GFP transfection efficiency of nanocomplexes following concentration. Notes: One anionic PRL formulation was concentrated (300 g/L dextran) and then transfected Neuro-2A cells in serum-containing media. GFP expression was observed by epifluorescence microscopy 48 hours later. Representative cells are shown in (A) phase contrast and (B) transfected cells appear green (10× magnification). Abbreviations: GFP, green fluorescent protein; PRL, peptide Y or RVG-9R, siRNA, liposome L^AP2.

Figure 5

Nanocomplexes achieve silencing ex vivo, but not in the brain following intravenous administration. Notes: Mice brain explants were transfected ex vivo with peptide/siRNA complexes (PR: RVG-9R/BACE1siRNA; 4:1 weight ratio), anionic PEGylated PRL nanocomplexes with BACE1 siRNA, L2K, or complexes with irrelevant control siRNA (all at 100 nM), and then 48 hours post-transfection, the tissues were processed for analysis (A) by qRT-PCR and (B) by Western blot analysis of the BACE1 protein. Protein silencing was calculated with densitometric analysis using tubulin as loading control. (C) Anionic PRL nanocomplexes containing RVG-9R and BACE1 siRNA were concentrated using 300 g/L dextran over 3.5 hours, and this concentrated nanoparticle formulation was used (D) in intravenous injections. Mice were injected with 100 μL of anionic PRL nanoparticles containing 16 μg or 50 μg BACE1 siRNA or IRR siRNA, and 48 hours later, brains were processed for qRT-PCR analysis. The values are the means of three animals ± standard deviation. Asterisks indicate comparisons of specific formulations with statistical significance (*P<0.05; **P<0.01). Abbreviations: mRNA, messenger RNA; IRR, irrelevant control; siRNA, small interfering RNA; L2K, Lipofectamine^® 2000; PRL, peptide Y or RVG-9R, siRNA, liposome L^AP2; RVG, rabies virus glycoprotein targeting peptide; PR, RVG-9R/BACE1siRNA; PEG, polyethylene glycol; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure 6

In vivo silencing of BACE1 following CED administration of PEGylated concentrated nanoparticles or control saline into rat striatum. Notes: Concentrated anionic PEGylated PRL nanoparticles containing RVG-9R peptide and BACE1 siRNA or irrelevant control siRNA were administered by CED in the striatum of rats, and 48 hours postadministration, tissues were removed for qRT-PCR analysis of siRNA-induced silencing of the BACE1 gene. Values are the means of five animals ± standard deviation (n=3 for the untreated control animals and n=4 for the saline control animals) with one-way analysis of variance and Bonferroni’s post hoc analysis performed to calculate significant differences (*P<0.05; **P<0.01; ***P<0.001). Abbreviations: mRNA, messenger RNA; siRNA, small interfering RNA; CED, convection-enhanced delivery; PEG, polyethylene glycol; PRL, peptide Y or RVG-9R, siRNA, liposome L^AP2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; n, number.