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

Tagalakis AD, Castellaro S, Zhou H, Bienemann A, Munye MM, McCarthy D, White EA, Hart SL - Int J Nanomedicine (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental and Personalised Medicine Section, University College London (UCL) Institute of Child Health, London, UK.

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.

No MeSH data available.


Related in: MedlinePlus

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 LAP2; RVG, rabies virus glycoprotein targeting peptide; PR, RVG-9R/BACE1siRNA; PEG, polyethylene glycol; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
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f5-ijn-10-2673: 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 LAP2; RVG, rabies virus glycoprotein targeting peptide; PR, RVG-9R/BACE1siRNA; PEG, polyethylene glycol; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Mentions: Ex vivo silencing studies were first performed with brain explant tissue transfected with 100 nM siRNA in anionic PRL nanoparticles containing RVG-9R or PR nanoparticles containing RVG-9R and siRNA (RVG-9R/siRNA). The analysis of silencing by qRT-PCR revealed about 40% silencing of BACE1 with PRL nanoparticles (P<0.05) and no silencing was achieved with RVG-9R/BACE1R complexes (Figure 5A). L2K, which was used as a positive control, achieved 54% silencing compared to its irrelevant siRNA control (P<0.01). Western blotting revealed 32% silencing of BACE1 protein (Figure 5B). We then progressed to in vivo studies with just RVG-9R-targeted anionic PRL nanoparticles.


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

Tagalakis AD, Castellaro S, Zhou H, Bienemann A, Munye MM, McCarthy D, White EA, Hart SL - Int J Nanomedicine (2015)

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 LAP2; RVG, rabies virus glycoprotein targeting peptide; PR, RVG-9R/BACE1siRNA; PEG, polyethylene glycol; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4388080&req=5

f5-ijn-10-2673: 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 LAP2; RVG, rabies virus glycoprotein targeting peptide; PR, RVG-9R/BACE1siRNA; PEG, polyethylene glycol; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
Mentions: Ex vivo silencing studies were first performed with brain explant tissue transfected with 100 nM siRNA in anionic PRL nanoparticles containing RVG-9R or PR nanoparticles containing RVG-9R and siRNA (RVG-9R/siRNA). The analysis of silencing by qRT-PCR revealed about 40% silencing of BACE1 with PRL nanoparticles (P<0.05) and no silencing was achieved with RVG-9R/BACE1R complexes (Figure 5A). L2K, which was used as a positive control, achieved 54% silencing compared to its irrelevant siRNA control (P<0.01). Western blotting revealed 32% silencing of BACE1 protein (Figure 5B). We then progressed to in vivo studies with just RVG-9R-targeted anionic PRL nanoparticles.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental and Personalised Medicine Section, University College London (UCL) Institute of Child Health, London, UK.

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.

No MeSH data available.


Related in: MedlinePlus