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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

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 LAP2.
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f4-ijn-10-2673: 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 LAP2.

Mentions: The transfection efficiency of an anionic polyethylene glycol (PEG)ylated PDL formulation was evaluated with the plasmid expressing enhanced GFP in Neuro-2A cells. Fluorescent microscopy images provided evidence of the high transfection efficiency of PDL anionic nanoparticles following concentration (Figure 4).


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)

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 LAP2.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ijn-10-2673: 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 LAP2.
Mentions: The transfection efficiency of an anionic polyethylene glycol (PEG)ylated PDL formulation was evaluated with the plasmid expressing enhanced GFP in Neuro-2A cells. Fluorescent microscopy images provided evidence of the high transfection efficiency of PDL anionic nanoparticles following concentration (Figure 4).

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