Limits...
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

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 LAP2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; n, number.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4388080&req=5

f6-ijn-10-2673: 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 LAP2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; n, number.

Mentions: We then evaluated direct brain administration in rats of anionic PRL nanoparticles (with RVG-9R) incorporating either BACE1 siRNA or irrelevant control, administered by CED38,39 into rat striata, and BACE1 expression was examined 48 hours later. A significant reduction in BACE1 mRNA was observed (Figure 6) between the BACE1-treated group and the control groups (P<0.05 compared to the irrelevant control group; P<0.01 compared to the saline group; and P<0.001 compared to the untreated control group).


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)

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 LAP2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; n, number.
© Copyright Policy
Related In: Results  -  Collection

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

f6-ijn-10-2673: 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 LAP2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; n, number.
Mentions: We then evaluated direct brain administration in rats of anionic PRL nanoparticles (with RVG-9R) incorporating either BACE1 siRNA or irrelevant control, administered by CED38,39 into rat striata, and BACE1 expression was examined 48 hours later. A significant reduction in BACE1 mRNA was observed (Figure 6) between the BACE1-treated group and the control groups (P<0.05 compared to the irrelevant control group; P<0.01 compared to the saline group; and P<0.001 compared to the untreated control group).

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