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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

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 LAP2; conc, concentrated; PDL, peptide Y, DNA, liposome LAP1; P, peptides; R, siRNA; D, DNA; h, hours.
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f3-ijn-10-2673: 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 LAP2; conc, concentrated; PDL, peptide Y, DNA, liposome LAP1; P, peptides; R, siRNA; D, DNA; h, hours.

Mentions: LYD nanoparticles were concentrated using different amounts of dextran (100–300 g/L), and were then used for the transfection of Neuro-2A cells (Figure 3A). There was no statistical difference in the transfection efficiency between the formulations irrespective of the amount of dextran used, or if they were concentrated or nonconcentrated. The same was true when the LYD nanoparticles that were concentrated against 300 g/L of dextran were used for the transfection of HBE cells (Figure 3B).


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 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 LAP2; conc, concentrated; PDL, peptide Y, DNA, liposome LAP1; P, peptides; R, siRNA; D, DNA; h, hours.
© Copyright Policy
Related In: Results  -  Collection

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

f3-ijn-10-2673: 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 LAP2; conc, concentrated; PDL, peptide Y, DNA, liposome LAP1; P, peptides; R, siRNA; D, DNA; h, hours.
Mentions: LYD nanoparticles were concentrated using different amounts of dextran (100–300 g/L), and were then used for the transfection of Neuro-2A cells (Figure 3A). There was no statistical difference in the transfection efficiency between the formulations irrespective of the amount of dextran used, or if they were concentrated or nonconcentrated. The same was true when the LYD nanoparticles that were concentrated against 300 g/L of dextran were used for the transfection of HBE cells (Figure 3B).

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