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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

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.
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f1-ijn-10-2673: 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.

Mentions: Concentration of LYD nanoparticles19 by reverse dialysis was first performed through cellulose dialysis membrane (MWCO: 14,000) against solutions of dextran (MW: 150,000) dissolved in a range of concentrations. The high osmotic pressure of the dextran solutions leads to the displacement of water from the tubing into the surrounding dextran solution. Figure 1A shows the change in concentration of nanocomplexes achieved over time from a starting concentration of 0.2 mg/mL with respect to the DNA with varying dextran concentrations. The lowest dextran concentration of 100 g/L resulted in a 30% increase in nanocomplex concentration after 9 hours of dialysis, whereas the 150 g/L, 200 g/L, 250 g/L, and 300 g/L concentrations resulted in 3.9-fold (in 8.5 hours), 2.7-fold (in 9 hours), 8.2-fold (in 6 hours), and 23.9-fold (in 5 hours and 15 minutes) increases, respectively. When longer time points were assessed for dextran at 100 g/L (24 hours) and 150 g/L (15 hours and 15 minutes), 2.25-fold and 10.6-fold increases in DNA concentration were achieved, respectively. At dextran concentrations of >150 g/L nanoparticle concentrations of 0.59–4.6 mg/mL were achieved, which is in the target range for possible clinical applications.10,29


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f1-ijn-10-2673: 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.
Mentions: Concentration of LYD nanoparticles19 by reverse dialysis was first performed through cellulose dialysis membrane (MWCO: 14,000) against solutions of dextran (MW: 150,000) dissolved in a range of concentrations. The high osmotic pressure of the dextran solutions leads to the displacement of water from the tubing into the surrounding dextran solution. Figure 1A shows the change in concentration of nanocomplexes achieved over time from a starting concentration of 0.2 mg/mL with respect to the DNA with varying dextran concentrations. The lowest dextran concentration of 100 g/L resulted in a 30% increase in nanocomplex concentration after 9 hours of dialysis, whereas the 150 g/L, 200 g/L, 250 g/L, and 300 g/L concentrations resulted in 3.9-fold (in 8.5 hours), 2.7-fold (in 9 hours), 8.2-fold (in 6 hours), and 23.9-fold (in 5 hours and 15 minutes) increases, respectively. When longer time points were assessed for dextran at 100 g/L (24 hours) and 150 g/L (15 hours and 15 minutes), 2.25-fold and 10.6-fold increases in DNA concentration were achieved, respectively. At dextran concentrations of >150 g/L nanoparticle concentrations of 0.59–4.6 mg/mL were achieved, which is in the target range for possible clinical applications.10,29

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