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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

Electron microscopy of nanocomplexes.Notes: Negative staining transmission electron microscopy was used to visualize (A) LYD nanoparticles before and after concentration, (B) PDL nanoparticles before and after concentration, and (C) PRL nanoparticles before and after concentration. Scale bar =500 nm for all nanoparticles. 300 g/L dextran was used to concentrate all three different nanoparticle formulations.Abbreviations: LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA; PDL, peptide Y, DNA, liposome LAP1; PRL, peptide Y or RVG-9R, siRNA, liposome LAP2.
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f2-ijn-10-2673: Electron microscopy of nanocomplexes.Notes: Negative staining transmission electron microscopy was used to visualize (A) LYD nanoparticles before and after concentration, (B) PDL nanoparticles before and after concentration, and (C) PRL nanoparticles before and after concentration. Scale bar =500 nm for all nanoparticles. 300 g/L dextran was used to concentrate all three different nanoparticle formulations.Abbreviations: LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA; PDL, peptide Y, DNA, liposome LAP1; PRL, peptide Y or RVG-9R, siRNA, liposome LAP2.

Mentions: The sizes and zeta potentials (Figure 1B) of LYD nanocomplexes were determined before and after concentrating with different dextran solutions (100–300 g/L). There was no statistical difference in either the size or the zeta potential before and after concentration (size ranges: 98–137 nm and zeta potential: +64–69 mV), indicating that this procedure does not alter these biophysical properties. All PDI measurements before and after concentration were <0.3, indicating monodisperse nanoparticle populations. Cationic (LYD) and anionic (PDL and PRL) nanoparticle formulations16,21 were further characterized by negative staining TEM to determine their shape and morphology before and after concentration using 300 g/L of dextran (Figure 2). Most nanocomplexes were spheres (Figure 2A–C), but with some rods (in LYD and PDL; Figure 2A and B) and some toroidal structures (Figure 2A and B). The majority of the spherical particles measured by TEM for each formulation were in the range determined by DLS with no obvious differences between formulations before and after concentration.


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)

Electron microscopy of nanocomplexes.Notes: Negative staining transmission electron microscopy was used to visualize (A) LYD nanoparticles before and after concentration, (B) PDL nanoparticles before and after concentration, and (C) PRL nanoparticles before and after concentration. Scale bar =500 nm for all nanoparticles. 300 g/L dextran was used to concentrate all three different nanoparticle formulations.Abbreviations: LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA; PDL, peptide Y, DNA, liposome LAP1; 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

f2-ijn-10-2673: Electron microscopy of nanocomplexes.Notes: Negative staining transmission electron microscopy was used to visualize (A) LYD nanoparticles before and after concentration, (B) PDL nanoparticles before and after concentration, and (C) PRL nanoparticles before and after concentration. Scale bar =500 nm for all nanoparticles. 300 g/L dextran was used to concentrate all three different nanoparticle formulations.Abbreviations: LYD, liposome 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), peptide Y, and DNA; PDL, peptide Y, DNA, liposome LAP1; PRL, peptide Y or RVG-9R, siRNA, liposome LAP2.
Mentions: The sizes and zeta potentials (Figure 1B) of LYD nanocomplexes were determined before and after concentrating with different dextran solutions (100–300 g/L). There was no statistical difference in either the size or the zeta potential before and after concentration (size ranges: 98–137 nm and zeta potential: +64–69 mV), indicating that this procedure does not alter these biophysical properties. All PDI measurements before and after concentration were <0.3, indicating monodisperse nanoparticle populations. Cationic (LYD) and anionic (PDL and PRL) nanoparticle formulations16,21 were further characterized by negative staining TEM to determine their shape and morphology before and after concentration using 300 g/L of dextran (Figure 2). Most nanocomplexes were spheres (Figure 2A–C), but with some rods (in LYD and PDL; Figure 2A and B) and some toroidal structures (Figure 2A and B). The majority of the spherical particles measured by TEM for each formulation were in the range determined by DLS with no obvious differences between formulations before and after concentration.

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