A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

Jeffs, Lloyd B.; Palmer, Lorne; Ambegia, Ellen; Giesbrecht, Cory; Ewanick, Shannon; MacLachlan, Ian

doi:10.1007/s11095-004-1873-z

Cited by 274 publications

(259 citation statements)

References 45 publications

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“…C12-200-siRNA formulations were prepared using a method adapted from Jeffs et al (34) Briefly, C12-200, distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG were solubilized in 90% ethanol at a molar ratio of 50∶10∶38.5∶1.5. The siRNA (or pool of siRNAs) was solubilized in 10 mM citrate, pH 3 buffer at a concentration of 0.4 mg∕mL.…”

Section: Methodsmentioning

confidence: 99%

Lipid-like materials for low-dose, in vivo gene silencing

Love

Mahon

Levins

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

835

803

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Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

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Section: Methodsmentioning

confidence: 99%

Lipid-like materials for low-dose, in vivo gene silencing

Love

Mahon

Levins

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

835

803

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“…The encapsulation efficiency of plasmid DNA contained in the prepared PEG-CLPs was determined by following a previously described protocol [26]. Briefly, we used PicoGreen (Molecular Probes, Eugene, OR, USA) a membrane impermeable dye that specifically binds to double-stranded DNA and is fluorescent.…”

Section: Measurement Of Pdna Encapsulation Efficiencymentioning

confidence: 99%

Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery

Kibria

Hatakeyama

Ohga

et al. 2011

Journal of Controlled Release

190

139

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“…The resulting particle size was ,200 nm and was monodispersed. 19 LNPs have been evaluated in several pipelines of clinical trials as stable nucleic acid lipid particles (SNALP). 20, 21 Rodrigueza et al reported the preparation of lipid-based nanoparticle (PNT2258) loading of a 24-base DNA oligonucleotide using a one-step method that is similar to the method of SNALP preparation, 22 and such nanoparticles have been evaluated in clinical trials.…”

Section: Introductionmentioning

confidence: 99%

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

Kubota¹,

Onishi²,

Sawaki³

et al. 2017

IJN

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Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment.

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A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

Abstract: This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.

Cited by 274 publications

References 45 publications

Lipid-like materials for low-dose, in vivo gene silencing

Lipid-like materials for low-dose, in vivo gene silencing

Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

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