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

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Purpose.

A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.

Methods.

Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.

Results.

Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.

Conclusions.

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.

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Abbreviations

Chol:

cholesterol

DODAC:

dioleyldimethylammonium chloride

DODAP:

1,2-dioleoyl-N,N-dimethyl-3-aminopropane

DODMA:

1,2-dioleyloxy-N,N-dimethylaminopropane

DOPE:

dioleoylphosphatidylethanolamine

DOPG:

dioleoylphosphatidylglycerol

DSPC:

distearoylphosphatidylcholine

EPC:

egg phosphatidylcholine

HBS:

Hepes buffered saline

3H-CHE:

tritium-labeled cholesteryl hexadecyl ether

OGP:

octylglucopyranoside

PBS:

phosphate buffered saline

PEG-CerC20:

1-O-(2′-(ω-methoxypolyethyleneglycol)2000)-2-N-arachidoylsphingosine

PEG-S-DSG:

3-O-(2′(ω-methoxypolyethyleneglycol)2000)-1,2-distearoyl-sn-glycerol

QELS:

quasi-elastic light scattering

SPLP:

stabilized plasmid lipid particles

SVF:

spontaneous vesicle formation

TE:

Tris EDTA buffer

TEM:

transmission electron microscopy

TNS:

potassium 2-(p-toluidino)-6-naphthalenesulfonic acid

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Authors and Affiliations

  1. Protiva Biotherapeutics Inc., Burnaby, British Columbia, Canada, V5G 4Y1

    Lloyd B. Jeffs, Lorne R. Palmer, Ellen G. Ambegia, Cory Giesbrecht, Shannon Ewanick & Ian MacLachlan

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  1. Lloyd B. Jeffs
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  2. Lorne R. Palmer
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  3. Ellen G. Ambegia
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  6. Ian MacLachlan
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Correspondence to Ian MacLachlan.

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Jeffs, L., Palmer, L., Ambegia, E. et al. A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA. Pharm Res 22, 362–372 (2005). https://doi.org/10.1007/s11095-004-1873-z

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