Pharmaceutical Research

, Volume 22, Issue 3, pp 362–372

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

Authors

  • Lloyd B. Jeffs
    • Protiva Biotherapeutics Inc.
  • Lorne R. Palmer
    • Protiva Biotherapeutics Inc.
  • Ellen G. Ambegia
    • Protiva Biotherapeutics Inc.
  • Cory Giesbrecht
    • Protiva Biotherapeutics Inc.
  • Shannon Ewanick
    • Protiva Biotherapeutics Inc.
    • Protiva Biotherapeutics Inc.
Research Papers

DOI: 10.1007/s11095-004-1873-z

Cite this article as:
Jeffs, L., Palmer, L., Ambegia, E. et al. Pharm Res (2005) 22: 362. doi:10.1007/s11095-004-1873-z

No Heading

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.

Key Words:

DNAliposomeplasmidsystemic gene delivery

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

Copyright information

© Springer Science+Business Media, Inc. 2005