A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA
Abstract
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 elongated 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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The ethanol dilution process produces small uniform lipid particles with a high RNA encapsulation efficiency.
“An ethanol dilution process (28) was used to produce small uniform lipid particles with a high RNA encapsulation efficiency.”
From Nonviral delivery of self-amplifying RNA vaccines (2012) · cites this paper as [28]
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- Authors
- Jeffs, L. B., Palmer, L. R., Ambegia, E. G., Giesbrecht, C., Ewanick, S., & MacLachlan, I.
- Corresponding author
- MacLachlan, Ian
- Venue
- Pharmaceutical Research
- Published
- 2005-03
- Volume
- 22
- Issue
- 3
- Pages
- 362-372
- ISSN
- 0724-8741 (ISSN-L)
- 1573-904X
- OpenAlex
- W1966334833
- Cited by
- 307
- References
- 48
- Citations by year
- 2026: 9 · 2025: 19 · 2024: 20 · 2023: 18 · 2022: 23 · 2021: 24 · 2020: 13 · 2019: 18 · 2018: 14 · 2017: 10 · 2016: 12 · 2015: 11 · 2014: 16 · 2013: 15 · 2012: 20
- FWCI
- 3.78
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Pharmaceutical Research (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advanced Drug Delivery Systems
- Keywords
- liposome encapsulation, plasmid DNA, non-viral gene therapy, systemic gene delivery, liposome stability, vesicle morphology
- MeSH terms
- Animals, DNA, Liposomes, Plasmids, Technology, Pharmaceutical, Mice
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