Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025:2965:355-365.
doi: 10.1007/978-1-0716-4742-4_17.

Optimization of DOTAP/Cholesterol Cationic Lipid Nanoparticles for Nucleic Acid Delivery

Mengwei Sun  1 , Anthony J Di Pasqua  2
Affiliations

Optimization of DOTAP/Cholesterol Cationic Lipid Nanoparticles for Nucleic Acid Delivery

Mengwei Sun et al. Methods Mol Biol. 2025.

Abstract

Effective delivery methods are essentiel for the successful clinical application of nucleic acids. This chapter outlines a detailed protocol for the preparation, optimization, and characterization of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) lipid nanoparticles (LNPs) as efficient delivery systems for mRNA, plasmid DNA (pDNA), and oligonucleotides. We describe the formulation of LNPs using a thin-film hydration technique, optimization of key parameters (e.g., DOTAP/Chol ratios, PEGylation, and lipid-to-nucleic acid ratios), and their evaluation using physicochemical and biological assays. This protocol includes methodologies for gel retardation, particle size and zeta potential characterization, in vitro transfection assays, cytotoxicity testing, and long-term stability studies. By following this step-by-step guide, researchers can achieve reproducible results in designing LNPs for various therapeutic applications.

Keywords: Cholesterol; Cytotoxicity; DOTAP; Lipid nanoparticles; Nucleic acid delivery; PEGylation; Transfection efficiency.

PubMed Disclaimer

Similar articles

See all similar articles

References

    1. Nayerossadat N, Maedeh T, Ali PA (2012) Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 1:27 - PMC - PubMed
    1. Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15(8):541–555 - PubMed
    1. Hou X, Zaks T, Langer R, Dong Y (2021) Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6(12):1078–1094 - PMC - PubMed
    1. Cullis PR, Hope MJ (2017) Lipid nanoparticle systems for enabling gene therapies. Mol Ther 25(7):1467–1475 - PMC - PubMed
    1. Templeton NS (2009) Nonviral delivery for genomic therapy of cancer. World J Surg 33(4):685–697 - PubMed

LinkOut - more resources