For the past years, the CRISPR-cas9 system has been studied as a new genome editing tool that could lead to new gene therapy strategies and improve human health. However, the delivery of such a system to a cell has remained a challenge.
Lipid-based nanoparticles (LNPs) are versatile, have low immunogenicity, and are easy to manufacture – all these characteristics make liposomes an attractive potential delivery vector for CRISPR/Cas9 transportation through cell membranes [1]. This research project investigated stability and protein encapsulation efficiency of different liposome sizes and compositions.
Based on previous studies there were chosen 4 different LNP compositions [2]. All LNPs were made using these lipids: Dipalmitoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), cholesterol, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioctadecenoyl-sn-glycero-3- Phosphoserine (DOPS), PEG-lipids and ALC-0315 [2]. The size and stability of LNPs were measured using the dynamic light scattering technique (DLS, ZetaSizer). Then, different sizes of LNPs were prepared using membranes with 40nm, 200nm, and 400nm pores [3]. The encapsulation efficiency of these LNPs was investigated by measuring the fluorescence intensity of encapsulated model protein BSA- Alexa Fluor 488 or Cas9 – GFP protein by Fluorometer (Spectrofluorometer FS5) and HPLC.
The results showed that the concentration of PEG and cholesterol changes the stability and size of LNPs. Composition DOPC/DOPE/chol/DOTAP/ALC-0315/DMG-PEG 20/10/20/15/5 was determined as the most stable one. LNPs prepared with 200nm pores showed the best encapsulation efficiency – 70% and had the smallest polydispersity index.