Formulation of Lipid-Peptide-DNA Complexes for Gene Delivery

Jesper Clausen & Jesper Ernst

Studenteropgave: Speciale


Gene therapy is a promising therapeutic treatment that has the potential to cure many genetic disorders. DNA delivery into cells utilizing liposomes has been an area of interest for the last few decades. An emerging strategy is to formulate liposomes with specific peptides, such as cell-penetrating peptides, which has been shown to improve gene delivery. The aim of this research was to formulate lipid-peptide-DNA (LPD) complexes to enhance the transfection efficiency and cell viability. This was accomplished by a synthesis of two novel cationic lipids, having long hydrophobic tails (C14) and structurally different headgroups, such as a 1,3-propanediol or dimethylamine. These lipids, together with a previously studied cationic lipid with short hydrophobic tails (C11), but the same 1,3-propanediol headgroup, and the commercial co-cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) were tested with different peptides and the co-lipid 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE). Two of the peptides, KFF and SynB3, were synthesized and tested together with Penetratin and Neurotensin in this study. All formulations had a fixed molar ratio 3:2 (cationic parts:co-lipid), which were combined with plasmid DNA (pDNA) at four molar charge ratios and tested for their in vitro delivery into Chinese hamster ovary (CHO-K1) cells. We investigated both the formulation method and different formulations of lipoplexes and LPD complexes. All formulations were characterized by using particle sizing and gel electrophoresis, comprising the binding with and the protection of pDNA. The transfection efficiency and cell viability were evaluated by Beta-Glo® and MTS assays, respectively. An increase in transfection was observed when the peptide was added after hydration of the liposome in comparison with adding all components before hydration. Different lipoplexes and LPD complexes displayed synergistic effects and were able to compete with the positive control Lipofectamine® 2000. Formulations based on lipids with longer hydrophobic tails (C14) were generally associated with better transfection efficiency, but lower cell viability, compared to formulations based on lipids with shorter hydrophobic tails (C11). Overall, formulating liposomes with peptides has, in this instance, not been shown to improve transfection when compared with the lipoplexes without peptides, but it does still remain an area of potential since improvement has been shown in former cases, but further research into LPD complexes is needed.

UddannelserMolekylærbiologi, (Bachelor/kandidatuddannelse) KandidatKemi, (Bachelor/kandidatuddannelse) Kandidat
Udgivelsesdato2 jun. 2019
Antal sider59
VejledereWilliam Goldring & Håvard Jenssen