Liposome transfection efficiency

To bridge this gap, liposomal transfection efficiency can be dramatically enhanced by the inclusion of peptides into the complex without increasing immunogenicity. Peptides can be selected to assist lipofection at each key stage of the process complex formation, cell targeting and uptake, endosomal disruption, and nuclear targeting. The purpose of this chapter is... 

Gregoriadis G, Saffle R, Hart SL. High yield incorporation of plasmid DNA within liposomes effect on DNA integrity and transfection efficiency. J Drug Target 1996 3 469 75. 

Obika S, Yu W, Shimoyama A, et al. Properties of cationic liposomes composed of cationic lipid YKS-220 having an ester linkage adequate stability, high transfection efficiency, and low cytotoxicity. Biol Pharm Bull 1999 22(2) 187-190. 

Peptide modification of liposomes offers the potential for enhancing the packaging process and for enhancing each stage of the lipofection process, to ultimately improve transfection efficiency. 

Integrin receptor-binding peptides have been used to enhance liposome binding, uptake, and expression (25,47 9). The inclusion of an 0(5pi integrin-targeted peptide into a liposomal complex enhanced transfection efficiency four- to five-fold in Jurkat cells and 10- to 13-fold in TF-1 cells (48). Confocal and electron microscopy revealed that the mechanism of cell entry conferred by RGD peptides on liposomes is predominantly by clathrin-coated endocytosis rather than by phagocytosis (50). 

Peptides can also help overcome the most significant drawback to using liposome vectors when compared to viral vectors, which is lower transfection efficiency. Additional benefits include promotion of compaction, assisting cellular uptake of the DNA. Even peptides derived from viruses themselves can be used to compensate this deficit (e.g., adenovirus p protein and the HIV TAT). 

It was believed that the main factors affecting transfection efficiency were the structure of the cationic lipid, the type of helper lipid used and their susceptibility to disruption by serum proteins. For gene transfer in vivo, apart from DOTMA-based liposomes, other complexes (in equimolar ratios) are also used—such as dioctade-cylamidoglicylspermidin (DLS)/DOPE (137), DOPE/DOTMA (1 1), DOPE/DOTAP (1 1) (138, 139), dimethyloctadecylammonium bromide (DDAB), and DOTAP with cholesterol (1 1) (mol/mol) (139). 

Spermine has been found to enhance the transfection efficiency of DNA-cationic liposome complexes in cell culture and in animal studies this biogenic polyamine at high concentrations caused liposome fusion most likely promoted by the simultaneous interaction of one molecule of spermine (four positively charged amino groups) with the polar head groups of two or more molecules of lipids. At low concentrations (0.03-0.1 mM) it promoted anchorage of the liposome-DNA complex to the surface of cells and enhanced significantly transfection efficiency. 

A number of factors for DOTAP-cholesterol/DNA complex preparation including the DNA/liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery maximal gene expression was obtained when a homogeneous population of DNA/liposome complexes (200-450 nm) was used. Cryoelectron microscopy showed that the DNA was condensed on the interior of liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (150). 

Modification of depolymerization kinetics and release Endosomal escape Nonviral gene delivery Boron neutron capture therapy Fusogenic liposomes, increase transfection efficiency... 

Matsui, H., Johnson, L.G., Randell, S.H. and Boucher, R.C. (1997) Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells. J. Biol. Chem., 272, 1117-1126. 

Liposomes used for transfection are either large unilamellar vesicles (LUVs) of 100 to 200 nm in diameter or small unilamellar vesicles (SUVs) of 20 to 100 nm. Liu et al.124 have reported that for a given liposome composition, multilamellar vesicles (MLVs) of 300 to 700 nm in diameter exhibit higher transfection efficiency than SUVs. However, more recent studies on the nature of the liposome-DNA complex (or lipoplex) revealed that lipoplexes from SUVs or MLVs do not differ significantly in size. On the other hand, the composition of the medium, not the type of the liposome used in the preparation of the lipoplex, plays a key role in determining the final size of the complex. And the transfection efficiency is also shown to depend on the final size of the complexes but not the type of the liposome.125... 

We then examined the effect of phospholipid composition on the transfection activity. Liposomes containing various combinations of phospholipids were tested for transfection activity on BHK-21 and HeLa-S3 cells. As described in HVJ-AVE liposomes, the cationic liposomes containing all of ePC, DOPE, and eSph in equal molar amounts showed the highest transfection efficiency both with BHK-21 and HeLa-S3 cells. The same results were obtained with Ltk-, HEK 293, and NB-1 cells. We also examined other phospholipids, but none was observed to be more effective. We then examined the effect of the cholesterol/phospholipid ratio on the transfection efficiency. The phospholipid composition (ePC DOPE eSph = 1 1 1) and DC-Chol content (10% of total... 

With the optimized lipid composition (opDC ePC DOPE eSph Choi DC-Chol = 5 5 5 12 3), the HVJ-cationic liposomes showed 100 to 800 times greater transfection efficiency in vitro compared with the conventional HVJ-PS liposomes. The presence of serum (10% FCS) in the transfection mixture did not decrease luciferase activity significantly. Even 70% FCS reduced the activity by less than 40%. LacZ gene expression showed that transfection efficiency of BHK-21 cells by optimized HVJ-cationic liposomes (opDC) and by conventional HVJ-cationic liposomes (DC) was 90-100% and 50-60%, respectively. With conventional HVJ-anionic liposomes (PS), LacZ expression was found in only 1-3% of the cells. The optimized HVJ-cationic liposomes were also much more effective for the transfer of FITC-labeled ODNs to cultured cells [16]. 

---