Transfection efficiency serum effect

Chemg, J.Y., Van de Wetering, R, Talsma, H., Crommelin, D.J.A., Hennink, W.E. (1996). Effect of size and serum proteins on transfection efficiency of poly ((2-dime-thylamino)ethyl methacrylate)-plasmid nanoparticles. Pharm. Res., 13, 1038-1042. 

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]. 

Fig. 3. Property of gene delivery with BLs and US exposure (a) Schema of transfection mechanism by BLs and US. The mechanical effect based on the disruption of BLs by US exposure, which results in generation of some pores on plasma membrane, is associated with direct delivery of extracellular plasmid DNA into cytosol, (b) Luciferase expression in COS-7 cells transfected by BLs and US. COS-7 cells (1x10 cells/500 pLAube) were mixed wifh pCMV-Luc (5 pg) and BLs (60 pg). The cell mixture was exposed with US (Frequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/ cm. Time 10 s). The cells were washed and cultured for 2 days. Affer fhaf, luciferase acfivify was measured, (c) Effecf of US condition on transfection efficiency with BLs. COS-7 cells were exposed with US (Frequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/cm Time 0,1, 5,10 s) in the presence of pCMV-Luc (0.25 pg) and BLs (60 pg). Luciferase activity was measured as above, (d) Effect of serum on transfection efficiency of BLs. COS-7 cells in the medium containing EBS (0,10, 30, 50% (v/v)) were treated with US (Erequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/cm, Time 10 s), pCMV-Luc (0.25 pg) and BLs (60 pg) or transfected with lipoplex of pCMV-Luc (0.25 pg) and lipofectin (1.25 pg). (e) In vitro gene delivery to various types of cell using BLs and US. The method of gene delivery was same as above. S-180 mouse sarcoma cells, Colon26 mouse colon adenocarcinoma cells, B16BL6 mouse melanoma cells, Jurkat human T cell line, HUVEC human umbilical endothelial cells. Luciferase activity was measured as above. <10 RLU/mg protein, <10 RLU/mg protein Each data represents the mean S.D. n=3). L PEG-liposomes, LF Lipotectin...

Kim J K, et al. (2003). Enhancement of polyethylene glycol (PEG)-modified cationic liposome-mediated gene deliveries Effects on serum stability and transfection efficiency. J. Pharm. Pharmacol. 55 453-460. 

On the other hand, PEI nanocarriers have some toxicity problems. They are reported to show two types of toxicity one of them is immediate toxicity due to free PEI. Free PEI interacts with serum proteins which have a negative charge and also erythrocytes. This interaction results in precipitation in huge clusters, adherence to the cell membrane and damage to the plasma membrane. The other type is delayed toxicity as a consequence of cellular processing of the PEI polyplexes. Another toxicity problem also arises from the linear structure of PEI. When PEI polyplexes were administered via the intravenous route to mice, lethal side effects were observed. On the other hand, linear PEIs have higher transfection efficiency and lower cytotoxicity than branched PEIs, according to several studies. ... 

Sato, T, Ishii, T., and Okahata, Y. 2001. In vitro gene delivery mediated by chitosan. Effect of pH, serum, and molecular mass of chitosan on the transfection efficiency. Biomaterials 22 2075-2080. 

The presence of fetal calf serum (FCS) did not affect the transfection efficiency of the chitoplexes, whereas the transfection efficiency of DOTAP-DNA complexes was decreased. Cells remained 100% viable in the presence of chitosan oligomers whereas viability of DOTAP treated cells decreased to 50% in both cell lines. Both DOTAP-DNA lipoplexes and chitoplexes resulted in less transfection efficiency in Caco-2 cell cultures than in COS-1 cells however, quaternized chitosan oligomers proved to be superior to DOTAP. The effects on the viability of Caco-2 cells were similar to the effects observed in COS-1 cells. This report is also in line with the earlier one suggesting that chitosan-DNA complexes present suitable characteristics and less cytotoxic compared to lipid gene carriers and thus has the potential to be used as gene delivery vectors. 

The effect of serum on transfection efficiency is very important for the practical applications of gene therapy in vivo because transfection efficiency in vivo is inhibited by serum (Goldman et al. 1997). Sato et al. (2001) studied the effect of serum on the transfection efficiency of the complexes in vitro. The results showed that the transfection efficiency of the complexes was increased at np to 20% serum due to the increased cell function by the serum, and was decreased at 50% serum owing to cell damage, although PEl-mediated transfection was decreased at 10% semm (Erbacher et al. 1998). 

Sato et al. studied the effect of pH on transfection of A549 cells because the pH of the culture medium affects the transfection efficiency of chitosan/DNA complexes [21]. The results showed that the transfection efficiency at pH 6.9 was higher than that of pH 7.6 because chitosan/DNA complexes at pH 6.9 are positively charged (due to the chitosan pKa value of 6-6.8) and can bind to negatively charged cells. Nimesh et al. reported enhanced transfecticMi efficiency in HEK293 cells when transfection was initiated at pH 6.5 with 10% serum for 8-24 h and continued at pH 7.4 with 10% serum for an additional 24—40 h [22]. 

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