Serum Stability Of Optimized Nucleic Acidliposome Complexes For Use In Vivo

Serum stability of cationic complexes is complicated and cannot be assessed by simply performing studies at a random concentration of serum. Figure 8 shows results from serum stability studies of DNA-liposome complexes that have been optimized in our laboratory for systemic delivery. Serum stability of these complexes was studied at 37°C out to 24 h at concentrations of serum ranging from 0% to 100%. Two different serum stability assays were performed. The first assay measured the OD400 of BIV DOTAP:Chol-DNA liposome complexes added into tubes containing a different concentration of serum in each tube, ranging from 0% to 100%. The tubes were incubated at 37°C, and small aliquots from each tube were removed at various time points out to 24 h. The OD400 of each aliquot was measured on a spectrophotometer calibrated to accurately measure turbidity. Previous work in our laboratory demonstrated that the OD400 predicted both the stability of the complexes and the transfection efficiency results obtained for multiple organs after intravenous injections (14,28). Percent stability for this assay is defined as the transfection efficiency that is obtained at a particular OD400 of the complexes used for intravenous injections. Therefore, this assay is rigorous because slight declines in OD400 of these complexes result in obtaining no transfection in vivo. Declines in the 0D400 also measure precipitation of the complexes.

A second assay was performed to support the results obtained from the OD400 measurements described above. A different concentration of serum, ranging from 0% to 100%, was placed into each well of a 96-well microtiter dish. BIV D0TAP:Chol-DNA liposome complexes were added to the serum in the wells, and the plate was incubated at 37°C. The plate was removed at various time points out to 24 h and complexes in the wells were observed under the microscope. Precipitation of complexes in the wells was assessed. Stability (100%) was set at no precipitation observed. Results from this assay were compared with those obtained in the first assay. Stability (100%) of complexes was set at no decline of 0D400 in assay #1 and no observed precipitation in assay #2 at each % serum concentration, and the results were plotted (Fig. 8).

1. Unmasking of targeted liposome-DNA complex

2. Fusion of

Figure 7 Optimized strategy for delivery and gene expression in the target cell. Optimization of many steps is required to achieve targeted delivery, deshielding, fusion with the cell membrane, entry of nucleic acids into the cell and to the nucleus, and production of gene expression of a cDNA cloned in a plasmid.

Figure 7 Optimized strategy for delivery and gene expression in the target cell. Optimization of many steps is required to achieve targeted delivery, deshielding, fusion with the cell membrane, entry of nucleic acids into the cell and to the nucleus, and production of gene expression of a cDNA cloned in a plasmid.

0 0

Post a comment