336. Impact of Convective Flow on the Cellular Uptake and Transfection Activity of Lipoplex and Adenovirus
Authors: Takahiro Fujiwara, Hidetaka Akita, Katsuko Furukawa, Takashi Ushida, Hideyoshi Harashima
Journal: Molecular Therapy (2006)

[Purpose] Targeting of gene vector to various organs via systemic circulation is an ideal concept for the non-invasive gene therapy. Although currently developed gene vectors represent high transfection activity in vitro, the expression is barely detectable in various organs except for the lung and liver in vivo. This discrepancy of transfection activity between in vivo and in vitro was derived from the differences of extracellular environment of target cells, such as blood flow. However, it is difficult to estimate the effect of fluid flow on the transfection activity, since current in vitro transfection studies have been carried out under the static condition. In the present study, I established endothelial cell culture system under convective flow and analyzed the impact of blood flow on cellular gene uptake and expression. As a model vectors, I selected LipofectAMINE PLUS (LFN) and adenovirus (Ad) which are taken up via absorptive-mediated and receptor-mediated endocytosis, respectively. [Methods] A parallel flow model was established for cell culture under convective flow. Mouse brain capillary endothelial cells (MBEC4) were cultured on the slide until reaching confluence. The slide was mounted on the parallel plate chamber, and was perfused by Krebs-Ringer bicarbonate medium. Under convective flow or stationary state, cells were incubated with pDNA/LFN complex or fiber modified Ad (RGD-Ad) encoding luciferase driven by CMV promoter for 30 minutes. After incubation, cells were detached from slide slip and DNA was purified from cell lysates for quantifying the number of gene copies by real time PCR. Luciferase activity was measured at 6 h post-transfection. [Results] Concerning the cellular uptake process, compared with static condition, uptake was increased by less than 10-fold whereas convective flow increased the total amount of vector passing through the chamber by 3-order of magnitude, suggesting that the flow severely inhibited cellular uptake by reducing the retention time in the chamber and/or by diminishing the affinity between the cell and vector. Furthermore, the extent of the stimulation of cellular uptake was comparable between LFN and Ad, suggesting that the effect of flow on the cellular uptake was not significant. The transfection activity of Ad was stimulated in parallel with the cellular uptake in shear stress dependent manner. In contrast, the gene expression of LFN was drastically increased (53.3-fold) under flow whereas cellular uptake increased slightly (1.90-fold). The comparable luciferase activity of stable transfectants expressing CMV-driven luciferase-encoding pDNA between the static and flow condition indicated that the intrinsic activities of endogenous transcription factors associated with the CMV promoter may not be affected by shear stress. Collectively, altered intracellular trafficking may be responsible for the improvement of lipoplex-mediated transfection activity in response to the shear stress.