Transfection, mechanism

The transfection mechanism of plasmid-chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas-Red-labeled chitosan. Several factors affect transfection activity and cell uptake, for example the molecular mass of chitosan, stoichiometry of complex, seriun concentration and the pH of the transfection medium. The level of transfection with plasmid-chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, the ratio of chitosan nitrogen to DNA phosphate was 5, and serum at pH 7.0 was 10%. Plasmid-chitosan complexes most likely condense to form large aggregates (5-8 p,m), which absorb to the cell surface. After this, plasmid-chitosan complexes are endocytosed, and accumulate in the nucleus [97]. 

Three families of polymers have been used to study transfection mechanisms polyamines, polyamides, and polyvinyl type polymers. The transfection efficiencies achievable with these systems vary widely, so an in-depth analysis of each polymer family and subsequent comparison of what affects gene delivery will be discussed in this chapter. In addition to high transfection efficiency, it is important for the polymeric systems to be relatively nontoxic to cells in vitro and not to elicit an immune response in vivo. Thus, the effect of transfection parameters on cytotoxicity and immunogenicity will also be examined. 

The Effect of CL-DNA Complex Structure on Transfection Mechanism and Efficiency... 

The non-lamellar DL/DOPC-DNA complexes exhibit enhanced TE (over lamellar complexes of the same aM) in the aM regime where release of DNA from the complex is thought to be limiting TE. The structure of the Hj0 complexes gives a clue as to its possible role in the transfection mechanism and high TE. In contrast to the Lyc phase, both the H c phase and the distorted hexagonal phase exhibit a continuous sub-structure of DNA within the complexes. The DNA bundle phase... 

Therefore, improved chemical synthesis strategies for the design of tailored polymeric gene vectors and more information on transfection mechanisms will be needed for the optimization of DNA and siRNA vehicles. 

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...

To develop an efficient gene delivery system, it seems necessary to understand the extra- and intracellular processes involved in the overall transfection mechanism. This will lead to understanding the mechanism, which is necessary for developing novel lipid-based non-viral vectors. For this purpose, cationic liposomes and pDNA are used widely to understand the cellular mechanism involved in the transfection (Fig. 13.3). 

Ishii et al. [64] explained the transfection mechanism of chitosan/DNA complexes in relation to cell uptake. They used fluorescein isothiocyanate-labeled plasmid DNA and Texas Red-labeled chitosan. They found that the transfection of chitosan/DNA complexes was higher with the molecular weight of chitosan at 40 or 84 kDa and N/P ratio at 5 (Figure 20.4). The transfection... 

DNA molecules (polyplexes). Although there are a lot of reports on chitosan-based DNA nanocarriers varying in sizes (20-250 nm and higher), studies regarding effects and the chitosan-specific transfection mechanisms remain insufficient. It is shown that the level of transfection depends upon several factors, for instance chitosan molecular weight (from 22 kDa [65] to 400 kDa [66]), the ratio of chitosan nitrogen to DNA phosphate (N/P ratio), as well as serum concentration and pH of transfection medium, and finally on cell line [67],... 

The first idea to consider is the effect of receptor density on sensitivity of a functional system to agonists. Clearly, if quanta of stimulus are delivered to the stimulus-response mechanism of a cell per activated receptor the amount of the total stimulus will be directly proportional to the number of receptors activated. Figure 5.8 shows Gi-protein-mediated responses of melanophores transiently transfected with cDNA for human neuropeptide Y-l receptors. As can be seen from this figure, increasing receptor expression (transfection with increasing concentrations of receptor cDNA) causes an increased potency and maximal response to the neuropeptide Y agonist PYY. 

The process by which cells take up large molecules is called endocytosis. Some of these molecules (eg, polysaccharides, proteins, and polynucleotides), when hydrolyzed inside the cell, yield nutrients. Endocytosis provides a mechanism for regulating the content of certain membrane components, hormone receptors being a case in point. Endocytosis can be used to learn more about how cells function. DNA from one cell type can be used to transfect a different cell and alter the latter s function or phenotype. A specific gene is often employed in these experiments, and this provides a unique way to smdy and analyze the regulation of that gene. DNA transfection depends upon endocytosis endocy-... 

The study of active transport mechanisms has grown substantially in recent years, with transport proteins such as P-gp, BCRP, and MRP-2 among the most studied [59]. Several types of in vitro assays to assess substrates of transporters have been established these include assays directed toward intestinal and biliary efflux [60]. Assays that measure passive and active transport are also used to assess penetration of the blood-brain barrier. In addition to the assays described above, transfected cell lines that overexpress transporters present in the blood-brain barrier are also employed [61]. 

Besides this purported action on DAT, amphetamine has also been suggested to act upon the vesicular transporter as well. Pifl et al.87 examined COS cells transfected with cDNA for either DAT or the vesicular transporter, or both. A marked increase in DA release was noted in cells that expressed both DAT and the vesicular transporter when compared to the release from cells that express only DAT or the vesicular transporter. The mechanism of action for amphetamine was further defined with the work of Giros et al.59 In transgenic mice lacking the DAT, amphetamine did not produce hyperlocomotion or release DA. 

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