Cellular Uptake

Adsorptive endocytosis is a common method of uptake for nonviral DNA vectors, as seen under in vitro conditions [49,85]. Unfortunately, this method only passively targets cells, resulting in gene carriers adsorbing onto cells with surface glycoproteins that provide proper adhesive environments [171,172]. Viral DNA vectors, on the other hand, enter cells specifically and efficiently through the use of receptor-ligand interactions [173]. To impart nonviral DNA vectors with cellular uptake capabilities similar to viruses, various ligands have been attached to the vector surface, both covalently and noncovalently [86]. Attached ligands bind to their complementary cell surface receptors, resulting in enhanced rates of uptake and improved target cell specificity [174]. Examples of ligands used for active targeting include transferrin [175,176], epidermal growth factor (EGF) [174], and the integrin-binding RGD sequences [177,178]. RGD peptides associated with cationic liposomes were used to transfer genes into human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in vitro [179]. Use of the RGD sequence enhanced uptake via endocytosis and resulted in a 10-fold increase in gene expression.

The ability to modify the surface of gene carriers with relative ease is an advantage of nonviral systems. Viruses have a specific repertoire of surface receptors that may induce endocytosis [173], and it is not trivial to attach foreign ligands to viral gene carriers to modify their target cell specificity. Recently, however, bifunctional molecules that bind adenoviruses at one end and integrins at the other have been used to alter the specificity of the viruses [180].

Ligand attachment to DNA carriers may not only affect cellular uptake but may also have important implications on their intracellular trafficking [181]. Ligand choice should, therefore, consider intracellular trafficking in addition to target cell specificity and increased uptake.

High levels of intracellular DNA do not always correlate with high levels of protein production [182], suggesting that cellular uptake is not always a major bottleneck in gene delivery. However, this may be specific to cell and/or gene carrier type. For example, differentiated airway epithelial cells exhibited lower uptake than their poorly differentiated counterparts and, therefore, had lower expression of the cationic liposome-delivered gene [65]. Another study showed that adenovirus-mediated gene delivery into well-differentiated cultured airway epithelial cells was inefficient, due to a lack of adenovirus fiber-knob receptors and aVb3/5 integrins on the cells as well as to the low level of apical plasma membrane uptake [183]. Limited apical membrane uptake was also implicated as a potential explanation for low efficiencies in cationic lipid-mediated gene delivery to human primary cultures of ciliated airway epithelia [184].

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