Elucidating the mechanism of cellular uptake
Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific.Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake.When contemplating NP delivery into cells, the main cell uptake routes are: (i) specific uptake, such as receptor-mediated endocytosis, including both clathrin- and caveolin-mediated and (ii) non-specific uptake, typically pinocytosis (or ‘cell drinking’) .Caveolae are typically 50-100 nm sized flask- shaped membrane pits, formed by the inward budding of plasma membrane, that are found on the surfaces of smooth muscle cells, endothelial cells and fibroblasts, and are involved in clathrin-independent endocytosis .
Dowdy suggested in 2001 that they are basically charged , with 48-60 amino acid residues being mainly responsible for the translocation properties of the membrane .
Due to their small size they have the capability to cross the highly electrically resistant blood brain barrier as well as tight junctions .
Recent advances in nanotechnology allow functionalisation of NPs with ligands for various therapeutic and diagnostic applications including bio-sensing, magnetic resonance imaging (MRI), site-specific drug delivery, stem cell tracking and treatment of hyperthermia .
Other, naturally occurring CPPs include the 60 amino acid homeodomain of Antennapedia [17,18] and herpes simplex virus type 1 protein , both of which are able to translocate the cell membrane.
The discovery of these natural peptides was followed by the development of various synthetic analogues of tat peptides such as penetratin, a peptide of 16 amino acids, derived from the DNA binding domain of the Antennapedia homeoprotein, which is currently the second most commonly used CPP in NP research after the HIV tat peptide .
Therefore, this paper employs two different blockers of endocytosis, Pitstop 2 and Dyngo 4a, in a bid to determine the cellular uptake route.