Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Abstract

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.

Octaarginine- and Octalysine-modified Nanoparticles Have Different Modes of Endosomal Escape

“…Previously, it was confirmed that cell surface-bound Cps can be effectively removed using a heparin wash (14,29). Four different concentrations of DNAcoated particles were tested, two of which were lower, and one of which was higher than the pDNA dose used for transfection, as shown in Fig.…”

“…The plasma membrane label PKH67 was used to label different endocytic vesicles. PKH67 stably incorporates a green fluorescent dye into the lipid region of cell membranes (29); thus, most internalized vesicles should be labeled regardless of the internalization pathway. Co-localization was quantified by counting free particles and particles co-localized with vesicles, including macropinosomes, in live cells after a 3-h incubation.…”

Arginine‐rich cell‐penetrating peptides

“…The diffuse staining was not due to the release of peptide from endosomes as it appeared first and the punctate staining later, and it continued to occur at 4°C when endocytosis is inhibited. Several other studies have also reported cellular uptake when the cells have been incubated at 4°C [26,[51][52][53]. It has also been observed that blocking specific endocytotic pathways does not affect the ability of TAT peptide to enter cells.…”

“…The TAT protein is an 86 amino acid long protein that is released by infected cells and is an essential regulatory gene for HIV replication [8]. In 1997, Vives et al [9] found that a 11-amino acid sequence, TAT (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57), now known as the TAT peptide or TAT PTD, can not only enter cells but is more efficient than the full length protein. It was observed that the chirality of the peptide backbone has no effect on cellular uptake of TAT peptide; inverse and retro forms were able to enter cells as efficiently as the native peptide, suggesting uptake does not require a specific binding site.…”

Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes

“…To quantify the nuclear pDNA, the fraction was further purified as previously described [21,22]. The collected cells were further washed twice with PBS (-) supplemented with heparin (20 units/mL), and incubated with 250 μL of CellScrubBuffer (Gene Therapy Systems Inc., San Diego, CA, USA) for 30 min on ice.…”