The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-β-cyclodextrin (MβCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MβCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.

Introduction Membrane ruffling and the formation of the macropinosome Rabs and macropinocytosis The fate of the macropinosome on the endocytic pathway A role for nexins in macropinosome sorting Cell penetrating peptides as vectors for drug delivery The case for and against macropinocytosis Conclusions Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short‐lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides.