Endocytosis macropinocytosis

Several mechanisms of endocytotic pathways exist. These include clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and non-clathrin receptor-mediated endocytosis (Table 4). All of these pathways can be exploited for drug trafficking into resistant cancer cells. For example, the... 

Eukaryotic cells take up extracellular material by a variety of different mechanisms that are collectively termed as endocytosis. Endocytosis is an uptake mechanism for molecules from the extracellular area by invagination of the cell membrane.There are four routes for the uptake of nanoparticles clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and clathrin- and caveolae-independent endocytosis. 

Previous work has shown that the majority of cells internalize liposomes through an endocytic pathway (4,5). There are multiple pathways for internalization involving vesicles of 50 300 nm in diameter. These include clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis, macropinocytosis, and nonclathrin- noncaveolae-dependent endocytosis (6). 

Figure 1 Endocytosis of liposomes five different routes into the cell. Multiple pathways can be used by the cell to internalize liposomes. Besides the well-characterized clathrin-mediated endocytosis, other pathways can be applied by the cell. Possible alternative pathways include phagocytosis or macropinocytosis—two pathways that internalize by an actin-driven protuberance of the plasma membrane. Other routes include the involvement of caveolae where substances are taken up into the cell bypass the traditional endosome/lysosome system (particles might escape from being degraded in lysosomes). Finally there exists an ill-defined mechanism that is neither mediated by caveolae nor by clathrin. In a single cell type, two or more of these mechanisms can coexist. Source Adapted from Ref 8.

In addition to the well characterized roles of clathrin-caveolae-mediated endocytosis and macropinocytosis/phagocytosis, an ill-defined route of nonclathrin-noncaveolae mediated endocytosis still exists (31,32). It seems that all of the until now poorly understood mechanisms of internalization can be summarized in this topic. 

Follow the procedure in Subheading 3.2. However, in step 4 just prior to the addition of the ligand solution, add 100 ig/mL transferrin-AlexaFluor-647 or 300 (rg/mL dextran-Alexa Fluor 647 to the peptide/DNA mixture to visualize clathrin-dependent endocytosis or macropinocytosis, respectively. Analyze the cells either live or fixed and counter-stained. If the cells are fixed and counter-stained, omit adding TO-PRO-3 under step 18, as the fluorescence of TO-PRO-3 is in the far-red spectrum just as Alexa Fluor 647. 

Cell-surface receptors are involved in both phagocytosis and pinocytosis. At least four distinct mechanisms of pinocytosis have been characterized macropinocytosis, clathrin-mediated endocytosis, raft/caveolae-mediated endocytosis, and clathrin-and caveolae-independent endocytosis (1). Selected receptor-mediated aspects of these mechanisms are outlined below. 

Neurotrophin-Trk receptor complexes are internalized and retrogradely transported from distal axons to the neuronal cell body where they signal to the soma to mediate target-dependent survival, growth and gene expression. The neurotrophin-Trk receptor complex is internalized by four mechanistically diverse and highly regulated pathways macropinocytosis clathrin-mediated endocytosis caveolae-mediated endocyto-sis and Pincher-mediated endocytosis. The kinase activity of Trk is probably required for receptor internalization. 

Human peripheral blood-derived DCs take up therapeutic proteins via macropinocytosis or receptor-mediated endocytosis, degrade them proteolyti-cally, and load the resulting peptide fragments onto HLA class II molecules, provided that the latter contain an appropriate epitope and resist downstream quality control by the peptide editor HLA-DM (Vogt et al., 2005). The second step foresees cell lysis, affinity extraction of HLA-peptide complexes, and acid elution of bound peptides. This procedure provides a mixture of both selfepitopes from the DC proteome and potential epitopes derived from the therapeutic protein. To separate mixtures of 1500-2000 distinct peptides with maximal resolution, the so-called Multidimensional Protein Identihcation Technology is employed (Kropshofer and Spindeldreher, 2005). Peptides leaving the HPLC capillary upon separation are sprayed directly into the orifice of an ion trap mass spectrometer for sequence determination. 

TAT-fusion proteins have been suggested to be internalized by endocytosis (lipid raft-dependent macropinocytosis) [E. L. Snyder, S. F. Dowdy, Pharm. Res. 2004, 21, 389 J. S. Wadia, S. F. Dowdy, Adv. Drug Deliv. Rev. 2005, 57, 579]. 

Caveolae-mediated endocytosis is involved in viral transfection. This route can therefore be used for the delivery of oncolytic genetic materials by a viral vector [120]. Macropinocytosis is a relatively non-specific process which allows uptake of large particles up to the micron size range [121]. It is likely useful for the delivery of systems like solid lipid nanoparticles and PLN, which are... 

Fig. 1 Proposed model for the internalization of PTD-conjugated macromolecules into cells. Interaction of positively charged PTDs with negatively charged proteoglycans and glycosa-minoglycans plays an important role in the internalization. The electrostatic interaction is followed by energy- and temperature-dependent endocytotic pathways. This involves phago-cytotic and pinocytotic pathways clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis...

Cellular uptake is an important process by which cells internalize substances such as nutrients, proteins and foreign materials, and therefore plays an important role in ceU-nanomaterial interactions. The endocytic pathways for the internalization of substance include phagocytosis, macropinocytosis, clathrin-mediated endocytosis, and non-clathrin-mediated endocytosis [54,55], all of which are closely related to the host responses addressed in the previous section. However, cellular uptake of a material does not necessarily implicate toxicity. But due to exceptionally small size and large surface area, cellular uptake of nanomaterials is different from that of conventional micron materials, which probably induce altered toxicological effects and, thus, needs to be studied. 

Cell culture substrate can also the influence gene delivery by affecting the occurrence of endocytosis and, thereby, the uptake of DNA-bound NPs by the cells. Hsu et al. [139] observed that the culture of MSCs on chitosan or HA-modified chitosan membranes increased the intracellular uptake of iron oxide NPs ( 5 nm) as well as naked DNA (3.3 kb, 5 mn) by more than fivefold. The increased internalization of NPs was associated with an increase in clathrin-mediated endocytosis on chitosan ( 50%) and in caveolae-mediated endocytosis on chitosan-HA ( 30-40%). In the case of naked DNA, but not iron oxide NPs, macropinocytosis also occurred on both substrates. 

Nano-sized polymers and particles adhere on the surface of tumor cells, and are taken up into tumor cells by endocytosis or macropinocytosis. In the endocytic pathway, pH gradually decreases from 7.4 (physiological pH) to 6 (endosomal pH) and to 5 (lysosomal pH). Eor intracellular release of pDNA, poly[Asp(Hyd-PEG)]-block-p[Asp(diaminoethane)] (Eigure 10.1(b)) has been reported to show high... 

FIGURE 2 Diagraimnamtic presentation of (A) Clathrin-mediated endocytosis (B) Caveolae-mediated endocytosis (C) Macropinocytosis. 

Macropinocytosis is another type of clathrin-independent endocytosis pathway [29], occurring in many cells, including macrophages [24]. It... 

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