Endocytosis cytoplasmic delivery

Anionic polyelectrolytes have been used in the development of new intracellular delivery systems by membrane destabilizing mechanisms (Wasungu and Hoekstra, 2006).These polymers can be tailored to interact actively with phospholipid membranes upon external stimulation, such as acidification of the surrounding medium. This strategy has been exploited to improve the cytoplasmic delivery of biomolecules (DNA, proteins) that enter cells by endocytosis and end up in acidic organelles (Gupta et al, 2005). 

Attempts to study the entry of ES products into cells using markers of fluid phase endocytosis yielded unexpected results. When larvae browse resistant IEC-6 cells in the presence of extracellular fluorescent dextran, dextran enters the cytoplasm of a significant proportion of the cells in the mono-layer (Butcher et al., 2000). The parameters of dextran entry are most compatible with the conclusion that larvae wound the plasma membranes of IEC-6 cells that is, they create transient breaches in the membrane that allow impermeant markers to enter the cell (McNeil and Ito, 1989). Wounding is considered to be a common occurrence in intestinal epithelia (McNeil and Ito, 1989). Injured cells are able to heal their wounds by recruiting vesicles to seal the breach (Steinhardt et al., 1994). In an experimental system, healing allows the injured cell to retain cytoplasmic dextran. In epithelial cell cultures inoculated with T. spiralis larvae, the relationship between glycoprotein delivery and injury of plasma membranes is not clear, i.e. dextran-laden cells do not always stain with Tyv-specific antibodies and... 

Figure 14.10 Overview of cellular entry of (non-viral) gene delivery systems, with subsequent plasmid relocation to the nucleus. The delivery systems (e.g. lipoplexes and polyplexes) initially enter the cell via endocytosis (the invagination of a small section of plasma membrane to form small membrane-bound vesicles termed endosomes). Endosomes subsequently fuse with golgi-derived vesicles, forming lysosomes. Golgi-derived hydrolytic lysosomal enzymes then degrade the lysosomal contents. A proportion of the plasmid DNA must escape lysosomal destruction via entry into the cytoplasm. Some plasmids subsequently enter the nucleus. Refer to text for further details...

Viruses are complex particles, entering the cells by fusion of their envelope to the plasma membrane or by endocytosis followed by the escape of the capsid by membrane fusion or lysis (Sodeik, 2000). The diameter of the viral particle could be several hundred nanometers, implying a very inefficient diffusional movement in the cytoplasm, based on those physicochemical considerations that were discussed above (Kasamatsu and Nakanishi, 1998). Despite these limitations, those viruses that replicate in the nucleus have evolved sophisticated mechanisms to ensure a highly efficient nuclear delivery of their genetic material. Since these mechanisms may provide a conceptual framework to design novel non-viral delivery systems, we shall review some of the key elements that account for the nuclear targeting of certain viruses. 

Gene delivery systems can distribute plasmids to the desired target cells, after which the plasmid is internalized into the cell by a number of mechanisms, such as adsorptive endocytosis, receptor-mediated endocytosis, micropinocytosis, caveolae-mediated endocytosis and phagocytosis (see Section 1.3.3.2). The intracellular fate of plasmids depends on the means by which they are internalized and translocated to the cytoplasms and then to the nucleus. In coated-pit endocytosis, DNA complexes first bind to the cell surface, then migrate to clathrin-coated pits about 150 ran in diameter and are internalized from the plasma membrane to form coated vesicles. 

The acidification of endosomal compartments, as they evolve toward lysosomes is a well-described phenomenon (1) that can be exploited to design drug delivery systems capable of releasing their contents after endocytosis. Enhanced cytoplasmic drug concentrations can therefore be achieved with smart formulations, which are sensitive to acidic pHs. For this purpose, liposomal formulations are attractive, because their deformable phospholipid bilayers can be rapidly disrupted to trigger drug release. In this section, ionizable copolymers of ISTisopropylacrylamide (NIPAM) are anchored in the phospholipid membrane and used to destabilize the bilayer upon acidification of the environment. 

Appropriate cellular adhesion, endocytosis, and intracellular trafficking to allow therapeutic delivery or imaging in the cytoplasm or nucleus. 

Fig. 10.17 Cytoplasmic drug delivery using lysosomal-pH-responsive fast-release nanoparticles a the nanoparticle is internalized by endocytosis b transferred to a lysosome c the PDEA core is protonated at the lysosomal pH (4-5) and the nanoparticle dissolves, releasing the drug into the lysosome d the continuous PDEA (poly[2-(JV,A-diethylamino)ethyl methacrylate]) protonation causes an osmotic imbalance across the lysosome membrane, which finally ruptures the lysosome and hence releases the drug into cytoplasm...

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