Membrane polyplexes

Endolysosomal Escape. Polyplexes bind to the surface of cells via nonspecific electrostatic interactions and are internalized via adsorptive pinocyto-sis. Alternatively, polyplexes derivatized with targeting ligands may bind to specific cell-snrface receptors, in which case they are often internalized by receptor-mediated endocytosis. In either case, the polyplexes become localized within endocytic vesicles, which isolate the polyplex from the rest of the cell. The endocytic pathway represents a hostile environment for polyplexes. The first vesicle, termed the early endosome, fuses with sorting endosomes from which the internalized material may be transported back to the membrane and ont of the cell by exocytosis. More generally, however, polyplexes are believed to be trafficked into late endosomes, vesicles that rapidly acidify to pH 5-6 becanse of the action of an ATPase proton-pump enzyme in the vesicle membrane. Polyplexes may subsequently be trafficked into lysosomes, which further acidify to... 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

A highly stable and shielded polyplex should circulate in the blood stream without undesired interactions until it reaches the target cell. At that location, specific interactions with the cell surface should trigger intracellular uptake. While lipid membrane interaction is undesired at the cell surface, it should happen subsequently within the endosomal vesicle and mediate polyplex delivery into the cytosol. During or after intracellular transport to the site of action, the polyplex stability should be weakened to an extent that the nucleic acid is accessible to exert its function. 

Fig. 1 Bioresponsive polyplexes. (a) Systemic circulation of shielded polyplexes in blood stream and attachment to cell surface receptor (b) endocytosis into endosomes, deshielding by cleavage of PEG linkers and activation of membrane-destabilizing component by acidic pH or other means (c) endosomal escape into cytosol (d) siRNA transfer to form a cytosolic RNA-induced silencing complex complex (e) cytosolic migration and intranuclear import of pDNA (/) presentation of pDNA in accessible form to the transcription machinery...

As outlined in previous sections, escape of polyplexes from endosomes to the cytosol can be a major bottleneck in delivery. Membrane-active polymer domains or other conjugated molecules can help to overcome this barrier (see Sect. 2.3), but they may trigger cytotoxicity when acting extracellularly or at the cell surface. Therefore membrane-crossing agents either have to be inherently specific for endo-somal compartments (for example by pH-specificity), or they have to be modified to be activated in endosomes. For example, the reducing stimulus of intracellular vesicles has been used to activate formulations containing less active disulfide precursors of LLO [163] or Mel [170]. 

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...

These anionic peptides can be linked covalently to polylysine conjugates, but that may cause a decrease of their fusogenic features once the polyplexes are formed. For instance, we have found impairment in permeabilization of the plasma membrane upon couphng E5CA to bovine serum albumin (Midoux et al., 1995). 

Once internalized, the essential step for the polyplex is to escape rapidly the endosomal vesicle in order to release the nucleic acid in the cytosol and prevent its lysosomal degradation. As the endosomal and lysosomal pH presents values between 4.5 and 6.5 and therefore differs from the neutral pH of 7.4 in other biological compartments [58], some polycations containing protonable residues like PEI facilitate this step by the proton sponge effect [59, 60]. As not all cationic polymers display this attribute, another effective method for enhanced endosomal polyplex release is incorporation of specific endosomal membrane disrupting or pore-forming domains, such as lytic lipid moieties or endosomolytic peptides. 

This is in contrast to viruses, where the virus particles also show active transport when present in the cytosol after fusion with the plasma membrane or endosomal membrane [60-62], This is due to the ability of specific proteins of the virus particle to bind motor proteins. Single-particle tracking reveals that the quantitative intracellular transport properties of internalized non-viral gene vectors (e.g., polyplexes) are similar to that of viral vectors (e.g., adenovirus) [63]. Suk et al. showed that over 80% of polyplexes and adenoviruses in neurons are subdiffusive and 11-13% are actively transported. However, their trafficking pathways are substantially different. Polyplexes colocalized with endosomal compartments whereas adenovirus particles quickly escaped endosomes after endocytosis. Nevertheless, both exploit the intracellular transport machinery to be actively transported. 

Whether the components of the gene carriers actually remain associated during import into the nucleus or enter individually cannot be answered by optical methods as their resolution is limited. A possible technique to study the complexation of DNA within cells is fluorescence correlation spectroscopy (FCS). Clamme et al. studied the intracellular fate of PEI after transfection with polyplexes by two-photon fluorescence FCS [54]. They showed that PEI binds to the inner membrane of endosomes and lysosomes and shows free diffusion in the cytosol as well as the nucleus. However, they did not detect any PEI/DNA complexes inside the nucleus. 

Lipoplexes and polyplexes are DNA-cationic molecular complexes, formed, respectively, by DNA interaction with lipids or polymers. The main property of these complexes is to allow easier passage of DNA through the cell plasma membrane, by means of two mechanisms DNA charge neutralization and plasmid condensation, which reduces its size. Such complexes are formed by an excess of positive charges to neutralize DNA phosphate groups, resulting in transfecting particles with a net... 

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