Nucleic acid polyplexes

Figure 7.3-2. Matrix-based DNA delivery can be divided into encapsulation and release approaches, where the nucleic acid is encapsulated for later release, and matrix-tethered delivery, where nucleic acid polyplexes or lipoplexes are immobilized directly to a matrix that also supports cell adhesion. These approaches are typically used for applications in tissue engineering where the delivery of nucleic acids is used to augment tissue formation.

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. 

Nanosized objects perform various functions in the biomedical field. In the human body, nanosized particulate substances behave very differently from larger particles. In 1986, Maeda et al. found that the stained albumin, having a size of several nanometers, naturally accumulates in the region of cancerous tissues, which is now well known as the enhanced permeability and retention (EPR) effect. Many studies in the field of nanoparticles are based on this finding. Another application of nanoparticles is the delivery system using various polyplexes that are composed of carrier molecules and plasmid DNA or nucleic acid drugs such as antisenses and siRNA. In addition, nanofibers are mainly used for biodegradable scaffolds in tissue... 

Abstract Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific dehvery is also briefly... 

As previously discussed, the protection of pDNA against degrading enzymes is a critical parameter for a non-viral carrier. Such ability is needed for the polyplex to protect the nucleic acid for an extended period of time in the blood while the polyplex circulates and distributes. Research conducted in 1999 by Richardson and coworkers [101] to study the ability of chitosan to protect against DNase degradation revealed that incubation of polyplexes prepared at NIP ratio of 3/1 in the presence of DNase I (8 U, 1 h incubation) protected pDNA from degradation. Other studies of chitosans as gene delivery vehicles confirm that the NIP ratio has to be at least 3/1 to 5/1 in order to provide a sufficient protective effect against DNases. 

Systemic delivery of cationic polyplexes is a challenging strategy in gene therapy. Polyplexes carrying a positive net charge due to condensation of nucleic acid by polymers such as PEI, oligo(ethylene imine OEI), or PLL encounter several... 

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. 

A magnetic nanoparticle-to nucleic acid w/w ratio of 0.5 to 1 has proven useful for both DNA and siRNA lipoplexes and polyplexes with a variety of magnetic nanoparticle types. To determine the optimal weight ratio for an unknown particle type, it is useful to also carry out this protocol with magnetic nanoparticle stock suspensions resulting in w/w ratios of 0.25, 0.75, 1 and 1.25 or higher. 

Nonviral formulations are generally more efficient and more toxic as the polymer-to-nucleic acid ratio is increased (higher N/P ratios), suggesting a role of free uncomplexed polymer in the solution in efficient delivery and toxicity. A recent report has shown that polyplexes of DNA and PEI contain an average of 3.5 plasmids (5800 base pairs) and 30 PEI (25 kDa) molecules when prepared at N/P ratios of 6 and 10, assuming that the DNA is completely complexed [267]. Based on these calculations, there is 86% of free PEI in the complex mixture [267,268]. Purification of the PEl/DNA complexes by dialysis has shown a reduction of toxcicity however, it also reduced transfection efficiency [269]. Efficient gene transfer was restored when free PEI was added to the mixture [269]. 

Fluorescence Microscopy. This microscopic approach is used to study the trafficking of either the vector or the nucleic acid. By a fluorescent labeling of the structure of interest (with fluorochrome such as FITC or TRITC), it is possible to visualize the internalization and trafficking processes using fluorescence microscopy or confocal microscopy. Using this approach, Midoux s group studied the internalization of polyplexes in HepG2 cells [11]. An interest of this last technique is that both the vector and the nucleic acid can be labeled. The complex evolution (and its disruption) can be observed. 

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