Chitosan/DNA polyplexes

The receptor ligand transferrin has been incorporated into chitosan/DNA polyplexes which enhanced gene transfer up to four-fold compared to immod-ified chitosan [67]. In a similar fashion, incorporation of C-terminal domain of adenovirus fiber knob protein enhanced transfection up to 130-fold in HeLa cells. Further modifications include the incorporation of hydrophobic moieties to generate dodecylated chitosan, deoxychoHc acid modified chitosan. Urocanic acid-modified chitosan [68] was reported to mediate efficient gene delivery it was hypothesized that the imidazole ring plays a crucial role for enhancing the release of internalized polyplexes from endosomal vesicles. 

Supaprutsakul S, Chotigeat W, Wanichpakom S et al (2010) Transfection efficiency of depolymerized chitosan and epidermal growth factor conjugated to chitosan-DNA polyplexes. J Mater Sci Mater Med 21 1553-1561... 

The size of the polyplex depends not only on the chemical structure of chitosan but also on the ratio between chitosan and DNA used for polyplex formulation, the concentrations of polymers, and formulation technique. This is commonly described in terms of N/P ratio, the ratio of protonatable polymer amines to... 

Cationic polymers, such as poly(L-lysine) (PEL), polyethylenimine (PEI), chitosan, polyamidoamine (PAMAM) dendrimers, poly(2-dimethylamino) ethyl methacrylate, and polyphosphoesters, condense DNA to form compacted polyplexes. ° The size and the stability of polyplexes depend on the ratio of cations vs. anions, temperature, ionic strength, and the solvent. Stability of polyplexes can be enhanced by conjugating PEG to the polycations or by using PEG-containing block or graft polymers that form micelles. Small cationic peptides are also able to condense DNA, however, six-consecutive-cations is the minimal requirement to achieve this effectively. 

In another interesting report, Jiang et al. showed the successful liver transfection after IP administration of galactosylated chitosan-graft-PEI (GC-g-PEI). Significant GFP expression was observed after IP administration of the polyplexes prepared from GC-g-PEI which was in agreement with the results of the successful in vivo biodistribution using Tc-GC-g-PEI by the same route of administration." Apart from successful DNA delivery by injectable carriers. 

Quaternized chitosan was developed by the reaction of chitosan with methyl iodide under basic conditions. After quaternization, the obtained N,N,N-trimethyl chitosan chloride (TMC) was soluble at neutral pH. The TMC makes it possible to prepare gene polyplexes over a wide pH range compared with the unmodified chitosan. The protonation of the amino group was pH independent when it was trimethylated, which favored the solubility of chitosan derivatives at physiological pH and the formation of polyplexes with DNA and siRNA. The presence of more positive charges contributed to better colloidal stability arising from both electrostatic stabilization and static effects. The size of the gene polyplexes obtained with trimethyl chitosan was reduced when compared with those formed with the... 

DNA molecules (polyplexes). Although there are a lot of reports on chitosan-based DNA nanocarriers varying in sizes (20-250 nm and higher), studies regarding effects and the chitosan-specific transfection mechanisms remain insufficient. It is shown that the level of transfection depends upon several factors, for instance chitosan molecular weight (from 22 kDa [65] to 400 kDa [66]), the ratio of chitosan nitrogen to DNA phosphate (N/P ratio), as well as serum concentration and pH of transfection medium, and finally on cell line [67],... 

However, being a natural polymer, chitosan has aU disadvantages of natural materials mentioned earlier (see Section 11. A). Presently, a lot of other polyplexes based on cationic synthetic polymers have been described in the literature. Among others, we could mention rather new DNA delivery nanoparticles based on poly(2-dimethylamino)ethyl methacrylate-co-poly(ethylene glycol) (PDMAEMA) [70,71] and PEGylated polyethylenimine (PEI/DNA) [72]. The last polyplexes have been encapsulated in PLGA microparticles and proposed for mucosal (oral) polyplex-based vaccination of Wistar rats. 

The strong complexation of chitosan and DNA arises due to the strong electrostatic interaction between the positively charged protonated chitosan and the negatively charged DNA and ensnres the entry of the intact polyplexes into the cell. 

Over the last four decades many different polycations have been employed in polyplexes, including natural DNA binding proteins such as histones, the synthetic amino acid polymers such as polylysine, polyornithine, and other cationic polymers such as polyamidoamine dendrimers, polyethylenimines, chitosan, polyphosphoramidates, or poly (dimethylaminoethyl) methacrylates. The use of these and other polymers has been previously reviewed in [7-9,29,30]. The characteristics of polymers which have been previously most commonly used are discussed below (Sect. 3.1), followed by the review of strategies to optimize these polymers (Sect. 3.2) or novel biodegradable polymers (Sect. 3.3). 

Danielsen, S., Strand, S., Davies, C.L., Stokke, B.T. Glycosaminoglycan destabilization of DNA—chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties. Biochim. Biophys. Acta 1721, 44-54 (2005)... 

Exceedingly high stability constants of polymer-DNA complexes appear to be a major difficulty for intracellular release of DNA. It was reported that the ability of polymer-DNA complexes to escape the endolysosomal compartment could be correlated to the buffering capacity of the polycation in the pH range of 5-7 (Lu et al, 2008). More specifically, optimal transfection efficiency of chitosan polyplexes can be achieved between pH 6.8 and 7.0. Above pH 7.5, DNA was shown to dissociate from the complex, thus preventing cellular uptake and transfection efficiency. Below pH 6.5, cellular uptake was significant but transfection efficiency was low, possibly due to hindered endosomal release (Ishii et al, 2001 Sato et al., 2001 Mintzer Simanek, 2009). 

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