No abstract

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (F(A)) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing F(A) of the chitosan, indicating that the charge density of chitosan, proportional to (1 - F(A)), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles.

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.

The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular plasmid DNA, and xanthan were investigated using tapping mode atomic force microscopy followed by quantitative image analysis. A shape factor was calculated for each of the observed polyelectrolyte complexes and used as a basis for dividing the structures into ensembles of morphologically linear, toroidal, and globular structures for subsequent quantitative analysis. Compaction of polyanions with chitosan yielded a small fraction of the torus morphology when the persistence length, L p, of 25 nm (acetan) was reached. For both DNA, L p = 50 nm, and xanthan, L p = 120 nm, it was found that the toroids make up a substantial fraction of the complexed structures formed by the given chitosan and at room temperature. Rodlike complexes were additionally observed within DNA−chitosan complexes, whereas they do not appear as a significant fraction of chitosan-complexed high-molecular-weight xanthan. The average height of the condensates was observed to be ∼2 nm for the compacted xanthan toroids, while it was determined to be ∼5 nm for compacted DNA toroids. Reducing the degree of polymerization of xanthan yielded a decrease in the fraction of toroids. Compacted xanthan at room temperature displays a number of racquets and other morphologies similar to the reported intermediate, metastable states by simulations. The reduced abundance of such structures following annealing supports the interpretation of their metastable nature.