Biodegradable Polymers for Gene Delivery.

The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.

Studies on the condensation of depolymerized chitosans with DNA for preparing chitosan-DNA nanoparticles for gene delivery applications.

High molecular weight chitosan (CS) was depolymerized by oxidative degradation with NaNO(2) at room temperature to get 11 samples of CS derivatives of varying molecular weights with a view to assessing their effective molecular weight range for gene delivery applications. Viscosity studies indicated that the molecular weight of the depolymerized CS was proportional to the CS/NaNO(2) ratio. The condensation behavior of DNA/CS complexes at various charge ratios was studied using UV spectroscopy, FTIR, CD, SEM, and AFM. The results indicated that CSs having very low molecular weights and high charge density exhibited strong binding affinity to DNA compared to high molecular weight CSs. However, the very low molecular weight (1.9-7.7 kDa) CSs were found to form aggregates easily even at very low charge ratios. On the other hand, CSs having medium molecular weight (49-51 kDa) and high degree of deacetylation (DD) gave stable uniform-sized nanoparticles. Biological studies carried out with the spherical nano-sized polyplexes formed between CS of 50 kDa (DD of 94%) and pEGFP plasmid DNA at N/P ratio of 5.0 showed excellent gene transfection efficiency at pH 6.5 in HeLa cells without cytotoxicity indicating their potential as gene delivery carriers.

Liquid crystalline phase behavior of high molecular weight DNA: a comparative study of the influence of metal ions of different size, charge and binding mode.

The ability of Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Cu(2+), Cd(2+), Al(3+), V(4+), Hg(2+), Pd(2+), Au(3+), and Pt(4+) to provoke liquid crystalline (LC) phases in high molecular weight DNA was investigated. The alkali and alkaline earth metal ions provoked typical cholesteric/columnar structures, whereas transition metal ions precipitated DNA into solid/translucent gel-like aggregates. Heavy metal ions reduced viscosity of DNA solution, disrupting rigid, rod-like DNA structure necessary for LC textures. Three-layer quantum mechanical-molecular mechanical (QM/MM) studies of Li(+), Na(+), K(+), Mg(2+), and Ca(2+) binding DNA fragment suggested several possible binding modes of these ions to the phosphate groups. The dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allowed for higher DNA binding affinity of the alkaline earth metal ions. These results have implications in understanding the biological role of metal ions and developing DNA-based sensors and nanoelectronic devices.

Role of Mg2+ and Ca2+ in DNA bending: evidence from an ONIOM-based QM-MM study of a DNA fragment.

The binding of hydrated Mg2+ and Ca2+ ions with a DNA fragment containing two phosphate groups, three sugar units, and a G.C base pair is modeled in the anion and dianion states using a three-layer ONIOM approach. A monodentate binding mode was the most stable structure observed for both the ions in the anion model. However, the interactions of Mg2+ and Ca2+ with the dianion model of the DNA fragment gave rise to a large structural deformation at the base pair region, leading to the formation of "ring" structures. In both anion and dianion models, Mg2+-bound structures were considerably more stable than the corresponding Ca2+-bound structures. This feature and the formation of ring structures in the dianion models strongly supported the higher coordination power of the Mg2+ toward DNA systems for its compaction. The charge of the DNA fragment appeared to be crucial in deciding the binding strength as well as the binding mechanism of the metal ions. To the best of our knowledge, this is the first theoretical investigation of the interaction of a comparatively larger DNA model system with the biologically important Mg2+ and Ca2+ ions.

Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics.

DNA undergoes condensation, conformational transitions, aggregation and resolubilization in the presence of polyamines, positively charged organic molecules present in all cells. Under carefully controlled environmental conditions, DNA can also transform to a liquid crystalline state in vitro. We undertook the present work to examine the ability of spermidine, N4-methylspermidine, spermine, N1-acetylspermine and a group of tetramine, pentamine and hexamine analogs of spermine to induce and stabilize liquid crystalline DNA. Liquid crystalline textures were identified under a polarizing microscope. In the absence of polyamines, calf thymus DNA assumed a diffused, planar cholesteric phase with entrapped bubbles when incubated on a glass slide at 37 degrees C. In the presence of spermidine and spermine, the characteristic fingerprint textures of the cholesteric phase, adopting a hexagonal order, were obtained. The helical pitch was 2.5 micro m. The final structures were dendrimeric and crystalline when DNA was treated with spermine homologs and bis(ethyl) derivatives. A cholesteric structure was observed when DNA was treated with a hexamine at 37 degrees C. This structure changed to a hexagonal dendrimer with fluidity on prolonged incubation. These data show a structural specificity effect of polyamines on liquid crystalline phase transitions of DNA and suggest a possible physiological function of natural polyamines.