N-alkylated Chitosan Transgene Vectors

Chitosan (2-amino-2-deoxy-?-D-glucose) is linear cationic polysaccharide with a series of unique chemical properties and biofunctions. Chitosan can be modified with its active hydroxyl and amino groups. Its derivatives have a great potential in the application of biomedical engineering.Based on the biocompatibility, degradability and fine transmembrance of chitosan, N-alkyl chitosan have bee synthesized to be a transgenic vector of noncell toxicity and regulability on hydrophilicity and hydrophobibity . We have explore the effect of chitosan molecular weight, alkylsubstituent and different alkylateralchain to the formation and structure of ligand. And then, proceed to study the effect to transfectrate of gene, by which, study deeply in transmembrance machanism of alkylate chitosan and inprovement on slowreleastrate and substituent.1. N-alkyl chitosan were prepared with alkyl halides with different chain length. The analysis of IR and X-ray Diffractiong of N-alkyl chitosan derivatives confirms that the hydrogen bonds and crystallinity are weakened by the incorporation of pendant alkyl moieties.2. Complex formation between N-alkyl self-aggregates and DNA was confirmed by electrophoresis of complexes on an agarose gel. The amount of free DNA which does not participate in the complex formation decrease with increasing the charge ratio and the total retention occurs above a charge ratio of 4/1. Dynamic light scattering indicate that Self-aggregate in the solution has the mean diameter of 150 nm with an unimodal size. The transfection of C2C12 cells with N-alkyl chitosan /DNA complexes was studied using the plasmid PC DNA 3.1+CAT. The transfection efficiency of N-alkyl chitosan/DNA complexes are higher than that achieved by DNA alone and by calcium phosphate and nearly the same level achieved by commercially available cationic liposome (Lipofectamine ). An efficient transfection of MCF7 cells was achieved by N-alkyl chitosan /PIRES2-EGPF complexes.3. The characteristics of self-aggregates of chitosan and N-alkyl chitosan were examined by fluorescence probe techniques and the polarity parameter(I1/I3) was also calculated. The critical aggregation concentration (cac) of self-aggregates was determined from the fluorescence emission spectra and the cac value was about 8× 10－3mg/ml. These results indicate that the introduction of alkyl chains leads to the formation of self-aggregates.The interactionbetween chitosan/N-alkyl chitosan and dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)bilayer was examined by differential scanning calorimetry(DSC).Under the influence of chitosan/N-alkyl chitosan, the calorimetric enthalpy of DPPC was reduced in a concentration-dependent manner.