[Feasibility of chitosan as gene therapy vehicle].
OBJECTIVE
Recently, chitosan, as a nonviral vehicle for transferring DNA molecules into the cells, has attracted much attention because of its cationic properties. This study was to investigate characteristics and transfection activity of chitosan-pDNA microparticles, and confirm the feasibility of chitosan as gene therapy vehicle, which may be employed in further in vivo study.


METHODS
Plasmid DNAs were amplified in Eschericha Coli JM109 with transfection of enhanced green fluorescence protein (EGFP) gene, and isolated according to the protocol of Qiagen plasmid midi kit. Chitosan-pDNA microparticles were prepared by the coacervation method, and observed under transmission electronic microscope. In vitro releasing experiment and ultraviolet spectrophotometry were used to measure DNA loading capacity and loading efficiency. The location of pDNA in the microparticles was analyzed by gel retardation assay. Transfection efficiency of chitosan-pDNA microparticles was evaluated by detecting gene expression of EGFP in HEK293 cells after transfection.


RESULTS
Chitosan-pDNA microparticles were global, and the size ranged 100-200 nm, with the average of (138 +/- 43) nm. DNA loading capacity and loading efficiency of the microparticles were (46.8 +/- 9.0)% and 100%, respectively. Gel retardation assay confirmed that DNA was wholly encapsulated in the microparticles. In vitro study revealed that the release of DNA from chitosan-pDNA microparticles included two-step process, namely burst effect with a slow second phase. Most of DNA could be released at the time of 48 h. In vitro transfection results indicated that chitosan could efficiently deliver pEGFP into HEK293 cells and stably express green florescence protein.


CONCLUSIONS
Chitosan can efficiently transfer target DNA molecules into mammalian cells, continuously release DNA, and stably express DNA. It can be potentially employed as a gene therapy vehicle.
State Key Laboratory of Oncology in Southern China, Guangzhou, Guangdong, 510060, PR China.
Yi-Yuan
YY
Wan
Xing
X
Zhang
You-Jian
YJ
He
Wen-Qi
WQ
Jiang
D002460:Cell Line; D048271:Chitosan / Q000008:administration & dosage* / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004337:Drug Carriers; D016503:Drug Delivery Systems; D005240:Feasibility Studies; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008863:Microspheres; D010316:Particle Size; D010957:Plasmids; D014162:Transfection
D004337:Drug Carriers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D048271:Chitosan