TY - JOUR
T1 - Chitosan-DNA nanoparticles as gene carriers
T2 - Synthesis, characterization and transfection efficiency
AU - Mao, Hai Quan
AU - Roy, Krishnendu
AU - Troung-Le, Vu L.
AU - Janes, Kevin A.
AU - Lin, Kevin Y.
AU - Wang, Yan
AU - August, J. Thomas
AU - Leong, Kam W.
N1 - Funding Information:
The authors would like to thank Dr Barry J. Byrne (Department of Pediatrics and Powell Gene Therapy Center, University of Florida) for helpful discussion on the preparation of recombinant KNOB protein. This project was partially supported by the Cystic Fibrosis Foundation and NIH grant CA68011.
PY - 2001/2/23
Y1 - 2001/2/23
N2 - Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of 100 μg/ml, the size of particles was optimized to ∼100-250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by Lipofectamine™-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.
AB - Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of 100 μg/ml, the size of particles was optimized to ∼100-250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by Lipofectamine™-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.
KW - Chitosan
KW - Complex coacervation
KW - Gene delivery
KW - Nanoparticles
KW - Receptor-mediated
UR - http://www.scopus.com/inward/record.url?scp=0035936972&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035936972&partnerID=8YFLogxK
U2 - 10.1016/S0168-3659(00)00361-8
DO - 10.1016/S0168-3659(00)00361-8
M3 - Article
C2 - 11182210
AN - SCOPUS:0035936972
SN - 0168-3659
VL - 70
SP - 399
EP - 421
JO - Journal of Controlled Release
JF - Journal of Controlled Release
IS - 3
ER -