Transfection of HEK cells via DNA-loaded PLGA and P(FASA) nanospheres.
HEK cells were transfected with the GFP gene using various vectors: naked DNA, lipofectamine, and both PLGA and P(FASA) plasmid-loaded nanospheres. All methods were assessed alone and with the use of chloroquine, a lysosomal enzyme inhibitor. Transfection efficiencies were determined and compared at various times post-incubation using a fluorescence standard curve. Neither naked DNA alone nor naked DNA and chloroquine were capable of transfecting cells. No differences were evident between lipofectamine with chloroquine and lipofectamine alone which transfected cells with a constant increase in efficiency up to 2 weeks. While transfection was not feasible with polymeric nanospheres alone, the addition of chloroquine allowed DNA released from nanospheres within cells to escape endosomal degradation and transfect the cells. The increase in transfection efficiency via nanospheres over time was exponential up to 1 week, as compared to the constant rate seen for the bolus-type administration of lipofectamine, indicating that nanospheres delivered DNA to the cells by a controlled release mechanism. Additionally, the effective dose delivered to cells via nanospheres was approximately 25% that of lipofectamine, indicating that transfection via PLGA and P(FASA) nanospheres might actually be more efficient.
Sandor
M
M
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Brown University, Providence, RI 02912, USA.
Mehta
S
S
Harris
J
J
Thanos
C
C
Weston
P
P
Marshall
J
J
Mathiowitz
E
E
D002460:Cell Line; D004247:DNA / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics; D006801:Humans; D019344:Lactic Acid / Q000008:administration & dosage* / Q000493:pharmacokinetics; D008863:Microspheres; D036103:Nanotechnology / Q000379:methods*; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics; D011100:Polyglycolic Acid / Q000008:administration & dosage* / Q000493:pharmacokinetics; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers / Q000008:administration & dosage* / Q000493:pharmacokinetics; D014162:Transfection / Q000379:methods*
D011108:Polymers; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D004247:DNA