Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles.
We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles gamma of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm(2) on the surface of a horn tip. Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t' from 0 to 60 s. HeLa-S3 viability decreased as a single exponential function of the total exposure time t = gammat' with a common time constant tau = 3.8 s for three duty cycles. Transfection was evaluated by counting the number of beta-galactosidase(beta-Gal)-positive cells relative to the total number of cells. Pulsed ultrasound provided an enhanced transfer of the beta-Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control. The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t = tau with gamma = 10, 20 and 30%, respectively. The number ratio of beta-Gal-positive cells to the surviving cells after exposure increased with t' according to a modified logistic equation. The degree of hemolysis also increased exponentially with t' at a time constant tau' = tau(0)/gamma for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with tau(0) = 0.9 s. Thus the total exposure time for the optimal transfection efficiency was tau, that is, nearly four times of tau(0). Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Japan.
Y
Y
Liu
H
H
Uno
H
H
Takatsuki
M
M
Hirano
A
A
Sakanishi
D000818:Animals; D002463:Cell Membrane Permeability / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006461:Hemolysis / Q000502:physiology* / Q000528:radiation effects; D006801:Humans; D011829:Radiation Dosage; D013552:Swine; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics