Hector Guzman

Ultrasound-mediated disruption of cell membranes. I. Quantification of molecular uptake and cell viability.

Ultrasound-mediated drug delivery is a nonchemical, nonviral, and noninvasive method for targeted transport of drugs and genes into cells. Molecules can be delivered into cells when ultrasound disrupts the cell membrane by a mechanism believed to involve cavitation. This study examined molecular uptake and cell viability in cell suspensions (DU145 prostate cancer and aortic smooth muscle cells) exposed to varying peak negative acoustic pressures (0.6-3.0 MPa), exposure times (120-2000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. With increasing pressure and exposure time, molecular uptake of a marker compound, a calcein, increased and approached equilibrium with the extra cellular solution, while cell viability decreased. Varying pulse length produced no significant effect. All viability and molecular uptake measurements collected over the broad range of ultrasound conditions studied correlated with acoustic energy exposure. This suggests that acoustic energy exposure may be predictive of ultrasounds nonthermal bioeffects.

Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells through a mechanism believed to involve cavitation. Because cavitation is both temporally and spatially heterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects of ultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145 prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures (0.6-3.0 MPa). exposure times (120-2,000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. Cell-to-cell heterogeneity was observed at all conditions studied and was classified into three subpopulations: nominal uptake (NUP), low uptake (LUP), and high uptake (HUP) populations. The average number of molecules within each subpopulation was generally constant: 10(4)-10(5) molecules/cell in NUP, approximately 10(6) molecules/cell in LUP, and approximately 10(7) molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strong dependence on both acoustic pressure and exposure time. Varying pulse length produced no significant effect. The distribution of cells among the three subpopulations correlated with acoustic energy exposure, which suggests that energy exposure may govern the ability of ultrasound to induce bioeffects by a nonthermal mechanism.

Intracellular Drug Delivery Using Low-Frequency Ultrasound: Quantification of Molecular Uptake and Cell Viability

AbstractPurpose. To determine the dependence on acoustic parameters of molecular uptake and viability of cells exposed to low-frequency ultrasound. Methods. DU145 prostate cancer cells bathed in a solution of calcein were exposed to ultrasound at 24 kHz over a range of different acoustic pressures, exposure times, pulse lengths, and duty cycles. Flow cytometry was employed to quantify the number of calcein molecules delivered into each cell and levels of cell viability. Results. Both molecular uptake and cell viability showed a strong dependence on acoustic pressure and exposure time, weak dependence on pulse length, and no significant dependence on duty cycle. When all of the data were pooled together, they exhibited good correlation with acoustic energy exposure. Although molecular uptake showed large cell-to-cell heterogeneity, up to ∼15% of cells achieved an intracellular calcein concentration approximately equal to its extracellular concentration. Conclusions. Large numbers of molecules can be delivered intracellularly using low-frequency ultrasound. Both uptake and viability correlate with acoustic energy, which is useful for design and control of ultrasound protocols.

A high-throughput approach towards a novel formulation of fenofibrate in omega-3 oil

A novel lipid formulation containing fenofibrate in omega-3 oil was developed using a novel high-throughput screening platform. The optimized formulation combines the cardiovascular health benefits from omega-3 oil with the potent lipid-regulating effect of fenofibrate. When tested against the current marketed product Tricor in healthy human volunteers, the new formulation was shown to be equivalent to Tricor.

Acoustic spectrum and sonoluminescence as indicators of cell membrane disruption by ultrasound

Ultrasound has been shown to reversibly disrupt cell membranes and thereby drive drugs and genes into cells by a mechanism believed to involve cavitation. We have measured the number of molecules delivered into cells and levels of cell viability following exposure to a broad range of ultrasound conditions. Many cellular responses are possible, including uptake of up to millions of molecules per cell and cell viability ranging from 0 to 100%. Because these effects are believed to occur due to acoustic cavitation, we measured features of the acoustic spectrum known to be associated with cavitation and measured sonoluminescence light output during ultrasound exposures. We found that these measures of cavitation correlate with both the number of molecules taken up by cells and levels of cell viability. This observation strengthens the hypothesis that ultrasound’s bioeffects are mediated by cavitation and suggests a noninvasive means to assess these bioeffects which could be used as part of a real‐time feedback loop. [Work supported by the NSF, NIH, and Whitaker Foundation.]