BMC Cancer 2002, 2 http://www.biomedcentral.com/1471-2407/2/20 s t n e v E f o r e b m u N Fluorescence Intensity Figure 3 A histogram of fluorescent events vs. fluorescent intensity for HL-60 cells. Left: control cells with no fluorescent probe added. Center: cells that have been incubated with the fluo- rescent probe in the presence of 10% P105 micelles for one hour. Right: cells that have been incubated with the fluores- cent probe in a solution of PBS for one hour. Using Lysosen- sor Green and flow cytometry, more fluorescence is observed when the probe is introduced in a solution of PBS than when encapsulated in P105. hanced uptake does not appear to involve low-pH compartments. Discussion and conclusions The results of these and of previously reported experi- ments allow evaluation of several critical hypotheses re- garding the mechanisms of this ultrasonically enhanced drug delivery system. We have previously established the fact that ultrasound releases Dox and other drugs from micelles [2,5–10]. The following discussion pertains to if and how ultrasound also enhances uptake of the drug into the cells. We will consider both passive transport via dif- fusion through the cell membrane, and active transport in the cell via endocytosis. Hypothesis #1 Ultrasound perturbs the cell membrane and/or cell wall to render it more permeable to the drug. Ultrasound creates stress on cells by at least two mechanisms. The first is acoustic streaming in which momentum from directed propagating sound waves is transferred to the liquid, caus- ing the liquid to flow in the direction of the sound prop- s t n e v E f o r e b m u N Fluorescence Intensity Figure 4 A histogram showing that no difference in fluorescence is observed between HL-60 cells incubated and ultrasonicated for one hour (at 1 W/cm2 and 70 kHz) in the presence of 10% P105 and Lysosensor Green (histograms with incubated cells overlap with histograms of ultrasonicated cells). The his- togram on the left is a control with no fluorescent probe added. The 6 histograms on the right are incubated and ultra- sonicated HL-60 cells (3 replicates each) in the presence of 10% P105. agation [11]. Acoustic streaming happens to varying degrees at any intensity of ultrasound. The second mech- anism is microconvection created by oscillating gas bub- bles in the liquid. The cycles of low and high acoustic pressure cause the micron-sized gas bubbles to expand and shrink, which in turn creates shear flow around the oscillating bubbles. Stable cavitation results when the acoustic intensity is sufficiently low that the bubbles do not collapse completely during their contraction cycle. Collapse or transient cavitation is produced when the bubble collapses during the contraction cycle [12]. Both stable and collapse cavitation create high shear stresses in the fluid which can damage the cell membrane. The sud- den collapse produces a shock wave, and the adiabatic compression of the gas produces temperatures on the or- der of 5000 K, which in turn fragments water and other molecules into free radicals [12]. The general phenome- non of creating holes or pores in cell membranes via these intense acoustic forces is called sonoporation. Tachibana et al. have shown that the exposure of HL-60 cells to 255 kHz of ultrasound and merocyanine 540 (a photosensitive durg) for 30 seconds formed pores in the cell membrane [13]. Electron micrographs showed that the cytoplasm of some cells seemed to have extruded through the pores formed in the cell membrane as a result of sonoporation. When cells were exposed to ultrasound alone, the cell membrane showed minor disruptions. Page 4 of 6 (page number not for citation purposes)