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 1 Figure 5 A histogram of HL-60 cells incubated (middle, 3 replicates) and ultrasonicated (right, 3 replicates) with the non-pH sensi- tive probe Cell Tracker Orange CMTMR. The histogram on the left represents the control with no fluorescent probes added. Saito et al. showed that exposure to ultrasound increased the permeability of corneal endothelium cells [14]. The increase in permeability appeared to be reversible and the cells regained their membrane integrity after several min- utes. The experiments reported herein using flow cytometry have shown that more of the non-pH sensitive probe (C- 2927) accumulates in HL-60 cells during the application of ultrasound (Figure 5). This is consistent with our previ- ous results showing that more Dox and Ruboxyl accumu- late in the cells during ultrasonic exposure [1,6,10,15]. We postulate that the shear events associated with ultrasound and accompanying cavitation events leads to stress on the cell membrane and/or the cell wall and renders the cell more permeable to the passive transport of the drugs or the fluorescent probes. In addition to rendering the mem- brane more permeable, ultrasound simultaneously releas- es the drug (or other molecules) from the micelles, thus increasing the external local concentration. None of our data nor of literature reports leads us to reject hypothesis #1, and in fact supports it. Hence, ultrasound of the inten- sity and frequency used herein appears to make cell mem- branes more permeable. Hypothesis #2 Ultrasound only increases the external drug concentra- tion, but does not change the permeability of the cell membrane. If this alternative hypothesis were true, the in- creased drug concentration outside the cells (and in- creased driving force for diffusion) would lead to faster and greater drug uptake into the cells by the same passive routes as without ultrasound. It may also be argued that ultrasound can reduce the mass transport boundary layer around the cells (due to microconvection currents) and hence enhance the diffusion of the drug to the cell mem- brane. If this small mass transfer boundary layer were the limiting diffusive barrier to membrane transport, then the reduction in boundary layer thickness would indeed in- crease transport into the cell. However, transport into the cell is a serial process, and the external boundary layer is only one step, and for water-soluble molecules this resist- ance is probably much smaller than the resistance of transport across the lipid membrane of the cell. We posit that the controlling resistance is that of the cell mem- brane, and therefore ultrasonic reduction of the external mass transfer boundary, without any decrease in the cell membrane resistance would not measurably increase the transport into the cell. Thus we dismiss the possibility that solely the decrease in the mass transfer boundary layer surrounding the cell (due to insonation) would cause a significant increase in drug uptake. It is true that ultrasound releases drug from the micelles, and thus the external concentration of free drug is in- creased. To test the second hypothesis we need another tracer besides Dox that crosses the cell membrane, a tracer that does not change its external concentration upon in- sonation. Rapoport showed that ultrasonication increased the amount of fluorescently labeled P-105 that entered the cell (along with increased Dox) [16]. Since the exter- nal P-105 concentration does not change upon insona- tion, the only way for ultrasound to increases its uptake is if the cell membrane were rendered more permeable to P- 105 transport. Thus ultrasound increases the membrane permeability, and we can reject hypothesis #2 and accept hypothesis #1. Hypothesis #3 Ultrasound enhances endocytosis or other activated up- take of the entire micelle (with the encapsulated drug) into the cancer cells. Previous research has established that endocytosis of drug-containing Pluronic micelles oc- curs [16]. However, the question remains as to whether ultrasound triggers an increase in the endocytotic process that transports micelles in the cells. In order to test the postulate that the cells are taking the drug in by endocytosis, a probe that fluoresces more strongly in acidic environments was used, namely Lyso- sensor Green. During normal endocytosis, the great ma- jority of endosomes fuse with a primary lysosome to form a secondary lysosome. The pH inside lysosomes is about 4.8 while the pH outside these compartments is around 7.1; therefore Lysosensor Green would show an increase in fluorescence when taken up in an endosome and trans- ferred to a lysosome. Page 5 of 6 (page number not for citation purposes)