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 1 A histogram of fluorescence events vs. fluorescent intensity for HL-60 cells. When cells were incubated at pH 5.0 encap- sulated in 10% P105 (right side histograms), the fluorescence of Lysosensor Green in the cells was greater than that at pH 7.0 in the presence of 10% P105 (left side histograms). docytotic activity, and HeLa cells which have a relatively high amount of non-receptor mediated endocytosis [3]. Figure 1 shows a positive control, in which the pH of the HL-60 cells exposed to Lysosome Green encapsulated in 10% P105 was reduced to 5.0 and compared with cells in- cubated in the presence of 10% P105 at neutral pH. The x- axis shows the amount of fluorescence emitted from the cells and the y-axis is the number of cells that exhibited the amount of fluorescence shown on the x-axis. At the lower pH, HL-60 cell fluorescence increased substantially, indicating that the probe associated with the acidified cells is in an acidic environment; therefore the probe ap- pears to be responsive to low pH such as found inside a low-pH lysosome. HeLa cells were also used as another positive control. At normal pH (7.1–7.4) the pH-sensitive probe encapsulated in 10% P105 showed stronger fluorescence in the HeLa cells than the non pH sensitive probe in the presence of 10 % P105, indicating that HeLa cells display strong endocy- totic activity and incorporate the probe into acidic vesicles (Figure 2). Figure 3 shows the amount of fluorescence for negative control HL-60 cells (left), cells incubated with Lysosensor s t n e v E f o r e b m u N Fluorescence Intensity Figure 2 A histogram of emissions from HeLa cells incubated with pH sensitive probe and 10% P105 (right) and the pH independ- ent probe encapsulated in P105 (left). Green encapsulated in 10% P105 (middle), and cells in- cubated with Lysosensor Green in PBS (right). As was found previously for Dox [1], less Lysosensor Green is as- sociated with the cells when the hydrophobic probe is en- capsulated in the Pluronic micelle; much less encapsulated probe was able to accumulate in low-pH compartments in the cells than free probe introduced from a solution of PBS. This is consistent with our convic- tion that hydrophobic molecules partition to the micellar core and are not taken up as readily by cells. Figure 4 shows that ultrasound produces no difference in Lysosensor Green fluorescence in HL-60 cells when the probe is encapsulated in P105 micelles. The cells were ei- ther incubated or ultrasonicated for 1 hour in the presence of Lysosensor Green in 10% P105. For each condition, three replicates are shown in the figure. The observation that the incubated and ultrasonicated cell histograms overlap closely (see Figure 4) suggests that the ultrasound does not cause the probe to partition to a more acidic en- vironment anymore than it does without ultrasound. Figures 4 and 5 show the results with the HL-60 as a result of incubating and sonicating these cells with the pH sen- sitive probe (Figure 4) and the non-pH sensitive probe (Figure 5). Figure 5 shows a slight increase in the amount of fluorescence as a result of ultrasonication, while Figure 4 shows no increase in the amount of fluorescence as a re- sult of ultrasonication. These data indicate that ultra- sound enhances the uptake of a fluorescent probe into the cells as shown with the non-pH sensitive probe in Figure 5, but does not enhance the amount of the probe present in a low-pH compartment as shown with the pH-sensitive probe in Figure 4. This suggests that the ultrasound en- hances the uptake of hydrophobic molecules, but the en- Page 3 of 6 (page number not for citation purposes)