BMC Cancer 2002, 2 http://www.biomedcentral.com/1471-2407/2/20 increase in the active endocytosis of the micelles (with drug) into the cell. Methods Cells HL-60 cells were cultured in Roswell Park Memorial Insti- tute medium, RPMI-1640 (HyClone, Logan, UT, USA), supplemented with 10% fetal calf serum, 6 mM L- glutamine and 7.5% sodium bicarbonate. The culture was maintained in 75-ml plastic tissue flasks at 37°C in hu- midified 5% CO2 and was passaged every 3 to 4 days. HeLa cells were cultured in Minimum Essential Medium, α MEM (HyClone, Logan, UT, USA), supplemented with 10% fetal calf serum, L-glutamine, non-essential amino acids and sodium pyruvate. The culture was also main- tained in 75-ml plastic tissue flasks at 37°C in humidified 5% CO2 and was passaged every 2 to 3 days. HeLa cells have a high rate of non-receptor mediated endocytosis [3]. Fluorescent probes In order to test the hypothesis that endocytosis is involved in micelle uptake, a probe that fluoresces more strongly in acidic environments was used, namely Lysosensor Green (Molecular Probes, L-7535, Eugene, OR). Lysosensor Green has a pKa of 5.2, which gives it a very low fluores- cence until it enters inside acidic compartments of the cell. The pH inside endosomes is about 4.8, and therefore this probe will show an increase in fluorescence when it is lo- cated in the endosome. As a control in some experiments, a non-pH sensitive flu- orescent probe was used, namely Cell Tracker Orange CMTMR (C-2927, Molecular Probes, Eugene, OR). Measuring the partition coefficient Lysosensor Green is hydrophobic, which indicates that in aqueous Pluronic solutions it will partition to the poly (propylene oxide) core of the Pluronic micelles, similar to the manner in which Doxorubicin and Ruboxyl partition to the core [4]. The partition coefficient of Lysosensor Green between phosphate buffered saline (PBS) (pH = 7.4) and poly (propylene oxide) (PPO, MW = 4500 g/mol, Sigma Chemicals) was determined using the fluorescent characteristics of the probe. Solutions with a known con- centration of Lysosensor Green in both PBS and PPO were prepared and placed in a fluorometer. The pH was kept at 7.4. The samples were excited at 443 nm, and the emis- sion at 505 nm was measured. From these data, a calibra- tion plot of probe concentration vs. emission intensity was prepared in both PPO and PBS. Next, Lysosensor Green (final concentration 1 µ M) was placed into a vial containing both PBS and PPO and was magnetically stirred overnight in order to allow equilibri- um to be established. This produced an emulsion that was broken by centrifugation. The water and PPO phases were separated, and the emission intensity was measured for each sample. The concentration of the probe in each phase was determined from calibration plots, from which the partition coefficient was calculated. Flow cytometry One ml of HL-60 cells (1 × 106) was mixed with 1 ml of solutions of a fluorescent probe (final concentration of the probe was about 1 µ M). After one hour of incubation or ultrasonication, the cell fluorescence was determined using a flow cytometer (Coulter EPICS-XL cytometer, Coulter Corp., Hialeah, FL), and the fluorescence was compared between the ultrasonicated and non-ultrasoni- cated cells. Ultrasonication Ultrasound power was generated by a Sonicor SC-100 ul- trasonicating bath (Sonicor Instr., Copaique, N. Y.) oper- ating at 70 kHz. The power density was controlled by adjusting the input voltage using a variable A.C. trans- former (variac). The insonation intensity as a function of applied voltage was determined using a calibrated hydro- phone (Bruel and Kjaer model 8103, Decatur, GA). The acoustic intensity generated by the Sonicor bath increased with applied voltage. Following calibration experiments, the variac voltage was adjusted to produce an ultrasonic intensity of 1.2 W/cm2. Since we were not studying the ef- fect of hyperthermia induced by ultrasonication, the tem- perature of the bath was maintained at 37°C using a recirculating thermostatic bath. Results The partition coefficient of the Lysosensor Green between the PPO and PBS phases was measured to be 13; i.e., for every 13 molecules present in the PPO, one molecule is present in the surrounding aqueous solution. The meas- urement of the partition coefficient was not completely accurate, because some of the PPO molecules were still present in the water phase even after the sample was cen- trifuged three times. This causes the amount of the probe measured in the water phase to be larger than if the water were pure, indicating that the true partition coefficient is larger than 13. The high value of the partition coefficient indicates that the Lysosensor Green probe is fairly hydro- phobic, which suggests that it will accumulate inside the poly (propylene oxide) core of the P105 micelle, similarly to the accumulation of Dox and other hydrophobic drugs. Flow cytometry was used to determine if the mode of drug uptake was related to endocytosis. To do so, two cell lines were used, HL-60 cells with a relatively low amount of en- Page 2 of 6 (page number not for citation purposes)