For any tissue examined, nuclear staining was only detected in cells in close proximity to the particles and not in cells separate from the particles (Figures 7(c), 7(d), 7(e), 7(f), and 7(g)). We used an additional fluorescent dye, Dio, to label the PLGA
particles themselves. Dio-labeling facilitated the detection of the particles in tissue sections. Although the emission spectra of Hoechst 33342 and Dio partly overlap, the pattern of nuclear staining appears to be minimally affected because of the differential emission peak wavelength (461nm for Hoechst 33342; 501nm for Dio) and their respective affinities to distinct cellular components (Hoechst 33342, high affinity for nuclear DNA; Dio, high Inhibitors,research,lifescience,medical affinity for the plasma membrane). In practice, we did not observe any nuclear staining in situ when the Dio-labeled particles without Hoechst 33342-incorporation were used (data not shown). Figure 7 Frozen tissue sections of the femoral muscle, Inhibitors,research,lifescience,medical liver, lung, and spleen. The Dio-labeled and Hoechst 33342-incorporated PLGA particles were locally
injected into the femoral muscle or introduced intravenously through the caudal vein. The femoral muscle … Finally, we simulated Inhibitors,research,lifescience,medical characterization of cells isolated from mice after administration of Hoechst 33342-incorporated PLGA particles. We hypothesized that the particles gradually released Hoechst 33342 after peritoneal injection, resulting in a time-dependent increase in the concentration of Hoechst 33342 and enhancement of nuclear staining intensity of peritoneal macrophages in the peritoneal cavity. To test this hypothesis we isolated macrophages from the peritoneal cavity of mice injected with the control Inhibitors,research,lifescience,medical and Hoechst 33342-incorporated particles and then compared their staining pattern to that of U-937 cells incubated Inhibitors,research,lifescience,medical with serial amounts of Hoechst 33342. We divided the range of fluorescence
intensity into the four segments. We defined P1, P2, P3, and P4 segments as the range corresponding to the fluorescent intensity of U-937 cells incubated with 0, 10, 100, or 1000ng/mL Hoechst 33342, Selleck Epigenetic inhibitor respectively (Figure 8(a)). The cells from mice receiving the control particles showed similar cell distribution to that of U-937 cells without Hoechst 33342 (Figure 8(b)). Over 90% of the cells were Terminal deoxynucleotidyl transferase included in the P1 segment (Figure 8(c)). When we examined the cells 20hrs after the injection of the Hoechst 33342-incorporated particles, the peak in cell number shifted to the right and a large population of the cells (70%) fell into the P2 segment. We next examined the cells isolated 60hrs after injection. Two peaks were observed in the P3 segment with the majority of cells (70%) falling into this segment (Figures 8(b) and 8(c)). From the data we calculated the mean Hoechst 33342 concentration to which the isolated cells had been exposed in the peritoneal cavity.