Table 1.
Composition of nanoemulsions.
Figure 1.
Proposed nanoemulsion droplet.
Droplet carrying celecoxib, perfluoropoly (ethylene glycol) ether (PFPE) and near-infrared fluorescence (NIRF) dye. Cremophor EL® (CrEL) and Pluronic® P105 (P105) are the nonionic surfactants. Miglyol 810N is the hydrophobic oil phase.
Figure 2.
Shelf life of nanoemulsions with average droplet diameter (nm) at 4°C and 25°C.
(A) Representative size distribution by intensity of nanoemulsions A (black) and B (red). (B) Stability of nanoemulsion A. (C) Stability of nanoemulsion B. Error bars represent half width of polydispersity index (PDIw/2).
Figure 3.
Macrophage cell viability post labeling.
(A) Nanoemulsion A (B) Nanoemulsion B. Each data point represent mean of at least three replicates and the error bars are standard deviation of the mean. Values are reported as percent control (0 mg/mL PFPE).
Figure 4.
19F NMR and NIRF imaging of nanoemulsion B labeled macrophages.
(A) 19F NMR of cells labeled with nanoemulsion B. 0.02% v/v aqueous TFA set at −76.00 ppm was used as reference for 19F NMR. (B) NIRF image (at 800 nm) of cells labeled with nanoemulsion B in NMR tube.
Figure 5.
19F NMR-NIRF correlation of labeled macrophage cells.
Data points represent cells labeled with different concentrations of nanoemulsion B (0–1.4 mg/mL PFPE).
Figure 6.
Fluorescence images of macrophages.
(A) Cells labeled with anti-CD45 (FITC) green and incorporated nanoemulsion C containing celecoxib and Cellvue® Burgundy dye represented as red. (B) Cells not exposed to the nanoemulsion C exhibit CD45 labeling with FITC (green) but no red signal. Transmitted light DIC image acquired simultaneously shows field of view (Bar = 30 µm). The microscope image acquisition parameters were identical between the experimental and control, and the images were all acquired within 15 min of one another.
Figure 7.
Magnified fluorescence images of individual macrophages exposed to nanoemulsion C.
(A) Cells labeled with CD45-FITC (green) and incorporated nanoemulsion C containing celecoxib and Cellvue® Burgundy dye exhibit broad expression of CD45 as well as localized points of fluorescent signal indicating internalization of CD45 protein. (B) The same cell and focal plane as viewed in panel A reveals the internalized Cellvue® Burgundy labeled nanoemulsions as discrete particles. (C) The transmitted light DIC view of the cell reveals the black refractive droplets, coincident with the red and green fluorescent signals (Bar = 5 µm). (D) A different cell labeled with CD45-FITC (green), (E) internalized Cellvue® Burgundy (red) and (F) transmitted light DIC view reveals discrete droplets (Bar = 5 µm). (G) The cell shown in panel D was imaged in serial section and rendered by maximum-projection to represent all of the Cellvue® Burgundy labeled particles viewed from above and (H) in 90° cross-section, to reveal that the droplets are distributed throughout the cell cytoplasm.
Figure 8.
Production of PGE2 in macrophages assessed after LPS treatment.
LPS treatment was performed post cell labeling with nanoemulsion B, free drug dissolved in DMSO and DMSO. Cells not exposed to LPS were designated as untreated. * # $ represents statistical significance comparisons (p<0.0001) between treatments. Each data point represents the average of four independent measurements, where the error bars are the standard error of the mean (SEM).