Fig 1.
Schematic description of empty and NC containing PEG-modified lipid micelles without terminal groups and with COOH or NH2 moieties, respectively and corresponding short hand notation.
Fig 2.
Preparation and characterization of ‘as synthesized’ CdSe@ZnS NCs.
TEM (A) and HRTEM micrographs (B), absorption (E, black line) and PL (E, red line) spectra of organic capped CdSe@ZnS NCs dispersed in CHCl3. Picture of the sample under visible (C) and UV (D) light illumination.
Fig 3.
Effect of organic capped CdSe@ZnS NCs on cell viability of astrocytes.
Confluent astrocytes, plated in 96 well plates, were treated with luminescent organic capped CdSe@ZnS NCs or CHCl3 at the indicated concentrations. The control (CTRL, A) was obtained from untreated astrocytes in serum-free DMEM. After treatment for 24 h at 37°C, 5% CO2 the cells were subjected to the cell viability test with MTT as described in Experimental section. Micrographs show representative results of cell morphology observed under phase-contrast microscope (50X magnification) after 24 h of treatment with NCs (C, left panel) or CHCl3 (C, right panel). The graphs represents the cell viability expressed as percentage of cell survival in comparison with control (CTRL) (B). A dose of NCs or CHCl3 that determined a cell viability < 60% was considered toxic. Data represent the mean values ± SD of three different experiments performed on different cell populations.
Fig 4.
ζ-Potential measurements of empty PEG-modified lipid micelles without terminal groups (MIC, blue line) and with COOH (MIC-COOH, green line) or NH2 groups (MIC-NH2, red line) (A). Size distribution of empty PEG-modified lipid micelles described in terms of average hydrodynamic diameter by intensity and polydispersity index (PDI). Surface charge of empty PEG-modified lipid micelles by ζ-potential measurements (B). TEM micrograph with positive staining of MIC (C). Hydrodynamic diameter distribution by intensity of MIC dispersed in PBS at pH 7.4 (D).
Fig 5.
Effect of empty PEG-lipid micelles on cell viability of astrocytes.
Confluent astrocytes, plated in 96 well plates, were treated with the three different preparation of PEG-lipid micelle represented by PEG-lipid micelle, bare, i. e. without terminal groups (MIC), and COOH- (MIC-COOH) or NH2- (MIC-NH2) terminated, respectively, at the indicated phospholipid concentration. The control (CTRL) was obtained from untreated astrocytes in serum-free DMEM. After treatment for 24 h at 37°C, 5% CO2 the cells were subjected to the cell viability test with MTT as described in Experimental section. Micrographs show representative results of cell morphology observed under phase-contrast microscope (50X magnification) after 24 h of treatment. The graphs represent the cell viability expressed as percentage of survival cells in comparison with control (CTRL). The dose of MIC, MIC-COOH and MIC-NH2 determining a cell viability < 60% was considered toxic. Data report the mean values ± SD of three experiments performed on different cell populations.
Fig 6.
Preparation and characterization of NC/MIC-COOH.
Absorption (black line), PL (red line) spectra (A), hydrodynamic diameter distribution by intensity (B) and TEM micrograph with positive staining (D) of NC/MIC-COOH dispersed in PBS at pH 7.4. Schematic sketch of NC/MIC-COOH (C).
Fig 7.
Effect of CdSe@ZnS NC containing PEG-lipid micelles on cell viability of astrocytes.
Confluent astrocytes, plated in 96 well plates, were treated, at the indicated phospholipid concentrations, with two different preparation of PEG-lipid micelle functionalized with COOH-groups and containing CdSe@ZnS NCs at concentration of 1.2 μM (Prep. Low) and 6 μM (Prep. High) respectively (NC/MIC-COOH). The control (CTRL) was obtained from untreated astrocytes in serum-free DMEM. After treatment for 24 h at 37°C, 5% CO2 the cells were subjected to the cell viability test with MTT as described in Experimental section. Micrographs show representative results of cell morphology observed under phase-contrast microscope (50X magnification) after 24 h of treatment (A and C). The graphs represents the cell viability expressed as percentage of survival cells in comparison with control (CTRL) (B). The doses of NC/MIC-COOH that determined a cell viability < 60% were considered toxic. Data represent the mean values ± SD of three experiments performed on different cell populations.
Fig 8.
Intracellular visualization of NC/MIC-COOH nanoparticles by live cell epi-fluorescence microscopy.
Representative bright field (A, C) and fluorescence (B, D) images of living astrocytes after 9 h of incubation with NC/MIC-COOH at the NC concentration of 0.2 nM (A, B) or with serum-free medium alone (negative control) (C, D).
Fig 9.
Evaluation of cellular uptake of NC/MIC-COOH by confocal microscopy.
Confocal differential interface contrast and fluorescence micrographs of fixed astrocytes. Cells images after 1 h of incubation time with NC/MIC-COOH at NC concentration of 0.2 nM. Cell images in the differential interference contrast (Panel A), blue (Panel C) and red (Panel D) detection channel. Overlay of blue and red fluorescence detection channels with (Panel B) and without differential interface contrast (Panel E). Scale bar 25 μm.
Fig 10.
Concentration of emitting CdSe@ZnS NCs in cell lysates.
Confluent astrocytes plate in 6 well plates, were treated for 1h with NC/MIC, NC/MIC-COOH or NC/MIC-NH2, at the reported concentrations of phospholipids and NCs. Negative control, obtained from untreated astrocytes in serum free DMEM, was set at zero. After incubation, the concentration of fluorescence CdSe@ZnS NCs in cell lysates was determined by spectrophotometric assay as reported in experimental section. The histograms represent the NC concentration in cell lysates, expressed as mean values ±SD of three experiments performed on different cell populations.
Fig 11.
Effect of NCs, MIC-COOH and NC/MIC-COOH on the production of ROS in astrocytes.
PL spectrum of the lysates from astrocytes treated only with DCFH-DA (CTRL)(A); pretreated with DCFH-DA and then treated with: 100 μM of H2O2 ((B); MIC-COOH at lipid concentration of 5 μM (C) and 100μM (D); ‘as synthesized’ NCs at concentration of 1.8 nM (E) and 35 nM (F); NC/MIC-COOH at lipid concentration of 5 μM and NC concentration of 1.8 nM (G) or NC/MIC-COOH at lipid concentration of 100 μM and NC concentration of 35 nM (H). The PL peak centred at 525 nm is ascribable to DCF, while the peak centred at 605 nm is due to luminescent CdSe@ZnS NCs. Histograms represent ROS production, reported as relative percentage of PL intensity in comparison with the negative control (I). Data are mean values ± SD of three separate experiments performed on different cell populations (one-way ANOVA followed by Student-Newman-Keuls; *p < 0.05 and **p < 0.001).