Uptake and Intracellular Trafficking Studies of Multiple Dye-Doped Core-Shell Silica Nanoparticles in Lymphoid and Myeloid Cells

Figures & data

Table 1 Summary of Physicochemical Features of NT_B700 Nanoparticles

Fluorophores	TMB, DEB, Rhod, Cy5, Cy5.5
λ Maximum absorption wavelength	498 nm
λ Maximum emission wavelength	713/2 nm
FRET efficiency	78,8%
Concentration	6,4 mg/mL
Size (NTA)	97,7 ± 1.2 nm
Size (TEM)	30 ± 15 nm

Abbreviations: Cy5, cyanine 5; Cy5.5, cyanine 5.5; DEB, diethynylbenzene; FRET, fluorescence resonance energy transfer; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; TMB, tetramethylbenzidine; Rhod, rhodamine.

Figure 1 Synthesis, physicochemical characterization of NT_B700 nanoparticles and pH reactivity. (A) Schematic representation of the synthesis of the core-shell silica nanoparticles, through a micelle-assisted method, where a surfactant is used to create a nanoreactor within which all reagents arrange. The base-catalysed hydrolysis of a silane precursor, along with different dyes functionalized with a trialkoxysilane group, able to covalently link to the silica matrix, leads to the formation of fluorescent monodisperse NPs. (B) Absorption (dotted grey line) and emission spectra (solid red line) of NT_B700 nanoparticles. (C) NT_B700 diluted 1:1000 analysed using a nanoparticle tracking analysis (NTA), results are represented as mean ± sd; n = 5. (D) Transmission electron microscopy (TEM) analysis of NT_B700 diluted 1:100 (above) and 1:1000 (below), insert are enlargements of NP aggregates. Scale bar = 500 nm; insert scale bar = 100 nm. (E) NT_B700 diluted 1:100 in buffers at different pH (2.5, 4.5, 5.5 and 7.2) and analysed by flow cytometry at three time points (0, 30 and 240 min) for FSC increase to investigate the presence of aggregates.

Figure 2 Kinetics NT_B700 nanoparticle uptake in U937 cells. (A) U937 were incubated with 64 µg/mL NT_B700 at several times (0, 30, 60 and 120 min). Dot plots (FSC vs. SSC) representing U937 treated with 64 µg/mL NT_B700 nanoparticles during time. (B) FSC slightly decreased during time (above); cellular granularity (SSC values) increased in a time-dependent manner (below). (C) Time-dependence of NT_B700 nanoparticle uptake in U937 cells. Mean Fluorescence Intensity (MFI) from the flow cytometry histograms is shown as a function of time. (D) Flow cytometry histogram overlay for cells treated for 1 h with different concentrations of NT_B700 nanoparticles (0, 6.4, 64 and 640 µg/mL) (E) Cell viability of U937 loaded with increasing doses of NT_B700 nanoparticles (0, 6.4, 64 and 640 µg/mL) determined by membrane permeability assay using Propidium Iodide (PI) and flow cytometry analysis to calculate the percentage of PI positive cells, indicating cell death. (F) U937 incubated for 1 h at 37°C with NT_B700 nanoparticles (64 µg/mL), turned out best experimental conditions. Representative confocal images of Ctrl and NT_B700 NPs-treated U937 cells. NT_B700 in red. Scale bar = 10 µm. At least 10,000 events were analysed by flow cytometry for each experimental condition.

Figure 3 Effect of temperature and energy depletion on NT_B700 nanoparticle internalization in U937. Cells were incubated with NT_B700 (64 µg/mL) under different condition: control (NPs incubated at 37°C), +4°C and sodium azide (NaN₃). The percentage of nanoparticle uptake was measured by MFI, determined through flow cytometry (2 independent replicas of 3 experiments), normalized with NP MFI values in normal condition at 37°C (Ctrl is 100% uptake). Asterisks denote a statistically significant difference effect of temperature and energy with their respective control (***p < 0.001, ****p = < 0.0001). At least 10,000 events were analysed by flow cytometry for each experimental condition.

Table 2 Uptake Pathways and Inhibition Conditions

Pathway	Inhibitor	Conc.	Mechanism	References
Clathrin-mediated endocytosis	Chlorpromazine hydrochloride	5 µg/mL	AP2 inhibitor; blocks endosome recycling	Francia et al (2019)^Citation53
Caveolae-mediated endocytosis	Genistein	20 µg/mL	Tyrosin-kinase inhibitor and blocks the recruitment of dynamin II, by interfering with lipid rafts	Vercauteren et al (2010)^Citation54, dos Santos et al (2011)^Citation7
Macropinocytosis	Amiloride	30 µg/mL	Na^+/H^+ exchanger pump inhibitor and prevents Rac1 and Cdc42 signaling	Iversen et al (2011)^Citation55, Francia et al (2019)^Citation53
Microtubules	Nocodazole	7.5 µg/mL	Binds to tubulin, blocking microtubule polymerization	dos Santos et al (2011)^Citation7, Francia et al (2019)^Citation53

Note: The table shows the transport inhibitors selected for this study, their mechanism of action, pathways involved and condition of use.

Figure 4 Effect of pharmacological inhibitors on cell viability and NT_B700 endocytosis in U937 cells. (A) Cell viability assay performed through propidium iodide (PI) staining, analysed by flow cytometry, after 2 h 30 min of exposure to each one of the different inhibitors. Percentage of PI positive cells was calculated respect to control (untreated). (B) Effect of inhibitors on NT_B700 internalization in U937. Cells were pre-treated with each one of the inhibitors (chlorpromazine, genistein, amiloride, nocodazole) for 30 min, followed by 2 h of exposure to NPs, in the presence of the same inhibitor, then washed and analysed by flow cytometry. Normalized MFI (mean fluorescence intensity) was calculated compared to “NPs” which is reported as 100% uptake (cells incubated with NPs in normal condition, at 37°C without inhibitors). Mean values and sd of 2 independent replicas of 3 experiments. Asterisks denote a statistically significant difference (*p < 0.05) between nocodazole and NPs. At least 10,000 events were analysed by flow cytometry for each experimental condition.

Figure 5 Lysosomes and mitochondria involvement in NT_B700 uptake in U937 cells. (A) Representative confocal images of lysosomal (above) and mitochondria (below) involvement in U937 cells incubated for 1 h at 37°C with NT_B700 (64 µg/mL) and then stained with LTG and TMRE. Figure shows LTG (green), TMRE (blue), NT_B700 nanoparticles (red) and merged images (on the right); orange and violet indicate colocalization. Scale bars: 10 µm. Insert: binary version of merged images. The colocalization mask was generated by Image J software to show colocalization pixel. (B) Pearson’s colocalization coefficient (PCC) of LTG and TMRE with NT_B700 in U937 cells. Pearson’s coefficients were derived from three completely independent experiments with >10 fields per experiment contributing to the cumulative result. Each value is expressed as PCC ± sd; asterisks denote a statistically significant difference (****p < 0.0001) between strains. (C) Flow cytometry analysis of lysosomes (LTG), mitochondria (MTG), ROS production (DCF) and autophagolysosomes formation (MDC) expressed as fold of change calculated as the ratio between cells incubated with NPs (64 µg/mL) and control cells. 1 value indicates no differences between treated and untreated. (D) Flow cytometry analysis of O₂- production (MitoSOX) expressed as fold of change calculated as the ratio between cells incubated with NPs (64 µg/mL) and control cells at different time points (1, 4 and 24 h). Asterisks denote a statistically significant difference (***p < 0.001) between strains. At least 10,000 events were analysed by flow cytometry for each experimental condition.

Figure 6 Kinetics of NT_B700 uptake and confocal images of NP internalized in peripheral blood mononuclear cells (PBMCs). (A) PBMCs were incubated at 37°C with 64 µg/mL NT_B700 at different time points (1, 4, 24 and 48 h). Lymphocyte (red) and monocyte (light blue) populations were selected through a gating strategy on FSC vs. SSC dot plot (Figure S1). Mean Fluorescence Intensity (MFI) from the flow cytometry histograms is shown as a function of time in hours. (B) PBMCs were incubated at 37°C with 64 µg/mL NT_B700 at different time points (1, 4 and 24 h), washed and then stained with several mAbs to identify four PBMC subsets: CD5+ (T lymphocytes), CD19+ (B lymphocytes), CD16+ (NK cells) and CD14+ (monocytes). Asterisks denote a statistically significant difference (*p < 0.05) between 24 h strains. (C) Light and fluorescent images of PBMCs incubated for 1 h at 37°C with NT_B700 nanoparticles (64 µg/mL). Representative confocal images of NT_B700 nanoparticles uptake in PBMCs: brightfield (on the left), fluorescent nanoparticles inside cells (in the middle) and merged images (on the right). NT_B700 in red. Scale bar = 10 µm. At least 10,000 events were analysed by flow cytometry for each experimental condition. Error bars represent sd.

Figure 7 Assessment of lysosomes and mitochondria involvement in NT_B700 uptake in PBMCs. (A) Pearson’s colocalization coefficient (PCC) of lysosomes (LTG) and mitochondria (TMRE) with NT_B700 in PBMC cells. Pearson’s coefficients were derived from three completely independent experiments with >10 fields per experiment contributing to the cumulative result. Each value is expressed as PCC ± sd; (***p < 0.001). Below representative confocal images of lysosome (left) and mitochondria (right) colocalization in PBMC cells incubated for 1 h at 37°C with NT_B700 nanoparticles (64 µg/mL) and then stained with LTG and TMRE. Figure shows LTG (green), TMRE (blue), NT_B700 nanoparticles (red) and, respectively, orange and violet indicate colocalization. (B) Flow cytometry analysis of PBMCs incubated at 37°C with 64 µg/mL NT_B700 at different time points (1, 4, 24 and 48 h) washed and then stained LTG, TMRE, DCF and MDC. Fold of change, calculated as the ratio between cells incubated with NPs and control cells (w/o NPs), from the flow cytometry histograms is shown as a function of time in hours. 1 value indicates no differences between treated and untreated. (C) Flow cytometry analysis of O₂- production (MitoSOX) expressed as fold of change calculated as the ratio between cells incubated with NPs (64 µg/mL) and control cells at different time points (1, 4 and 24 h). Mean values and sd of 2 independent replicas of 3 experiments. Asterisks denote a statistically significant difference (***p < 0.001) between strains. (D) Effect of temperature and energy depletion on NT_B700 nanoparticle internalization in PBMC. Cells were incubated with NT_B700 (64 µg/mL) under different condition: control (NPs incubated at 37°C), +4°C and sodium azide (NaN₃). The percentage of nanoparticle uptake was measured by MFI, determined through flow cytometry (2 independent replicas of 3 experiments), normalized with NP MFI values in normal condition at 37°C (Ctrl is 100% uptake). In red (on the left) lymphocyte data and in blue/light blue (on the right) monocyte data. Asterisks denote a statistically significant difference (***p < 0.001; ****p < 0.001) compared to control. At least 10,000 events were analysed by flow cytometry for each experimental condition.

Figure 8 NT_B700 release studies in U937 and PBMC. U937 were incubated with NT_B700 (64 µg/mL) for 1 h, harvested and then seeded again with fresh medium (NP-free). The supernatants of all conditions (treatment of 1 h and 24 h after incubation in fresh medium following 1 h of NT_B700 treatment) have been analysed by flow cytometry. (A) Histogram shows the quantification of extracellular vesicles (EVs) counted by using Dako CytoCount^TM beads in fresh medium and released by the cells in 1 h of treatment or after 24 h of incubation in fresh medium following 1 h of NT_B700 treatment or control cells, in the same experimental conditions. Asterisks denote a statistically significant difference (***p < 0.001) between 24 h strains. (B) Flow cytometry histograms showing the fluorescence of particles in NT_B700 channel (violet peak represents Dako Cyto Count^TM beads, light blue peak represents particles released based on gates in FSC vs SSC dot plot). Cells incubated with NPs presented a marked peak in NT_B700 fluorescence channel (histogram on the right) compared to fresh medium and control cells (without NPs), which showed the ability of cells to excrete NPs after their internalization. (C) Flow cytometry contouring plots (fluorescence of particles in NT_B700 channel vs. CD63 fluorescence) of supernatants of control (above) and treated cells (below) after 24 h in fresh medium, presenting in green CD63+ particles and in blue NT_B700+particles. (D) Non-vesicle and vesicle-related secretion. PBMCs were incubated with NT_B700 (64 µg/mL) for 1 h, harvested and then seeded again with fresh medium (NP-free). The supernatants have been analysed by flow cytometry. Histogram shows the quantification of extracellular vesicles (EVs) counted by using Dako CytoCount^TM beads and CD63+ particles released by the cells after 24 h of incubation in fresh medium following 1 h of NT_B700 treatment or control cells in the same experimental conditions w/o NPs. (E) Pearson’s colocalization coefficient (PCC) of LTG with NT_B700 in PBMC varying during time to investigate lysosome particle secretion. Pearson’s coefficients were derived from three completely independent experiments with >10 fields per experiment contributing to the cumulative result. Each value is expressed as PCC ± sd; Asterisks denote a statistically significant difference (*p < 0.5, ****p < 0.0001) between strains. At least 50,000 events were analysed by flow cytometry for each experimental condition.

Figure 9 Representative scheme of NT_B700 uptake, intracellular trafficking pathways and release.

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