Selective and long-term staining of mitochondria

Chapter 3. Mixed-Charge Nanocarriers Allow for Selective Targeting of Mitochondria by Otherwise

3.3 Results

3.3.3 Selective and long-term staining of mitochondria

sequential assembly in aqueous solution – when combined with disulfide-based fluorescein ligands, yielded nanoparticles targeted to mitochondria with high specificity. However, since the simpler sequential assembly did not provide an easy way to separate free ligands and often resulted in aggregated nanoparticle assemblies, we used co-assembled fluorescein nanoparticles in most subsequent experiments.

Figure 3.4. Functional group requirement for mitochondria targeting. (a) Representative confocal microscopy images of various fluorescein nanoparticle assemblies incubated with MCF10A cells for 1 h. Fluorescein nanoparticles were prepared by the sequential assembly in which 50 nM 80:20 NPs were mixed with various FL derivatives either for 20 min or for 18 h in aqueous solution. The concentrations of FL derivatives were such that yielded assemblies with ~100 fluorophores per nanoparticle: 2.5 µM for 3b and 5 µM for all other compounds. No background subtraction was made. Scale bars = 10 μm.

(b) Graph showing the signal-to-background ratios (S/B) for free fluorescein derivatives (white bars) and sequentially-assembled particle assemblies shown in a (patterned bars). S/B > 3 corresponds to specific mitochondrial localization (described in Methods section). Data are mean ± s.d.. (c) The sizes of sequentially-assembled nanoparticles from fluorescence correlation spectroscopy (FCS) mea surements show that all fluorescein derivatives were associated with the particles (Table 3.3).

Sizes of the particles with associated fluorescein derivatives are indicated in color; free ligand s in grey; percentages indicate proportions of free to particle-associated ligands; grey dashed line shows the sizes of 80:20 NPs without fluorescein ligands obtained from dynamic light scattering (DLS). nd = undetectable with FCS. NA = data not available; FCS measurements for symmetric disulfide were not possible due to high FRET-based quenching. Data are mean ± s.d..

Figure 3.5. Additional control experiments with fluorescein derivatives. Representative confocal images (fluorescence channel – upper panel, differential interference contrast, DIC, channel – lower panel) for localization of 5-aminofluorescein (5-FAM), fluorescein isocyanate (FITC), and FL-C5 on their own or when preincubated with 80:20 NPs for 18 h. 5-FAM, FITC, and FL-C5 were added at a concentration of 5 μM, and 80:20 NPs at 50 nM. MCF10A cells were incubated with NP-fluorescein assemblies for 1 h and imaged immediately. Control cells contained no fluorescent dye. No preferential mitochondrial localization was observed for any of these smaller fluorescein derivatives. Images are shown without background subtraction, and brightness was adjusted equally. Scale bars = 10 μm.

Characterization of mitochondrial localization of mixed-charge fluorescein nanoparticles.

Intracellular localization of fluorescein nanoparticles prepared by co-assembly with FL-C10-SS-C10- COOH ligands (Figures 1.1 and 3.3) was examined in detail by live-cell imaging using confocal microscopy. Short-term incubation (~1 h) of HT1080 fibrosarcoma cells with different types of fluorescein nanoparticles (50 nM) resulted in varying degrees of mitochondrial localization (Figure 3.6). All fluorescein nanoparticles, except for the purely anionic [-]/FL NPs, localized to mitochondria with excellent specificity as evidenced by the Pearson’s correlation coefficient, PCC, between fluorescein/ FL NPs and MitoTracker Red images being close to unity (PCC ~0.8-0.9, Figure 3.6a,d).

The brightness of mitochondria labeling (quantified as fluorescence intensity in mitochondria) (Figure 3.6a) reflected the numbers of ligands per nanoparticle detected in ex vivo experiments (Figure 3.3c) with [+/-]9:3/FL nanoparticles yielding the brightest nano-dye, and, at [+/-] to FL ratio of 60:1, even surpassing [+]/FL NPs. Surprisingly, even nanoparticles with net-negative surface charge [+/-]3:9/FL NPs (violet, Figure 3.6a), albeit with less intense fluorescence, localized to mitochondria.

Importantly, the fluorescence intensity of fluorescein (λex = 488 nm) in lysosomal organelles was almost undetectable. To establish this selectivity, we compared the time-course of co-localization of mixed-charge [+/-]9:3/FL NPs with the mitochondria versus the LysoTracker-labeled acidic cellular compartments (Figure 3.6b,c). The localization of these particles to mitochondria (solid lines, Figure 3.6c) was rapid (within ~1 h), while no co-localization with lysosomal organelles was observed (dashed lines, Figure 3.6c).

We also show that mitochondrial targeting of fluorescein nanoparticles was general in both cancer and normal cells of various types. As shown in Figure 3.6e, Pearson’s correlation coefficient (PCC) coefficients between [+/-]9:3/FL NPs and MitoTracker Red images were high (PCC > 0.75) for HT1080, MDA-MB-231, PC3M, MCF10A, Rat2 cells and only slightly lower (PCC ≈ 0.47) for mouse embryonic fibroblasts, MEFs.

Figure 3.6. Selective localization of fluorescein nanoparticles to mitochondria. (a) Fluorescence intensity (a.u. = arbitrary units) of fluorescein in the mitochondria following short exposure of HT1080 fibrosarcoma cells to various FL NPs (50 nM,1 h) prepared by the co-assembly method with FL-C10- SS-C10-COOH (compound 3a; Figure 3.3). Representative confocal images are shown in d. (b) Time- course of the co-localization of [+/-]9:3/FL NPs (50 nM) (magenta) with mitochondria (stained with MitoTracker Red = MTR, solid lines) or with lysosomal organelles (stained with LysoTracker Red = LT, dashed lines) was quantified as Pearson’s correlation coefficient (PCC). Localization of [+/-]9:3/FL NPs to mitochondria was rapid (~1 h) and persistent (here, up to 6 h). At the same time, there was no overlap between fluorescence from FL NPs and LT. Data shown are mean ± s.d.. (c) Quantification of the fluorescein’s fluorescence intensity in mitochondria (FL in Mito; empty boxes) versus lysosomal organelles (FL in Lyso; patterned boxes). Data in a and c are displayed as box-and-whisker plots (see Methods section). (d) Localization of different types of FL NPs to mitochondria in HT1080

fibrosarcoma cells (fluorescence intensity quantification shown in a), representative merged confocal microscopy images. Data are mean ± s.d.. (e) Representative images for additional cancer (MDA-MB- 231, PC3M) and non-cancerous cell types (MCF10A, Rat2, and mouse embryonic fibroblasts, MEF) treated with [+/-]9:3/FL NPs. Data are mean ± s.d.. MitoTracker Red (red), FL NPs (green), co- localization (overlap in yellow). Images are shown after background subtraction. The brightness and contrast for individual color channels were adjusted (see in Methods section). Scale bars, 10 µm.

Uptake mechanism and the fate of MCNP carriers. On order to determine the mechanism of the cellular internalization of mitochondria-targeted fluorescein nanoparticles, MCF10A cells were incubated with [+/-]9:3/FL NPs in the presence of various endocytosis inhibitors (Figure 3.7). These experiments revealed that overall cellular uptake (assessed by quantifying gold amounts in cells by inductively coupled plasma atomic emission spectroscopy, ICP-AES, Figure 3.7a) and mitochondrial localization (quantified from the confocal images as fluorescein’s fluorescence intensity in mitochondria, Figures 3.7b,f) of fluorescein nanoparticles was mainly an energy-dependent process that proceeded, in part, through non-endocytic mechanisms. Moreover, internalization via multifarious endocytic pathways, including the caveolin pathway, clathrin-mediated endocytosis, and even micropinocytosis, was essential for specific and bright staining of mitochondria.

TEM images confirmed non-endocytic and endocytic uptake and revealed presence of nanoparticle “endosomal escape” in ~18% of endosomes observed in MCF10A cells (Figure 3.7d,e).

In HT1080 fibrosarcoma cells, more prominent endosomal disruption (~37% of endosomes) was observed for [+]/FL NPs (associated with higher toxicity), and again more modest effects (~20% of endosomes) for [+/-]9:3/FL NPs suggesting gradual “endosomal escape” of the latter particles. At the same time, gold nanoparticles were not detected in the mitochondrial matrix or membranes with TEM (see “empty” mitochondrion next to the multi-vesicular body MVB containing nanoparticles in Figure 3.7d, iii).

Dark-field microscopy observations were consistent with TEM, inasmuch we detected formation and dispersion of perinuclear aggregates of Au nanoparticles (likely corresponding to early and/or late endosomes) over time, but we did not detect nanoparticles and/or their aggregates inside the mitochondria (Figure 3.7c).

Additionally, we used live-cell imaging to identify nanocarrier-containing vesicles. Au NPs aggregates were imaged with label-free confocal reflection mode. Since co-assembled [+/-]9:3/FL NPs had less identifiable Au NP clusters than sequentially assembled NPs, we performed the experiment at later time points (Figure 3.7g). Two observations were made: (1) By 1 h, nanocarriers occur in Rab7a or Lamp1-positive vesicles, but not Rab5a-positive vesicles; (2) 24 h after labeling, nearly all Au NPs

cluster with Lamp1-vesicles (96 ± 4% of sequentially assembled and 90 ± 11% of co-assembled). 56 ± 14% of sequentially assembled and 78 ± 11% of co-assembled Au NPs clusters colocalized with Rab7- vesicles, demonstrating that a high fraction of vesicles carrying nanocarriers were Rab7a and Lamp1 positive. Both Lamp1- and Lamp1/Rab7-vesicles are considered as endo-lysosomal terminal vesicles51 (Figure 3.7g,h). These results match those described in Chapter 2. However, endo-lysosomal damage assessed with galectin puncta assay and the swelling of lysosomes were not observed (Figure 3.8).

These results suggest that fluorescein ligand shells may limit particle aggregation in cancer lysosomes also reducing their cytotoxicity towards cancer cells.

All together, these results reflect nanoparticles acting as the carriers delivering fluorescent ligands to mitochondria, but not necessarily permeating mitochondrial membranes.

Figure 3.7. The role of endocytosis and endo-lysosomal reservoirs in mitochondria staining with fluorescent ligands. Effects of various endocytosis inhibitors on (a, b) mitochondrial localization of fluorescent ligands (here, FL-C10-SS-C10-COOH) delivered by nanocarriers in MCF10A cells. No inhibitors added in ‘Control’. Inhibitors (pathway inhibited): NaN3+2DOG = 0.1% sodium azide and 50 mM 2-deoxyglucose (all ATP-dependent pathways); Amiloride, 50 µM (Na⁺/H⁺ exchangers/macropinocytosis); Filipin, 5 µg/mL (caveolin-mediated endocytosis); Chlorpromazine, 20 µM (clathrin-mediated endocytosis); 4 ºC (all endocytic processes). Data in b are displayed as box-and-

whisker plots (see Methods section). Statistical comparisons were made for each inhibitor group vs.

NP-treated control group (without inhibitors), *p < 0.05, **p<0.00001, one-way ANOVA, Tukey’s post-hoc test. Confocal images in a are shown after background subtraction and equal brightness adjustment. Scale bar = 10 µm. (c) The effects of the same endocytosis inhibitors on the uptake of [+/-]9:3/FL nanocarriers in MCF10A cells. Data are mean ± s.d., *p < 0.05, **p<0.00001, one-way ANOVA, Tukey’s post-hoc test. (d) Dark-field microscopy images showing [+/-]9:3/FL nanoparticle aggregation (yellow-orange spots at t = 1, 24 h after labeling) in MCF10A cells. Control cells don’t contain NPs, and blue/green spots in the control cells correspond to the light scattered by organelles.

Scale bar = 10 µm. (e) TEM images show non-endocytic (defined by single NPs or small aggregates in peripheral cytoplasm, blue arrows in i and iii) and endocytic (ii-iii) uptake of [+/-]9:3/FL NPs. Endo = early endosome; MVB = multivesicular body a.k.a late endosome; Mito = mitochondrion. Red arrows indicate endosomal escape of nanoparticles; in iii, NPs escape endosomes very close to “empty”

mitochondrion not harboring NPs. MEF in ii, and MCF10A in i, iii are shown here. Scale bars = 200 nm. (f) Quantification of endosomal escape observed in TEM images shown in e. (g, h) Colocalization of Au NP clusters with vesicles marked with: Rab5a-TagRFP (early endosome), Rab7a-TagRFP (late endosome/lysosome), Lamp1-TagRFP (lysosome/autolysosome). The data is displayed as split violin plots: blue lines show median values, and dashed lines indicate the 25^th and 75^th percentiles of the data.

For merged images shown in h, the brightness/contrast for individual color channels was adjusted. Scale bars: large images, 10 µm; insets, 5 µm. Fluorescent ligands stain mitochondria (blue), while nanocarriers (imaged label-free with confocal reflection microscopy, green) accumulate in endo- lysosomal vesicles (Tag-RFP, red) in contact with mitochondria.

Figure 3.8. Absence of endo-lysosomal damage and swelling of lysosomes. (a) U2OS-mCherry- galectin3 cells were untreated (control) or treated with Siramesine (10 µM, 12 h), a cationic amphiphilic drug that permeabilizes lysosomes, as a positive control. Mitochondria were stained by incubating

U2OS-mCherry-galectin3 with 100 nM [+/-]9:3/FL NPs in antibiotic and FBS-free DMEM for 1 h, after which media was replaced with complete cell culture media. Control and siramesine-treated cells were unstained. Despite the higher concentration of FL NPs (used to observe maximal effect), specific and bright mitochondrial staining was achieved in the absence of galectin3 puncta, indicating the absence of endo-lysosomal damage. (b) HT1080 fibrosarcoma cells were incubated for 1 h or 6 h with 50 nM fluorescein nanoparticles ([+]/FL, orange, empty box; [+/-]9:3/FL, magenta, empty box), pure-TMA NPs ([+] = 100:0; orange, patterned box), or standard mixed-charge 80:20 NPs ([+/-]9:3 = 80:20; magenta, patterned box). Cells cultured in the absence of NPs served as a control (black box). Cells were co- stained with 50 nM LysoTracker Red for the last 30 min of NP incubation, and lysosome diameters were quantified as previously described²⁶. Data on graphs is displayed as box-and-whisker plots (see Methods section). *p < 0.05, one-way ANOVA, Tukey’s post-hoc test. ‘ns’ indicates that the differences between the means for the treatment groups are not statistically significant from the means for the control group at the 0.05 level.

Long-lasting staining of mitochondria with mixed-charge fluorescein nanoparticles. The characteristic that sets our approach apart from most current mitochondria-targeting efforts is the specific and long-lasting retention (over days) of bright fluorescence in the mitochondria. To demonstrate this, we performed a series of long-term experiments in which we compared the persistence of mitochondrial labeling for the brightest co-assembled mixed-charge fluorescein nanoparticles [+/-]9:3/FL NPs and corresponding sequentially-assembled particles (80:20 NPs + FL-C10-SS-C10- COOH or FL-C10) with commercially available MitoTracker Green dye (Figure 3.9). MCF10A cells, plated at low density to allow for cell division, were exposed to the dyes for short time (1 h), followed by the removal of the dyes, replacement of media, and imaging of live cells daily (up to 72 h, a time when cells grew to confluent monolayer). The continuous cell division evidences the excellent biocompatibility of fluorescein nanoparticles. The pattern of bright and specific labeling of mitochondria achieved by brief exposure to [+/-]9:3/FL NPs remained unchanged at least up to 72 h, while sequentially-assembled NPs showed bright, specific labeling up to 48 h. Moreover, despite the ongoing cell division, which would typically “dilute” fluorescent probes, there was a considerable (~20%

of initial fluorescence intensity) increase in the fluorescence intensity 24-48 h after the labeling with [+/-]9:3/FL NPs. In contrast, the fluorescence intensity of mitochondria labeled with MitoTracker Green decreased by ~50% within the first 24 h and was close to background levels by 72 h (Figure 3.9d).

Similarly, the initially bright, but somewhat non-specific staining of cellular membranes with sequentially-assembled 80:20 NP + FL-C10 particles or free FL-C10 molecule decreased by ~62% or 82%, respectively, within 24 h reflecting a failure to maintain long-lasting membrane staining (Figure

3.9c and Figure 3.10). Furthermore, we showed that nanocarriers containing on-NP ligands with alkyl linkers are crucial for long-lasting mitochondria staining with asymmetric disulfides. Nanocarriers with hydrophilic ethylene-glycol linkers, on the other hand, produce specific but short-lived staining (Figure 3.11).

We also examined the suitability of fluorescein nanoparticles for tracking the dynamic rearrangements of mitochondria networks³ in living cells. Due to the larger mitochondria size (d > 1 µm), and the extended range of mitochondria dynamics (sec to min), their tracking does not necessarily require advanced super resolution approaches. As elsewhere in this paper, we used confocal laser scanning microscopy (CLSM) to image mitochondria in cells labeled with fluorescein nanoparticles ([+]/FL or [+/-]9:3/FL in MCF10A, and [+/-]9:3/FL or [+/-]6:6/FL NPs in more sensitive U2OS fibrosarcoma cells) every ~3 sec for up to ~10-min duration, the temporal resolution similar to other studies⁴. These movies showed the continual remodeling of mitochondrial networks. Peripheral mitochondrial tubules showed fast expansions and sideways sliding/displacements followed by retractions and/or fission events. Some expanded tubules joined to another mitochondrion, producing new network nodes that were rapidly modified by fusion and/or fission. Most prior investigations that documented mitochondrial network dynamics with time-lapse imaging (requiring repeated exposures of the same cells) employed genetically-encoded fusion proteins (for example, mEmerald-Tomm20 outer membrane protein)^2–4 to mark mitochondria. Small-molecule dyes are more likely to cause photo- induced mitochondrial swelling and fragmentation (preventing network dynamics), which can be reduced by adding oxygen scavengers and/or antioxidants, such as Trolox, to the imaging medium^17,18. Importantly, we imaged in native media conditions without such additives and did not see phototoxicity.

Specifically, cancer cells, such as U2OS, were more sensitive to more cationic [+/-]9:3/FL NPs, but were successfully imaged by using less cationic [+/-]6:6/FL particles (see Figure 3.9f).

Figure 3.9. Long-term mitochondrial labeling after short exposure to fluorescein nanoparticles.

MCF10A cells were incubated for 1 h with (a) co-assembled nanoparticles: 50 nM [+/-]9:3/FL NPs where FL ligand is FL-C10-SS-C10-COOH, or (b,c) with sequentially-assembled nanoparticles: 80:20 NPs (50 nM) mixed with 5 µM (b) 3a, FL-C10-SS-C10-COOH or (c) 3d, FL-C10 (18 h assembly time), or with (d) 200 nM MitoTracker Green dye. Next, dyes were removed, media was replaced, and cells were imaged immediately (1 h = Control), 24 h, 48 h, or 72 h after the labeling. Images are shown after background subtraction (see Methods section). Note, that cells continue to divide. (e) The graph shows normalized fluorescein’s fluorescence intensity in mitochondria (expressed as % of control/1 h) for co-assembled [+/-]9:3/FL NPs (magenta), sequentially-assembled FL-C10-SS-C10-COOH + 80:20 NPs (green), sequentially assembled FL-C10 + 80:20 NPs (purple), and MitoTracker Green (grey) corresponding to images shown in a-d. Data are mean ± s.d.. (f) Time-lapse confocal images of living U2OS cells with mitochondria stained using [+/-]6:6/FL FL-C10-SS-C10-COOH NPs (100 nM, 2 h).

Red arrows indicate a rapidly growing mitochondrial tubule; asterisks indicate fission. Scale bars = 10 µm.

Figure 3.10. Cell staining with C10-alkyl fluorescein derivative. MCF10A cells were incubated with 5 µM FL-C10 for 1 h after which media were replaced, and cells were imaged immediately (1h/Control) and as indicated up to 72 h. Images are shown after background subtraction and equal brightness adjustment (see Methods section). Scale bars = 10 µm. The graph shows normalized fluorescence intensity (expressed as a percent of the initial 1 h/Control levels) of FL-C10 in mitochondria. Data are shown as mean ± s.d..

Figure 3.11. Long-term mitochondria staining of nanocarriers with ethylene glycol linkers. (a) Structure of on-NP ligands used to generate hydrophilic 80:20 (EG3/EG3) and 80:20 (EG5/EG5) nanocarriers. All particles were sequentially assembled with asymmetric disulfide FL-C10-SS-C10- COOH ligands. (b-d) Mitochondrial staining with asymmetric disulfides delivered with hydrophilic nanocarriers is specific but short-lived (solid lines in g), as opposed to longer-lasting staining with standard 80:20 NPs (dashed line in g, data from Figure 3.9 shown for comparison). Scale bars = 10 µm.

Data are shown as mean ± s.d..

Asymmetric disulfides covalently bind to cellular proteins. SDS-PAGE examination under non- reducing conditions was used to test if FL-C10-SS-C10-COOH asymmetric disulfides could co valently bind to biological proteins through disulfide bonds⁴⁵. We demonstrate that asymmetric fluorescein ligands were conjugated to cellular proteins with a wide range of molecular weigh ts, both in cell lysates and when delivered to mitochondria in living cells with 80:20 mixed-c harge nanocarriers (although the profiles of bound proteins were not identical, see Figure 3.1 2, top panel). The FL-C10 ligand lacking a disulfide bond did not interact to biological prote ins, as hypothesized.

Figure 3.12. Asymmetric fluorescein disulfides covalently bind to cellular proteins. MCF10 A cell lysates (10 µg protein) were incubated with each fluorescein derivative (2 nmol) at 37

°C for 1 h (lanes 1,2). Alternatively, 80:20 NPs (50 nM) were mixed with FL-C10 or FL-C1 0-SS-C10-COOH (5 µM) for 18 h at rt; to label mitochondria, the mixture was incubated wit

h MCF10A cells at 37 °C for 6 h, followed by cell lysis (lanes 4,5). MCF10A cell lysate wi thout fluorescent dyes/ NPs (lane 6). The samples were resolved by SDS-PAGE (10 µg/lane f or 1,2,6 or 30 µg/lane for 4,5; lane 3 was empty). Std is protein standards. The arrow points at free fluorescein ligands running at the front of the gel, Fl.int (a.u.) = fluorescence intensity, arbitrary units.

Lastly, we asked if mitochondria staining is resistant to fixation and mitochondria depolarization.

Very similar results have been previously reported MitoTracker dyes, which is retained in mitochondria by thiol-reactive chloromethyl group(s) forming covalent bonds with cysteine residues in mitochondrial proteins^12,15, mitochondria staining with asymmetric disulfides is compatible with formaldehyde fixation (Figure 3.13). We observed that ~66% of fluorescence intensity from [+/-]9:3/FL NPs remains in the mitochondria after dissipation of the negative potential across the inner mitochondrial membrane with a protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (Figure 3.14).

Dalam dokumen Diana Kolygina (Halaman 91-109)