Chapter 2. Targeted crystallization of mixed-charge nanoparticles in lysosomes induces selective death
2.3 Results
2.3.7 Accumulation and clearance of mixed-charge NPs
The LAMP1+ organelles housing the [+/-] NPs aggregates include low-pH lysosomes (pHL- NORMAL ~4.8; pHL-CANCER < 5.5)22,23, as well as higher-pH autolysosomes (ALs, pHAL/MLB < 6.1) formed by lysosome fusion with autophagosomes (APs). Here, we tracked ALs and APs by using LC3B-eGFP- tagRFP autophagy sensor (see Methods section) to show that in the MEF non-cancerous cells [+/-]
80:20 NPs were predominantly found in autolysosomes whereas, in HT1080 cancer cells [+/-] 80:20 NPs mostly localized in both autophagosomes and autolysosomes (Figure 2.16). Specifically, live cell imaging revealed localization of [+/-] 80:20 NPs to acidic LC3B-positive vesicles (ALs) in non- cancerous MCF-10A cells (Figure 2.17), whereas only few [+/-] 80:20 NP aggregates overlapped with LC3B-positive vesicles of any type (both, acidic/ALs or neutral/APs) in cancerous MDA-MB-231 cells (Figure 2.17). Localization to ALs in non-cancerous cells was further supported by TEM analyses showing small NPs aggregates inside organelles containing concentric membrane rings, so called multilamellar bodies (MLBs), characteristic of ALs (Figure 2.16b). In non-cancerous cells, the overall number of autolysosomes increased while the number of autophagosomes decreased over time, indicating activation of the autophagy process; however, in cancer cells, the numbers of autophagosomes and autolysosomes remained roughly constant, with a higher number of autophagosomes than autolysosomes, indicating inhibited autophagic flux (Figure 2.16d,f). Finally, normal cells (MEFs, MCF10A) showed quick clearance of [+/-] 80:20 NPs: ~ 50% of internalized NPs were released into cell medium in (Figure 2.16h,i). However, only 20% of [+/-] 80:20 NPs were released by cancer cells (HT1080) in the same time period (Figure 2.16h). In contrast to [+/-] 80:20 NPs, pure-TMA NPs (
TMA:
MUA = 100:0), besides endocytosis entered cells possibly through direct membrane penetration by single NPs, then accumulated inside the lysosome-like organelles in less extend and undergo potential endosomal /lysosomal escape of NPs into the cytoplasm (Figure 2.18).33
Figure 2.16. Mixed-charge NPs accumulate in autophagic vesicles and are excluded from non- tumorigenic cells through exocytosis. (a) Cancer cells (left) and normal cells (right) illustrating potential aggregation pathways. As presented on the left, in cancerous cells NPs form small clusters
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first in late endosomes and then subsequently combine into larger NP aggregates in lysosomes (Lys).
After this, autophagosomes (APs) merge with Lys, containing NPs, to form autolysosomes (ALs). AP- NP-induced osmotic flows and an imbalance between fusion and budding events inhibit AP-AL 'swelling' and perinuclear clumping, restricting clearance and exocytosis of [+/-] NP aggregates from cancerous cells. (b) TEM images of HT1080 (left) and MEF (right) cells after 24 hours of incubation with 80:20 NPs. Most NP-containing organelles in HT1080 were non-lamellar and had intraluminal vesicles (ILVs). NPs in MEFs, on the other hand, were restricted to ALs containing MLBs. ALs carrying NP made contact with early Lys, simulating 'kiss-and-run' transitory vesicle fusion1 (red arrows). (c–f) APs and ALs were labeled with the eGFP-TagRFP-LC3B autophagy sensor, then cells were incubated with 80:20 NPs (50 nM) for the indicated times. (c) Shows representative merged images (NPs in ALs are magenta; NPs in APs are white). (d, f) Quantification of the number of APs and ALs per cell based on the images in c. (e) In HT1080, NPs are found in both APs and ALs, whereas in MEFs, they are exclusively found in ALs. (g) Table shows that lysosomal membrane permeabilization (LMP) was visible in TEM pictures at 24 hours. (h) Analysis of endocytosed/cleared 80:20 NPs in cancer (left) and normal (right) cells after 14 hours incubation with 50nM. (i) Quantification of Au amounts left inside normal cells (MEF and MCF10A) after NP internalization (50 nM, 24 h and 48h, respectively) reveals that 80:20 NPs have been cleared from non-tumorigenic cells within days. The data in h and i are presented as mean s.d..
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Figure 2.17. Accumulation of mixed-charge nanoparticles in autophagic vesicles: MDA-MB-231 vs MCF-10A. After marking autophagosomes (APs) and autolysosomes (ALs) with the eGFP-TagRFP- LC3B autophagy sensor, cells were incubated for the indicated times with 80:20 NPs (50 nM).
Representative merged confocal images are shown in (a). APs are shown in yellow, ALs are shown in red; NP/AL overlap in magenta and NP/AP overlap in white. Scale bars are 10 µm. (b, d) From the images in a, the number of vesicles per cell was calculated. Plot in (c) shows proportions of cells with NPs localizing only in yellow (AP), only in red (AL), in both types of vesicles (AP + AL), or in neither type of vesicle (see Methods section). In MDA-MB-231 cells, aggregation in autophagic organelles is slower than in HT1080 cells, which is consistent with other experiments. Nonetheless, the colocalization pattern to both APs and ALs is identical to that of HT1080. NP aggregates were exclusively found inside ALs and not inside APs in MCF-10A, similar to MEFs. In b and d, data is presented as mean ± s.d..
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Figure 2.18. Additional TEM images of cancer vs normal cells: Controls and cationic nanoparticles. (a) and (b) show TEM images of H1080 and MEF cell treated with 50 nM of pure-TMA NPs (
TMA:
MUA = 100:0) for 6 h. Images at different magnifications show, loose cell surface aggregation, single pure-TMA NPs entering the cell possibly through direct membrane penetration (green arrowheads), cytoplasmic aggregation (green arrows), limited accumulation inside the lysosome- like organelles and potential endosomal /lysosomal escape of NPs into the cytoplasm (yellow arrowheads).37