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Targeted crystallization of mixed-charge nanoparticles in lysosomes induces selective death of cancer cells

Abstract

Lysosomes have become an important target for anticancer therapeutics because lysosomal cell death bypasses the classical caspase-dependent apoptosis pathway, enabling the targeting of apoptosis- and drug-resistant cancers. However, only a few small molecules—mostly repurposed drugs—have been tested so far, and these typically exhibit low cancer selectivity, making them suitable only for combination therapies. Here, we show that mixed-charge nanoparticles covered with certain ratios of positively and negatively charged ligands can selectively target lysosomes in cancerous cells while exhibiting only marginal cytotoxicity towards normal cells. This selectivity results from distinct pH-dependent aggregation events, starting from the formation of small, endocytosis-prone clusters at cell surfaces and ending with the formation of large and well-ordered nanoparticle assemblies and crystals inside cancer lysosomes. These assemblies cannot be cleared by exocytosis and cause lysosome swelling, which gradually disrupts the integrity of lysosomal membranes, ultimately impairing lysosomal functions and triggering cell death.

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Fig. 1: Summary of how crystallization of mixed-charge NPs in cancer lysosomes leads to selective killing of cancer cells.
Fig. 2: Structure and pH-dependent aggregation of mixed-charge NPs.
Fig. 3: Engineering cancer-specific cytotoxicity by tuning the balance of surface charges on mixed-charge NPs.
Fig. 4: Cellular uptake and differential intracellular aggregation of mixed-charge NPs in cancerous versus normal cells.
Fig. 5: The impact of intracellular aggregation of mixed-charge NPs on lysosome organelles.
Fig. 6: Mixed-charge NPs accumulate in autophagic vesicles and are excluded from non-tumorigenic cells through exocytosis.

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Data availability

The data used to generate results in the current study are available from the corresponding authors upon reasonable request.

Code availability

Code used to compute osmotic pressures and NP volume fractions in lysosomes described in Supplementary Note 3 is available in GitHub repository (https://doi.org/10.5281/zenodo.3570320). Code for spatial analysis of lysosome distributions (from Lysotracker images) along with the raw data are available from the GitHub repository (https://doi.org/10.5281/zenodo.3570315).

References

  1. Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat. Rev. Mol. Cell Biol. 10, 623–635 (2009).

    CAS  Google Scholar 

  2. Maxfield, F. R. W., Willard, J. M. & Lu, S. (eds) Lysosomes: Biology, Diseases and Therapeutics (Wiley, 2016).

  3. Kirkegaard, T. & Jaattela, M. Lysosomal involvement in cell death and cancer. Biochim. Biophys. Acta 1793, 746–754 (2009).

    CAS  Google Scholar 

  4. Aits, S. & Jaattela, M. Lysosomal cell death at a glance. J. Cell Sci. 126, 1905–1912 (2013).

    CAS  Google Scholar 

  5. Domagala, A. et al. Typical and atypical inducers of lysosomal cell death: a promising anticancer strategy. Int. J. Mol. Sci. 19, 2256 (2018).

    Google Scholar 

  6. Petersen, N. H., Kirkegaard, T., Olsen, O. D. & Jaattela, M. Connecting Hsp70, sphingolipid metabolism and lysosomal stability. Cell Cycle 9, 2305–2309 (2010).

    CAS  Google Scholar 

  7. Petersen, N. H. et al. Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase. Cancer Cell 24, 379–393 (2013).

    CAS  Google Scholar 

  8. Atkins, J. H. & Gershell, L. J. Selective anticancer drugs. Nat. Rev. Drug Discov. 1, 491–492 (2002).

    CAS  Google Scholar 

  9. Pagliero, R. J. et al. Discovery of small molecules that induce lysosomal cell death in cancer cell lines using an image-based screening platform. Assay Drug Dev. Technol. 14, 489–510 (2016).

    CAS  Google Scholar 

  10. Kalsin, A. M. et al. Electrostatic self-assembly of binary nanoparticle crystals with a diamond-like lattice. Science 312, 420–424 (2006).

    CAS  Google Scholar 

  11. Kalsin, A. M. & Grzybowski, B. A. Controlling the growth of ‘ionic’ nanoparticle supracrystals. Nano Lett. 7, 1018–1021 (2007).

    CAS  Google Scholar 

  12. Bishop, K. J. M., Wilmer, C. E., Soh, S. & Grzybowski, B. A. Nanoscale forces and their uses in self-assembly. Small 5, 1600–1630 (2009).

    CAS  Google Scholar 

  13. Pillai, P. P., Kowalczyk, B. & Grzybowski, B. A. Self-assembly of like-charged nanoparticles into microscopic crystals. Nanoscale 8, 157–161 (2016).

    CAS  Google Scholar 

  14. Elci, S. G. et al. Surface charge controls the suborgan biodistributions of gold nanoparticles. ACS Nano 10, 5536–5542 (2016).

    CAS  Google Scholar 

  15. Liu, X. S. et al. Enhanced retention and cellular uptake of nanoparticles in tumors by controlling their aggregation behavior. ACS Nano 7, 6244–6257 (2013).

    CAS  Google Scholar 

  16. Liu, X. S., Li, H., Jin, Q. & Ji, J. Surface tailoring of nanoparticles via mixed-charge monolayers and their biomedical applications. Small 10, 4230–4242 (2014).

    CAS  Google Scholar 

  17. Nel, A. E. et al. Understanding biophysicochemical interactions at the nano–bio interface. Nat. Mater. 8, 543–557 (2009).

    CAS  Google Scholar 

  18. Pillai, P. P., Kowalczyk, B., Kandere-Grzybowska, K., Borkowska, M. & Grzybowski, B. A. Engineering gram selectivity of mixed-charge gold nanoparticles by tuning the balance of surface charges. Angew. Chem. Int. Ed. 55, 8610–8614 (2016).

    CAS  Google Scholar 

  19. Verma, A. et al. Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles. Nat. Mater. 7, 588–595 (2008).

    CAS  Google Scholar 

  20. Forest, V., Cottier, M. & Pourchez, J. Electrostatic interactions favor the binding of positive nanoparticles on cells: a reductive theory. Nano Today 10, 677–680 (2015).

    CAS  Google Scholar 

  21. Frohlich, E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int. J. Nanomed. 7, 5577–5591 (2012).

    Google Scholar 

  22. Arvizo, R. R. et al. Effect of nanoparticle surface charge at the plasma membrane and beyond. Nano Lett. 10, 2543–2548 (2010).

    CAS  Google Scholar 

  23. Lin, J. Q., Zhang, H. W., Chen, Z. & Zheng, Y. G. Penetration of lipid membranes by gold nanoparticles: insights into cellular uptake, cytotoxicity, and their relationship. ACS Nano 4, 5421–5429 (2010).

    CAS  Google Scholar 

  24. Leroueil, P. R. et al. Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers. Nano Lett. 8, 420–424 (2008).

    CAS  Google Scholar 

  25. Jiang, Y. et al. The interplay of size and surface functionality on the cellular uptake of sub-10 nm gold nanoparticles. ACS Nano 9, 9986–9993 (2015).

    CAS  Google Scholar 

  26. Dykman, L. A. & Khlebtsov, N. G. Uptake of engineered gold nanoparticles into mammalian cells. Chem. Rev. 114, 1258–1288 (2014).

    CAS  Google Scholar 

  27. Kim, B. et al. Tuning payload delivery in tumour cylindroids using gold nanoparticles. Nat. Nanotechnol. 5, 465–472 (2010).

    CAS  Google Scholar 

  28. Xia, T., Kovochich, M., Liong, M., Zink, J. I. & Nel, A. E. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACS Nano 2, 85–96 (2008).

    CAS  Google Scholar 

  29. Pillai, P. P., Huda, S., Kowalczyk, B. & Grzybowski, B. A. Controlled pH stability and adjustable cellular uptake of mixed-charge nanoparticles. J. Am. Chem. Soc. 135, 6392–6395 (2013).

    CAS  Google Scholar 

  30. Pillai, P. P., Kowalczyk, B., Pudlo, W. J. & Grzybowski, B. A. Electrostatic titrations reveal surface compositions of mixed, on-nanoparticle monolayers comprising positively and negatively charged ligands. J. Phys. Chem. C 120, 4139–4144 (2016).

    CAS  Google Scholar 

  31. Damaghi, M., Wojtkowiak, J. W. & Gillies, R. J. pH sensing and regulation in cancer. Front. Physiol. 4, 370 (2013).

    Google Scholar 

  32. Rao, A. et al. Regulation of interparticle forces reveals controlled aggregation in charged nanoparticles. Chem. Mater. 28, 2348–2355 (2016).

    CAS  Google Scholar 

  33. Marques, M. R. C., Loebenberg, R. & Almukainzi, M. Simulated biological fluids with possible application in dissolution testing. Dissolut. Technol. 18, 15–28 (2011).

    CAS  Google Scholar 

  34. Sandin, P., Fitzpatrick, L. W., Simpson, J. C. & Dawson, K. A. High-speed imaging of Rab family small GTPases reveals rare events in nanoparticle trafficking in living cells. ACS Nano 6, 1513–1521 (2012).

    CAS  Google Scholar 

  35. Paddock, S. Confocal reflection microscopy: the ‘other’ confocal mode. Biotechniques 32, 276–278 (2002).

    Google Scholar 

  36. Kim, C. S. et al. Cellular imaging of endosome entrapped small gold nanoparticles. MethodsX 2, 306–315 (2015).

    Google Scholar 

  37. Adler, J. & Parmryd, I. Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytom. A 77, 733–742 (2010).

    Google Scholar 

  38. Kirkegaard, T. et al. Hsp70 stabilizes lysosomes and reverts Niemann–Pick disease-associated lysosomal pathology. Nature 463, 549–553 (2010).

    CAS  Google Scholar 

  39. Petersen, N. H., Kirkegaard, T. & Jäättelä, M. Lysosomal stability assay. Bio Protoc. 4, e1162 (2014).

    Google Scholar 

  40. Aits, S. et al. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay. Autophagy 11, 1408–1424 (2015).

    CAS  Google Scholar 

  41. Maier, O., Marvin, S. A., Wodrich, H., Campbell, E. M. & Wiethoff, C. M. Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape. J. Virol. 86, 10821–10828 (2012).

    CAS  Google Scholar 

  42. Lajoie, P., Guay, G., Dennis, J. W. & Nabi, I. R. The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies. J. Cell Sci. 118, 1991–2003 (2005).

    CAS  Google Scholar 

  43. Xu, H. & Ren, D. Lysosomal physiology. Annu. Rev. Physiol. 77, 57–80 (2015).

    CAS  Google Scholar 

  44. Asakura, S. & Osawa, F. On interaction between two bodies immersed in a solution of macromolecules. J. Chem. Phys. 22, 1255–1256 (1954).

    CAS  Google Scholar 

  45. Lekkerkerker, H. N. & Tuinier, R. Colloids and the Depletion Interaction (Springer, 2011).

  46. Catapano, E. R., Natale, P., Monroy, F. & Lopez-Montero, I. The enzymatic sphingomyelin to ceramide conversion increases the shear membrane viscosity at the air–water interface. Adv. Colloid Interface Sci. 247, 555–560 (2017).

    CAS  Google Scholar 

  47. Zhao, X. et al. Switchable counterion gradients around charged metallic nanoparticles enable reception of radio waves. Sci. Adv. 4, eaau3546 (2018).

    CAS  Google Scholar 

  48. Yan, Y., Warren, S. C., Fuller, P. & Grzybowski, B. A. Chemoelectronic circuits based on metal nanoparticles. Nat. Nanotechnol. 11, 603–608 (2016).

    CAS  Google Scholar 

  49. Longmire, M., Choyke, P. L. & Kobayashi, H. Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. Nanomedicine 3, 703–717 (2008).

    CAS  Google Scholar 

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Acknowledgements

We thank Y.-K. Jeong for TEM imaging of [+/−] NPs in vitro. We also thank M.-S. Jeong at the Korea Basic Science Institute for the TEM analyses of [+/−] NPs in cells. This work was supported by the Institute of Basic Science, Republic of Korea (award no. IBS-R020-D1 to B.A.G.).

Author information

Authors and Affiliations

Authors

Contributions

M.B., M.S., D.K. and K.K.-G. designed and performed experiments and analysed the data. Y.S. performed osmotic pressure calculations and wrote the codes for the analysis of microscopy images. S.K. and Y.-K.C. performed dark-field microscopy experiments and analysed data. S.L. characterized small-molecule ligands and prepared selected figures for publication at early stages of manuscript preparation. K.K.-G. and B.A.G. conceived and supervised research, designed experiments and wrote the paper. All authors read and corrected the manuscript.

Corresponding authors

Correspondence to Kristiana Kandere-Grzybowska or Bartosz A. Grzybowski.

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Competing interests

Invention disclosure (Patent No. 10-2020-0021515) describing this research has been made to the Institute for Basic Science, which sponsored this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–30, Tables 1 and 2, Videos 1–10, Notes 1–5 and refs. 1–48.

Reporting Summary

Supplementary Video 1

Impact of mixed-charge nanoparticles on lysosomes in HT1080 fibrosarcoma cells.

Supplementary Video 2

Impact of mixed-charge nanoparticles on lysosomes in MDA-MB-231 adenocarcinoma cells.

Supplementary Video 3

Impact of mixed-charge nanoparticles on lysosomes in MCF7 breast carcinoma cells.

Supplementary Video 4

Impact of mixed-charge nanoparticles on lysosomes in non-cancerous mouse embryonic fibroblasts.

Supplementary Video 5

Impact of mixed-charge nanoparticles on lysosomes in non-cancerous epithelial MCF-10A cells.

Supplementary Video 6

Mixed-charge nanoparticle transport through endo-lysosomal system in non-cancerous MCF-10A cells: early events.

Supplementary Video 7

Mixed-charge nanoparticle transport through endo-lysosomal system in non-cancerous MCF-10A cells: late events.

Supplementary Video 8

Mixed-charge nanoparticle transport through endo-lysosomal system in MDA-MB-231 cancer cells: early events.

Supplementary Video 9

Mixed-charge nanoparticle transport through endo-lysosomal system in MDA-MB-231 cancer cells: late events.

Supplementary Video 10

Localization of mixed-charge nanoparticles to autolysosomes in non-cancerous MCF-10A cells.

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Borkowska, M., Siek, M., Kolygina, D. et al. Targeted crystallization of mixed-charge nanoparticles in lysosomes induces selective death of cancer cells. Nat. Nanotechnol. 15, 331–341 (2020). https://doi.org/10.1038/s41565-020-0643-3

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