Unexpected Intracellular Biodegradation and Recrystallization
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Unexpected intracellular biodegradation and recrystallization of gold nanoparticles Alice Balfouriera, Nathalie Luciania, Guillaume Wangb, Gerald Lelongc, Ovidiu Ersend, Abdelali Khelfab, Damien Alloyeaub, Florence Gazeaua,1,2 , and Florent Carna,1,2 aLaboratoire Matiere` et Systemes` Complexes, CNRS, Universite´ de Paris, Paris 75205 Cedex 13, France; bLaboratoire Materiaux´ et Phenom´ enes` Quantiques, CNRS, Universite´ de Paris, Paris 75205 Cedex 13, France; cMuseum´ National d’Histoire Naturelle, CNRS, Institut de Recherche pour le Developpement´ (IRD), Institut de Mineralogie,´ de Physique des Materiaux´ et de Cosmochimie, Sorbonne Universite,´ Paris 75005, France; and dInstitut de Physique et Chimie des Materiaux´ de Strasbourg, Universite´ de Strasbourg, CNRS, Strasbourg 67087, France Edited by Catherine J. Murphy, University of Illinois at Urbana–Champaign, Urbana, IL, and approved November 18, 2019 (received for review July 10, 2019) Gold nanoparticles are used in an expanding spectrum of biomed- processes have been described for metal or metal oxide nanopar- ical applications. However, little is known about their long-term ticles, but only few concern GNPs (23–25). As a noble metal, gold fate in the organism as it is generally admitted that the inert- is more inert, less sensitive to acidic environment, and less reac- ness of gold nanoparticles prevents their biodegradation. In this tive than most other metals and metal oxide (26). Therefore, the work, the biotransformations of gold nanoparticles captured by current dogma is that the inertness of gold prevents biodegra- primary fibroblasts were monitored during up to 6 mo. The combi- dation of gold implants or GNPs that could be left indefinitely nation of electron microscopy imaging and transcriptomics study intact in tissues. Sustained integrity of GNPs can indeed be an reveals an unexpected 2-step process of biotransformation. First, asset to maintain their nanoscale-related optical properties for there is the degradation of gold nanoparticles, with faster dis- a long time in the body. This inertness of gold would also pre- appearance of the smallest size. This degradation is mediated by vent the release of potentially toxic bioactive uncontrolled ionic NADPH oxidase that produces highly oxidizing reactive oxygen forms of gold. So far, to our knowledge, only 1 study from our species in the lysosome combined with a cell-protective expres- group has reported in vivo degradation of 5-nm gold nanocrys- sion of the nuclear factor, erythroid 2. Second, a gold recrystalliza- tals to 3-nm size, which was observed in mice spleen 3 mo after tion process generates biomineralized nanostructures consisting systemic administration of gold iron oxide heterostructures and of 2.5-nm crystalline particles self-assembled into nanoleaves. dissolution of the iron oxide shell (27). Although numerous stud- BIOPHYSICS AND COMPUTATIONAL BIOLOGY Metallothioneins are strongly suspected to participate in build- ies have been conducted in vitro on the influence of GNPs on ings blocks biomineralization that self-assembles in a process cultured cells, only few exceed a few days, and no clue indicates that could be affected by a chelating agent. These degrada- GNP degradation on electron microscopy (28–32). tion products are similar to aurosomes structures revealed 50 y Here, we revealed the unexpected degradation of GNPs of dif- ago in vivo after gold salt therapy. Overall, we bring to light ferent sizes in lysosomes up to 6 mo in fibroblasts and evidenced steps in the lifecycle of gold nanoparticles in which cellular path- a surprising phenomenon of recrystallization and self-assembly ways are partially shared with ionic gold, revealing a common gold metabolism. MEDICAL SCIENCES Significance gold nanoparticles j biodegradation j biomineralization j nanoparticles fate While gold nanoparticles are at the core of an increasing range of medical applications, their fate in the organism has barely old has been used in various forms in medicine since antiq- been studied so far. Because of their chemical inertness, com- Guity (1). Therapeutic use of gold in modern medicine began mon belief is that gold nanoparticles remain endlessly intact in in 1890 with the discovery that gold (AuI) cyanide was bacterio- tissues. We show that 4- to 22-nm gold nanoparticles are actu- static to the tubercle bacillus in vitro (2). This result initiated ally degraded in vitro by cells, with a faster degradation of the the development of chrysotherapy in modern medicine to treat smallest size. Transcriptomics studies reveal the active role of rheumatoid arthritis (3, 4). The use of those therapies have cell lysosome into this biodissolution. Furthermore, we point declined since the 1980s due to long-term adverse effects and the out that the released gold recrystallizes into biopersistent development of alternative treatments (5, 6). Gold nanoparticles nanostructures. Interestingly, these degradation products are (GNPs), which were only exploited from the 1950s as a source similar to previously observed gold deposits in human tissues after gold salts treatment for rheumatoid arthritis, underly- of gamma ray (i.e., radioactive Au198 seeds) for cancer treatment ing a common metabolism between gold nanoparticles and (7), raised new interest for their plasmonic and radiosensitizing ionic gold. properties in an expanding spectrum of biomedical diagnostic [photoacoustic imaging (8), 2-photon luminescence (9), surface Author contributions: A.B., N.L., D.A., F.G., and F.C. designed research; A.B., N.L., F.G., enhance Raman spectroscopy (10)] and therapeutic applica- and F.C. performed research; A.B., N.L., G.W., G.L., O.E., A.K., and D.A. contributed new tion [photothermal therapy (11), photodynamic therapy (12), reagents/analytic tools; A.B., N.L., D.A., F.G., and F.C. analyzed data; and A.B., N.L., D.A., radiosensitization (13)] to cite but a few. F.G., and F.C. wrote the paper.y A wide variety of GNPs with different sizes, shapes, and coat- The authors declare no competing interest.y ings has been studied in vitro and in vivo so far to evaluate This article is a PNAS Direct Submission.y their distribution (14, 15) and toxicology (16–18). It is estab- Published under the PNAS license.y lished that GNPs are internalized mostly in the liver and spleen Data deposition: The transcriptomic data and script reported in this paper are available by macrophages (19, 20) and sequestrated inside their lysosomes at the Zenodo repository, https://zenodo.org/record/3530617#.XfFNDht7ncs.y (21, 22), the organelles responsible for the degradation and 1 F.G. and F.C. contributed equally to this work.y recycling of exogenous (xenobiotics, bacteria) or endogenous 2 To whom correspondence may be addressed. Email: florence.gazeau@univ-paris- compounds (defective organelles, protein aggregates). Lyso- diderot.fr or fl[email protected] somes are characterized by an acidic environment (pH around This article contains supporting information online at https://www.pnas.org/lookup/suppl/ 4.5), which is regulated by proton pumps, and contain spe- doi:10.1073/pnas.1911734116/-/DCSupplemental.y cific enzymes, namely acid hydrolases. Lysosomal degradation First published December 18, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1911734116 PNAS j January 7, 2020 j vol. 117 j no. 1 j 103–113 Downloaded by guest on September 29, 2021 of their degradation products. Ultrastructural observations of face proportion decreasing with time and second, an increasing GNPs in cells were combined with transcriptomic analysis to proportion of more diffuse structures (Fig. 1 D–K). Interestingly, unravel the biotransformations of GNPs kept in active lysosomes this phenomenon of apparent GNPs transformation shows het- and to shed light on the mechanisms of lysosomal process- erogeneous aspects. At the cell level, 2 wk after exposure to ing of gold species. To address this challenge, we implemented GNPs, some lysosomes are the seat of GNP transformations, a methodology that enables long-term cell culture and high- while others show only unchanged GNPs. At the lysosome level, resolution detection of GNPs and of their degradation products when diffuse structures are present, they are often, but not nec- up to 6 mo after internalization in cells. essarily, in contact with a domain containing intact GNPs. The Primary human fibroblasts were chosen for their ubiquitous electronic contrast, spatial concentration, and aspect of the dif- character in the body, their potential intended or acciden- fuse structures as well as the original GNPs do not seem to evolve tal exposition to GNPs, and their low proliferation rate that over time on our study timescale. However, the surface propor- enhances the residence time of nanoparticles in the same cell, tion of the diffuse structures relative to the total surface occupied enabling culture over several months and long-term follow-up by electron-dense objects increases from 43% after 2 wk to 89% of nanoparticles. Spherical GNPs (diameter [D] = 4, 15, and after 2 mo and did not change between 2 and 6 mo (Fig. 1L). 22 nm) (SI Appendix, Fig. S1) covered with noncytotoxic cit- The nature of these appearing diffuse structures was then further rates were chosen because of their narrow size distribution, easy explored with high-resolution electron-based tools. synthesis, and wide use. GNP-labeled fibroblasts’ viability was High-resolution scanning transmission electron microscopy maintained for 6 mo, with curbed proliferation to limit the dilu- (STEM) observations performed 2 wk after exposure to GNPs tion of GNPs on cell division and enable us to monitor their in reveal that the diffuse structures are composed of discrete situ biotransformation over time (SI Appendix, Fig. S2). nanoparticles with a characteristic size of 2.5 ± 0.4 nm (Fig. 2 A–D). Interestingly, these individual elements are most often Results and Discussion aligned along curved trajectories forming lash-like architectures The 6-Mo Biotransformations of GNPs in Lysosomes. The biotrans- with similar thickness (t = 5.3 ± 1.4 nm) and radius of curva- formation of 4-nm GNPs in fibroblasts was monitored by trans- ture (Rc = 43.5 ± 13.2 nm).