Downloaded by guest on September 29, 2021 ntttd Min de Institut Universit CNRS, a Balfourier Alice nanoparticles gold of recrystallization and biodegradation intracellular Unexpected .) hc srgltdb rtnpms n oti spe- contain www.pnas.org/cgi/doi/10.1073/pnas.1911734116 and degradation Lysosomal pumps, hydrolases. proton acid by namely around regulated enzymes, (pH cific is environment Lyso- acidic which an aggregates). 4.5), by and characterized endogenous protein are degradation or organelles, somes the bacteria) (defective for (xenobiotics, compounds responsible exogenous of organelles evaluate recycling the lysosomes spleen estab- to their 22), and inside is far liver sequestrated (21, the and It so 20) in (16–18). (19, vivo mostly macrophages internalized toxicology by in are and and GNPs that 15) vitro lished (14, in distribution studied their been has ings (12), few. therapy a but applica- photodynamic cite to therapeutic (11), (13)] radiosensitization and therapy (10)] [photothermal tion spectroscopy surface diagnostic Raman (9), biomedical luminescence enhance 2-photon of (8), spectrum imaging radiosensitizing expanding [photoacoustic and an plasmonic their in for properties source interest a new as raised 1950s Au (7), radioactive the (i.e., from ray gamma exploited of have only were therapies nanoparticles which Gold those 6). (GNPs), the (5, and of treatments effects alternative adverse use of long-term development to The due 1980s the treat 4). since to declined (3, medicine modern arthritis in rheumatoid chrysotherapy of development tubercle the (Au the gold to that discovery static the with 1890 in G fate nanoparticles common nanoparticles gold a revealing light gold, to ionic bring path- with metabolism. cellular we shared gold which partially in Overall, nanoparticles are therapy. gold degrada- ways of salt y lifecycle These 50 gold the revealed in agent. after steps structures vivo chelating aurosomes in a to process ago similar by a are affected products in tion be self-assembles could build- that in that biomineralization participate nanoleaves. blocks to into suspected ings self-assembled strongly are particles Metallothioneins crystalline consisting 2.5-nm nanostructures of biomineralized recrystalliza- gold generates expres- a process Second, cell-protective 2. tion a erythroid factor, with nuclear the oxygen combined of reactive sion lysosome oxidizing the highly in by dis- mediated produces species faster is that degradation with oxidase This nanoparticles, size. NADPH smallest gold the of of degradation appearance First, the biotransformation. is of process there study 2-step transcriptomics unexpected and an reveals imaging combi- microscopy The electron mo. of 6 by to nation inert- up captured during the nanoparticles monitored were that gold this fibroblasts primary In of admitted biodegradation. biotransformations generally their the is prevents work, nanoparticles it gold as long-term of organism ness their the about 2019) known 10, in July is fate review little for biomed- (received However, of 2019 spectrum applications. 18, expanding November ical an approved in and used IL, are Urbana, nanoparticles Urbana–Champaign, Gold at Illinois of University Murphy, J. Catherine by Edited Mat Alloyeau Damien aoaor Mati Laboratoire ievreyo Nswt ifrn ie,sae,adcoat- and shapes, sizes, different with GNPs of variety wide A raxd tabug Universit Strasbourg, de eriaux ´ iy() hrpui s fgl nmdr eiiebegan medicine modern antiq- in since gold medicine of in use forms Therapeutic (1). various uity in used been has old rlge ePyiu e Mat des Physique de eralogie, ´ ePrs ai 50 ee 3 France; 13, Cedex 75205 Paris Paris, de e ´ r tSyst et ere ` | a biodegradation ahleLuciani Nathalie , b lrneGazeau Florence , bacillus msCmlxs NS Universit CNRS, Complexes, emes ` eSrsor,CR,Srsor 78,France 67087, Strasbourg CNRS, Strasbourg, de e ´ nvto() hsrsl initiated result This (2). vitro in | biomineralization 198 ed)frcne treatment cancer for seeds) a ulam Wang Guillaume , raxe eCsohme obneUniversit Sorbonne Cosmochimie, de et eriaux ´ a,1,2 I ynd a bacterio- was cyanide ) n lrn Carn Florent and , c Mus | u ainldHsor auel,CR,Isiu eRcecepu eD le pour Recherche de Institut CNRS, Naturelle, d’Histoire National eum ePrs ai 50 ee 3 France; 13, Cedex 75205 Paris Paris, de e ´ ´ b eadLelong Gerald , a,1,2 1 doi:10.1073/pnas.1911734116/-/DCSupplemental. at online information supporting contains article This 2 is ulse eebr1,2019. 18, December published First available are paper this in repository, reported Zenodo script the and at data transcriptomic The deposition: Data the under Published Submission.y Direct PNAS a is article This interest.y competing no declare D.A., authors N.L., The A.B., and new paper. data; y contributed the analyzed F.C. F.G., D.A. wrote F.C. and and N.L., F.G., and F.G., A.K., D.A., A.B., O.E., N.L., research; G.L., A.B., G.W., designed tools; N.L., F.C. reagents/analytic A.B., and research; F.G., performed F.C. D.A., and N.L., A.B., contributions: Author self-assembly and evidenced recrystallization and fibroblasts of in phenomenon mo 6 surprising to a up lysosomes in sizes ferent indicates on clue no GNPs (28–32). and microscopy of days, electron influence few on the a degradation GNP exceed on few vitro only in cells, cultured conducted stud- been numerous Although have after (27). ies shell mo and oxide 3 heterostructures iron the spleen oxide of mice iron dissolution in gold nanocrys- observed our of was gold administration from which 5-nm systemic study size, of 1 3-nm degradation only to vivo knowledge, tals in our pre- reported to also has far, ionic would group So uncontrolled gold gold. bioactive of of toxic inertness potentially forms This of release body. the the for an vent in properties be time optical indeed long nanoscale-related can indefinitely a their GNPs left maintain of be to integrity could asset biodegra- Sustained that tissues. prevents GNPs in gold or intact of implants inertness gold of the the dation that Therefore, (26). is oxide reac- dogma metal less and current and metals environment, other acidic most to than tive sensitive gold metal, less noble inert, a more As (23–25). is GNPs nanopar- concern oxide few metal only but or ticles, metal for described been have processes ..adFC otiue qal oti work. this to equally contributed F.C. and F.G. owo orsodnemyb drse.Eal florence.gazeau@univ-paris- Email: addressed. be may fl[email protected] or diderot.fr correspondence whom To n omnmtbls ewe odnnprilsand nanoparticles gold between underly- gold. metabolism arthritis, ionic common rheumatoid a for treatment ing tissues salts human in gold deposits after biopersistent gold observed into previously to are recrystallizes similar products gold degradation these released Interestingly, nanostructures. point the we that Furthermore, biodissolution. out of this role into active lysosome the cell the reveal of studies degradation Transcriptomics faster a size. with smallest actu- cells, are by vitro nanoparticles in gold degraded 22-nm ally to 4- that in intact show endlessly We com- remain tissues. nanoparticles inertness, gold chemical that their barely is belief of has mon Because organism far. the so in studied fate been their range increasing applications, an medical of core of the at are nanoparticles gold While Significance ee ervae h nxetddgaaino Nso dif- of GNPs of degradation unexpected the revealed we Here, c vduErsen Ovidiu , ,Prs705 rne and France; 75005, Paris e, ´ NSlicense.y PNAS PNAS b https://zenodo.org/record/3530617#.XfFNDht7ncs.y aoaor Mat Laboratoire | d aur ,2020 7, January beaiKhelfa Abdelali , d ntttd hsqee hmedes Chimie et Physique de Institut raxe Ph et eriaux ´ y y https://www.pnas.org/lookup/suppl/ | o.117 vol. b enom ´ , vlpeet(IRD), eveloppement ´ | nsQuantiques, enes ` o 1 no. | 103–113

MEDICAL SCIENCES BIOPHYSICS AND COMPUTATIONAL BIOLOGY 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). These dimensional characteristics mission electron microscopy (TEM) observation on microtome are conserved for at least 6 mo (SI Appendix, Fig. S3). Occasion- sections of 70 nm at 1 d, 2 wk, 2 mo, and 6 mo after exposure ally, straight linear self-assemblies (length from 50 to 650 nm; to GNPs (Fig. 1). One day postlabeling, the original GNPs are thickness from 5 to 80 nm) are also observed (SI Appendix, densely packed into intracellular organelles showing the ultra- Fig. S4). Furthermore, the size distributions of the nanoparti- structural features of lysosomes (33). Two weeks after exposure cles composing the dense areas are shown to depend on their to GNPs and up to 6 mo, we observed 2 types of electron-dense proximity to diffuse areas. In lysosomes presenting only dense objects in lysosomes (dark and light orange arrows in Fig. 1 A–C): areas, nanoparticles diameters are close to the original GNPs, first, the very same GNPs as those observed at day 1 with a sur- remaining arounds 4 nm over time. Surprisingly, at the interface

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Fig. 1. TEM observations on microtome sections of human fibroblasts exposed to 4-nm GNPs observed 1 d to 6 mo after GNPs incubation, evidencing the existence of dense and diffuse electron-dense areas. (A–C) Images of 1 cell 2 wk after GNPs exposure presenting 3 electron-dense areas identified as lysosomes constituted of dense areas (dark orange arrows) and diffuse areas (light orange arrow). (D–K) Representative lysosomes observed 1 d (D and H), 2 wk (E and I), 2 mo (F and J), and 6 mo (G and K) after exposure at 2 magnifications. L, Surface proportion of dense and diffuse areas from 1 d to 6 mo after exposure; 5 to 10 images have been analyzed for each condition, with at least 1 electron-dense area per image.

104 | www.pnas.org/cgi/doi/10.1073/pnas.1911734116 Balfourier et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 iprinsetocp ES ttennsae(.0 to (0.009 nanoscale the at (EDS) spectroscopy dispersion smaller almost of of structures. presence diffuse composed the in domains areas with crystalline compatible the fewer and is and in which intense one GNPs, less the had intact than nanoleaves of peaks area broader diffuse the in 3 obtained (Fig. crystal gold 4.079 to = (a cal structure nanoparticles lattice cubic tiny face-centered that a the diffraction shows electron in by and confirmed GNPs leaves, into original self-assembled the in both lattices they axis. thickness optical beam, higher the their electron along of the because lashes to high-contrast parallel like orientations. appear are various nanoleaves with these nanoleaves, When like objects, curved platelet (2D) in a shown like tomogram more 3D looks S1 calculated it The 1- range, edges. angular rough this specific with nanostructure angular this a series, of narrow When out tilt nanostructure. a is individual the in is each observed structure of for only range lash-like frame is the each architecture If dimensional sam- on beam. the electron observed between the frequently angle and tilt holder the ple on depending 2 varies angles. 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(A–C nanoparticles. 2.5-nm assembled 2. Fig. afuire al. et Balfourier AC TMHAFiae xrce rmtetl eisaqie uigtmgah xeiet.Tetl nl ftesml odrwt epc to respect with holder sample the of angle tilt The experiments. tomography during acquired series tilt the from extracted images STEM-HAADF –G) ial,eeetlaaye efre ySE energy STEM by performed analyses elemental Finally, atomic crystalline similar revealed also TEM High-resolution curved common most the of character (3D) 3-dimensional The 500 nm eel htls-ieacietrsaei at2-dimensional fact in are architectures lash-like that reveals TMosrain fhmnfirbat katrepsr o4n Nssoigta ifs tutrsae2 aoevscmoe fself- of composed nanoleaves 2D are structures diffuse that showing GNPs nm 4 to exposure after wk 2 fibroblasts human of observations STEM E 200 nm h rjce hp ftediffuse the of shape projected the E–G, .Hwvr h ifato pattern diffraction the However, A–G). B TMiae facl oana ifrn anfiain,soigtelclsrcueo h ifs ra previously areas diffuse the of structure local the showing magnifications, different 3 at domain cell a of images STEM ) 100 nm 96° - 39.6 oi S1. Movie FG 200 nm )identi- A) ˚ Movie 50 nm 0.04 ls hyas togyrifretehptei htsuch that nanoparti- hypothesis larger the to reinforce generalized strongly be also could They described GNPs process cles. biodegradation 4-nm the 4D). that for (Fig. prove GNPs appeared 4-nm results with and labeled These observed cells with frequently comparison lyso- in less later within were GNPs structures 22-nm or diffuse 4 15- of (Fig. aggregates somes with com- side 2.6 nanoleaves by of of same nanocrystals side sizes gold very larger individual the for of observed posed occur GNPs indeed also of We could degradation particles. recrystallization lysosomal situ if in investigate and to GNPs coated atoms. gold newly of reactivity the the increasing to effects surface governed addition mech- by likely effect in nontrivial degradation/recrystallization the highlights GNPs of This anisms original products. the process degradation of recrystallization formed and surface degradation with transfer the interface gold at the a at gold size suggests GNPs from products the of results Furthermore, increase novo species. the de sulfur that by of mediated degradation self-organization probably nanocrystals, and GNPs recrystallization, release, of ions same the products in to are nanoleaves 4 with from coexist increase they to when the found mo time, lysosomes. 6 is same in GNPs the nm original In 10 of number. size in orig- the decreased average of which expense sulfur. nanocrys- the GNPs, at with inal 2.5-nm time associated over of increased and occurrence made nanoleaves Their structures into self-assembled gold tals of appearance the for atoms sulfur 1.2 not atom). to but gold (1.1 1 GNPs nanoleaves untransformed with of structures proximity in diffuse within 3 concentration detected (Fig. lower also nanoleaves significantly diffuse a the in in but areas electron-dense irbat eete aee ih1-ad2-mcitrate- 22-nm and 15- with labeled then were Fibroblasts nanoleaves the that conclude to us led observations These reveals cells GNP-labeled of monitoring 6-mo the Overall, µm 2 × 0° 0n)uabgosycnre oddtcinin detection gold confirmed unambiguously nm) 70 200 nm A–C PNAS and E–H | D

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Fig. 3. High-resolution TEM observations, electron diffraction, and STEM-EDS performed on human fibroblasts 2 wk after exposure to 4-nm GNPs reveal the crystallinity of nanoparticles composing diffuse areas and a specific signal of sulfur. (A–D) TEM imaging of dense (A and B) and diffuse (C and D) area, presenting crystalline lattice highlighted in red. (E and F) The 2D diffraction patterns obtained on representative regions of dense (E) or diffuse (F) areas. (G) Scattered intensity obtained after radial integration of E and F.(H and K) STEM images of noncontrasted (H; red frame), diffuse (H; light orange frame), and dense (K; dark orange frame) areas analyzed by EDS. (I, J, and L) EDS spectra of noncontrasted (I), diffuse (J), and dense (L) areas and (Inset) enlargement of the 2- to 3-keV area, with Gaussian fits for gold, sulfur, and chloride peaks. The slight horizontal contrast modifications seen at the bottom of the STEM-HAADF image are due to sample charging.

self-organized gold nanostructures are the result of size- heterodimers (SI Appendix, Fig. S7). Although these struc- dependent GNP lysosomal degradation taking place faster for tures were previously attributed to the erosion and further small GNPs with higher surface/volume ratio and reactivity than self-assembly of initial gold core, their compelling morpho- for large GNPs (34). logical similarities with the structures witnessed here in vitro strongly suggest a general mechanism of gold dissolution fol- GNPs Degradation Products Are Similar to Aurosomes Observed In lowed by in situ recrystallization to form 3-nm GNPs organized Vivo after Gold Salts Treatment. It is worth noting that similar into suprastructures with assistance of endogenous lysosomal self-assembled gold nanostructures have been observed before proteins. in at least 2 different contexts in vivo. In the previously pub- This hypothesis is further supported by the second phys- lished study on iron oxide-coated GNPs intravenously admin- iopathological condition in which similar gold nanoassemblies istrated in mice, the intralysosomal degradation of iron oxide were observed in the so-called aurosomes. Based on elec- crystals and the transformations of 5-nm gold crystalline core tron microscopy observation by Ghadially (33), aurosomes were were observed sequentially (27). Organized assemblies of 3-nm described early in the 1970s as lysosomes containing gold GNPs forming plates or curved structures were also seen in nanoassemblies formed in various organs after administration spleen and liver from 3 mo postinjection of iron oxide@gold of gold salts for the treatment of rheumatoid arthritis. Striking

106 | www.pnas.org/cgi/doi/10.1073/pnas.1911734116 Balfourier et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 os(tagto uld,rdlk,o aelrelectron-dense S8 lamellar Fig. or Appendix, rod-like, filamen- containing curled), same profiles the or by (straight supported tous is highlighted products presently degradation the GNPs and have images aurosomes 10 between to 5 resemblance GNPs; 22-nm to exposure after mo 2 (E–H to magnifications. image. 2 d per at 1 area observed from electron-dense areas areas 1 diffuse ( C diffuse least and mo and at dense 2 dense with presenting of or condition, each proportion (B), for Surface wk analyzed (D) 2 been arrow). (A), orange d (light 1 areas observed diffuse lysosomes representative of images forclua oe,w huh opromatranscrip- a perform to al. et thought Balfourier we model, advantage cellular Taking mechanism. our biological of degradation the GNPs’ on clearance and fate GNP term. control long to and avenues opens understand This better (38). salts formation gold aurosome with presenting treated patients and for observed of been elimination to has renal species translated gold Interestingly, tissues, is aurosomes. and GNPs of cells of elimination in clearance the gold or of elimination of form aurosomes concern stable If the ultimate aurosomes. an as be form to the when recrystallized proved organism on its the GNPs from under of clearance is can their toxicity gold also, conclusion the and This evaluate term long long to vivo. very importance the in core- great in or or of GNPs, same vitro pure be the salt, in (gold is heterostructures) ions ions of shell conclusion gold origin important the of whatever the fate term called to intracellular be us the will leads that products analogy degradation This GNPs aurosomes. for parts, Hence, study. the following this to in the observed similar GNPs very of products are size, degradation (i.e., composition) pro- features chemical aurosome biotransformation the and that this morphology, clear colloidal is of and it generality Overall, salts (33). the cess gold to various different attesting for for gold, human), and and routes, (37). rabbit, (rat, administration origin species numerous biological several in in endogenous found were organs, of composition and be morphology same should revealed The sulfur been of has Au part +1 nonsulfur degree from produced oxidation were aurosomes from Importantly, gold (36). ratios spectroscopies a absorption atomic X-ray and the by Au/S contrast, 2.2 with By to sulfur (35). 1.5 and time that gold at of established colocalization fully not morpholo- was these S9). forming gies “granules” Fig. the Appendix, of (SI nature study crystalline similarities our The in high observed shows and nanoleaves the ultrastructure curvature, with their of of radius thickness the average aurosomes, these composing ticles 4. Fig. 2n D=22nm D =22nm AB D =15nm E oee,arsm omto ean lsv swl as well as elusive remains formation aurosome However, E bevtoso ua bolssepsdt 2 r1-mGP eeltepeec fdfueaesascae odgaain (A–C degradation. to associated areas diffuse of presence the reveal GNPs 15-nm or 22- to exposed fibroblasts human of observations TEM .Dmninlaayi ftesz ftepar- the of size the of analysis Dimensional ). -mgl atce ihnlssms( lysosomes within particles gold <5-nm 6 months 500 nm 200 nm202 1 day 0 0 nmn F m III at,sgetn htalor all that suggesting salts, D =22nm 200 nm SI fe 2n Nsepsr,peetn es ra dr rnearw)and arrows) orange (dark areas dense presenting exposure, GNPs 22-nm after ) 2 weeks D =15nm 200 nm G rne epn nFRqvletrsoda .1 nln with line In 0.01. at ref- threshold as q-value point FDR time an each keeping for used erence, were samples control unlabeled (SI phase G0 at maintained S2). were response Fig. mea- Appendix, previous cells with stress that line in showing mo, diverse 2 surements and wk response) as 2 protein at cellular down-regulated well unfolded were stress, (metabolism, as oxidative functions response, appeared repair) (immune related It DNA S2). and and machinery, division S1 Tables cell Appendix , q-value that (SI FDR 0.01 an using at correspond- (SI genes threshold functions expressed mo differentially biological these 2 the to (41) and/or ing identify (GSEA) (FDR) to wk analysis performed enrichment rate 2 set was Gene discovery 4,129 at S12). criteria, expressed Fig. false these Appendix, differentially to a According were 0.01. and genes at using set reference analysis threshold as q-value expression gene sample differential 1-d performing by ples GNPs 5B). and (Fig. control points and time mo, 2 2 these and for wk condition 2 discrim- at respectively, obtained variance, fourth samples of and inate 2.4% third and 4.6 the with Finally, dimensions, 1-d samples. the GNP-treated between and differences the control highlights variance, 5A of (Fig. 13.5% points ing time later from samples and from account 1-d come into differences separating main reproducibil- and takes the time that good dimension showing first with variance, of be The dimensions, 73.5% could triplicates. 4 samples between first high- between ity the variance the the with with Appendix of captured genes (SI 94% compo- 500 input that principal the as appeared ratio using by expression variance-to-mean (PCA) analyzed gene est (40) first in analysis were Differences fibro- nent of samples (39). not or all GNPs incubation after between 4-nm mo) 2 wk, with 2 microarray d, blasts DNA (1 using points time genes 3 18,537 at Pathways. on performed Detoxification was analysis Metal to Response Involving Oxidative GNPs Long-Term Reveals GNP Analysis in Transcriptomics involved pathways the on light shed biotransformation. to analysis tomic nodrt ihih ifrnilgn xrsindet GNPs, to due expression gene differential highlight to order In sam- control on evaluated was time of effect dominant The .Tescn ieso,recapitulat- dimension, second The S11). 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MEDICAL SCIENCES BIOPHYSICS AND COMPUTATIONAL BIOLOGY AB ● ● 1 day ● 2 weeks 2 months ● ●● ● ● ● ● ● ● CTL ● GNPs Dim 4 (2.37%) Dim 2 (13.48%) ● ● −10 −5 0 5 10 −20 −10 0 10 20 −40 −30 −20 −10 0 10 20 30 −10 −5 0 5 10 Dim 1 (73.54%) Dim 3 (4.63%) C 2 weeks Reactive oxygen species 1549 16 54 Glutathione metabolism Xenobiotic metabolism 13 Cytochrome P450 39 38 mTORC1 signaling Reactive oxygen species Protein secretion Glutathione metabolism Ribosome Xenobiotics metabolism 56 Cytochrome P450 Cell division 2 months Ribosome

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Fig. 5. Transcriptomics analysis reveals a time-dependent answer to 4-nm GNPs and the activation of 3 main pathways at the longer times of study (NADPH production, ROS detoxification, and metal chelation). (A and B) PCA of transcriptomics data performed with the 500 most variable genes. (C) Venn diagram of GNPs samples differential expression compared with unlabeled control at the same time (FDR q value lower than 10−3 only). Lists and arrows summarize highest hits of GSEA (FDR q value lower than 10−4 only). (D) Expression heatmap of up-regulated genes 2 wk after GNPs exposure. Only genes that are attributed to identified pathways are displayed (38 genes on 124). Expression was considered as significantly different for FDR q value below 0.01. CTL, control; TNF, tumor necrosis factor.

PCA result, the Venn diagram shows that 88% of the modi- 2 mo (highest hits in Fig. 5C; SI Appendix, Tables S4 and S5), fied genes only concern the early time point (Fig. 5C). The most revealing a long-lasting response of interest to understand GNPs impacted biological functions were identified by GSEA, keep- degradation pathways. ing an FDR q-value threshold at 0.01 (highest hits in Fig. 5C; Five up-regulated pathways are common to the 2 later time SI Appendix, Table S3). At day 1 postlabeling, it reveals a down- points: reactive oxygen species (ROS) pathway, xenobiotics regulation of cell division, DNA repair, and cellular machinery and drug metabolism by cytochrome P450, glutathion (GSH) for GNP-labeled cells. These down-regulated functions were metabolism, interferon (IFN)-alpha response, and adipogenesis. identified previously as a general nonspecific and fast response Activation of ROS has already been reported for small GNPs (D to stress, whether it is induced by xenobiotics or other stress < 5 nm) and for different cell types (43). Likewise, the activation factors (42). In contrast, gene set corresponding to oxidative of cytochrome P450 pathway was already described in spleen and stress, exogenous stress, and immune response was enriched at liver 1 and 2 mo after GNPs (D = 20 nm) injection in vivo (44). It 1 d (SI Appendix, Table S3) and was still affected at 2 wk and suggests that cytochrome activation might be a late response to

108 | www.pnas.org/cgi/doi/10.1073/pnas.1911734116 Balfourier et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 afuire al. et Balfourier eoe h pcfi epnet N lodcessfo wk 2 from decreases also GNP to response can- mentioned specific As GNPs the assessments. to time before, short response 1. with specific day appraised fully the at be that up-regulated not fact not are the points reinforces time This later the at expressed originated for ions MTs in metallic 56). place shown (55, take for gold be could process matrix already similar More- recrystallization a has nanoparticles, 54). a from ferritin (36, as protein act structures storage to iron aurosomes time the of long as a a part with over, for be were gold, able and complex to as be effect, to suspected chelate hand, to likely to other more owing known the stability are is higher on MTs GSH (53); protein, hand, clusters storage prod- a gold for one numerous degradation responsible On stabilize be sulfur. as to could of location MTs signal or same this sulfur- GSH the reveals Either at ucts/aurosomes. which compounds STEM-EDS, ized per obtained response of composition kinetics between h, a same 24 again the after once was indicating not GNPs. points, to gold but time GNPs internalized 2 to of the up- exposure amount were of MTs the of h while types with 72 7 cells that after Caco-2 evidenced of regulated was exposure It presence (46). after GNPs in reported 5-nm up-regulation been MTs has GNPs 52). functions. of (51, sulfur gold different bind role including their to able key metals, proteins via detox- storage a metal metal metal play cystein-rich of are of to mediator MTs chelation as appear ROS GNPs through MTs to of as ification response well production sustained as the likely GSH in the cell. the despite inside here, are (CAT) up-regulated catalase and not family), (SOD), ROS (GPXs dismutase well-known peroxidases superoxide the glutathione on that , Appendix relying underline (SI pathway to important detoxification NADPH is consume It S14). that Fig. reactions destruction be ROS redox in could implied through are relationship quinone, responses HMOX1, strong thioredoxin S13). and informa- Fig. no GSH, Appendix, of because (SI lack them or between a roles found of their because up-regulated on either Other tion commented 5D). not (Fig. are production produc- genes thioredoxin NADPH MTs and sets: GSH and 50), quinone, gene of responses, (49, (HMOX1), main 1 reactions 38 6 oxidase of heme S6); identify tion, cascades to Table same us Appendix, the enable (SI which to transcription belong targets (MT) genes gene metallothionein these (MTF1) transcription to 1 (NFE2) 17 factor 2 (66%) and erythroid genes pathway up-regulated factor factor 2-wk nuclear the to of contribute 80 gen- Indeed, a scheme. revealing iRegulon, eral using the factor in transcription studied their further of were light wk 2 at genes when overexpressed nificantly mo, 2 aurosomes. at into transformed level already basal are their GNPs 4-nm to most return energy would for Accordingly, functions recrystallization. need these and the degradation reflect GNPs could process pro- it to energy metabolism), acid to fatty path- linked way, phosphate penthose [mTORC1] are gluconeogenesis, 1 functions glycolysis, complex signaling, rapamycin these at of to of target only related (mammalian several functions up-regulated duction As contrary, are wk. division the metabolism cell On 2 and/or and mo. secretion points, 2 time at protein longer only both affected at is affected is bolism unclear. although still (48), are GNPs mechanisms of the pre- (46, activity the labeling immunological with GNPs line reported after in viously h is 72 it up-regulation, to IFN-alpha 24 Regarding GNPs (45). 47). previ- 13-nm detoxification reported and been 5- metal for has in ously metabolism GSH and of impli- stress up-regulation is oxidative and The cell a in the is both of GSH state points. cated redox time the early for the responsible at tripeptide detected be cannot that GNPs neetnl,ecpigfrHO1 oto h ee over- genes the of most HMOX1, for excepting Interestingly, chemical the on light shed results transcriptomics our Here, sig- the degradation, GNPs in implicated genes the identify To meta- ribosome pathways, down-regulated the Concerning hi erdto naii odtos hsi ossetwith up consistent speed is to This and conditions. respec- pH acidic 2, neutral in and the at degradation observe 7 GNPs to their contrast of needed = in are process (pH ions 12), degradation chloride environments = Moreover, acidic (pH (63). degradation tively) conditions and no basic neutral that in to shown observed have be studies could OH TEM liquid likely beam- of Previous (most ability the ROS reveals dissolution generated GNPs of monitoring situ bev Nsdgaaina hw nFg 6 specific Fig. GNPs, to in of required shown case are as the concentration degradation In chloride observed GNPs (62). and observe were pH cells of living that car- conditions in damages multiwalled weeks recapitulating of for degradation nanotubes, ROS-triggered bon to the radicals, energy of monitor source input generation illumination subsequent OH an and including as the Indeed, radiolysis and as 63). water imaging both (62, induce nanoparticle water serve real-time in can TEM for beam liquid generation electron by morphol- achieved the ROS nanoparticles be of Tunable can observation ogy dynamic GNPs. the of to coupled structure crystalline degradation the intralysosomal the in NOX5 GNPs. and inhibitor of NOX4 NOX area of without gold-containing sibly, condition electron-dense 6 control whole (Fig. the the with of from products compared 32% decreased NOX degradation to unequivocally of with fibroblasts 45 proportion was and supplemented GNPs, the structures) endosomes medium wk, (aurosome-like 4-nm in 2 NOX culture After to active the in inhibitor. be exposure of cultured to After protein was were shown types only choice (61). been 3 This the lysosomes has on (60). is that active NOX5) NOX1 family is and because that NOX4, motivated (NOX1, inhibitor NOX NOX of a GKT137831, use therefore, transcriptional by (59). and confirmed results directly level, be posttranslational not could a be activation their on could regulated ROS NOX is Nevertheless, of gold. activity ionic production into degradation NOX GNPs in metallic reaction, implied or this during ions dized metal HO of radical presence H in nanoparticles, Somehow, H water. peroxisome, the into H in in CAT SOD, or via or spontaneously dismute, will that O ical NOX and (NOX). NADPH oxidase NADPH consumes mem- called by assemblies or compounds protein undesirable extracellularly brane damage generated to be lysosome the can inside ROS degradation. GNPs Recrystallization to and Chelation. Degradation MT GNPs through Activates Production ROS on MTs– chelator metal of a structure of the structures. impact degradation the 3) the 4) GNPs, 2) finally, degrade degradation, and GNPs to complexes, gold in ROS production of ROS following ability of the the test role studying To the of MTs. 1) ROS thought points: by by we recrystallization mechanisms, GNPs and putative of this capture degradation gold a possi- by suggests and followed It elimination binding. ROS NFE2 metal by directs bly, directed which mostly factor, is transcription latter a The and and process. degradation GNP uptake recrystallization to GNP linked likely to more related response long-lasting stress nonspecific process early degradation identified complete almost the mo. with 2 at production-related line NADPH and in GSH, genes, MTs, especially mo, 2 to O 2 enx vlaeteaiiyo O ooiieaddegrade and oxidize to ROS of ability the evaluate next We we degradation, GNPs in role NOX assess to Therefore, microarrays DNA by analysis transcriptomic the conclude, To nai eim hn hog P cini h lysosome the in action GPX through Then, medium. acid in .Ti lal niae oeo O1adpos- and NOX1 of role a indicates clearly This A–C). · i etnrato 5,5) stemtli oxi- is metal the As 58). 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MEDICAL SCIENCES BIOPHYSICS AND COMPUTATIONAL BIOLOGY ABC2 weeks 2 weeks NOX inhibitor

Dense area

Diffuse area Surface proportion (%) 0 20406080100

itor b 500 nm 500 nm 2 weeks 2 weeks

+ NOX inhi D E F G H

IJ 3.0 2.5 1:0 1:7 1:18 1:12 ● 293 nm −1:1 −1:8 −1:21 2.5 −1:2 −1:9 −1:24 2.0 ● −1:3 −1:10 −1:44 ● 2.0 −1:4 −1:11 −1:64 ● Slope: −1:5 −1:12 −1:124 1.5 ● −1:6 −1:15 −1:144 2.34 x ●● 1.5 − − − ● ●● 1.0 ● 1.0 ●● ● Absorption at 293 nm ●

Corrected absorption 0.5 ● 0.5 ● ● ● ●● 0.0 0.0 200 250 300 350 400 450 01234567 Wavelength (nm) Sodium aurothiomalate concentration (mmol/L) K MT:Au = 1:120 L

5nm 10 nm MNO

50 nm 200 nm 50 nm

Fig. 6. GNPs degradation is mediated by NOX-created ROS followed by a probable recrystallization inside MTs and a self-assembly process impacted by BAL. (A and B) TEM observations of human fibroblasts 2 wk after exposure to 4-nm GNPs with (A) or without (B) the NOX inhibitor GKT137831. Dense and diffuse areas are indicated by dark orange and light orange arrows, respectively. (C) Proportion of dense and diffuse areas with or without GKT137831; 30 images or more have been analyzed, with at least 1 electron-dense area per image. (D–H) Liquid TEM snapshot performed in aqueous solution ([HCl] = 0.02 mol/L, [NaCl] = 1 mol/L) under constant radiation at 200 keV. Observations were performed on citrate-coated GNPs and gold nanorods (D = 25 nm, l = 11 nm, L = 45 nm). (I) Ultraviolet (UV)-visible spectroscopy performed steadily during MT filling with sodium aurothiomalate. The legend indicates the ratio MT:Au. (J) UV-visible signal evolution at 293 nm during MT filling. (K and L) High-resolution TEM imaging of MT–gold complex at the MT:Au ratio of 1:120 observed at 2 magnifications. (M–O) STEM observation of human fibroblasts 2 wk after exposure to 4-nm GNPs and cultured with BAL. Diffuse areas of 2 types were observed (N): an aurosome-like one (green frame and M) and a new one (blue frame and O).

110 | www.pnas.org/cgi/doi/10.1073/pnas.1911734116 Balfourier et al. 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MEDICAL SCIENCES BIOPHYSICS AND COMPUTATIONAL BIOLOGY Overall, the degradation step seems to be mediated by NOX DNA Microarray Preparation. For RNA extraction, cells were washed twice proteins that can produce highly oxidizing ROS in the lysosome, with PBS, lysed, and treated using the NucleoSpin RNA Kit (Macherey resulting in GNPs dissolution. In a second step, released ionic Nagel) according to manufacturer’s protocol, including a DNAse treatment. gold would then be crystallized and sequestrated, probably inside The following steps concerning DNA microarray were performed by the an MT-composed shell, owing to the morphology similarities platform Genomics (Institut Cochin, UMR8104, National Center for Scien- between aurosome building blocks and MT–gold complexes. The tific Research [France]/National Institute of Health and Medical Research [France]/Universite´ Paris Diderot, Paris, France). After validation of the RNA nature of self-assembly template is much more puzzling, and we quality with Bioanalyzer 2100 (using the Agilent RNA6000 nanochip kit), establish a list of criteria that it should fulfill according to our 75 ng of total RNA is reverse transcribed following the GeneChipWT Plus observations. It has also been shown to be affected by BAL, Reagent Kit (Affymetrix). Briefly, the resulting double-strand complemen- which created self-assembled patterns, possibly by disrupting tary DNA is used for in vitro transcription with T7 RNA polymerase (all of disulfide or gold–sulfur bonds. these steps are included in the wild-type complementary DNA synthesis and amplification kit of Affymetrix). After purification according to Affymetrix Conclusion protocol, 5.5 µg of Sens Target DNA is fragmented and biotin labeled. After control of fragmentation using Bioanalyzer 2100, complementary DNA is We have studied the biotransformation of GNPs in primary ◦ human fibroblast during 2 to 6 mo. First, TEM observations then hybridized to GeneChipClariom S Human (Affymetrix) at 45 C for evidence the intracellular degradation of GNPs with a size- 17 h. After overnight hybridization, chips are washed on the fluidic sta- tion FS450 following specific protocols (Affymetrix) and scanned using the dependent dynamic. This degradation is induced by ROS, gen- GCS3000 7G. The scanned images are then analyzed with Expression Con- erated by NOX, that oxidized GNPs. This oxidation occurs sole software (Affymetrix) to obtain raw data (cel files) and metrics for together with an upregulation of NFE2 pathway, which creates quality controls. a cell-protective environment. This result invalidates the current dogma of intracellular gold inertness and highlights the need for Electron Microscopy. GNPs TEM images were obtained with a Tecnai long-term studies to capture the slow process of nanoparticles 12 microscope operating at 80 kV (Platform Imagoseine, Institut degradation. Jacques Monod, UMR7592, National Center for Scientific Research Second, we show that the released gold undergoes a biominer- [France]/Universite´ Paris Diderot, Paris, France) after deposition of a droplet alization process and ends up in well-defined structures consist- of NPs solution on a hydrophilized 400-mesh grid. For MT–Au complexes, ing of 2.5-nm crystalline particles self-assembled into nanoleaves. 3 µL of a solution of sodium aurothiomalate (9.9 mM) and MTs (83 µM ) was Reviewing of the literature dedicated to therapeutic gold salts deposed onto a 400-mesh grid 15 min after mixing. For TEM imaging, cells were washed with PBS, detached with 0.5% trypsin, and centrifuged. After evidences that similar structures, called aurosomes, have pre- 3 washes with PBS, cells were fixed with 5% glutaraldehyde (Sigma Aldrich) viously been described in vivo in different species and organs. in a 0.1 mol/L sodium cacodylate buffer (Sigma Aldrich) for 1 h at ambient It supports the original idea that ionic and crystallized gold temperature, washed with cacodylate buffer, and kept in this buffer until have a common intracellular fate that could be named as inclusion. Samples were then contrasted with Oolong Tea Extract (0.5% in gold metabolism. The morphological features of aurosomes and cacodylate buffer), osmium tetroxyde (1% in cacodylate buffer), and potas- the transcriptomics results designate MTs as credible candi- sium cyanoferrate (1.5% in cacodylate buffer). They were then gradually dates to capture gold and form the ubiquitous crystalline clus- dehydrated in ethanol (25 to 100%). The samples were then substituted ter. If more understanding on the process of self-assembly is gradually in propylene oxide, a mix of propylene oxide and Epon, and still needed, BAL, a thiolated chemical compound, has been finally, embedded in pure Epon (Delta microscopie, Labege,` France). Thin sections (70 nm) of samples were collected on 200-mesh grids. Observa- shown to affect aurosome and to generate biomineralization tions were performed on either a Tecnai 12 microscope (Platform Imago- patterns. seine, Institut Jacques Monod, UMR7592, National Center for Scientific Fig. 7 summarized these 2 steps in GNPs lifecycle that could in Research [France]/Universite´ Paris Diderot, Paris, France) or a Hitachi HT7700 the future be extended to other cell types (such as macrophages), microscope (Platform Microscopie et Imagerie des micro-organismes, ani- in vivo, or to other persistent nanoparticles (such as quantum maux et aliments 2 [MIMA2], UMR1313, Institut National de Recherche dots). Furthermore, this process can potentially impact other Agronomique [INRA]/AgroParis Tech, Jouy-en-Josas, France), both operat- GNPs-related concerns, such as their toxicity or their elimination ing at 80 kV. Further TEM investigations, STEM-HAADF, high-resolution from the organism. TEM, electron tomography, EDS, and electron diffraction were performed on an aberration-corrected JEOL ARM 200-F microscope operating at 80 kV (Laboratoire Materiaux´ et Phenom´ enes` Quantiques, UMR7162, National Materials and Methods Center for Scientific Research [France]/Universite´ Paris Diderot, Paris, Cell Culture. Deidentified human skin primary fibroblasts were obtained France). with consent from a healthy individual with no known metabolic dis- ease at Necker–Enfants Malades Hospital. All experiments have been done Data Availability. Transcriptomic data and script are available at the Zenodo with cells between 15 and 19 passages. All medium, antibiotics, and repository, https://zenodo.org/record/3530617#.XfFNDht7ncs. serum were purchased from Life Technologies (Thermo Fischer Scientific). Cells were cultivated in Dulbecco modified Eagle medium (DMEM) high ACKNOWLEDGMENTS. A.B. received a PhD fellowship from the doc- glucose supplemented with 1% penicillin/streptomycin. Prior to GNPs inter- toral school Physique en Ile de France. We acknowledge the financial nalization, 10% fetal bovine serum (FBS) was added to this medium, support of the National Center for Scientific Research (France; CNRS); while only 2% was added after cell exposure to nanoparticles. Cells were Region´ Ile-de-France Convention SESAME E1845 for the JEOL ARM 200- ◦ maintained at 37 C with 5% CO2. For the culture with GKT137831 F electron microscope installed at the Paris Diderot University; Agence (Cayman Chemical), 2% FBS culture medium was supplemented with 50 Nationale de la Recherche (ANR) “Investissements d’Avenir” Program and µM GKT137831 previously dissolved in water with 2% DMSO. For the Grants Labex SEAM ANR-11-LABX-086, ANR-11-IDEX-05-02, ANR CarGold- 16-CE09-026, ANR CycLys-18-CE09-0015-01, and ANR Coligomere-18-CE06- culture with BAL (TCI Chemicals), 2% FBS culture medium was supple- 0006; French National Research Program for Environmental and Occupa- mented with 50 µM BAL. In both cases, the medium was changed twice tional Health of Agence Nationale de Securit´ e´ Sanitaire, de l’Alimentation, a week. de l’Environnement et du travail (ANSES) Grant 2018/1/007; and European Union’s Horizon 2020 Research and Innovation Program Grant 801305. GNPs Uptake. GNPs were diluted in Roswell Park Memorial Institute medium We thank the team Membrane Dynamics (Saints Peres` Institute for the with glutamine containing 10% FBS for NPs stabilization. The cells were Neuroscience, UMR8003, CNRS/University Paris Descartes, Paris, France) for incubated at 80% of confluence with this medium during 24 h at 37 ◦C. cell line provision and discussion; Christine Pechoux´ (Platform MIMA2, UMR1313, INRA/AgroParis Tech, Jouy-en-Josas, France) for the electron The medium was then removed, and the cells were washed with phosphate microscopy preparation and observations; Remi´ Leborgne, Nina Fekonja, buffer saline (PBS) and conserved for 2 h in 10% FBS medium; then, this and the Imagoseine core facility of the Institut Jacques Monod (UMR7592, medium was removed, and the cells were washed again in PBS. Finally, cells CNRS/Universite´ Paris Diderot, Paris, France); a member of the France were conserved in 2% FBS DMEM for up to 6 mo. Medium was replaced BioImaging (ANR-10-INBS-04) for the electron microscopy preparation and once or twice a week. observations; Angeline Duche´ and Sebastien´ Jacques (Platform Genomics,

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Espinosa A. 28. inor- of stability cellular Sabella (Intra) S. Manshian, 25. B. Rejman, J. Parak, J. into W. Soenen, nanoparticles J. S. gold 24. engineered of Uptake Feliu N. Khlebtsov, 23. G. N. Dykman, A. L. probing 22. molecular vivo In Kneipp, K. Brown, D. McLaughlin, M. Kneipp, H. Kneipp, J. nanopar- gold 21. engineered of toxicity and Biodistribution Dykman, L. Khlebtsov, N. 20. Jan M. nanoparticles 15. gold colloidal of Biodistribution Makino, K. Tomoda, enhance K. to nanoparticles Sonavane, gold G. of use The 14. Smilowitz, M. H. Slatkin, N. D. Hainfeld, F. J. 13. Nanotechnology-based Chorilli, M. Fontana, C. Freitas, de L. Bernegossi, J. 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