Alessandra Lo Cicero

Manoubia Saidani

Jennifer Allouche

Anne Laure Egesipe

Lucile Hoch

Celine Bruge

Sabine Sigaudy

Annachiara De Sandre-Giovannoli

Nicolas Levy

Christine Baldeschi

Xavier Nissan
email: xnissan@istem.fr

Pathological modelling of pigmentation disorders associated with Hutchinson-Gilford Progeria Syndrome (HGPS) revealed an impaired melanogenesis pathway in iPS-derived melanocytes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that leads to premature aging. In its classical form, this syndrome is caused by a de novo single-base substitution c.1824 C > T in the LMNA gene [START_REF] Sandre-Giovannoli | Lamin a truncation in Hutchinson-Gilford progeria[END_REF][START_REF] Eriksson | Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome[END_REF] . Even though the mutation is silent (G608G), it activates an alternative cryptic splicing site and the production of a truncated and constitutively farnesylated form of lamin A, called progerin [START_REF] Cau | Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective[END_REF] . In patients with progeria, the accumulation of this toxic protein triggers a spectrum of symptoms that resemble aging, with lipodystrophy, dermal and bone abnormalities and cardiovascular alterations, leading to a shortened lifespan [START_REF] Merideth | Phenotype and course of Hutchinson-Gilford progeria syndrome[END_REF] . Over the past 10 years, several therapeutic strategies have been proposed to treat such patients, either by inhibiting progerin farnesylation with farnesyl transferase inhibitors [START_REF] Toth | Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes[END_REF][START_REF] Gordon | Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome[END_REF][START_REF] Gordon | Impact of farnesylation inhibitors on survival in Hutchinson-Gilford progeria syndrome[END_REF] , statins and bisphosphonates [START_REF] Varela | Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging[END_REF] , or by decreasing its production using morpholinos [START_REF] Osorio | Splicing-directed therapy in a new mouse model of human accelerated aging[END_REF] , rapamycin [START_REF] Cao | Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells[END_REF] , retinoids [START_REF] Pellegrini | All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype[END_REF][START_REF] Kubben | A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds[END_REF][START_REF] Lo Cicero | A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells[END_REF] or, more recently, with metformin 14 and MG132 [START_REF] Harhouri | MG132-induced progerin clearance is mediated by autophagy activation and splicing regulation[END_REF] .

Nuclear lamins are the major structural proteins of the nuclear lamina [START_REF] Fawcett | On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates[END_REF][START_REF] Goldman | Nuclear lamins: building blocks of nuclear architecture[END_REF] . Since the discovery of the molecular mechanism underlying this syndrome, several studies have highlighted the major role of progerin in the dysregulation of key biological processes, such as cell proliferation, cell differentiation, the DNA repair process or mitochondrial activities [START_REF] Cau | Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective[END_REF] . The development of relevant cellular models for HGPS to study these biological dysfunctions has been a major challenge over the past decade. In this context, it is noteworthy that the breakthrough discovery of induced pluripotent stem cells (iPS cells) by Shinya Yamanaka has been a unique tool in the discovery of new dysregulated pathways and the identification of biological targets [START_REF] Takahashi | Induction of pluripotent stem cells from fibroblast cultures[END_REF] . Thanks to their pluripotency and self-renewal properties, iPS cells allowed access to cell types that were relevant, but still unexplored in specific diseases [START_REF] Maury | Human pluripotent stem cells for disease modelling and drug screening[END_REF] . In 2011, several research groups demonstrated the capacity of iPS cell lines to recapitulate some aspects of HGPS after differentiation into vascular smooth muscle cells (VSMCs) [START_REF] Liu | Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome[END_REF][START_REF] Zhang | A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects[END_REF] and mesenchymal stem cells (MSC). In 2012, our group used the same model to report the absence of progerin expression in neurons and the role of a neuron-specific microRNA, miR-9 in the preservation of cognitive capacities in patients [START_REF] Nissan | Unique preservation of neural cells in Hutchinson-Gilford progeria syndrome is due to the expression of the neuralspecific miR-9 microRNA[END_REF] . More recently, two studies have revealed the effect of progerin on adipogenic [START_REF] Xiong | An inhibitory role of progerin in the gene induction network of adipocyte differentiation from iPS cells[END_REF] and osteogenic differentiation [START_REF] Lo Cicero | A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells[END_REF] processes of MSCs derived from HGPS iPS cells.

In this study, we used iPS cells derived from HGPS patients to study pigmentation disorders associated with this premature aging syndrome. To do so, we took advantage of the recent development of an efficient protocol of differentiation, allowing the generation of a pure population of functional melanocytes from pluripotent stem cells. The first method of differentiation was described in 2006, reporting an efficient method for differentiating hESCs into a melanocyte population within 4-6 weeks through the generation of embryoid bodies and their treatment using three growth factors: Wnt3a, endothelin-3, and stem cell factor [START_REF] Fang | Defining the conditions for the generation of melanocytes from human embryonic stem cells[END_REF] . In 2011, our group reported an alternative 2D protocol of differentiation, allowing the generation of melanocytes from both hESC and iPSC [START_REF] Nissan | Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis[END_REF] . In addition, we reported that these cells could be used to study pathological conditions, as demonstrated for neurofibromatosis type 1 [START_REF] Allouche | In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells[END_REF] . In the present study, we have used this model to investigate the role of progerin in the depigmentation observed in HGPS patients.

Results

Dermatological observations on the skin of HGPS patients reveal global hypopigmentation, associated with a typical mottled pigmentation (Fig. 1A). To investigate the molecular mechanism underlying these symptoms, two iPS cell lines derived from two HGPS patients (HGPS1 and HGPS2) and two healthy patients (WT1 and WT2) were differentiated into melanocytes using a protocol of differentiation based on the ones described by Allouche et al. [START_REF] Allouche | In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells[END_REF] , Gledhill et al. [START_REF] Gledhill | Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells[END_REF] and Mica et al. [START_REF] Mica | Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs[END_REF] (Fig. 1B). Briefly, embryoid bodies (EBs) were generated in low attachment conditions and committed for 4 days in the neural crest lineage. Once generated, EBs were seeded on dishes coated with 0.1% gelatin for 3 days and in a melanogenic differentiation medium for 11 days. At this stage, differentiating cells appeared with a morphology typical of a melanocytic lineage migrated out of adherent EBs by day 9. At day 18, iPS-derived melanocytes (Mel-iPSC) present a melanocyte-like morphology and can be cultivated in melanocyte culture medium for several passages. No difference was observed in HGPS and WT cells in terms of neural crest commitment or differentiation rate. Progerin expression was monitored by qPCR during the process of melanogenic differentiation, revealing no significant difference between WT and HGPS cell lines before 8 days of differentiation (Fig. 2A). Kinetics of differentiation revealed an increase in expression of progerin over time and stabilization after four passages of Mel-iPSC (Day 35) in culture (Fig. 2B). All further experiments were carried out in this passage. Progerin expression in Mel-HGPS was confirmed by western blot at the protein level (Fig. 2C, uncropped blots in sup Fig A andB). Nuclear shape disorganization of Mel-HGPS was measured by quantifying the percentage of abnormal lamin A/C-positive nuclei, revealing a significant increase in abnormal nuclei in the two HGPS cell lines (Fig. 2D andE). Observations of the cells obtained using this method revealed no difference between HGPS, WT and adult melanocytes in terms of morphology (Fig. 3A). Immunostaining of lamin A/C confirmed that all the differentiated cells express lamin A/C (Fig. 3A). Finally, molecular characterization of the expression of the key melanogenic markers, MITF, PMEL17 and TYRP1, in these cells revealed no differences in terms of expression of these proteins (Figs 2B and3A) and mRNA (Fig. 3C,D andE).

Macroscopic observations of cell pellets containing HGPS and WT melanocytes revealed lower pigmentation in HGPS cells (Fig. 4A). Melanin quantification confirmed these observations, demonstrating that HGPS melanocytes produced five times less melanin than WT melanocytes, 21 µg/ml and 25 µg/ml vs 4 µg/ml and 6 µg/ ml, respectively (Fig. 4B). The melanosome maturation process was analysed by electron microscopy in HGPS and WT melanocytes to investigate the cause of this deficient process of melanogenesis. Whereas all the stages of melanosome maturation were observed in all cell lines, whatever the genetic background (Fig. 4C), a higher proportion of non-mature melanosomes (type I and II) vs mature pigmented melanosomes (type III and IV) was observed in HGPS cells (Fig. 4C). Quantitative analysis of melanosome maturation stages revealed that the two HGPS melanocyte cell lines exhibit a significantly lower percentage of mature melanocytes (38% and 43%) than in WT cells (65% and 82%, respectively) (Fig. 4D). Conversely, a higher percentage of non-mature melanosomes was observed in HGPS cells (62% and 57%) than in WT cells (35% and 18%). Overall, these results reveal a deficient process of melanin production in HGPS melanocytes, suggesting a role of progerin in the regulation of melanosome maturation. In order to confirm this hypothesis, knockdown experiments were conducted using a siRNA that has previously been described to target progerin. While this sequence of siRNA was efficient up to 95% in skin fibroblasts, progerin was only partially decreased in melanocytes (40% in HGPS1 and 60% in HGPS2) (sup Fig C). Although incomplete, analysis of nuclear shape abnormalities confirmed a functional rescue of this phenotype (sup Fig D ) following siRNA treatment. Analysis of the melanosome maturation process in HGPS melanocytes treated with the siRNA revealed a significant rescue of the hypopigmented phenotype (Fig. 5A), with a percentage of melanosome type III and IV close to that for WT melanocytes (Fig. 5B). In addition, measurements of melanin content confirmed this result, with a 60 to 70% increase in melanin in both HGPS melanocyte cell lines following 48 h of treatment with the siRNA (Fig. 5C) but no significant effect on the expression of MITF, TYRP1 and PMEL17 (sup Fig E andF).

Discussion

We report here on a novel pathological mechanism associated with HGPS, showing that progerin regulates melanin synthesis and melanosome maturation in melanocytes derived from pluripotent stem cells. Our results shed new light on our understanding of the mechanisms underlying age-related skin dyspigmentation and describe Pathological modelling of HGPS using iPS cells. The pathological mechanism of HGPS and related premature aging has been extensively investigated over the past decade, mainly through the use of animal models or via the comparison of healthy vs HGPS primary cells (for a review, see [START_REF] Gordon | Progeria: a paradigm for translational medicine[END_REF] ). These pioneer studies revealed that progerin accumulation disorganizes nuclear shape, leading to major consequences for cell proliferation, cell differentiation, the DNA repair process and chromatin organization. However, with a few exceptions, almost all of these observations were made on skin fibroblasts, thus limiting the exploration of their consequences for relevant tissue-specific functions. The use of iPS cells has hereby been the source of breakthrough discoveries for numerous genetic disorders, giving the research community access to an unlimited resource of cells based on the clinical features of the disease. In HGPS, the death of patients occurs at a mean age of 13 years due to cardiovascular problems caused by vascular dysfunction [START_REF] Merideth | Phenotype and course of Hutchinson-Gilford progeria syndrome[END_REF] . Analysis of vascular smooth muscle cells derived from iPS cells revealed premature senescence, increased apoptosis, an increase in calcification, a defective DNA repair process through defective DNAPK trafficking and suppression of PARP1 in HGPS VSMC that, jointly, could explain the loss of these cells in patient's vessels [START_REF] Liu | Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome[END_REF][START_REF] Zhang | Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1[END_REF][START_REF] Atchison | A Tissue Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome Using Human iPSC-derived Smooth Muscle Cells[END_REF] . Similarly, studies were performed on MSC derived from HGPS iPS cells to investigate the molecular mechanisms causing the loss of adipogenic and osteogenic tissues, describing a lipid storage defect in HGPS cells [START_REF] Xiong | An inhibitory role of progerin in the gene induction network of adipocyte differentiation from iPS cells[END_REF] and an increase in the osteogenic differentiation process [START_REF] Lo Cicero | A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells[END_REF] , respectively. Here we describe that the presence of progerin leads to a decrease of melanin content in melanocytes derived from HGPS iPS cells suggesting an additional key role of progerin on the regulation of skin pigmentation. Although, there is no evidence that this effect is direct or indirect, our study revealed that melanosomes maturation process was partially rescued following progerin knockdown suggesting a functional relationship. Since 2011, the identifications of several other functional parameters in HGPS iPS cells derivatives has successively allowed to investigate the benefits of drug candidates on non-structural readouts, as demonstrated with rapamycin, lonafarnib, zoledronate or pravastatin [START_REF] Blondel | Induced pluripotent stem cells reveal functional differences between drugs currently investigated in patients with hutchinson-gilford progeria syndrome[END_REF] or, more recentl with metformin 14 and MG132 [START_REF] Harhouri | MG132-induced progerin clearance is mediated by autophagy activation and splicing regulation[END_REF] . Here, we describe a novel model associated with a new functional readout that can used for the further evaluation of new therapeutic strategies for patients with HGPS.

HGPS, a model of physiological aging?. Aging is a complex process that results from several intrinsic mechanisms and exogenous factors operating simultaneously. Over the past decade, progerin has become a potential candidate for the regulation of this process, even though it cannot clearly be allocated to one single gene [START_REF] Burtner | Progeria syndromes and ageing: what is the connection? Nature reviews[END_REF][START_REF] Lopez-Otin | The hallmarks of aging[END_REF] . This hypothesis was proposed in 2006 because of experimental results showing the presence of progerin in cells [START_REF] Rodriguez | Increased expression of the Hutchinson-Gilford progeria syndrome truncated lamin A transcript during cell aging[END_REF][START_REF] Scaffidi | Lamin A-dependent nuclear defects in human aging[END_REF] and tissues of aged individuals, as described in skin [START_REF] Mcclintock | The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin[END_REF] and vessels [START_REF] Olive | Cardiovascular pathology in Hutchinson-Gilford progeria: correlation with the vascular pathology of aging[END_REF] . These observations, in conjunction with the similarity of symptoms between HGPS and aged patients, highlight a potential link between ageing and progerin. The role of progerin in physiological aging has been intensively investigated, mainly through overexpression experiments in healthy cells, revealing similar dysregulation to that observed in cells from elderly individuals, with reduced proliferation and replicative senescence [START_REF] Mcclintock | Hutchinson-Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody[END_REF] , a decrease in cellular DNA repair capacity that leads to genetic instability [START_REF] Benson | Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence[END_REF][START_REF] Musich | Genomic instability and DNA damage responses in progeria arising from defective maturation of prelamin A[END_REF] , a loss of telomeres [START_REF] Benson | Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence[END_REF] and increased oxidative stress [START_REF] Richards | The accumulation of un-repairable DNA damage in laminopathy progeria fibroblasts is caused by ROS generation and is prevented by treatment with N-acetyl cysteine[END_REF] . Taken together, these results and clinical observations suggest that progerin may at least contribute towards to physiological aging. In this context, the HGPS iPS cells and their derivatives offer a unique tool for consolidating the analogy between aging and HGPS and for modelling physiological aging.

HGPS melanocytes to study pigmentation disorders associated with aging.

Melanocytes are an essential cell type found in the skin and hair, providing pigmentation and photoprotection from ultraviolet (UV) radiation. The skin becomes mottled with hyper-and hypopigmentation areas as it ages, resulting in an irregular pattern of lighter and darker areas and spots called dyspigmentation and a reduced ability to tan [START_REF] Montagna | Structural changes in ageing skin[END_REF] . Even if the molecular mechanism underlying skin aging is complex, several studies have reported that individuals appear paler with age due to a reduction in the number of melanocytes (8 to 20% per decade) and a reduced capacity to tan in response to UV radiation [START_REF] Nordlund | The lives of pigment cells[END_REF][START_REF] Haddad | Aging in epidermal melanocytes: cell cycle genes and melanins[END_REF] . However, paradoxically, elderly patients usually present focal hyperpigmentation due to localized proliferation and aggregation of melanocytes [START_REF] Haddad | Aging in epidermal melanocytes: cell cycle genes and melanins[END_REF] . Even though progerin expression in aged skin melanocytes remains unexplored to date, our results open up a new perspective for the understanding of the reduced pigmentation observed in the skin of HGPS patients and, by extension, in elderly individuals. To date, levels of progerin expression in melanocytes from elderly individuals remain unknown. However, if this hypothesis is supported, it would favour new dermatological indications for several compounds recently reported to decrease progerin expression, such as metformin, MG132 or retinoids.

Material and Methods

Fibroblast culture and reprogramming. HGPS Fibroblasts (13-8243) used in this study was isolated from a patient biopsy taken in the Assistance Publique Hôpitaux de Marseille during diagnostic procedures (referred as HGPS_1 in this study). HGPS Fibroblasts (AG001972) used in this study was provided by Coriell Cell Repository (Camden, USA) (referred as HGPS_2 in this study). Informed consents were obtained from the parents of the patient included in this study, complying with the ethical guidelines of the institutions involved and with the legislation requirements of the the french ministry of health (Declaration number DC-2008-429). The HGPS cell lines explored in this study have been prepared and stored according to the French regulation by the labeled Biological Resource Center of Tissues, DNA, Cells (CRB TAC), Department of Medical Genetics, la Timone Hospital, Marseille (Dr A De Sandre-Giovannoli and Mrs K. Bertaux). The fibroblast cell lines used belong to a biological sample collection declared to the French ministry of Health (Declaration number DC-2008-429) whose use for research purposes was authorized by the French ministry of Health (authorization numbers AC-2011-1312 and AC-2017-2986). Two control cell lines were used in this study. First cell line (DM4603) was provided by Coriell Cell Repository (Camden, USA) (referred as WT_1 in this study). Cells were obtained from the NINDS Human Genetics Resource Center DNA and Cell Line Repository. NINDS Repository sample numbers corresponding to the samples used are DM4603. The second control cell line (IMR-90) was obtained from ATCC (referred as WT_2 in this study). Cultures were maintained in Dulbecco's modified Eagle's medium + GlutaMAX II + 4500 mg/L D-Glucose (Gibco), supplemented with 20% fetal bovine serum (research grade, Sigma) and 1% sodium pyruvate 100 mM (Life technologies). Cell cultures were maintained at 37 °C, in 5% CO2 in a humidified atmosphere, with the media changed every 2 days. Fibroblasts from the four cell lines were reprogrammed into iPS cells using Yamanaka's original method [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF] with OCT4, KLF4, SOX2, c-myc, and transferred using retroviral vectors. Molecular characterization of the four cell lines were already reported in a previous study [START_REF] Nissan | Unique preservation of neural cells in Hutchinson-Gilford progeria syndrome is due to the expression of the neuralspecific miR-9 microRNA[END_REF] .

Pluripotent stem cell culture and differentiation. WT and HGPS iPSCs were grown in colonies on mouse embryonic fibroblasts (MEF), inactivated with 10 mg/ml mitomycin C seeded at 30,000 cells/cm², and grown as previously described. For differentiation, embryonic bodies (EBs) are formed from hESC and iPSC clumps and grown on low attachment dishes in neural medium composed of 1/2 neurobasal and 1/2 HAM:F12 complemented with 2% of B-27 without VitA (Invitrogen) and 1% N-2 (Invitrogen). Induction of neural-crest differentiation was realized using CHIR-99021 (TOCRIS), LDN-193189 (Miltenyi) and SB 431542 (TOCRIS). Melanogenic commitment was realized with CHIR99021(TOCRIS), EDN3 (American peptide), SCF (peprotech), BMP-4 (peprotech) and ascorbic acid (Sigma-Aldrich). At day 7, EBs were plated on gelatine 0.1% and grown in MGM-4 medium supplemented with the same cytokines until around day 30.

Melanocytes culture. Melanocytes derived from HGPS iPSCs and WT iPSCs were cultured in melanocytes specific medium, MGM-4 (Lonza). Cell cultures were maintained at 37 °C, in 5% CO2 in a humidified atmosphere, with the media changed every 2 days. Human adult epidermal melanocytes (HEM) were used as control.

Quantitative PCR. Total RNA was isolated using an RNeasy Micro extraction kit (Qiagen, Courtaboeuf, France), according to the manufacturer's protocol. An on-column DNase I digestion was performed to avoid genomic DNA amplification. RNA level and quality were checked using the Nanodrop technology. A total of 500 ng of RNA was used for reverse transcription using the Superscript III reverse transcription kit (Invitrogen). Q-PCR analysis was performed using a QuantStudio 12 K Flex real-time PCR system (Applied biosystem) and TaqMan gene expression Master Mix (Roche), respectively, following the manufacturers' instructions. Quantification of gene expression was based on the DeltaCt Method and normalized to 18 S expression. PCR primers for 18 S, MITF, TYRP1, PMEL17 lamin A, lamin C and progerin were previously described [START_REF] Egesipe | Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells[END_REF][START_REF] Nissan | Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis[END_REF][START_REF] Allouche | In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells[END_REF] .

Western immunoblotting. Whole-cell lysates of melanocytes were collected, proteins were extracted in NP40 cell lysis buffer (Invitrogen) with a protease and phosphatase inhibitor cocktail (ThermoScientific). Lysates were sonicated 4 times for 15 sec each, each with an interval of 15 sec in between, and then centrifuged at 10,000 g for 10 minutes at 4 °C. Protein concentration was measured using the Pierce BCA Protein Assay Kit (ThermoScientific) and the absorbance at 562 nm was evaluated using a Clariostar (BMG Labtech). A total of 20 µg of protein was loaded and run on a 7% tris-acetate gel (Criterion ™ XT) using XT tricine running buffer (Biorad). Gels were then transferred onto polyvinylidene fluoride (PVDF) membranes (Biorad) using a Trans-Blot Turbo Transfer System (Biorad). Blots were blocked in Rockland blocking buffer (Millipore) diluted 1:2 in TBS 1× for 1 hour at room temperature. Membranes were incubated with primary antibodies diluted in blocking buffer with 0.1% Tween20 (VWR) overnight at 4 °C. The primary antibodies used here are a mouse anti-progerin 1:200 (SantaCruz, 13A4D4, sc-81611) and a mouse anti-actin 1:5,000 (Millipore, MAB1501R). Washing was carried out for 45 minutes at room temperature with TBS + 0.1% Tween20 and the membranes were incubated with an IR-Dye 800CW conjugated with a secondary donkey anti-mouse antibody at 1:10,000 in blocking buffer with 0.1% Tween20 and 0.01% SDS (Ambion). The detector was an Odyssey Infrared Imaging System (LI-COR Biosciences).

Immunocytochemistry. Cells were fixed in 4% paraformaldehyde (15 minutes, room temperature) before permeabilization in PBS supplemented with 0.1% triton X-100 (Sigma) (5 minutes, room temperature). They were then blocked during 30 minutes at room temperature using PBS with 1% BSA (Sigma-Aldrich, St. Louis, USA). The primary antibodies used are: Mouse anti-MITF (1:100, M362129-2, Dako), Rabbit anti-lamins A/C (1:200, ab26300, Abcam), Mouse anti-NKI (1:100, ab34165, Abcam), Mouse anti-TYRP1 (1 :500, LSC39939) were incubated for one hour at room temperature in blocking buffer. Cells were stained with the species-specific fluorophore-conjugated secondary antibody (Invitrogen) (one hour, room temperature) and nuclei were visualized with Hoechst 33342 (Invitrogen).

SiRNAs transfection.

Melanocytes were transfected with SiControl or SiLamninA/Progerin using Oligofectamine (Invitrogen). Cells were transfected a second time after 48 h and analysed after 96 h from the first shot of transfection. siRNA Lamin-Progerin : 5′-GCAUCUAUCUCAUCUAUCU-3′/ 5′-AGAUAGAUGAGAUAGAUGC-3′.

Melanin content analysis. 100 000 cells were lysed in 1 M NaOH (Sigma-Aldrich ® ), warmed at 65 °C during 2 H and then centrifuged. Absorbance of supernatants was measured at 405 nm using 96-well microplates. A standard synthetic melanin curve (0 to 50 μg/ml) was performed in triplicate for each experiment (Sigma-Aldrich ® ).

Electron Microscopy. For ultrathin cryosectioning, melanocytes were fixed with 2% PFA or with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Cells were processed for ultracryomicrotomy (70 nm of thickness) and stained using the method described for ultrathin cryosections [START_REF] Raposo | Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells[END_REF] . All samples were analysed using a FEI CM120 electron microscope (FEI Company), and digital acquisitions were made with a numeric camera (Keen View; Soft Imaging System, SIS, Germany).

Image Analysis and quantification. Quantification of melanosomes was determined using the iTEM software (Soft Imaging System, SIS, Germany).

Statistical analysis.

Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 1 .

 1 Figure 1. Pathological modelling of pigmentation disorders in HGPS patients. (A) Picture of the skin of a HGPS patient that shows global hypopigmented skin, with a typical mottled pigmentation that alternates between hypo-and hyper-pigmented skin areas. The patient also shows typically prominent nipples, due to altered subcutaneous adipose tissue distribution. (B) Schematic representation of the pathological modelling strategy to model pigmentation disorders in HGPS patients.

Figure 2 .

 2 Figure 2. Measurement of progerin expression and nuclear shape abnormalities in WT and HGPS melanocytes. (A) Measurement of progerin expression in HGPS iPS during the melanogenic differentiation process at 0, 7, 14, 21, 28 and 70 days (equivalent to melanocyte passage 4). Human epidermal melanocytes (HEM) were used as a control. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). Representative pictures of the process of differentiation of iPS cells into Mel-iPSC at 4, 9, 11, 14, 18 days. (B) Comparative gene expression analysis for progerin in two WT (WT_1 and WT_2) and two HGPS (HGPS_A and HGPS_2) Mel-iPSCs. HEMs were used as a control. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Western blot analysis of progerin expression in WT and HGPS Mel-iPSCs. Actin was used as a control. The grouping of gels/blots was done from different gels. Uncropped gels are presented in sup figure (A,B). (C) Quantification of nuclear shape abnormalities in WT and HGPS Mel-iPSCs. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Lamin A/C immunostaining in WT and HGPS Mel iPSCs, showing typical nuclear disorganization of nuclear shape abnormalities.

Figure 3 .

 3 Figure 3. Molecular characterization of melanocytes derived from iPS cells. (A) MITF, PMEL17, TYRP1 (green) and Lamin A/C (red) immunostaining in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) Mel-iPSC and HEM cells. (B) Confocal analysis of TYRP1 localization in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) Mel-iPSC and HEM cells. (C) Gene expression analysis of MITF in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) iPS, Mel-iPSC and HEM cells. HEMs were used as a control. (D) Gene expression analysis of PMEL17 in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) iPS, Mel-iPSC and HEM cells. HEMs were used as a control. (E) Gene expression analysis of TYRP1 in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) iPS, Mel-iPSC and HEM cells. HEMs were used as a control.

Figure 4 .

 4 Figure 4. Characterization of pigmentation disorders and the melanosome maturation process in HGPS melanocytes. (A) Cellular pellet observation of WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) Mel-iPSC. (B) Analysis of melanin content (optical density) in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) Mel-iPSC. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Electron microscopy analysis of WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) Mel-iPSC. An inset corresponding to a magnified area of the boxed region is shown on the left. (D) Quantification of melanosome stages (stage I/II and stage III/IV) in WT (WT_1 and WT_2), HGPS (HGPS_1 and HGPS_2) Mel-iPSC. Data are from three independent experiments. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5 .

 5 Figure 5. Progerin knockdown experiments revealed functional regulation of pigmentation in Mel-iPSC. (A) Electron microscopy analysis of HGPS Mel-iPSC transfected with siLaminA/Progerin and siControl. (B) Quantification of melanosome stages (stage I/II and stage III/IV) in HGPS (HGPS-1 and HGPS_2) melanocytes transfected with siLaminA/Progerin and siControl. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Analysis of melanin content (optical density) in HGPS (HGPS_1 and HGPS_2), Mel-iPSC transfected with siLaminA/Progerin, normalized to siControl. Statistical analysis was performed with one-way analysis of variance (ANOVA), using Dunnet's comparison test. Values of p < 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

SciEntific REPORtS | (2018) 8:9112 | DOI:10.1038/s41598-018-27165-y

SciEntific REPORtS | (2018) 8:9112 | DOI:10.1038/s41598-018-27165-ya new potential readout that can be targeted in the search for potential treatment for HGPS, photo-aging and hypopigmentation disorders.

Acknowledgements

I-Stem is part of the Biotherapies Institute for Rare Diseases (BIRD), supported by the Association Française contre les Myopathies (AFM-Téléthon). This research was supported by grants from INSERM, the domaine d'intéret majeur (DIM) Biothérapies, Genopole and the European Commission: the laboratoire d'Excellence Revive (Investissement d' Avenir; ANR-10-LABX-73), NeurATRIS: A Translational Research Infrastructure for Biotherapies in Neurosciences (Investissement d' Avenir -ANR-11-INBS-0011), INGESTEM: the National Infrastructure Engineering for Pluripotent and differentiated Stem cells (Investissement d' Avenir -ANR-11-INBS-0009). The authors thank Marc Peschanski, Gilles Lemaitre and Walter Habeler (I-stem) for helpful discussions, Drs Laure Chatrousse and Jerome Polentes for their help during the revision process, Ilse Hurbain, Maryse Romao, and Graca Raposo (Institut Curie Centre de Recherche,. UMR CNRS 144, Paris, France) for advice, skill, and expertise for electron microscopy analysis, and the BioImaging Cell and Tissue Core Facility of the Institut Curie (PICT-IBiSA), a member of the French National Research Infrastructure France-BioImaging (ANR-10-INSB-04).

Author Contributions

Additional Information

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-27165-y.

Competing Interests:

The authors declare no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Djadidi Toybou

Caroline Celle

Catherine Aude-Garcia

Thierry Rabilloud
email: thierry.rabilloud@cea.fr

Jean-Pierre Simonato
email: jean-pierre.simonato@cea.fr

A toxicology-informed, safer by design approach for the fabrication of transparent electrodes based on silver nanowires

Fabrication of silver nanowires (AgNWs) with fine and independent control of both the diameter (from 30 to 120 nm) and length (from 5 to 120 μm) by concomitant addition of co-nucleants and temperature control is demonstrated, and used for the preparation of size standards. Percolating random networks were fabricated using these standards and their optoelectronic properties were measured and compared with regard to the nanowire dimensions. The transparent electrodes appear suitable for various applications and exhibit excellent performances (e.g. 16 ohm sq -1 at 93% transparency), with haze values varying from 1.6 to 26.2%. Besides, in vitro toxicological studies carried out on murine macrophages with the same size standards revealed that AgNWs are weakly toxic (no toxicity observed below 50 μg mL -1 Ag), in particular compared to other silver nanoparticles. Short AgNWs (4 μm) appeared to be slightly more toxic than longer AgNWs (10 and 20 μm). Conversely, long AgNWs (20 μm) induced a more prolonged pro-inflammatory response in murine macrophages. These results contribute, in a safer by design approach, to promoting the use of short AgNWs. The global knowledge dealing with the combination of nanowire dimensions associated with optoelectronic performances and related toxicity should encourage the rational use of AgNWs, and guide the choice of the most adequate AgNW dimensions in an integrated approach.

Introduction

The recent development of nanomaterials has generated a wave of hope in many fields. The potential of nanomaterials appears boundless, either due to their intrinsic properties (e.g. quantum dots and fullerenes), their additional properties for bulk materials (e.g. nanocomposites), or when they are used in the form of assemblies (e.g. carbon nanotubebased cables). A characteristic and high-potential example of nanomaterial assembly is the fabrication of random networks of metallic nanowires, in particular, silver nanowires (AgNWs). [1][2][3] Simply put, it is possible to generate random networks of metallic nanowires, above the percolation threshold, which conduct electricity through the nanowire-based metallic lattice and concurrently let the light pass through the empty spaces between the nanowires. This has been widely studied during the last decade and has raised the maturity of this technology up to the industrial level. Many applications using AgNWs have been investigated, including touch screens, light-emitting devices, transparent film heaters and others. [4][5][6][7][8][9][10][11][12] This system has many advantages such as very low sheet resistance with high transparency, flexibility, and low-cost processing under ambient conditions.

Though many important advances have been realized in this field, some key points remain to be tackled, in particular, toxicity issues for safe industrial use. Since the expected properties are application-dependent and rely on the AgNWs' morphology, 13,14 it would be of great interest to be able to tailor the shape of the nanowires in order to obtain optimal optoelectronic performances for each use. Conventional parametric studies 15,16 as well as other more specific methods 17,18 have demonstrated efficient routes for modifying both the length and diameter, but not independently. An ultrasonic method was also proposed to select the desired length by cutting AgNWs; however, this method leads to poor control over size distribution and generates large amounts of by-products. 19 The fabrication of AgNW size standards would also allow toxicological studies to be performed on definite species, in particular to determine whether the two main dimensions (i.e. diameter and length) play a critical role in toxicity. This is of tremendous importance since AgNWs will increasingly be introduced in technological devices and consumer products, and thus data are awaited to assess the real toxicity of these nanomaterials. Although several reports have begun to tackle this topic, [20][21][22][23][24][25][26] much more remains to be done.

In this article, we propose a straightforward method to modulate independently the diameter and the length of nanowires in a wide range, allowing us to fabricate size standards, i.e. AgNW samples with definite lengths and diameters. It has permitted us to perform a direct comparison of the optoelectronic properties of transparent electrodes made with these different nanowires, and to realize comparative experiments on toxicity with macrophages.

Materials and methods

Synthesis and purification of AgNWs

In a typical synthesis, AgNO 3 (0.68 g) was dissolved in EG (40 mL) at a slow stirring rate in a round flask. In another flask, PVP (average mol. wt 40 000, 1.77 g), NaCl, and the conucleant were dissolved in EG (80 mL) at 120 °C. The solution was cooled to room temperature and then slowly added to the first flask within 8 min. The mixture was finally heated at the reaction temperature and cooled down at ambient temperature. The purification of the AgNWs was realized by decantation according to a published procedure. 27

Characterization of the mean dimensions of AgNWs

The morphology analysis of AgNWs was performed using a Leo 1530 SEM. Measurement of the diameter and length dimensions was realized with the ImageJ software. The statistical studies were performed on 100 to 200 nanowires for each sample.

Preparation of AgNW electrodes

The AgNW-based solutions were used for the fabrication of electrodes after dilution in methanol at 0.2 mg mL -1 concentration. The silver concentration was measured using an atomic absorption spectrometer (Perkin Elmer AAnalyst 400). The deposition was performed on 2.5 × 2.5 cm 2 heated substrates (70 °C) with an automatic SonoTek spray-coater. The performances of the electrodes were measured after cooling, without any post-treatment.

Performance measurements

Total transmittance and haze values were measured with a UV-visible spectrometer (Varian Cary 5000) equipped with an integrating sphere. The sheet resistance was set as the mean value of at least 5 measurements by using a four-pin probe with a Loresta resistivity meter (EP MCP-T360).

Toxicological assays

Nanomaterials. As control nanomaterials, spherical silver nanoparticles (<100 nm) coated with PVP40 were purchased from Sigma-Aldrich (catalogue number 758329) as a 5 wt% dispersion in ethylene glycol. Working solutions were prepared by dilution in deionized water. The characterization of these nanoparticles has been published previously by some authors of this article. 28 Additional controls consisted of silica-based nanomaterials, either colloidal silica 29 or crystalline silica (reference materials BCR66 and BCR70, purchased from Sigma-Aldrich).

Cell lines. The mouse macrophage cell line RAW 264.7 was obtained from the European Cell Culture Collection (Salisbury, UK). The cells were cultured in RPMI 1640 medium + 10% fetal bovine serum. The cells were seeded at 200 000 cells per ml and harvested at 1 000 000 cells per ml. For treatment with nanomaterials, the cells were seeded at 500 000 cells per ml. They were treated with nanomaterials on the following day and harvested after a further 24 hours in culture.

Neutral red uptake assay. This assay was performed according to a published protocol. 30 Cells grown in 12-well plates and treated or not with nanomaterials were incubated for 1 h with 40 μg mL -1 neutral red (final concentration, added from a 100× stock solution in 50% ethanol-water). At the end of the incubation period, the medium was discarded and the cell layer was rinsed twice with PBS (5 min per rinse). The PBS was removed, and 1 mL of elution solution (50% ethanol and 1% acetic acid in water) was added per well. The dye was eluted for 15 min under constant agitation, and the dye concentration was read spectrophotometrically at 540 nm. The results were expressed as % of the control cells (untreated).

Cytokine production and persistence experiments. The tumour necrosis factor (TNF-α), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) concentrations in the culture supernatants of cells exposed for 24 h to AgNWs and control nanomaterials were measured using the cytometric bead array (CBA) mouse inflammation kit (BD Pharmingen, France) according to the manufacturer's instructions. The measurements were performed on a FACSCalibur flow cytometer and the data were analysed using CellQuest software (Becton Dickinson). Bacterial lipopolysaccharide (LPS) was used as a positive control (200 ng mL -1 , 24 h). At the end of the exposure period, the medium was removed and saved for cytokine measurements. Fresh complete culture medium (without nanomaterials) was added to the cells and left for 36 h. The medium was then removed and saved, and fresh complete culture medium was added for a final 36 h period. Thus for each nanomaterial, 3 cytokine assays were performed, covering the 24 h exposure period and two post-exposure 36 h time windows.

Results and discussion

Controlled synthesis of AgNWs: key factors for independent tuning of length and diameter

The synthesis of AgNWs has been extensively studied since the first report of Xia et al. 31 Among different routes to fabricate these nanowires, the most studied process has been the in situ reduction of silver salts by the reductive polyol method. This solvothermal process usually includes the use of ethylene glycol as the main solvent, silver nitrate as a cheap silver source, a nucleant with or without co-nucleantIJs) to initiate the growth of nanowires and PVP (polyvinylpyrrolidone) as the capping agent which allows orientation of the uniaxial growth. 32 It has already been demonstrated that AgNWs with different lengths and diameters can be obtained "on demand" thanks to the conditions extracted from parametric studies; however, it has not been demonstrated so far that both the diameter and the length could be tuned independently. In other words, modification of the process to adjust one dimension has concomitantly a strong effect on the other dimension (e.g. protocol adjustment to modify the mean diameter will strongly impact the mean length of the AgNWs).

It has been previously reported in the literature that the use of a co-nucleant besides NaCl, the molar mass of PVP and synthesis temperature may strongly affect NW morphologies. 33 In particular, halide ions are known to modify the nanowire diameter. [34][35][36] Bromide ions proved to be efficient in reducing the diameter, but at the same time they also al-tered the length of AgNWs. [37][38][39] To begin this study, the influence of F -, Cl -, Br -and I -as co-nucleants was investigated and compared to a standard synthesis protocol. 40,41 The impact of each halide ion on the morphology of assynthesized AgNWs is presented in Fig. 1. Compared to the standard synthesis without a co-nucleant (Fig. 1a, pink cross, 63 ± 10 nm diameter/8 ± 3 μm length), each added halide ion (molar ratio, 1 : 1 NaCl : KX) has a noticeable effect on the final morphology of AgNWs. An exception is the case of iodidemodulated synthesis which leads only to irregular nanoparticles. The results obtained with KBr or NaBr as a conucleant appeared very close. To confirm this point, NaCl and KCl were also used as nucleants (standard conditions, 0.7 mmol L -1 ), and as shown in Fig. 1(a), AgNWs with similar morphologies (51 ± 7 nm diameter and 6 ± 1 μm length) were produced. This means that using KCl as a co-nucleant in an equimolar ratio with NaCl gives the same result as doubling the amount of NaCl; thus alkali counter-cations do not play a significant role in the reaction mechanism. Nevertheless, it must be noticed that both the diameter and length are affected in the same way when the chloride quantity changes, as already reported elsewhere. 42 Fluoride ions (Fig. 1a blue triangle) increased the length from 8 ± 3 μm to 12 ± 5 μm, and the diameter to 71 ± 10 nm. Whereas the effects of fluoride and iodide ions have not been reported extensively, diameter shrinkage with the use of a bromide-based conucleant is in fair agreement with the literature. [37][38][39]43 To further investigate the effect of bromide as a conucleant, we performed experiments with varying concentrations of KBr, from 0 to 0.7 mmol L -1 . The results are presented in Fig. 1b. It appears that the diameter decreases from 63 ± 10 nm down to 20 ± 3 nm with increasing amount of bromide ions, while the length increases from 6 ± 3 to 12 ± 5 μm. Nanowires with 15 nm diameter can also be obtained at higher concentrations of bromide, but the amount of metallic by-products becomes excessive. As shown here, and in agreement with literature reports, adjustment of the KBr quantity leads to a fine control of the diameters but changes significantly the lengths.

Thus, in our goal to fabricate "on demand" AgNW size standards with defined lengths and diameters, we carried out experiments by setting the KBr amount at 350 mmol L -1 to control the diameter, and we introduced NaCl in various amounts in order to modulate the length. The results plotted in Fig. 2a show that when the chloride concentration was varied between 0.5 and 2.5 mmol L -1 , we obtained a collection of AgNWs with lengths ranging from 28 ± 9 down to 4 ± 2 μm. For higher concentrations of chloride, nanoparticles were obtained as the sole product. These results demonstrate that by adequate choice of the nature of co-nucleants and by fine adjustment of their concentrations, the variation of length is actually possible while keeping the diameter almost unchanged (Fig. 2b). This is a straightforward route to select the desired length at constant diameter. The mean length modification can be ascribed to the increased (or decreased) number of seeds, which depends on the molar ratio between chlorine and silver. This is consistent with the report from Buhro and colleague 44 who demonstrated, in the early stage of the polyol synthesis, the impact of chloride concentration on the generation of nucleation sites.

Whereas diameters below 50 nm are desirable for most optoelectronic applications requiring a low haze value, in some specific uses like photovoltaics, larger diameters are expected. 5 In our effort to modulate length and diameter independently, we looked at a protocol to modify the diameter while keeping the length mostly unchanged. It was previously demonstrated by Unalan and colleagues that polyol synthesis carried out at low temperatures generates thick wires (with diameters higher than 300 nm) and that temperature plays a crucial role in the nanowire formation. 15 We used our standard protocol at various temperatures. The results are presented in Fig. 2c andd. We observed that the mean diameter decreased drastically, from 90 ± 10 nm down to 50 ± 8 nm, when the temperature was raised from 150 to 180 °C. At the same time, the mean length of the AgNWs remained almost constant. This demonstrates that it is also possible to change the diameter of AgNWs while keeping the length mostly unchanged by selecting the adequate reaction temperature. Fabrication of AgNW size standards. Thanks to the protocols presented hereinbefore, we were able to prepare different AgNW size standards, as shown in Fig. 3. Combinations of any lengths or diameters in the range of 5-120 μm and 30-120 nm, respectively, were achievable. This gives access to calibrated samples of nanowires. Histograms showing the distribution in diameters of three different samples of 10 μm long AgNWs with various mean diameters are presented in Fig. 3a, and histograms of three different populations with different lengths of 40 nm diameter AgNWs are shown in Fig. 3b. In each case, the overlap between two adjacent statistical populations was calculated to be below 15%. SEM images are also presented to illustrate the major differences obtained between the different samples in Fig. 3c; they show undoubtedly how both diameters and lengths can be tuned according to specific synthetic protocols. The main criteria to tune the synthesis of the AgNW standards are schematized in Fig. 3d. We also verified that the proposed conditions are scalable. We performed experiments using up to 5 L semibatch reactors, which allowed us to confirm that the synthesis of AgNWs with pre-determined dimensions can be carried out "on demand".

Optoelectrical performances of transparent electrodes: impact of AgNWs' dimensions on haziness. When assembled in the form of random networks above the percolation threshold, it is known that these materials can provide a relevant alternative to TCOs (transparent conductive oxides) for the fabrication of transparent conductive materials. In this case, the properties of interest (e.g. transparency and sheet resistance) are measured at the macroscopic scale, but depend strongly on the nanosized building blocks. We thus fabricated transparent electrodes using various AgNW standards with aspect ratios (i.e. length/diameter ratios) ranging from 55 to 1000. For the sake of clarity, the different AgNW mean dimensions are expressed as DaLb, with a and b indicating the mean diameter in nm and the mean length in μm, respectively. The transparency, conductivity and haze factor (i.e. the diffused part of the transmitted light) of the electrodes were measured and compared.

A typical plot of the transmittance as a function of the sheet resistance is presented in Fig. 4a. This series of dots was measured using D30L10 AgNWs. As expected, for these dimensions and for highly purified nanowires, excellent optoelectrical performances were obtained, with for instance 97% transparency for a 50 ohm sq -1 sheet resistance. This compares very well with the state-of-the-art for metallic nanowire-based technology, and also with ITO or any other kind of transparent electrode. We chose to limit the field of study to nanowires with at most 20 μm length because longer AgNWs are much more difficult to synthesize, to handle, to formulate and to deposit with high homogeneity on a large substrate area using low-cost solution deposition techniques. In Fig. 4b, the haze values are presented as a function of the total transmittance for AgNWs with different diameters and for different lengths (5, 10 and 20 μm long). This graph shows a linear relationship between the haze value and the total transmittance regardless of the length. The dimension effect is very important since the haze values vary from 16% down to less than 2%. Very low haziness, compatible with optoelectronic applications, is demonstrated for small diameters, which is consistent with previous studies, [45][46][47] and very interestingly, it is also shown that even with 90 nm diameter AgNWs, it is possible to achieve low haze values for long nanowires. The haze factor reported in the literature for sub-15 nm diameter silver nanowires reached very low values below 1% at 94.5% transmittance. 48 However, reducing the diameter of AgNWs below the mean free path of electrons (∼40 nm) is risky due to charge carrier scattering at the interface and to material instability under operational stress. The histogram in Fig. 4c shows haze values at given sheet resistances for AgNWs with various lengths and diameters. When the densities of AgNWs are decreased (i.e. increasing sheet resistances), the haze value is lowered drastically regardless of the size standards. The impact of the length is obvious since 20 μm long nanowires are much less light diffusive than 10 μm long nanospecies. For instance, in the case of 40 nm diameter AgNWs, the haze value is more than doubled when the length is reduced from 20 μm down to 10 μm. This is expected by percolation theory. Indeed, for finite size scaling in stick percolation dealing with large size systems, the critical number density (N c ) of sticks required for percolation is given by N c × L 2 = 5.637 where L is the length of nanowires. 49 This indicates that for a two-fold increase of the length of the AgNWs, the required N c to reach percolation is divided by four. 17 emphasized that in our examples, the electrodes are fabricated with densities far above N c , but the length effect is still clearly perceptible. The diameter effect is also visible for both 10 μm and 20 μm long AgNWs, and as expected, small diameters lower the haziness. Pictures of two electrodes are included in Fig. 4d to illustrate the pronounced optical difference (transparency and haziness) for different densities of AgNWs.

Toxicity of AgNW size standards. Even if silver toxicology is well known, 53,54 the toxicity of silver-based nanomaterials is difficult to predict because of their different shapes, which can contribute to different biological responses regarding the plurality of microorganisms. It is described in the literature that low-aspect ratio nanosilver species such as nanoparticles exhibit toxicity mainly relying on silver ion release, either extracellularly (for bacteria) or due to internalization and dissolution in the lysosomes for animal cells. 28,55,56 For high aspect ratio silver nanomaterials, like AgNWs, the toxicity effect may be driven by dissolution on the one hand; on the other hand, the form factor may contribute largely, as AgNWs exhibit a fibre shape having similarities with asbestos. Different toxicity approaches are currently ongoing to decipher possible mechanisms in order to avoid toxicity caused by the aspect ratio, and some research studies are carried out to establish safety thresholds like for asbestos. [57][58][59] In this study, we investigated the basic responses of murine macrophages in the presence of AgNWs. We chose macrophages because they are in charge of removing nanomaterials, and because they are also the main actors at play in insoluble fibre toxicity. Thanks to the fabrication of AgNW size standards described above, toxicological experiments were performed on macrophages to discriminate the effects of the diameter and length.

Previous studies dealing with the potential toxic effect of AgNWs on lung and macrophage models revealed a lengthdependent toxicity for both in vitro and in vivo studies, with short nanowires causing less inflammatory responses. 20,57,[60][61][62] These different studies compared various lengths; however, a possible diameter effect was not directly tackled by comparative experiments, or both diameters and lengths were changed at the same time.

Prior to toxicity experiments on macrophages, we evaluated the contribution of dissolved species. The dissolution of relevant morphologies of nanowires was monitored over a period of 24 h in medium only under working conditions. The AgNWs were compared to silver nanoparticles coated with PVP with a mean diameter close to standard AgNWs (≈60 nm) and citrate-coated silver nanoparticles which are known to dissolve quickly. The results are shown in Fig. 5a.

Regardless of their dimensions, AgNWs exhibit similar dissolution rates. Silver nanoparticles coated with PVP dissolved slightly faster than the nanowires. For all the AgNWs, the solubilized part of silver was found to be low (<10%), even after 24 h. The corresponding Ag + concentration was lower than the known silver ion cytotoxicity (1 μg mL -1 ); hence subsequent toxicity measurements on macrophages were considered relevant to evaluate the potential intrinsic toxicity of AgNWs.

AgNWs show low and length-dependent toxicity for macrophages. Cytotoxicity assays were performed on RAW264.7 murine cells, and the neutral red uptake assay was used to determine the potential adverse effects for each morphology of AgNWs. Various morphologies were chosen to check independently the potential diameter or length effects. For instance, we used AgNWs with the diameter dimension set at 40 nm with various lengths (4, 10 and 20 μm), and 10 μm long AgNWs with various diameters (40, 60 and 90 nm). No decrease of cellular activity up to high doses (50 ppm nanomaterials) was observed, as evidenced in Fig. 5b. The shortest AgNWs (length of 4 μm) presented the lowest IC50 at 100 ppm concentration, followed by the three other nanowires with 10 μm length. Longer AgNWs (20 μm length) did not present a cellular activity decrease under our experimental conditions. This length effect can be related to the uptake ability of macrophages. Phagocytosis of short nanowires (4 μm) can be easily completed, leading to silver ion release inside the cells, as with the well-described silver nanoparticle toxicity. The 10 μm long nanowires, with different diameters, show a similar inhibition behaviour. The quasi non-toxicity of long AgNWs (20 μm length) can probably be ascribed to the fact that the nanowires were not internalized, as the threshold length of phagocytosis was exceeded. These results suggest that AgNWs induce cytotoxicity in a length-typedependent manner, which is consistent with a reported study on RAW264.7 treated with carbon nanotubes of different lengths. 63 These results are in accordance with the literature, with cytotoxicity being correlated with the degree of cell uptake and the amount of ionic silver due to intracellular dissolution. 62,64 Long AgNWs induce a slight but persistent proinflammatory profile. Macrophages are known for their capacity of phagocytizing particulate substances, such as pathogenic agents but also particulate chemicals (e.g. oxidized lipoprotein particles in the case of atherosclerotic foam cells).

They also play a strong role in immune reaction modulation through their ability to release various signalling molecules such as pro-or anti-inflammatory cytokines, depending on the nature of the phagocytized particles. They also trigger these mechanisms upon frustrated phagocytosis (e.g. in the asbestos case), which makes them an attractive choice to study possible pro-inflammatory responses. To evaluate this response, we measured two major inflammatory cytokines: interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) at the highest non-toxic concentration (50 ppm). The inflammatory responses due to the nanowires were compared, on the one hand, to that of amorphous and crystalline silica particles (LS30, BCR66, and BCR70) known to induce a low but well-documented inflammatory response [65][66][67][68] and on the other hand, to bacterial lipopolysaccharide (LPS) as a positive control known to induce very strong pro-inflammatory responses.

Fig. 6a shows the relative amount of secreted TNF-α by macrophages in response to the different nanowires. After 24 h, we can notice a similar production rate for all the morphologies of AgNWs. However, D40L20 is the only AgNW that induces a slightly higher TNF-α production than silica. For IL-6 (Fig. 6b), the production induced by AgNWs was very low and always lower than those induced by silica. Overall, the bar charts for IL-6 and TNF-α show a globally low secretion for each cytokine after a 24 h exposure to nanomaterials, representing less than 1% of the LPS-induced production. These results show that the AgNWs induce a weak increase Fig. 6 Pro-inflammatory cytokine secretion after treatment with nanomaterials (R0) and persistence results at 36 h (R36) and 72 h (R72). The secretion level was compared to LPS as a positive control and silica-based nanomaterials (LS30, BCR66, and BCR70) as the nanomaterial control. (a) TNF-α secretion is very low (the same level as the control) for all AgNWs, and lower than the silica control (BCR66), except for D40L20. (b) The IL-6 secretion of cells treated with AgNWs is similar regardless of the length or diameter, and at the same level as the negative control. For both IL-6 secretion and TNF-α secretion, the comparative secretion with LPS is very low (<1%).

Environmental Science: Nano Paper in cytokine release, which correlated positively with the length of the wires, as expected for fibre-like materials. One of the major mechanisms at play in persistent fibre/ particle toxicity is the presence of a prolonged proinflammatory response over time. In order to test this possibility by our in vitro system, we studied the evolution of the production of TNF-α and IL-6 over time after a 24 h exposure to nanomaterials (Fig. 6). LPS-induced TNF-α release was both acute and very transient. Compared to the control, none of the short nanowires, the amorphous silica or the silver nanoparticles induced any significantly higher TNF-α release. Only the small crystalline silica (BCR66) and the long nanowires (20 μm) induced a weak but sustained TNF-α release. Although the shape of the curves is different for IL-6, with a prolonged LPS-induced release, the same trend can be observed, with only the small crystalline silica (BCR66) and the long nanowires inducing a prolonged IL-6 release compared to the control. These results suggest that the long nanowires have an inflammatory effect that is sustained over time. To the best of our knowledge, this is the first reported study on the persistence of the inflammatory response in vitro.

Our results are in line with those obtained using in vivo models 20,26,60 and suggest that simple in vitro models can be predictive of the inflammatory potential of nanomaterials. In addition, these in vitro systems are much cheaper and ethically more acceptable than in vivo experiments.

Taken collectively, all these data allow us to propose a "safer by design" approach for the use of AgNWs, depending on the applications. Different performances for both electrical and optical aspects (including haziness) can be achieved, depending on the density and on the dimensions of AgNWs. According to the toxicological results, a rational approach would be to foster the use of nanowires up to 10 μm length since they can combine high optoelectrical performances and minimal biological effects.

Conclusions

By careful modifications of the polyol process, in particular, precisely defined halide ion concentrations and temperature setting, independent fine tuning of the diameter and length of AgNWs has been successfully demonstrated. Size standards were prepared accordingly, and used for the fabrication of transparent electrodes. Excellent optoelectrical performances were obtained and the haze value was varied from 1.6 to 26.2%. In the meantime, we performed in vitro toxicological assays on murine macrophages. The AgNWs were found to be weakly toxic for macrophages and showed a length-dependent toxicity, with the toxicity decreasing with length. Conversely, the cytokine release assays showed a weak but significant proinflammatory potential for long nanowires (20 μm), and the observed persistence of the response, although at weak levels, calls for attention when devising the rules for the use of AgNWs in consumer products and in the recycling of such products. This study set out to take a holistic view of AgNWs, with a clear relationship between AgNW dimensions and both their optoelectronic performances, when used in the form of random percolating networks, and their toxicity and proinflammatory potential. Since AgNWs will certainly be increasingly introduced in commercial devices in the short term, it seems important to have a global vision of these nanomaterials, which will ineluctably encourage the rational use of AgNWs in a "safer by design" approach.

Environmental

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Fig. 1

 1 Fig. 1 Influence of several halide ions on the morphology of AgNWs. (a) Lengths of AgNWs synthesized by a standard protocol 41 (pink dot, NaCl as the only nucleant) and modified procedures with additional halide salts as co-nucleants (KBr, NaBr, KCl, NaCl, and KF). (b) Effect of KBr concentration on diameters and lengths of AgNWs.

Fig. 2

 2 Fig. 2 Tuning of the length of thin AgNWs by NaCl concentration and reaction temperature. (a) Length as a function of the chloride concentration, with a constant KBr concentration of 350 mmol L -1 . Increasing the quantity of chloride decreases the mean length of AgNWs. (b) The diameter of AgNWs remains constant around 40 nm while the length decreases from 28 μm to 3 μm. The KBr concentration is set at 350 mmol L -1 . (c) The length remains almost constant while the reaction temperature is modified. (d) Diameter tuning by reaction temperature modification, where increasing the temperature induces a diameter decrease.

Fig. 3

 3 Fig. 3 Independent tuning of the diameter and length of silver nanowires. (a) Histograms of three different mean diameters for 10 μm long AgNWs. (b) Histograms of three different mean lengths for 40 nm diameter AgNWs (c) SEM pictures of various lengths and diameters. Top, from left to right: mean diameters of 30 nm, 60 nm, and >100 nm; bottom, from left to right: mean lengths of >100 μm, 10 μm, and 5 μm. (d) Schematic diagram to control the morphology of AgNWs.

  ,50 As shown by De et al. and by Lagrange et al., percolation theory dominates the electrical behaviour of networks associated with low density.51,52 However, it should be

Fig. 4

 4 Fig. 4 Optoelectrical properties of transparent electrodes fabricated with various AgNW size standards. (a) Total transmittance as a function of the sheet resistance for D30L10 AgNWs. (b) Haze values for 30, 60 and 90 nm diameter AgNWs as a function of the total transmittance. The green part represents measurements made on 5 μm long AgNWs, the white part for 10 μm length and the pink part for 20 μm length. The solid lines correspond to linear regressions for each diameter. (c) Comparison of the haze level on given sheet resistances for standard AgNWs (D60L10), thin and short (D40L10), thick and long (D90L20) and thin and long nanowires (D40L20). (d) Left, a 96% transparent electrode with a very low haze value (1.6%); right, a 60% transparent electrode with high haziness (13.0%).

Fig. 5

 5 Fig.5Dissolution rate of the studied nanomaterials and cell viability of RAW264.7 murine cells treated with the nanomaterials. (a) Dissolution monitored for 24 h at 37 °C in RPMI medium containing only the nanomaterials at 2.5 μg mL -1 concentration except for less concentrated citratecoated nanoparticles (0.4 μg mL -1 ). PVP-coated nanoparticles (NP-PVP, red triangle) dissolve faster than nanowires (AgNWs, black dot). The total dissolution of all the nanomaterials is less than 10% in the medium. (b) The cells were incubated for 24 h with different concentrations of the nanomaterials (from 20 to 100 ppm). The shortest nanowires (D40L4) exhibit a higher toxicity, AgNWs with 10 μm length (D40L10, D60L10, and D90L10) exhibit a similar toxicity regardless of the diameter dimension, and long nanowires with 20 μm length (D40L20) and PVP-coated nanoparticles (NP-PVP) show a very low toxicity. The LC50 of the short nanowires, which exhibit the highest toxicity, is higher than 80 ppm.

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Acknowledgements

This work was supported by the Labex Serenade (no. ANR-11-LABX-0064) funded by the "Investissements d'Avenir" French Government program of the French National Research Agency (ANR) through the AMIDEX project (no. ANR-11-IDEX-0001-02). In particular, the authors thank Pr L. Charlet for coordinating the "nanosilver topic", and they are grateful for the funding of D. Toybou's PhD grant.

Conflicts of interest

There are no conflicts of interest to declare.