S Mura

N Tsapis

M Pallardy

H Hillaireau

Immunotoxicity of poly(lactic-co-glycolic acid) nanoparticles: influence of surface properties on dendritic cell activation

Keywords: Nanoparticles, poly(lactic-co-glycolic acid), immunotoxicity, dendritic cell

Immunotoxicity of poly (lactic-co-glycolic acid) nanoparticles: influence of surface properties on dendritic cell activation

Introduction

Nanotechnology applications in medicine, termed as nanomedicine, have opened doors to a plethora of possible answers to several impediments of conventional medicine.

Among the broad panel of available materials in nanomedicine, poly (lactic-co-glycolic) acid (PLGA) has been extensively investigated, as this copolymer is generally viewed as biocompatible and biodegradable [START_REF] Panyam | Biodegradable nanoparticles for drug and gene delivery to cells and tissue[END_REF][START_REF] Bala | PLGA nanoparticles in drug delivery: the state of the art[END_REF][START_REF] Mundargi | Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives[END_REF][START_REF] Lu | Current advances in research and clinical applications of PLGA-based nanotechnology[END_REF][START_REF] Xu | Intracellular drug delivery by poly(lactic-coglycolic acid) nanoparticles, revisited[END_REF]. It has been approved by the US FDA for drug delivery and other applications in clinics, and widely investigated for applications in cardiovascular diseases, cancer, vaccine and tissue engineering [START_REF] Lu | Current advances in research and clinical applications of PLGA-based nanotechnology[END_REF]. In this context, NPs of PLGA are explored as carriers for the controlled delivery of small molecules such as anticancer cytotoxic compounds, and macromolecular therapeutics such as proteins, peptides, vaccines, genes, antigens and growth factors [START_REF] Mundargi | Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives[END_REF]. Because of this increasing interest for these PLGA NPs, it is obvious that a careful assessment of their potential toxicity is crucial.

Despite such a need for risk assessment studies to be carried out, the current state of knowledge about biological effects and toxicity of PLGA NPs is well characterized for some aspects [START_REF] Bisht | PLGA nanoparticles for intravitreal peptide delivery: statistical optimization, characterization and toxicity evaluation[END_REF] but remains limited for most of exposure routes.

In the specific context of nanotoxicology, the field of immunotoxicology is gaining a growing interest, as the interactions between NPs and the immune system have proven pivotal in the response to NP exposure [START_REF] Dobrovolskaia | Immunological properties of engineered nanomaterials[END_REF][START_REF] Dobrovolskaia | Evaluation of nanoparticle immunotoxicity[END_REF][START_REF] Zolnik | Minireview: Nanoparticles and the Immune System[END_REF][START_REF] Blank | Opportunities and challenges of the pulmonary route for vaccination[END_REF][START_REF] Di Gioacchino | Immunotoxicity of nanoparticles[END_REF][START_REF] Oostingh | Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects[END_REF]. The immune response to foreign chemical and/or biological molecules as well as to particles is typically initiated through a protective innate inflammatory response. With the exception of some scarce studies, PLGA-based NPs would not seem to induce such an innate response, as shown by our group and others [START_REF] Dailey | Investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung[END_REF]Mura et al., 2011a, 2011b[START_REF] Aragao-Santiago | Compared in vivo toxicity in mice of lung delivered biodegradable and non-biodegradable nanoparticles[END_REF]. In particular, PLGA-based NPs induced moderate but significant suppression of of Tlymphocytes proliferation [START_REF] Aragao-Santiago | Compared in vivo toxicity in mice of lung delivered biodegradable and non-biodegradable nanoparticles[END_REF]. However, little is known about the effects of PLGA NPs on the activation of antigen-specific cells (APCs) such as dendritic cells (DCs). DCs play a major role in innate immunity by driving the specific reaction by the adaptive immune system. A myriad of cytokines (TNF-, GMCSF, IL-1, …) may be produced by our innate immune system in response to a xenobiotic allowing inflammation and contributing to immunotoxic effects [START_REF] Petrarca | Engineered metal based nanoparticles and innate immunity[END_REF].

Given the importance of DCs, NP impact on these cells raises growing concerns.

Among the deleterious effects of NPs on DCs currently expected, immunosuppression has been suggested [START_REF] Ilinskaya | Immunosuppressive and anti-inflammatory properties of engineered nanomaterials[END_REF]. More insidiously, NPs could act as immune adjuvants, inducing exacerbation or modification of the type of immune response to a given antigen, hence resulting in hypersensitivity or allergic reactions.

Possible impacts on the immune system stress the need for a careful immunotoxicological evaluation, particularly for regulatory purposes [START_REF] Dobrovolskaia | Evaluation of nanoparticle immunotoxicity[END_REF][START_REF] Dobrovolskaia | Current understanding of interactions between nanoparticles and the immune system[END_REF].

To elucidate PLGA NP-dependent outcomes on DCs, we herein present an in vitro study of the effect of 100-250 nm PLGA NPs on both human monocyte-derived DCs (MoDCs) and murine bone-marrow derived DCs (BMDCs). Since the performance of nanotechnology-based drug delivery systems is strongly influenced mostly by surface properties (charge and hydrophilicity), surfactants may have a strong impact on resulting biological effects of particles, especially in terms of uptake and intracellular fate [START_REF] Bhardwaj | Pharmaceutical aspects of polymeric nanoparticles for oral delivery[END_REF][START_REF] Yue | Surface Charge Affects Cellular Uptake and Intracellular Trafficking of Chitosan-Based Nanoparticles[END_REF]Sadat et al., 2016;[START_REF] Chen | Influences of surface coating of PLGA nanoparticles on immune activation of macrophages[END_REF]. We therefore conducted our study on PLGA NPs formulated with three different stabilizers: polyvinyl alcohol (PVA), chitosan (CS) or poloxamer 188 (commercially named Pluronic F68 [PF68]), resulting in neutral, positively or negatively charged particles, respectively. NP uptake by DCs was first examined. Viability, phenotypical changes and cytokine release of DCs were then assessed. To further understand underlying mechanisms involved in observed responses, intracellular signaling pathways were also investigated. We believe that this approach assessing the immune response to PLGA NPs may contribute to a better understanding of their biological fate and to a robust assessment for their use as pharmaceutical ingredients.

Methods

Preparation of nanoparticle suspensions

PLGA NPs were prepared by solvent evaporation-based methods (Mura et al., 2011a).

Positively and negatively charged as well as neutral NPs were tailored by coating their surface with chitosan (CS, Protasan® UP CL113, 75%-90% deacetylation, molecular weight 50-150 kD), poloxamer 188 (commercially named Pluronic F68 [PF68]), or polyvinyl alcohol (PVA, 87%-89% hydrolyzed, molecular weight 30-70 kDa), respectively.

Neutral NPs (PLGA/PVA NPs) were prepared by dissolving 100 mg of PLGA (75:25 Resomer RG756) in 5 mL of a dichloromethane/acetone (1/1 v/v) mixture. This organic solution was pre-emulsified with 20 mL of a 0.25 % (w/v) PVA (molecular weight 30-70 kDa) aqueous solution by vortexing for one minute. The pre-emulsion was kept on ice and sonicated for one minute using a VibraCell sonicator (Fisher Scientific, Illkirch, France) at 40% power. As for positively charged NPs (PLGA/CS NPs), the organic solution of PLGA was pre-emulsified with a 0.6 % (w/v) aqueous CS solution containing 0.5 % (w/v) of Mowiol 4-88 PVA by vortexing for one minute. This preemulsion was kept on ice and sonicated for 2 minutes using a VibraCell sonicator at 40 % power. Then, for both the neutral and positively charged NPs, the organic phase was allowed to evaporate at room temperature with magnetic stirring (600 rpm). NP dispersion was then completed to 20 mL. The excess of stabilizers was removed by centrifugation of NPs at 37,000 × g for one hour at 4 °C, and the pellet of NPs was resuspended in ultrapure water.

Negatively charged NPs (PLGA/PF68 NPs) were prepared using PF68 as stabilizer. PLGA was dissolved in 10 mL ethyl acetate and added dropwise into 20 mL of a 1 % (w/v) PF68 aqueous solution under vigorous magnetic stirring. The preemulsion was vortexed for one minute and then sonicated for another minute using a VibraCell sonicator at 40 % power. Water (20 mL) was added to the emulsion, in order to promote diffusion of the organic solvent into the external phase, leading to formation of the NPs. The organic phase was allowed to evaporate at room temperature with magnetic stirring (600 rpm).

Rhodamine B-labelled NPs were prepared as described earlier (Mura et al., 2011a) by dissolving in the organic solvent a 70/30 % (w/w) mixture of PLGA (Resomer RG756) and PLGA covalently bound to rhodamine B.

Nanoparticle suspension characterization

NPs were characterized in terms of size, zeta potential and surface chemical composition, confirming modifications provided by hydrophilic stabilizers. NP size (hydrodynamic diameter) and polydispersity were determined after dilution in MilliQ® water at 25°C by dynamic light scattering using a Nano ZS (Malvern Instrument, UK) set at a 173° scattering angle. The zeta potential was determined using the same instrument after dilution in 1 mM NaCl. All measurements were done in triplicate.

The amount of stabilizers associated to NPs after their purification was assessed through the formation of colored complexes of PVA and CS, or by 1H NMR in the case of PF68, as previously described (Mura et al., 2011a).

The morphology of NPs in aqueous suspension was studied by transmission electron microscopy (TEM) using a JEOL JEM-1400 microscope operating at 80 kV (filament current of 55 µA). 5 µL of NP aqueous suspensions was deposited onto copper grids covered with formvar film (400 mesh) for one minute. PLGA samples were then stained using 2% phosphotungstic acid (PLGA/PVA, PLGA/PF68) or 2% uranyl acetate (PLGA/CS) for 30 seconds. The excess liquid was blotted off using filter paper, and the grids were dried before observation. Images were acquired using a postcolumn high-resolution (11 megapixels) high-speed camera (SC1000 Orius; Gatan) and processed with Digital Micrograph (Gatan) and ImageJ.

Endotoxin level in NP suspensions was <0.05 EU/ml as tested by limulus amebocyte lysat (LAL) assay (GenScript, Piscataway, NJ, USA).

Generation of primary cultures of human and murine dendritic cells

The generation of human DCs derived from monocytes (MoDCs) was performed as described elsewhere [START_REF] Antonios | Mechanisms of IL-12 Synthesis by Human Dendritic Cells Treated with the Chemical Sensitizer NiSO4[END_REF] from human peripheral blood supplied by the French Blood Institute (EFS, Rungis, France). First, peripheral blood mononuclear cells (PBMCs) were sorted from phosphate buffer saline (PBS) diluted buffy coats by density centrifugation on a Ficoll gradient (650 × g, 20 min, 19 °C). Monocytes were then isolated through positive magnetic selection using MiniMacs separation columns and anti-CD14 + antibodies coated on magnetic beads (Miltenyi Biotec, Paris, France).

Finally, CD14 + cells were differentiated immature monocyte-derived DCs (MoDCs) for 5 days in RPMI 1640 supplemented with Glutamax-I, 10 % heat inactivated fetal calf serum (FCS), 550 U/mL granulocyte-macrophage colony-stimulating factor (rh-GM-CSF), 550 U/mL interleukin-4 (rh-IL4), 1 % sodium pyruvate, streptomycin and penicillin.

The generation of murine DCs (BM-DCs) was performed as described elsewhere [START_REF] El Ali | Dendritic cells' death induced by contact sensitizers is controlled by Nrf2 and depends on glutathione levels[END_REF]. In brief, DCs were derived from bone-marrow hematopoietic progenitors collected from the femurs and tibiae of 8-10-week-old C57BL/6 mice (Janvier SAS, Saint Berthevin, France). Cells were then differentiated to immature BMDCs for 7 days in Iscove's modified Dulbecco's medium (IMDM) containing 10 % FCS, 50 µM β-mercaptoethanol and 10 % culture supernatant from mouse GM-CSF-producing J558 cells (a kind gift of S. Amigorena, Institut Curie), coresponding to a final concentration of about 40 ng/mL GM-CSF. Mice were handled in accordance with the principles and procedures in accordance with the decree n°2013-1118 of 1 February 2013 on the protection of animals, used for scientific purposes Article R214.89.

In vitro exposure of immature dendritic cells to PLGA nanoparticles

After proper differentiation, immature DCs were collected, washed, and resuspended in fresh complete culture medium (RPMI or IMDM for human and murine cells respectively, supplemented with 10 % FCS, 1 % sodium pyruvate and antibiotics) at a final density of 10 6 cells/ml. Cells were then exposed to either PLGA NPs, their vehicle (distilled water, negative control), or any other chemical or biological molecule that would constitute an adequate positive control for a given analysis.

Nanoparticle internalization by DCs

For microscopy analysis, after exposure to NPs, cells were harvested, washed with cold PBS, fixed for 15 min in PBS supplemented with 2 % paraformaldehyde (PFA) and 1.5 % sucrose. Cells were then washed twice with PBS, incubated for 10 min in 50 mM ammonium chloride, and washed twice with PBS once again. Cells were then mounted onto microscope slides using an appropriate mounting medium (DakoCytomation mounting medium supplemented with 4′,6-diamidino-2-phenylindole (DAPI) for nucleus labeling) and examined under a confocal microscope equipped with epifluorescence.

For cytometry analysis, after exposure to rhodamine-labelled NPs, cells were harvested, washed with cold PBS, and analyzed on a FACSCalibur® cell analyzer using the CellQuest® software (BD Biosciences). Results were expressed as percentage of positive cells.

Cytotoxicity evaluation

Cell viability was first assessed by the MTT (methylthiazolyldiphenyl-tetrazolium bromide) test. Immature cells were seeded at 10 5 cells/100 µL/well into 96-well plates and exposed to 1-400 µg/mL PLGA NPs for 24 h (5 replicate wells per treatment group). At the end of the exposure, 10 µL of a 5 mg/ml MTT solution was added to each well. After 3 h at 37 °C, medium was replaced by 100 µL of dimethyl sulfoxide (DMSO) to dissolve the resulting formazan crystals and optical density was measured at 550 nm with the reference filter set to 620 nm. Cell viability values were finally determined as percent differences relative to untreated (negative) control values.

The trypan blue dye exclusion method was additionally used to assess cytotoxicity. Viable and dead cells were counted with a hemocytometer, distincted thanks to the use of trypan blue, a vital stain that only enters dead cells.

Obtained cytotoxicity curves were used to identify NP exposure conditions that do not induce more than 30 % cell death so as to investigate NP effects on DC maturation.

Phenotypic analysis

To assess the phenotypic effects of NP exposure on DCs, several surface maturation markers were studied. After 24 h of exposure to either 200 µg/mL PLGA NPs, distilled water (negative control) or 25 ng/mL lipopolysaccharide (LPS, positive control), DCs were collected, washed with PBS and incubated for 15 min in Fc Block (BD Biosciences, Le Pont de Claix, France) to avoid antibody preparations binding via their Fc portions to low-affinity receptors for complexed IgG. Cells were then incubated for 20 min either with fluorochrome-conjugated monoclonal antibodies (mAbs) or with appropriate isotype control antibodies (IgGs). For MoDCs, the following mouse antihuman mAbs were used: FITC-conjugated anti-CD40 (clone 5C3), APC-conjuguated anti-CD54 (ICAM-1,clone HA58), FITC-conjuguated anti-CD80 (clone L307.4), APCconjugated anti-CD83 (clone HB15e), FITC-conjugated anti-CD86 (clone 2331(FUN-1)), FITC-conjugated anti-CD274 (PD-L1, clone MIH1), PE-conjugated anti-CD197 (CCR7) and FITC-conjugated anti-HLA-DR (clone G46-6) (BD Biosciences). For BMDCs, the following rat anti-mouse mAbs were used: FITC-conjugated anti-CD40 (clone 3/23), FITC-conjugated anti-CD86 (clone GL1), PE-conjugated anti-CD197 (CCR7, clone 4B12) (Beckman Coulter -Immunotech, Marseille, France). After mAbs or IgGs incubation, DCs were washed twice with PBS supplemented with 0.5% BSA, then once with PBS, and finally fixed in PBS supplemented with 1 % PFA.

Cells unstimuled with NP were used to determine nonspecific staining. A minimum of 20,000 cells were analyzed on a FACSCalibur cell analyzer with CellQuest software (BD Biosciences). Living cells were identified on the basis of their physical characteristics [forward scatter and side scatter. Results were expressed as percentage of positive cells (i.e., cells that express the marker) as well as using the relative fluorescence intensity (RFI, i.e. mean fluorescence intensity of cells incubated with a given mAb divided by mean fluorescence intensity of cells incubated with its corresponding isotype) and the "fold increase RFI" (i.e. NP-or LPS-exposed cells RFI divided by control cells RFI). This latter parameter (fold increase RFI) therefore reflects the mean expression level of a given maturation marker within the total cell population.

Cytokine analysis

The BD Biosciences cytometric bead array (CBA) technique was performed to measure cytokine concentrations within DC supernatants at the end of NP exposure. This technique employs microbeads coated with a capture antibody specific for a given soluble protein. For human MoDC supernatants, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10, interferon-γ-induced protein (IP)-10 (also known as CXCL10), monocyte chemotactic protein (MCP)-1 (also known as CCL2) and RANTES (regulated upon activation, normal T-cell expressed and secreted, also known as CCL5) were tested. For murine BMDC supernatants, TNF-α, IL-6, IL-10, MCP-1 and CCL5 were tested.

Briefly, cell culture supernatants were either diluted or not and serial dilutions of each cytokine standard (from 10 to 2500 pg/mL) were prepared. Test samples and standard dilutions (50 µL/test) were then incubated with capture beads for 1 h at room temperature, protected from light. Then, PE detection reagents were added and an additional incubation step was performed (2 h, room temperature, protected from light).

After proper washes, a three-color cytometric analysis was carried out using a FACScalibur flow cytometer (BD Biosciences). Results were generated using the FCAP Array™ Software.

Intracellular signalling pathways

Western blot analyses of both mitogen activated protein kinases (MAPK) and NFκ-B (nuclear factor kappa-light-chain-enhancer of activated B cells) were performed as previously described (Larangé et al., 2012). Immature DCs (10 6 cells/mL) were either exposed to distilled water, PLGA NPs (200 µg/mL), or LPS (25 ng/mL) for 15, 30, 60 or 120 min. At the end of their stimulation, cells were harvested, washed with PBS and pelleted by centrifugation. Cell lysates were then prepared by resuspending cell pellets in lysis buffer (20 mM Tris pH 7.4, 137 mM NaCl, 2 mM EDTA pH 7.4, 2 mM sodium pyrophosphate, 1 % Triton X-100, 10 % glycerol, 1 mM PMSF, 1 mM sodium orthovanadate, 25 mM β-glycerophophate, 10 µg/mL aprotinin, 10 µg/mL leupeptin and 100 µg/mL pepstatin). Total protein extracts were then collected after a 20 min 15,000 rpm centrifugation. Fifty µg of denaturated protein were then loaded onto 12.5 % SDS-PAGE gel and transferred on polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences, Les Ulis, France). Membranes were then incubated with antibodies directed to the phosphorylated forms of p38MAPK (Thr180/Tyr182), JNK 1/2 (Thr183/Tyr185), ERK 1/2(Thr202/Tyr204), NFκ-B p65(Ser536) (all from Cell Signaling Technology, Ozyme, St-Quentin-en-Yvelines, France) or to IκB-α (C-21, Santa Cruz biotechnology, Santa Cruz, CA). Total p38MAPK was used as a loading control with an antibody raised against total p38MAPK (Cell Signaling Technology).

After proper washes and incubation with secondary HRP-linked antibody, immunoreactive bands were detected bychemiluminescence (ECL solution, Amersham Biosciences) using a ChemiDoc XRS Imager (Bio-Rad, Marnes-La-Coquette, France).

Bands were quantified by densitometry using theImageLab software.

T-lymphocyte proliferation

Statistical analysis

Significance of obtained results was calculated using nonparametric one-way analyses of variance on ranks approach (Kruskall-Wallis) combined with Mann-Whitney U tests for multiple comparisons (Statistica 7.0, Statsoft, Chicago, IL, USA). Data are expressed as means ± S.E.M. Effects were designated significant at p <0.05 (*) or at p <0.001 (**).

Results

PLGA nanoparticle properties

As shown in Table 1, the selection of different stabilizers allowed the modification of NP surface charge. PLGA/PF68 NPs had a mean diameter of 100 nm, while both PLGA/PVA and PLGA/CS NPs were around 200 nm. All formulations had a narrow size distribution, with a polydispersity index of 0.1-0.2. Transmission electron microscopic observations showed spherical NPs with a smooth surface. Zeta potential measurements confirmed that the stabilizers influence NP surface charge. CS-coated NPs exhibited a positive zeta potential (+32 ± 3 mV), whereas PVA-coated NPs were almost neutral (-5 ± 1 mV), and PF68-coated NPs exhibited a negative zeta potential (-24 ± 1 mV). The presence of stabilizers on the NP surface was quantified by spectrophotometric methods and 1 H NMR spectroscopy. Between 10 mg and 30 mg of stabilizer (per 100 mg of PLGA) was found to be associated with the NPs (Table 1).

The use of Rhod-PLGA to render fluorescent NPs did not influence their size distribution nor their surface charge. The only exception was Rhod-PLGA/CS NPs, the size and zeta potential of which slightly decreased (Table 1) due to the presence of rhodamine moieties on the NP surface. An important point is to monitor the possible aggregation of NPs in serum-containing media [START_REF] Hinderliter | ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies[END_REF], especially since the PLGA NPs used in this study are expected to be transported by sedimentation [START_REF] Deloid | Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials[END_REF]. The dispersion of the three NP types in fetal bovine serumcontaining cell culture medium resulted in only minor modifications of the mean hydrodynamic diameters for up to 72 h incubation, and the same trends in zeta potential values (Table S1), despite the complexity of the cell culture medium.

Cytotoxic effects of PLGA nanoparticles on dendritic cells

Our toxicological evaluation of PLGA NPs first consisted in the evaluation of the cytotoxic impact of NP exposure on DCs. Results obtained by both the MTT test (Figure 1) and the trypan blue dye exclusion assay (data not shown) showed that human and murine DCs underwent about 5 to 10 % cell death after 24 h of exposure to concentrations as high as 0.5 mg/ml NPs. Given such relatively high NP concentrations needed to induce only moderate cell death within the DC populations, these first results clearly indicate that PLGA NPs exert very low cytotoxic effects on DCs.

PLGA nanoparticle uptake and internalization into dendritic cells

NP uptake by DCs was assessed from a quantitative point of view using flow cytometry by monitoring rhodamine-labelled NP fluorescence associated to DCs. In human DC (MoDC), results show that PVA-coated PLGA NPs were the most efficiently taken up by human DCs (Figure 2A). With these particles, fluorescence measured after 24 h of exposure was about 70 times higher than cellular autofluorescence, while CS-and PF68-coated PLGA NPs entry resulted in a 40-fold increase of cellular fluorescence.

Interestingly, fluorescence measurements carried out at early time points indicated that for both PVA-and CS-coated NPs, the major part of the 24 h NP entry within cells occurred within the very first hours of exposure (about 75 % after 3 h and 90 % after 6 h). Conversely, PF68-coated NP uptake by cells went on according to a slower mechanism since only 40 % of the 24-h NP entry within cells occurred within the first 6 hours of exposure. In murine DC (BMDC), Rresults show the major part of the 24h NP entry within cells occurred after 3 hours of exposure. PVA-coated PLGA NPs were also the most efficiently taken up by BMDC (Figure 2B). With these particles, fluorescence measured after 24 h of exposure was about 50 times higher than cellular autofluorescence compared to 40 % with CS-coated NPs. Regarding PF68-coated NP, uptake by BMDC is very low.

To better understand the PLGA NP interaction with DCs, we then used rhodaminelabelled NPs to assess NP intracellular localization within DCs. Confocal microscopic observations showed that, after 24 h of exposure, very large quantities of PLGA NPs were encountered within the cytoplasm of both human DCs and murine (Figures 2A &B).

The herein demonstrated capacity of DCs to internalize large quantities of PLGA NPs is in good accordance with their constitutive ability to constantly sample their surrounding environment since they act as sentinels of the immune system.

Interestingly, surfactants exert a clear effect on NP internalization, which might be due to differences of the electric charge present at the surface of particles. According to our present results, PVA-coated NPs, which are uncharged particles, are the most rapidly and efficiently internalized. Regarding CS-and PF68-coated NPs, which are charged particles, similar quantities of NPs entered the cells after a 24-h exposure period.

However, negatively charged particles (i.e. PF68-coated ones) seemed to be internalized according to a slower kinetic pattern than both uncharged and positively charged particles.

Surface protein modifications in dendritic cells after PLGA nanoparticle exposure

To assess the phenotypic effects of NP exposure on DCs, we first studied the modification of membrane markers of maturation. Obtained results clearly indicate that a 24-h exposure to 200 µg/mL PLGA NPs induced moderate modifications of herein tested maturation markers. Regarding human DCs (Figure 3A), we noticed subtle (fold increases generally <2) but significant enhancement of the surface expression of the CD83 maturation marker, the CD54 adhesion molecule (also known as Inter-Cellular Adhesion Molecule 1, ICAM-1), as well as of costimulatory molecules such as the CD40 receptor that interact with the CD40L of T cells and several members of the B7 family ligands (CD80/B7-1, CD86/B7-2 and PD-L1/CD274/B7-H1) that bind to the CD28 family receptors of T cells. Similar results were obtained in murine DCs at the surface of which expressions of CD40, CD86 and CCR7 (a chemokine receptor) molecules were significantly increased (Figure 3B).

Both human and murine DCs can therefore be considered as in an activated state, able to be engaged in immunological synapses with T cells. However, it is noticeable that PVA-coated NPs generally exerted the most marked slight effects effects on murine DC phenotype compared to human DC. , while CS-and PF68-coated NPs induced only slight effects.

The effect of a soluble surfactant exposed to cells at an equivalent concentration to that associated to NPs was evaluated in the case of PVA vs. PVA-coated NPs (Figure S1A). For all human DC markers, no significant increased was noticed for free PVA.

Cytokine secretion profile of dendritic cells exposed to PLGA nanoparticles

As a second approach of the phenotypic impact of PLGA NP exposure on DCs, we evaluated the secretory activity of DCs (Figure 4). Eight cytokines and chemokines were tested in the culture medium supernatants of human MoDCs collected after 24 h of exposure to NPs. Results showed that the pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α were induced by human DCs. A similar induction was observed for the CXCL10 and CCL5 chemokines (Figure 4A). Conversely, in murine BMDCs, no effect on secretory activity of cells was observed as a result of NP exposure. Interleukine-6 and TNF-α cytokines even tended to be encountered in cell media at lower concentrations when cells were exposed to PLGA NPs. However, such tendency was not statistically significant (Figure 4B).

Our results therefore indicate that human MoDCs but not murine BMDCs enhanced their cytokine secretory activity as a result of NP exposure. In human CDs, the three categories of NPs induce the same cytokines (IL-6, IL-8 and CXCL10) in identical proportions. In murine CDs, the base level of cytokine production is higher, therefore no effect is reported on cytokine production by the NPs.

As for membrane maturation markers, surfactants had an impact on cytokine secretions since PF68-coated NPs generally exerted the slightest effects. As for maturation markers, the effects of PVA alone were tested (Figure S1B). With the exception of IL-8 secretion, no significant differences were observed compared to control conditions, suggesting that surfactant effect was only due to its direct association to NPs. In the case of IL-8, a slight but significant increase in its secretion was noticed as compared to controls. However, effects of PVA alone were lower than PVA-coated NP effects.

Investigation of signalling pathways activated as a result of PLGA nanoparticle exposure

Considering the results obtained regarding both surface maturation marker expressions and cytokine secretions, we then tried to elucidate the underlying signalling pathways involved in DC maturation. We first carried out western blot experiments to follow the time evolution of the intracellular contents of the phosphorylated forms of mitogen activated protein kinases (p38MAPK, JNK 1/2, ERK 1/2) and of the p65 subunit of NFκ-B, as well as the time evolution of IκB-α content, its cytoplasmic inhibitor (Figure 5). We showed that exposure to PVA-and PF68-coated PLGA NPs induce the significant activation of both JNK and ERK 1/2 pathways as indicated by the increase of the phosphorylated forms of these proteins in human DCs. Regarding CS-coated PLGA NP exposure, JNK pathway was not activated but both p38 and ERK 1/2 kinases were phosphorylated. NFκ-B pathway study did not show any significant activation.

T lymphocyte proliferation is not modulated in response to PLGA nanoparticle

MoDCs pre-loaded purified protein derivative (PPD) were treated with NPs, then washed and cultured with purified autologous CD4+ T lymphocytes CD4+ T-cells cells at 3 different ratio a 1:8, 1:4, 1:2 DC/T-cell ratio for 5 days. On day 5, CD4 cells CD4+ T lymphocytes cells proliferation was analyzed by flow cytometry and expressed as the percentage of CFSE low T cells. On day 5, <20% of total cells were CFSElow at ratio 1/4 in absence of NP. MoDC loaded with PPD and treated with LPS allow us to appreciate the maximal of proliferation of the cells, 50 % at ratio 1/4. MoDCs preloaded or not with PPD have a quit similar proliferation in regard to the ratio. NP did not affect the proliferation of lymphocytes in response to MoDC preloaded with PPD whatever the ratio tested (Figure 6). These results suggest that NP-stimulated MoDCs do not augment the proliferation of CD4 + T-cells lymphocytes. Collectively, these findings reveal that PLGA NPs do not induce the capacity of mature moDC to activate autologous CD4+ T cells proliferation.

Discussion

The use of NPs in medicine requires the understanding of their possible adjuvant effect in the immune response. In the particular case of PLGA materials, some questions regarding immune consequences of their use as NP carriers still remain unclear. It is currently accepted that charging professional APC with antigens loaded onto PLGA vehicles leads to a presentation of MHC class I-antigen complexes over an extended period [START_REF] Audran | Encapsulation of peptides in biodegradable microspheres prolongs their MHC class-I presentation by dendritic cells and macrophages in vitro[END_REF]. More generally, PLGA NP carriers are considered as efficient carriers of antigens in comparison with the use of soluble forms of antigens [START_REF] Diwan | Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses[END_REF]. However, to this day, professional APC activation has not been fully investigated in response to PLGA NP. The current study was therefore conducted to examine PLGA NP effects on DCs. A particular emphasis was placed on the impact of polyelectrolyte coatings on the particle uptake by cells, as well on the phenotype and secretory profile of DCs. To this purpose, PLGA NPs were coated either with CS, PVA, or PF68 stabilizers, resulting in significant differences in their zeta potential (the resulting particles being either cationic, neutral or anionic, respectively).

Regarding NP uptake, our results showed that, irrespective of their surface charge, all herein tested NP formulations were efficiently taken up and internalized by DCs, as evidenced by cytometry analysis and confocal microscopy observations, respectively. Uptake of PLGA nano-and micro-particles has previously been demonstrated in vitro by human peripheral blood-monocytes-derived DCs [START_REF] Walter | Hydrophilic poly(-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells[END_REF]Lustiak et al., 2002;[START_REF] Waeckerle-Men | Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy[END_REF]Yoshida & Babensee, 2006b;[START_REF] Fischer | The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles[END_REF][START_REF] Ma | Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy[END_REF], human cord-blood CD34+ stem cells-derived DCs [START_REF] Diwan | Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses[END_REF], mouse bone marrow-derived DCs [START_REF] Elamanchili | Characterization of poly(,-lacticco-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells[END_REF][START_REF] Ma | Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy[END_REF], and in vivo upon intradermal immunization [START_REF] Newman | Uptake of poly(D,Llactic-co-glycolic acid) microspheres by antigen-presenting cells in vivo[END_REF]. Some of these authors additionally reported that PLGA NPs were not detectable inside the DCs when cells were pretreated with cytochalasin, an inhibitor of actin polymerization, or when incubation with the fluorospheres was carried out at 4°C, therefore demonstrating that NP internalization is achieved by a phagocytosis phenomenon (Lustiak et al., 2002;Yoshida & Babensee, 2006b). This conclusion was supported by electron microscopy studies, where membrane ruffling around the phagocytosed particle was visualized [START_REF] Diwan | Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses[END_REF]. According to [START_REF] Hamdy | Targeting dendritic cells with nano-particulate PLGA cancer vaccine formulations[END_REF], the uptake of PLGA particles is affected by surface charge. Cationic particles would indeed be particularly effective for uptake by DCs. The ionic attraction between the positively charged particles and the negatively charged cell surface initiates efficient binding and facilitate particle internalization, even though the ossible formation of a protein corona around the NPs in serum-containing media should be taken into account [START_REF] Tenzer | Nanoparticle Size Is a Critical Physicochemical Determinant of the Human Blood Plasma Corona: A Comprehensive Quantitative Proteomic Analysis[END_REF]. In our study, cationic (CS-coated) NPs remained positively-charged in cell culture media, and were indeed rapidly engulfed by DCs since about 80 % of the particles that were encountered into the cells after 24 h of exposure actually entered the cells within the first 3 h of incubation. Anionic (PF68-coated) NPs were taken up by DCs according to a slower kinetics. Despite this difference, after 24 h of incubation, both anionic and cationic particles were internalized to a similar extent on a quantitative basis. Neutral (PVA-coated) NPs appeared as the most efficiently internalized particles since DC fluorescence was about twice as high compared to CS-or PF68-coated NP exposed cells after 24 h. Such a difference shows that electrostatic interactions cannot account alone for internalization by cells, and could be related to the fact that neutral particles are less susceptive to attract surrounding molecules such as proteins that are present in the culture medium.

As for their cytotoxic potential, PLGA NPs do not significantly affect the survival of DCs whatever their charge, even when the cells were loaded with very large amounts of NPs, which is in total agreement with others studies [START_REF] Walter | Hydrophilic poly(-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells[END_REF][START_REF] Waeckerle-Men | Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy[END_REF][START_REF] Fischer | The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles[END_REF][START_REF] Semete | In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems[END_REF]. Previous studies on PLGA NPs displaying positive, negative or neutral surface charge revealed that positively-charged NPs appeared to be more cytotoxic and genotoxic to different cell lines with ROS induction linked to high endocytosis and chromosomal aberrations, contrary to other NP types that appear to be relatively inert [START_REF] Platel | Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and endocytosis[END_REF].

Noticeable phenotypic changes induced in both human and murine DCs in response to PLGA NP exposure as evidenced by significant upregulations (CD86, CD80, CD40, CD83), are consistent with a change allowing the transition from an antigen-capturing to an antigen-presenting state of DCs [START_REF] Banchereau | Dendritic cells and the control of immunity[END_REF]. The observed effects are attributed to PLGA NP free of endotoxin since the endotoxin levels were below the USP prescribed content in water for injection (0.25 endotoxin units/mL, as tested by the LAL assay). Even if all tested PLGA NPs had an impact on DC maturation, PVA NPs were the most potent since they exerted a higher induction of all surface markers (i.e. CD40, CD54, CD80, CD83, CD86 and PDL-1 in MoDCs; CD40,CD86 and CCR7 in BMDCs). This maturation can be correlated with the higher and rapid phagocytosis of PVA-coated NPs. However, Human DCs are more sensitive than murine DCs in response to nanoparticles since many surface markers are induced, as are cytokines.

Although some literature describes the absence of DC maturation upon PLGA particle exposure, both in MoDCs [START_REF] Walter | Hydrophilic poly(-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells[END_REF][START_REF] Waeckerle-Men | Phenotype and functional analysis of human monocyte-derived dendritic cells loaded with biodegradable poly(lactide-co-glycolide) microspheres for immunotherapy[END_REF][START_REF] Fischer | The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles[END_REF][START_REF] Bivas-Benita | Pulmonary delivery of DNA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA-PEI nanoparticles enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice[END_REF] and BMDCs [START_REF] Sun | Analysis of the role of vaccine adjuvants in modulating dendritic cell activation and antigen presentation in vitro[END_REF][START_REF] Clawson | Delivery of a peptide via poly(d,l-lactic-co-glycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity[END_REF][START_REF] Ma | Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy[END_REF], our study confirms a large number of studies demonstrating phenotypical changes induced in both human and murine DCs exposed to PLGA films and particles [START_REF] Yoshida | Poly(lactic-co-glycolic acid) enhances maturation of human monocyte-derived dendritic cells[END_REF], 2006a;[START_REF] Babensee | Differential levels of dendritic cell maturation on different biomaterials used in combination products[END_REF][START_REF] Diwan | Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses[END_REF][START_REF] Sun | Analysis of the role of vaccine adjuvants in modulating dendritic cell activation and antigen presentation in vitro[END_REF]Elmanchili et al., 2004;[START_REF] Yoshida | Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells[END_REF].

Moreover, we demonstrate a relationship between PLGA particle charge and the acquired DC phenotype, which remained an open question in several studies [START_REF] Fischer | The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles[END_REF][START_REF] Jilek | Composition and surface charge of DNA-loaded microparticles determine maturation and cytokine secretion in human dendritic cells[END_REF].

Beside the expression of maturation markers, the secretion of cytokines is indeed crucial for T-cell activation and efficient antigen presentation. We therefore investigated the influence NPs on cytokine secretion of DCs. Our results clearly demonstrated that, upon exposure to PLGA NPs, MoDCs secrete significant amounts of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) and chemokines (CXCL10, CCL5), consistent with the maturation of human DCs upon exposure to PLGA NPs, and with other studies demonstrating the impact of PLGA particles on cytokine secretion by DCs. The induction of IL-6, IL-8 and TNF-α by PLGA-exposed MoDCs may control a TH1 response as suggested elsewhere (Yoshida & Babensee, 2006a,b). This result is in agreement with another study in which the delivery of a model antigen with PLGA particles resulted in a TH1 response as measured by production of IgG2 isotype antibody against the antigen [START_REF] Newman | Ovalbumin peptide encapsulated in poly(d,l lactic-co-glycolic acid) microspheres is capable of inducing a T helper type 1 immune response[END_REF]. The effect of the surfactant themselves have been raised in previous reports on PLGA NPs [START_REF] Guedj | Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide nanoparticles for protein delivery into macrophages[END_REF][START_REF] Menon | Effects of surfactants on the properties of PLGA nanoparticles[END_REF]. Our results show that the effects of PVA alone, exposed at an equivalent concentration to that associated to NPs, had no own effect on DC maturation. This can be related to previous observations that PVA remains strongly associated to PLGA NPs even after thorough purification [START_REF] Spek | Characterisation of PEGylated PLGA nanoparticles comparing the nanoparticle bulk to the particle surface using UV/Vis spectroscopy, SEC, 1H-NMR spectroscopy, and X-ray photoelectron spectroscopy[END_REF]. Regarding the contribution of the PLGA core on the cell response, previous reports by our group [START_REF] Grabowski | Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages[END_REF] showed that dendritic cell surrogates do not show any difference in terms of mitochondrial activity and necrosis induction following exposure of PVA-coated PLGA nanoparticles and surfactant-free PLGA nanoparticles, which were obtained by a specific preparation technique, at concentration ranges used in the present study (<1 mg/mL).

Phenotypic changes and concomitant cytokine secretion are maturation hallmarks that result from intracellular signaling cascades. Different signaling pathways such as MAPKs and NF-κB play important roles in DC maturation [START_REF] Neves | Signal transduction profile of chemical sensitisers in dendritic cells: An endpoint to be included in a cell-based in vitro alternative approach to hazard identification?[END_REF].

Regarding the maturation process, it has been proved that different chemicals could stimulated different signal transduction pathways and subsequently induced different phenotypic and functional changes in DC, such as MAPKs and NF-κB signalling pathways [START_REF] Antonios | Mechanisms of IL-12 Synthesis by Human Dendritic Cells Treated with the Chemical Sensitizer NiSO4[END_REF]. In our hands, MAPK pathways were activated in response to all PLGA NPs, but at different levels depending on the type of PLGA NPs.

Our results show a nice correlation between MAPKs and human DC maturation induced by PLGA NPs.

In addition, we show that PLGA NP do not affect the activation of T lymphocytes in response to an antigen such as PPD. This confirms these NP do not modify the adaptive immune response of DC even if the phenotype is slightly activated.

Conclusions

Our study indicates that PLGA-NPs induce clear consequences on the pivotal properties of DCs in vitro. Exposure of immature DCs to three PLGA NP types with different surface coatings and surface charge resulted in marked particle uptake by the cells, but no significant cell death. Nevertheless, these NPs were not as inert as expected since alterations in DC phenotype were reported, as assessed by the expression levels of cell surface markers of DC maturation. In addition, the functional secretory activity of DCs was largely altered upon phagocytosis of the different NPs. The study presented herein lays a foundation for studying the effects of PLGA nanomaterials on the immune system prior to their use as vehicles in nanomedicine. This study allowed us to indicate that interspecific differences are important to take into account since human activation of CDs is higher than that of murine CDs. Human DCs are and This is the first study that demonstrates MAPK activation by PLGA exposure in DCs, and the potential role of surfactants in the effectively taken pathways. Further studies are needed to study the role of encapsulated bioactive compounds on the effects of such PLGA nanocarriers, which are relevant for drug and vaccine delivery purposes.

  The capacity of MoDCs to activate T cells has been evaluated by the use of T CD4+ T-cells lymphocytes labelled with 0.5 μM CFSE following the manufacturer's instructions. T cells are isolated from PBMC by positive selection with the MACS CD4 isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). These T cells are confirmed to have purity greater than 95%, based on CD4 (555349, BD Biosciences) expression evaluated by flow cytometry. MoDCs are pre-loaded with purified protein derivative (PPD) or not and then stimulated with NPs. Treated MoDCs were washed and then cultured with autologous T cells at 3 different ratio a 1:8, 1:4, 1:2 DC/T-cell ratio for 5 days in RPMI 1640 Glutamax supplemented with 10% AB human serum, in round bottomed 96-well plates. On day 5, CD4+T-cells proliferation is analyzed by flow cytometry and expressed as the percentage of CFSE low T cells present in the sample.

  However, PLGA NP modules CD40 and CD86 in both DC models (BMDC or MoDC) equally. The differences mainly observed in cytokine production by MoDCs might be explained by the origin of DC. Human MoDCs are inflammatory monocytederived DC close to conventional DC while murine DCs are differentiated from CD34.

Figures captions Figure 1 .

 captions1 Figures captionsFigure1. Effects of PLGA NPs on cell viability. Human MoDCs (solid lines) and murine BMDCs (dashed lines) were exposed to PLGA NPs (0-500 µg/mL) for 24 h.Cell death was assessed by the MTT test.

Figure 2 .

 2 Figure 2. PLGA nanoparticle uptake and internalization. A. Confocal microscopy observations. Up. Human DCs were exposed for 24 h to rhodamine coated-PLGA NPs (200 µg/mL). From left to right: PLGA/PVA, PLGA/CS, PLGA/PF68. Lysosomes were labeled using a green LysoTracker® probe (InVitroGen). Flow cytometry fluorescence analysis Down. Human DCs were exposed for 3, 6 or 24 h to rhodamine-coated PLGA NPs (0-400 µg/mL). From left to right: PLGA/PVA, PLGA/CS, PLGA/PF68. Red fluorescence was then measured by flow cytometry. Fluorescence intensity of NP exposed cells was then divided by fluorescence intensity of control cells (i.e. autofluorescence). B. Confocal microscopy observations. Up. Murine DCs were exposed for 24 h to rhodamine coated-PLGA NPs (200 µg/mL). From left to right: PLGA/PVA, PLGA/CS, PLGA/PF68. Lysosomes were labeled using a green LysoTracker® probe (InVitroGen). Flow cytometry fluorescence analysis Down. Murine DCs were exposed for 3, 6 or 24 h to rhodamine-coated PLGA NPs (0-400 µg/mL). From left to right: PLGA/PVA, PLGA/CS, PLGA/PF68. Red fluorescence was then measured by flow cytometry. Fluorescence intensity of NP exposed cells was then divided by fluorescence intensity of control cells (i.e. autofluorescence).

Figure 3 .

 3 Figure 3. Phenotypic analysis of dendritic cells treated by PLGA NPs. Human MoDCs (A) and murine BMDCs (B) were exposed for 24 h to distilled water (unexposed cells, white), PLGA NPs (200 µg/mL, grey) , or lipopolysaccharide (LPS, 25 ng/mL, positive control, black). Phenotypic markers of maturation were then studied using flow cytometry analysis of fluorescent immunolabelling (statistically significant differences from unexposed cells: grey star, p<0.1; black star, *, p<0.05; **; p<0.01, n=5).

Figure 4 .

 4 Figure 4. Cytokine production by DCs. MoDCs (A) and murine BMDCs (B) were exposed for 24 h to distilled water (unexposed cells, white), PLGA NPs (200 µg/mL, grey), or lipopolysaccharide (LPS, 25 ng/mL, positive control, black). Cytokine concentrations were then measured in exposure medium using the cytometric bead array

Figure 5 .

 5 Figure 5. PLGA NPs modulate MAPK phosphorylation in MoDCs. Human MoDCs were exposed for 15 min, 30 min, 1 h or 2 h to distilled water (unexposed cells, white ), PLGA NPs (grey ,200 µg/mL), or lipopolyssacharide (LPS, 25 ng/mL, positive control, black). Cells were lysed and the level of phosphorylated p38MAPK, JNK and ERK was evaluated by western-blotting. Membrane was then probed with anti-p38MAPK Ab for loading control. A. Results of a representative experiment B. Quantitative time evolution of phosphorylated MAPKs. Folds represent the ratio of normalized intensity of specific band divided by the normalized intensity of unexposed cells (statistically significant differences from unexposed cells: grey star, p<0.1; black star, p<0.05; *, p<0.05; **; p< 0.01 n=5).

Figure 6 .

 6 Figure 6. T lymphocyte proliferation is not modulated in response to PLGA NPs.

TableTable 1 . PLGA nanoparticle characterization.

 1

	Data are presented as mean ± SD. Abbreviations: CS, chitosan; PF68, poloxamer 188;
	PLGA, poly(lactide-co-glycolide); PVA, poly(vinyl alcohol); rhod, rhodamine B; TEM,
	transmission electron microscopy.

Acknowledgements

This study was supported by the the ANSM "Emerging risks" program and the Agence Nationale de la Recherche (under reference Nanobiotox).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


Wei Wang

Bernard Bonello
email: bernard.bonello@insp.jussieu.fr

Bahram Djafari-Rouhani

Yan Pennec

Topological valley, pseudospin, and pseudospin-valley protected edge states in symmetric pillared phononic crystals

published or not. The documents may come   L'archive ouverte pluridisciplinaire

effect (QSHE) or quantum valley Hall effect (QVHE) is emulated by solely imposing different perturbations to the geometry. When occurring at the deep subwavelength scale, the valley-polarized edge state emulating QVHE becomes evanescent at the zigzag termination and can be enhanced by a locally resonant mode. When occurring at high frequency, the refracted patterns (refracted modes and angles) can be tailored. Furthermore, we demonstrate the occurrence of the pseudospin and pseudospin-valley combined edge states and the valley-dependent propagation at a T-junction constructed by PPnCs supporting QSHE and QVHE respectively.

The willingness to manipulate the propagation of elastic waves, especially Lamb waves, in phononic crystals (PnCs) by tailoring the band structures through either band folding or local resonances has been one of the most fruitful scientific pursuits in the past decades. Abnormal wave propagation phenomena, such as negative refraction, lensing, and clocking, unachievable in natural materials, have been demonstrated [1][2][3][4]. Recently, the emergence of topological insulators has provided a fascinating scheme to achieve defect-immune and lossless energy transport [5][6][7][8]. By analogy with quantum spin Hall effect (QSHE) [9][10][11][12][13][14] or quantum valley Hall effect (QVHE) [15][16][17], this concept was soon extended to PnCs to realize robust guiding of Lamb waves at the interface between two topologically distinct configurations.

In the former case, it explores the pseudospin degree of freedom (DOF) and requires intricate designs to obtain a double Dirac cone at either Γ or K (K') points of the Brillouin zone (BZ). Usually, a double Dirac cone is easily achieved at the Γ point owing to a band folding mechanism [18][19][20], whereas, its occurrence at K (K') points at high frequency scale can be obtained in specifically patterned plates [21]. It has been afterwards simplified with perforated macro holes [22] which significantly reduces the geometric complexity and further allows for the experimental observation of the pseudospin-protected edge states in continuous elastic plates. Regarding QVHE, it takes advantage of the valley DOF and only requires a single Dirac cone at the K or K' points of the BZ. This significantly simplifies both the design and the fabrication even though a large separation between two opposite valleys must be ensured to avoid the inter-valley scattering.

Very recently, the topologically protected edge state, that combines both pseudospin and valley DOFs, has gained more and more interest. Specifically, it has been reported that pseudospin-valley combined edge states can occur at the domain wall formed by two distinct configurations supporting QSHE and QVHE respectively [23][24][25][26][27][28][29]. Usually, these two configurations involve totally different geometries which renders their designs very complex. Therefore, designing a programmable platform that allows for swapping between QSHE and QVHE is of primary importance for this research field. Likewise, little attention has been paid to the refracted pattern of the valley-polarized edge state at the valley-conservation zigzag termination so far. Recently, an elastic phononic plate with perforated holes mimicking QVHE by either expanding or shrinking the holes and QSHE by using blind holes was reported [23]. These geometries allowed the authors to demonstrate the splitting of the equal-frequency helical edge waves in the highfrequency regime. In this work, we go a step further. By setting up pillar-shaped resonators on the perforated phononic crystal plate, the dispersion curves are significantly altered: Dirac cones in the deep subwavelength scale or at high frequency regime, and even a double Dirac cone when the dimensions are properly chosen, are achieved. We describe a symmetric double-sided PPnC where QVHE or QSHE can be emulated by merely imposing dissimilar perturbations to the geometries of the attached pillars. We demonstrate the occurrence of the valley protected, the pseudospin protected, and the pseudospin-valley combined edge states at different domain walls. When emulating QVHE, we show a variety of refracted patterns at the zigzag termination for the valley-polarized edge state including positive or negative refraction, and even evanescence. Then, we investigate the valley-dependent feature of the pseudospinvalley combined edge state and we demonstrate the valley based splitting of the pseudospin protected edge states in a T-shaped waveguide constructed by merging PPnCs supporting QSHE and QVHE respectively. Design of the system. The unit cell of the honeycomb lattice formed by assembling two identical periodic arrays of pillars over a thin plate, together with its first irreducible BZ, are shown in Fig. 1 The most suitable way to investigate the edge states is to consider the three-layer ribbon supercell shown in Fig. 2(a). It is built by sandwiching eight unit cells with perturbation Δd V = 10 µm, in between two sets of six unit cells, each of them with perturbation Δd V = +10 µm. This supercell features two different zigzag domain walls of type SDW and LDW with adjacent pillars having respectively small and large diameters. (Table 1 below summarizes the geometries we investigate in this work). The projection of the valleys K and K' on these domain walls is k x = -2π/3a and k x = 2π/3a, respectively. Three branches appear in the reopened band gap, as shown in Fig. 2(b). The eigenmodes at k x = 0.5π/a are also displayed in Fig. In contrast, there is no vibration enhancement if the resonant pillars (highlighted in blue in the zoom) are removed, as shown in Fig. 2(d). This well explains that the magnitude at the bottom interface is nearly zero. It should be noticed that such a behavior may be fairly different from the refraction scheme undergone by TE and TM electromagnetic modes at the outlet of a photonic crystal having a quite similar geometry. Actually, instead of the evanescence of the elastic modes observed here, the photonic structure made of metallic cylindrical rods in between two parallel plates described in Ref. [26] allows for the anomalous refraction, while in a different manner, of both TE and TM optical modes.

Note that another Dirac cone occurs at 5.916 MHz in the symmetric dispersion curves when the height of the pillars is reduced down to 80 µm. In that case, the edge state refracts into both S 0 and SH 0 Lamb waves (see Figs. The spin Chern numbers

(K/K') 1 2   S C m
were derived from the integration of the Berry curvature of the four constitutive branches around the valley K (Fig. S12 in [30]). Therefore, the juxtaposition with another PPnC whose height of pillars is reduced by Δh S = 3 µm (type-IV), allows for pseudospin protected edge states at the domain wall, as shown in Fig. 3(c). The spin Chern number difference 1 at each valley guarantees that two spin-locked pairs of edge state occur in the reopened band gap, namely, a forward spin-down (↓) pair (cyan dotted lines) and a backward spin-up (↑) pair (magenta dotted lines). Figure 3(d) shows the propagation of the spin-locked edge states at 1.782 MHz at the valley K, the harmonic source being located in the middle of the domains limit. Both the left-going (spin-up) and right-going (spin-down) waves are excited, which is consistent with the two available edge states. The refracted patterns at the left and right zigzag terminations predicted by EFCs analysis are depicted in Fig. 3 In conclusion, we have demonstrated in this Rapid Communication that the proposed symmetric doublesided PPnC can emulate both and QSHE by solely imposing different geometric perturbations.

When QVHE is emulated, the type of refracted Lamb modes as well as the angles of the valley-polarized edge states at the zigzag and armchair terminations can be tailored through a proper choice of the height of the pillars. Specifically, in the deep subwavelength scale, the edge state becomes evanescent and can be enhanced by the locally resonant mode at the interface. By optimizing the geometrical parameters of the

  (a). Four perforated holes are drilled at the corners of the unit cell. The lattice constant and the thickness of the plate are a = 400μm and e = 100μm. The diameter and the height of the pillars are d A,B,C,D = D = 120 μm and h A,B,C,D = H = 160μm, and the radius of the hole is R H = 140 µm. In what follows, both the plate and the pillars are made of steel whose Young's modulus, Poisson's ratio, and mass density are E = 200 GPa, v = 0.3, and ρ = 7850 kg/m 3 , respectively. conditions to the unit cell and solving the eigenvalue equations. Thanks to the symmetry about the midplane of the plate, it can be further decomposed into symmetric (red dotted lines) and antisymmetric (blue dotted lines) subcomponents (see the eigenmodes in Supplemental Material Fig. S1 in [30]). For comparison, the band structure of the single-sided PPnC, obtained by removing all the pillars on one side of the plate, is shown in Fig. 1(c). Clearly, two Dirac cones related to the antisymmetric branches that can be seen in Fig. 1(b) at K point of the BZ at 0.937 and 2.361 MHz persist, unlike the Dirac cone at 2.068 MHz created by the symmetric modes. In view of the wavelengths of both Lamb modes involving symmetric displacements, i.e. 2557 and 1513 μm for S 0 and SH 0 modes respectively, the latter Dirac cone occurs in the deep subwavelength scale and should be attributed to local resonances of the pillars. It should be noticed here that the position of the Dirac cones can be easily modified by adjusting the height of the pillars (Fig. S2 in [30]). In what follows, emphasis is placed on the Dirac cone formed by the symmetric branches, the other one being analyzed in [30].

FIG. 1 :

 1 FIG. 1: (a) Elementary unit cell and its first irreducible BZ. Band structures of (b) the double-sided, (c) the singlesided and (d) the perturbed PPnCs with Δd V = 4 µm (d). (e) Energy flux (black arrows) and phase distribution at the lower (bottom panel) and higher (top panel) bounding of the lifted Dirac cone at the valley K. (f) Evolution of the valley pseudospin states at the valley K against the perturbation. Red (blue) symbols represent the frequencies of the symmetric (antisymmetric) dispersion curves.

2

  (b). The magenta and cyan dotted lines represent the edge states occurring at SDW and LDW respectively whereas the black dotted line is the locally resonant mode at the bottom end where the pillars with a large diameter are placed at the extreme end of the supercell (see the zoomed view in the inset).Let us now consider the propagation of the K'-polarized edge state along SDW and its refraction at the zigzag termination. To this end, a left-going wave at 2.042 MHz is excited by two phase-matched sources[31] and the displacement field on the top surface of the plate is recorded. The in-plane component is plotted in Fig.2(c) (see Fig.S7in[30] for the out-of-plane component). The edge state, which is mainly SH polarized, turns downwards and remains mostly localized at the interface, even though some elastic energy is spread across the plate and a small portion propagates along the bottom interface. The equifrequency contours (EFCs) shown in the inset of Fig.2(d) allow one to interpret this mapping. The deformation associated to the edge state having the mirror symmetry, only the S 0 and SH 0 Lamb modes should be considered; the corresponding EFCs are represented in Fig.2(d) by the red and green solid lines respectively. The two cyan dashed lines represent the normal to the zigzag termination. The edge state is locked to three K' valleys (black dots) in the BZ (black solid line). The wave vector in the plate can be graphically obtained owing to the conservation of the component of the wave vector parallel to the interface. Obviously, it is imaginary and the refracted wave is thus evanescent. However, due to the coupling with the locally resonant mode occurring at the edges ending with pillars having a large diameter [see the zoomed view in the inset of Fig.2(c)], the magnitude of the evanescent wave is strongly enhanced.

6 FIG. 2 :

 62 FIG. 2: (a) Schematic of the three-layer ribbon supercell constructed by type-I (Δd V = 10 µm) and type-II (Δd V = 10 µm) PPnCs. (b) Symmetric dispsersion curves of the supercell and the eigenmodes of the edge states (magenta and cyan branches) and locally resonant mode (black branch) at k x = 0.5π/a with a zoomed view of the undeformed supercell at the bottom end. Plots of the magnitude of the in-plane displacement on the top surface of the plate (c) with and (d) without the resonant pillars at the interfaces. Inset in (c): zoomed view at the left bottom corner. Inset in (d): EFCs anlysis.

8 FIG. 3 :. 10 FIG. 4 :

 83104 FIG. 3: (a) Occurrence of the double Dirac cone for specific dimensions (see text). (b) Band structure of the perturbed PPnC with Δh S = 3 µm and the eigenmodes at two Dirac cones. (c) Dispersion curves of the two-layer ribbon supercell constructed by type-III and type-IV PPnCs and the eigenmodes of the edge states (magenta and cyan branches) at k x = 0.5π/a. (d) Plot of the out-of-plane displacement on the top surface of the plate and the refracted patterns at the left and right zigzag terminations predicted by EFCs analysis.

Acknowledgments: W. W. acknowledges support from the research scholarship No. 201608310130 provided by the China Scholarship Council.

unit cell, a double Dirac cone can be obtained. We put into evidence the occurrence of the pseudospin protected edge state, in analogy to QSHE. Furthermore, we have demonstrated the valley-dependent feature of the pseudospin-valley combined edge state and the valley based splitting of the pseudospinprotected edge states in a T-shaped wave guide.