Floriane Guyot

François Cheviré

Ronan Lebullenger

Nathalie Herbert

From highly reactive amorphous tantalum oxide to tantalum nitride. Impact of the nitridation temperature on the optical and photocatalytic properties


Sara Gouarderes

Camille Ober

Layal Doumard

Jany Dandurand

Patricia Vicendo

Isabelle Fourquaux

Alexander Golberg

Valérie Samouillan

Dr Laure Gibot

Pulsed electric fields induce extracellular matrix remodeling through MMPs activation and decreased collagen production

Keywords: reversible electroporation, electrochemotherapy (ECT), gene electrotransfer (GET), metalloproteinases (MMPs), skin, tissue engineering extracellular matrix, LP: long pulses protocol, MMPs: metalloproteinases, PEFs: pulsed electric fields, SP: short pulses protocol, ROS: reactive oxygen species

ABSTRACT. Impairment of extracellular matrix (ECM) remodeling is observed in the tumor microenvironment or fibrosis and results in excessive collagen production and/or decreased degradation by metalloproteinases MMPs. Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel ECM. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute was used to demonstrate that microsecond and millisecond pulsed electric fields induced 1) a rapid modulation (4h after electrostimulation) of mRNA's genes composing the matrisome, particularly a down-regulation of pro-collagens and ECM maturation's enzymes such as transglutaminase TG2 and LOX-like; 2) a transient decrease in pro-collagens production and hydroxyproline tissue content within a week after electrostimulation; 3) a long-lasting ROS-dependent over-activation of MMPs for at least 48h and 4) a downregulation of TGF-β. These observations underpin that pulsed electric fields, a technology already approved for clinical use combined with anti-cancer agents, are particularly promising to provide local and effective treatment of abnormal ECM.

INTRODUCTION.

The extracellular matrix (ECM) is a dynamic and complex mesh of structural proteins and carbohydrates that are essential for tissue development, structure, and functionality [START_REF] Frantz | The extracellular matrix at a glance[END_REF]. Impairment in its regulation, composition, biophysical properties and remodeling is observed in diseases such as cancers or fibrosis [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF]. ECM components and the proteins that regulate ECM remodeling appear as promising therapeutic targets for human healthcare [START_REF] Ivey | Improving cancer therapies by targeting the physical and chemical hallmarks of the tumor microenvironment[END_REF]. Physical strategies such as radiotherapy, hyperthermia, photodynamic therapy or emerging ones (plasma, sonoporation) appear to be a relevant strategic solution to induce ECM remodeling (Gouarderes et al. 2020b). Their local effect and space-time control application resonate with the ongoing quest to alleviate patients' adverse side effects from systemic pharmacological approaches.

In this study, we applied external pulsed electric fields (PEFs) that increase the cell membrane permeability, a phenomenon known as electroporation, presenting a wide variety of biomedical applications [START_REF] Geboers | High-Voltage Electrical Pulses in Oncology: Irreversible Electroporation, Electrochemotherapy, Gene Electrotransfer, Electrofusion, and Electroimmunotherapy[END_REF]. Electroporation can be either reversible or irreversible. While irreversible electroporation (IRE) and partial IRE lead to cell death and preservation of the structural integrity of surrounding tissue [START_REF] Davalos | Tissue Ablation with Irreversible Electroporation[END_REF][START_REF] Golberg | Preventing Scars after Injury with Partial Irreversible Electroporation[END_REF][START_REF] Golberg | Skin regeneration with all accessory organs following ablation with irreversible electroporation[END_REF], on the contrary, reversible electroporation transiently increase membrane permeability, enhancing concomitant delivery of therapeutics in drug delivery strategy, while preserving cell viability. In our work, we focused on reversible electroporation and applied similar electrical conditions to those applied in clinics for drug delivery. Thus, two distinct calibrated electric protocols already used in routine in human and veterinarian clinics for drug delivery to cure superficial and deep tumors were applied [START_REF] Nuccitelli | Application of pulsed electric fields to cancer therapy[END_REF][START_REF] Yarmush | Electroporation-Based Technologies for Medicine: Principles, Applications, and Challenges[END_REF]. They consist of millisecond length "long pulses protocol" (LP) which is used for nucleic acids delivery in DNA vaccination gene therapy, and microsecond length "short pulses protocol" (SP) used for anti-tumor drug delivery in electrochemotherapy [START_REF] Marty | Electrochemotherapy -An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study[END_REF][START_REF] Gehl | Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases[END_REF]. Our strategy lay on applying solely physical electrical stimuli, without the addition of any drugs, to modulate cell behavior. These calibrated electric parameters were applied to a sophisticated in vitro tissue-engineered human dermal substitute, produced through the self-assembly approach in which cells produce their own extracellular matrix [START_REF] Athanasiou | Self-organization and the self-assembling process in tissue engineering[END_REF]. Thus, the final tissue substitutes are rich in endogenous ECM proteins, as demonstrated by proteomic analyses [START_REF] Magnan | Characterization of a Cell-Assembled extracellular Matrix and the effect of the devitalization process[END_REF], demonstrating its complex composition and similarity to the native dermis.

Our findings indicate that applying a calibrated train of electric pulses as a purely physical stimulus tends to be a relevant therapeutic tool with a broad potential that can extend far beyond its actual clinical application in electrochemotherapy. A good understanding of the cell mechanisms induced by the electric field itself (no need for external molecules addition) will increase its applicability in a context where ECM is abnormal such as in fibrosis.

RESULTS AND DISCUSSION

Human dermal substitutes present a unique biological architecture rich in collagens.

Nowadays, researchers have the most benefit of hindsight with PEFs treatment of skin cancers. To demonstrate the high potential of PEFs in ECM remodeling, we turned to a model of human dermis substitute we have engineered. Scanning electron microscopy showed a polarization of the tissue model, with more cells on its top (culture medium side) and more ECM fibers on its bottom (Petri side) (Fig1A, B). Transmission electron microscopy of a whole transversal section confirmed this vertical gradient of cells and ECM (Fig1C). A zoom performed on the ECM part allowed to detect the characteristic banding pattern of collagens fibrils (Fig1D). Of note, fibril-forming collagens types I, III, and V, characterized by their ability to assemble into highly orientated supramolecular fibrils, are the main components of the human dermal extracellular matrix [START_REF] Gelse | Collagens-structure, function, and biosynthesis[END_REF] and of the human dermal substitutes as assessed by proteomics [START_REF] Magnan | Characterization of a Cell-Assembled extracellular Matrix and the effect of the devitalization process[END_REF]. This result correlated with the second-harmonic generation (SHG) signal, detected by two-photon microscopy, which confirms the proper organization of these fibrillar collagens within our tissue model (Fig1E). Fourier Transform Infrared-Attenuated Total Reflectance (FTIR-ATR) [START_REF] Sanden | The use of Fourier-transform infrared spectroscopy to characterize connective tissue components in skeletal muscle of Atlantic cod (Gadus morhua L.)[END_REF]) corroborated the polarization of the tissue model (Fig1F, FigS1, Table S1). PEFs induce a differential expression of matrisome's genes. Mean FTIR spectra pointed out that electrostimulation did not cause significant alteration of the global spectral signature of the different biological components but changes in bands intensities (FigS2). Interestingly, a focus on RNA 990 cm -1 band (Fig2A) evidenced a significant transient increase in RNA content of each side of the substitute 24h after electrostimulation. Transcriptomic analysis of differentially expressed genes in the whole tissue was led 4h after electrostimulation, within a time frame close to the stress, in order to not lose the dynamics following the application of PEFs. Results showed that among the 20,000 genes analyzed, 606 genes were differentially (±2 fold-changes and p-value p <0.01) expressed 4h after long pulses (LP) protocol (350 upregulated and 256 down-regulated) and 351 after short pulses SP protocol (258 up-regulated and 93 down-regulated) (Fig2B). All the genes modulated by SP protocol were also regulated in LP protocol, meaning that common pathways are activated independently of the pulse protocol applied. Gene ontology analysis was led using GOrilla tool [START_REF] Eden | GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists[END_REF]) in order to visualize the most significantly enriched biological processes following electrostimulation (table S2). It appeared that metabolic pathway processes at large were mainly affected. In the context of our study, we focused on genes involved in the composition of the matrisome defined as the list of all the genes coding for proteins entering in the composition of matrices, grouping the "core matrisome" and "matrisome associated proteins" [START_REF] Hynes | Overview of the Matrisome-An Inventory of Extracellular Matrix Constituents and Functions[END_REF][START_REF] Naba | The extracellular matrix: Tools and insights for the "omics" era[END_REF]. Exhaustive transcriptomic results are dispatched in tables S3 (core matrisome) and S4 (matrisome-associated proteins). Presented volcano plots indicated that most of the genes impacted by PEFs were assigned to matrisome-associated proteins sub-panel and consisted principally in secreted proteins such as growth factors, regulatory factors such as metalloproteases MMPs and their inhibitors, and enzymes responsible for ECM cross-linking (Fig2C). Previous in vitro studies confirmed the ability of PEFs to favor growth factors and the release of cytokines from different cell types at protein level [START_REF] Garner | Electrical stimulation of whole blood for growth factor release and potential clinical implications[END_REF]Gouarderes et al. 2020a). expressed genes 4h after electrostimulation in control (Ctrl) condition, long pulses (LP) and short pulses (SP) protocols among the 20,000 genes analyzed by transcriptomic analysis using Clariom S Affymetrix array. n=4. C. Volcano plots showing the significant impact on differentially expressed matrisome's genes 4h after pulsed electric field stimulation with LP, and SP protocols. ADAMTS1: ADAM metallopeptidase with thrombospondin type 1 motif 1; ADAMTS15: ADAM metallopeptidase with thrombospondin type 1 motif 15; ANGPTL1: angiopoietin like 1; BMP1: bone morphogenetic protein 1; CCL4L2: chemokine (C-C motif) ligand 4-like 2; CLCF1: cardiotrophin-like cytokine factor 1; CLEC2D: C-type lectin domain family 2, member D; COL1A1: collagen 1A1; CRISPLD2: cysteine-rich secretory protein LCCL domain containing 2; CRLF1: cytokine receptor-like factor 1; CTGF: connective tissue growth factor; CXCL8: chemokine (C-X-C motif) ligand 8; DDX26B: (Asp-Glu-Ala-Asp/His) box polypeptide 26B; ELFN1: extracellular leucine-rich repeat and fibronectin type III domain containing 1; F13B: coagulation factor XIII, B polypeptide; HBEGF: heparinbinding EGF-like growth factor; IFNE: interferon epsilon; IL12A: interleukin 12A; IL6: interleukin 6; INHBA: inhibin beta A; LOXL4: lysyl oxidase-like 4; NGF: nerve growth factor; PLXNA1: plexin A1; PLXND1: plexin D1; PPT2: palmitoyl-protein thioesterase 2; SERPINB2: serpin peptidase inhibitor member 2; TGM2: transglutaminase 2; TIMP3: TIMP metallopeptidase inhibitor 3; TNFAIP6: tumor necrosis factor, alpha-induced protein 6; VEGFA: vascular endothelial growth factor A.

PEFs induce a transient decrease in collagens production. mRNA modulation of the whole collagen family members 4h after the dermal tissue has been electrostimulated indicated a general trend towards a decrease in the expression of collagens (Table S3 andFigS3A). FTIR-ATR analyses of dermal substitutes 24h and 48h after electrostimulation indicated that the collagens 1338 cm -1 band was not altered on the top side, rich in cells, but decreased in the bottom side, rich in ECM, solely for SP protocol conditions (Fig3A). Because FTIR-ATR is a superficial probe (up to 3.6 µm depth), we performed differential scanning calorimetry (DSC) analyses in order to evaluate collagens integrity in the whole tissue (FigS4). No significant differences were observed for denaturation enthalpy ΔHd and denaturation temperature TMID after electrostimulation, indicating no major variation in collagens integrity after PEFs application. In parallel, we performed a whole tissue hydroxyproline quantification to estimate global collagen content, 48h, one week, and four weeks after the electrostimulation (Fig3B). Regardless of the electric protocol applied, a significant decrease ranging from 35% to 50% in tissue collagen's content was observed 1 week after electrostimulation. We verified that this decrease in hydroxyproline content one week after electrostimulation was not due to a decrease in the number of living cells capable of synthesizing collagens. No statistical difference between the control condition (110,000 cells ± 10,300) and the LP (116,000 cells ± 8,600) and SP (130,000 cells ± 6,600) conditions was observed. This decrease in tissue collagen's content was totally reversed after a long-term culture (4 weeks), demonstrating the character transient of the electrostimulation effects in an irrefutable way. To determine if this drop in global collagens content arises from a decrease in collagens production, the abundance of major dermal collagen (Ricard-Blum 2011), namely collagen I, was quantified in the supernatant (Fig3C). The analyses showed a significant decrease in pro-collagen Iα1 abundance in the supernatant of about 25% compared to the control condition as soon as 24h consecutively to electrostimulation, which lasted at least 48h. During their maturation phase, collagen fibrils and other ECM components are strengthened by covalent cross-linking by lysyl oxidases (LOXs), lysyl hydroxylases, and tissue transglutaminases (TGs) [START_REF] Griffin | Transglutaminases: Nature's biological glues[END_REF][START_REF] Gelse | Collagens-structure, function, and biosynthesis[END_REF][START_REF] Scott | Lysine post-translational modifications of collagen[END_REF]). In our experiments, analyses of LOXs mRNA family indicated a significant decrease for LOXL4 mRNA (Fig3D), and a major down-regulation (by -5 and -6-fold changes for LP and SP, respectively) of TG2 expression in transglutaminases mRNA family (Fig3E). TG2 is the most widely distributed and expressed member of the transglutaminase enzyme family [START_REF] Collighan | Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications[END_REF]. Our results on LOX and TG mRNA down-regulation are significant but would deserve further investigation, by quantifying not only the protein level of these enzymes (ELISA) but also their activity (functional assays). A better definition of the effect of PEFs on the effective modulation of their activities, whose deregulation is implicated in many diseases [START_REF] Trackman | Lysyl oxidase isoforms and potential therapeutic opportunities for fibrosis and cancer[END_REF][START_REF] Szondy | Transglutaminase 2 in human diseases[END_REF], would allow envisaging new therapeutic applications for this physical technology.

PEFs induce sustained activation of MMPs without overexpression of the proteins.

In order to understand the origin of collagen decrease after electrostimulation, we analyzed the mRNA modulation of the whole MMPs family members (FigS3B). 4h after electrostimulation, mRNAs of MMP-1 (-3.5-fold changes), and to a lesser extent MMP-2, -14, -16 were significantly down-regulated while MMP-19 mRNA was up-regulated. We then moved to the protein level, to quantify the main major cutaneous MMPs by ELISA, namely MMP-1, -2, -3, -9 [START_REF] Amălinei | Biology of metalloproteinases[END_REF]; Kähäri and Saarialho-Kere 1997) (Fig4A). ELISA analyses showed that MMP-1 and MMP-2 protein expression slightly and transiently decreased in the 6 first hours after electrostimulation, while no difference in protein quantity was observed for MMP-3 over 48h after treatment. By contrast, MMP-9 was significantly higher in abundance at both 24h and 48h after LP protocol was applied, but not after SP protocol. The cleavage of inactive zymogen (pro-MMPs) is a key step for their proteinase functions. In order to quantify the global amount of MMPs in the supernatant, we took advantage of the ability of organomercurial compounds to in vitro cleave pro-MMPs into their active form (Fig4B). Interestingly, no difference of global signal was observed 6h and 24h after electrostimulation, meaning that the quantity of MMPs in the supernatant is similar between control and electrostimulated conditions. However, a clear overexpression of global MMPs was noted in LP protocol 48h post-electrostimulation, which follows the same trends observed previously in the ELISA results for MMP-9 (Fig4A). To go further and be more realistic of the process involved in our experimental conditions, the activity of the solely endogenously active MMPs was quantified, reflecting the actual activity rate of MMPs in the model (Fig4C). A clear increase in MMPs activity was observed as soon as 6 hours after electrostimulation and maintained for at least 48 hours. Since this increase was not due to an overproduction of MMPs as quantified at the protein level (Fig4A and B), we hypothesized that its endogenous inhibitors, the TIMPs family, were down-regulated. The transcriptomic analysis of mRNA modulation of the TIMPs family members 4h after electrostimulation indicated that only TIMP3 was statistically down-regulated in LP protocol (Fig4D), an observation not confirmed at protein levels by ELISA (Fig4E).

Pharmacological MMPs' inhibitor partly restores collagens content. In order to estimate the degree of causality between MMP over-activity and the decrease in collagens content observed in tissues after the application of both LP and SP protocols, we added a broadspectrum pharmacological inhibitor, namely GM6001 or Ilomastat, to the cultures. As expected, after 48h of culture, MMPs activity was non-significantly different between conditions in the presence of the inhibitor (Fig5A). Moreover, total hydroxyproline content in tissues one week after electrostimulation was statistically restored when MMPs activity was inhibited with GM6001 (Fig5B). While inhibition of MMPs activity after SP protocol clearly normalizes whole tissue collagen content, it was less efficient after LP protocol. FTIR-ATR analyses performed on the bottom side (richer in ECM) of the dermal substitutes 24h and 48h after electrostimulation confirmed that collagens content was restored in the presence of GM6001 after SP protocol, while no effect was detectable for LP protocol (Fig5C). ECM remodeling is a subtle balance between synthesis/maturation/degradation which seems more complex for LP than SP protocol. Since pulses last longer in LP protocol, it seems reasonable to hypothesize that cells within the tissue are more affected and develop a more complex response to this stress. A key element to support this hypothesis is the higher number of modulated genes in the LP protocol compared to the SP protocols (606 versus 351 respectively) as determined by transcriptomic analyses. MMPs in supernatant 24h after electrostimulation and culture in the presence of 1mM and 5mM ROS inhibitor N-acetyl-L-cysteine (NAC). E. MMP-2 and -9 gelatinase activity was quantified by fluorimetry in presence or absence of 5mM NAC. F. MMPs activity against specific substrates was quantified by fluorimetry in presence or absence of 5mM NAC.

Control condition (Ctrl •), long pulses (LP ■), and short pulses (SP ▲) protocols.

Inactive zymogen pro-MMPs are known to be proteolytically activated in the extracellular space by endogenous proteases and other active MMPs or to be activated in response to exogenous signals such as low pH, heat, reactive oxygen species (ROS) [START_REF] Amălinei | Biology of metalloproteinases[END_REF][START_REF] Kähäri | Matrix metalloproteinases in skin[END_REF]. Low pH can reasonably be ruled out because of the buffering of both pulsing and cell culture medium. Heat is a parameter that can also be excluded as the pulsing buffer and electric parameters are designed to remain in athermal conditions [START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF]. In contrast, lipids peroxidation and ROS production have been shown to occur with PEFs [START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF]. Increasing concentrations (1mM and 5mM) of N-acetyl-L-cysteine (NAC), a potent ROS inhibitor that increases cellular pools of free radical scavengers [START_REF] Zhitkovich | N-Acetylcysteine: Antioxidant, Aldehyde Scavenger, and More[END_REF], were added to both pulsing buffer (during the application of PEFs) and in cell culture medium (Fig5D). Increasing concentration of the ROS scavenger led to a statistical decrease of MMPs over-activation. This observed dose-response clearly establishes the link between the over-activation of MMPs and the presence of ROS in the environment. At this stage of the investigations, no clear response can be provided concerning the origin and maintenance of these ROS. Given electrostimulation is a brief event (less than 12 seconds), abolished upon transfer of the dermal substitutes from the pulsation buffer to the fresh culture medium a few seconds after electrostimulation, and that ROS halflife is very short due to their inherent instability and high reactivity [START_REF] Dickinson | Chemistry and biology of reactive oxygen species in signaling or stress responses[END_REF], the cellular origin of these ROS is more likely to explain the sustained ROS-dependent over-activation of MMPs observed for at least 48h. In order to better discriminate the role of specific MMPs in ROS-dependant over-activation, we performed experiments with MMP-specific substrates (Fig5E, F). We demonstrated that gelatinase MMP-2 and -9 as well as stromelysin MMP-3 and -10 were not activated by electrostimulation, even if MMP-3 was undoubtedly the most abundant MMP at protein level (µg range compared to ng for MMP-1 and -9 and pg for MMP-2) (Fig 4A). It thus appears that the observed over-activation of ROSdependent MMPs was due to the activity of one or more of the following MMPs, namely MMP-1, -7, -8, -12, -13, -14, -15, -16, without being able to experimentally identify more precisely the responsible ones. Interestingly, the over-activity of the collagenases MMP-1, -8 and -13 can be linked with the observed decrease in tissue collagen's content (Fig 3A,B).

Pulsed electric fields down-regulated pro-fibrosis inducer TGF-β1. Fibrosis results from changes in collagen expression, biosynthesis, enzymatic cross-linking, and degradation by

MMPs [START_REF] Baffet | Molecular and tissue alterations of collagens in fibrosis[END_REF]. These molecular modifications leading to a fibrotic signature are primarily regulated by TGF-β1, considered the major pro-fibrogenic inducer [START_REF] Meng | TGF-β: the master regulator of fibrosis[END_REF]. Interestingly, our experiments show that PEFs significantly downregulated TGF-β1 both at mRNA and protein levels (Fig6A, B). To go further, we analyzed other factors known to be pro-fibrogenic and associated with fibrotic diseases, namely interleukin IL-4 and IL-13 [START_REF] Wynn | Fibrotic disease and the TH 1/TH 2 paradigm[END_REF]), but no change was observed at protein levels (Fig6C, D). To enrich the discussion on the therapeutic potential of PEFs in the context of fibrosis, it is essential to emphasize that they induced a drastic decrease in the gene expression of LOXL4 and transglutaminase TG2 (Fig 3D,E), which are known to play an important role in the fibrotic process when overexpressed [START_REF] Grenard | Transglutaminasemediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis[END_REF][START_REF] Benn | Tissue transglutaminase in fibrosis -more than an extracellular matrix cross-linker[END_REF][START_REF] Chen | Lysyl Oxidase (LOX) Family Members: Rationale and Their Potential as Therapeutic Targets for Liver Fibrosis[END_REF]. All these findings can be excitingly correlated with observations realized by clinicians using short pulse (SP) protocol to deliver anti-tumor drugs, whom commonly observed aesthetic and functional healing of sites treated with electrochemotherapy [START_REF] Glass | Intralesional bleomycin-mediated electrochemotherapy in 20 patients with basal cell carcinoma[END_REF][START_REF] Marty | Electrochemotherapy -An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study[END_REF][START_REF] Kis | Successful Treatment of Multiple Basaliomas with Bleomycin-based Electrochemotherapy: A Case Series of Three Patients with Gorlin-Goltz Syndrome[END_REF], and by patients themselves, who underlined an improvement in wound healing, and aesthetic impairment [START_REF] Campana | Bleomycin-Based Electrochemotherapy: Clinical Outcome from a Single Institution's Experience with 52 Patients[END_REF]). To conclude, PEFs application to locally and transiently remodel the extracellular matrix present a high and easy translational potential. Indeed, these types of electrical parameters are nowadays used in clinics as an anticancer treatment, meaning that validated apparatus (pulsed electric field generator, electrodes, dosimetry…) are available and clinicians are used to, and well-trained to apply this kind of protocol.

MATERIAL & METHODS

Human primary cell culture. Primary human dermal fibroblasts were isolated from 4-years old foreskin biopsy commercially available (Icelltis, France) [START_REF] Gibot | A Preexisting Microvascular Network Benefits In Vivo Revascularization of a Microvascularized Tissue-Engineered Skin Substitute[END_REF]. [START_REF] Gibot | A Preexisting Microvascular Network Benefits In Vivo Revascularization of a Microvascularized Tissue-Engineered Skin Substitute[END_REF]. Ethical approval has been obtained by the supplier from the French Ministry of Higher Education, Research and Innovation (#AC-2019-3444) and written, informed consents from the guardians of the children were obtained. Cells were grown in DMEM GlutaMAX (Gibco #10569010) supplemented with 10% of heat-inactivated fetal calf serum (Gibco #10270106), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco #15140122), at 37 °C in a humidified atmosphere at 5% CO2. Cells were regularly tested negative for mycoplasma (MycoAlert detection kit, Lonza #LT07-318).

Reconstruction of human 3D dermal substitutes. Human dermal tissues were generated by tissue-engineering as previously described [START_REF] Gibot | Tissue-engineered 3D human lymphatic microvascular network for in vitro studies of lymphangiogenesis[END_REF]. 30,000 cells/cm² primary human dermal fibroblasts were seeded in 24-well plates containing anchoring paper (Whatman #1004150). Cells were cultivated for 4 weeks in medium supplemented with 50 µg/ml ascorbic acid (Sigma #A4034, Merck KGaA, Darmstadt, Germany).

Pulsed electric field application to dermal substitutes to induce reversible electroporation. Two stainless steel, flat, parallel electrodes (0.8 cm between electrodes, 1 cm long) (Megastil, Ljubljana, Slovenia) were placed on the top of dermal tissue, covering all the cell area, and immersed in pulsing buffer (10 mM K2HPO4/KH2PO4, 1 mM MgCl2, 250 mM sucrose, pH 7.4 in sterile water) [START_REF] Madi | Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue[END_REF]Madi et al. 2016) (FigS5A). A βtech electro-cell S20 generator (LeroyBiotech, France) was used to deliver square-wave electric pulses at room temperature. Electric parameters applied were for the long-pulses protocol (LP): 10 pulses of 5 ms long, 1 Hz, 600 V/cm and for the short-pulses protocol (SP): 8 pulses of 100 µs long, 1 Hz, 1000 V/cm (FigS5B). Millisecond pulses (LP protocols) are classically used to improve nucleic acids delivery thanks to electrophoretic force, while microsecond pulses (SP protocol) are used for small antitumor drugs delivery. These electrical conditions were similar to those applied in clinics for drug delivery [START_REF] Gehl | Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases[END_REF][START_REF] Nuccitelli | Application of pulsed electric fields to cancer therapy[END_REF] and well-characterized to induce transient and reversible cell electroporation not inducing cell death. They induced cell permeabilization in the entire depth of the tissue (FigS5C, D) [START_REF] Madi | Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue[END_REF], while preserving cell viability (FigS5E).

Electron microscopy. For transmission and scanning electron microscopy characterization of 4-weeks old control (non-electrostimulated) tissues, dermal substitutes were fixed in 2% glutaraldehyde diluted in 0.1 M Sorensen phosphate buffer (pH 7.4) for 24 h at 4° C and processed as classically described in the literature for tissues. Observations were carried out using Hitachi HT7700 transmission electron microscope and FEG scanning electron microscope FEI Quanta 250.

Second-harmonic generation (SHG).

Organized fibrillar collagens bundles were imaged by SHG using a multi-photon microscope LSM 7MP (Carl Zeiss SAS, Jena, Germany). The total thickness of the dermal substitute (approximately 50 µm) was imaged.

Fourier-Transform Infrared-Attenuated Total Reflectance (FTIR-ATR). FTIR-ATR

spectra were performed using a Nicolet 5700 (ThermoFisher Scientific, Waltham, MA, USA) equipped with ATR accessory (Smart Orbit with a type IIA diamond crystal, refractive index 2.4) with a KBr beam splitter and a MCT/B detector. The penetration depth of the evanescent wave into biological tissue was estimated between 0.4 and 3.6 µm. Hydrated dermal substitutes were laid on the ATR device and dried for 20 min at ambient temperature before spectral acquisition. The top side and the bottom side were successively placed onto the crystal. Spectra were recorded over the region of 4000-450 cm -1 with a spectral resolution of 2 cm -1 and 32 accumulations. A background spectrum was recorded before each experiment and subtracted from the sample spectrum. Data were collected using Omnic 8.0 (ThermoFisher Scientific). Then spectra were baseline corrected, smoothed (11 points Savitzky Golay) and normalized to the amide II (1540-1550 cm -1 ). The spectra presented per group of samples were calculated by averaging the spectra of all samples within each group.

To quantify the different biological components, the areas of the specific absorption bands were computed from the individual spectrum of each sample, and the appropriate ratio of areas was used according to literature data [START_REF] Staniszewska | Rapid approach to analyze biochemical variation in rat organs by ATR FTIR spectroscopy[END_REF][START_REF] Wang | Infrared imaging of compositional changes in inflammatory cardiomyopathy[END_REF].

Transcriptomic analysis of electrostimulated dermal substitutes. RNAs were extracted 4 h after dermal substitutes electrostimulation using RNeasy Mini kit (Qiagen #74104). The yield and integrity of the extracted RNAs were confirmed on Bioanalyzer 2100 Expert (Agilent).

Differential gene expression was studied by transcriptomic using Clariom S Affymetrix chips on Genome and Transcriptome (GeT) platform of Genotoul (Toulouse, France). Data analysis was performed with TAC software, and expression analysis settings were: Gene-Level Fold Change <-2 or> 2; Gene-Level FDR <0,05; Anova Method: ebayes.

Quantification of hydroxyproline content. Total hydroxyproline content of whole dermal substitutes was measured using the Hydroxyproline Assay Kit (Sigma #MAK008). A specific set of experiments was led in the presence of 100µM of pan-MMPs pharmacological inhibitor GM6001 (Sigma #CC1010). GM6001 was added to the culture medium immediately after electric field applications, and renewed at each cell culture medium change.

Quantification of metalloproteinases (MMPs) activity. Quantification of global MMPs activity in the supernatant was carried out using SensoLyte520 Generic MMP Assay Kit (Anaspec #AS-71158) allowing detecting simultaneously the activity of 2,3,7,8,9,12,13,and 14. Two distinct strategies were led: 1) quantification of the total MMPs activity after chemical activation with 4-aminophenylmercuric acetate (APMA) for 4h at 37° according to manufacturer pieces of advices permits to quantify the total amount of (pro-)MMPs in supernatant, and 2) quantification of endogenously active MMPs in the supernatant. A set of experiments was carried out in the presence of 100 µM GM6001 in cell culture medium or in the presence of N-acetyl-L-cysteine (NAC, Sigma #A7250) in both pulsing buffer and cell culture medium. Specific MMPs activity was analyzed using commercial substrates according to manufacturer's instructions: MMP-2, -9 (Gelatin Degradation Assay Kit (#ab234057)), MMP-3, -10 (R&D systems #ES002) and other MMPs (R&D systems #ES001).

ELISA quantifications. Supernatants were harvested at 6h, 24h, 48h after electrostimulation and stocked at -80 °C to be used within a month. Following ELISA kits were used strictly according to manufacturer's protocols: Pro-Collagen I alpha 1 DuoSet ELISA kit (R&D systems, #DY008), TGF-β 1 DuoSet ELISA kit (R&D systems, #DY007), TIMP-3 (Elabscience, #E-EL-H1454), MMP-1 (ELISA Genie, #HUFI00205), MMP-2 (ELISA Genie, #HUFI00017), MMP-3 (ELISA Genie, # HUFI00208), MMP-9 (ELISA Genie, # HUFI00211), IL-4 and IL-13 (V-PLEX Proinflammatory Panel 1 Human Kit, MSD, # K15049D).

Statistical analyses. Data analysis was performed using GraphPad Prism 8 Software (La Jolla, CA, USA), and independent biological replicates were plotted and expressed as mean ± SEM (standard error to the mean). Data have been statistically analyzed using one-way ANOVA followed by Dunnett's post-test to compare every condition (LP, SP) to the control one (0 V/cm). Statistics are expressed relative to the control condition (0 V/cm). ns: nonsignificant, * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

DATA AVAILABILITY. The transcriptomic data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] LG.

Table S2: Visualization of GO (gene ontology) terms by processes in the target gene list using Gorilla tool. We performed enrichment analysis using the GOrilla platform in the transcriptomic dataset [START_REF] Eden | GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists[END_REF]. This analysis allowed to identify GO terms that are significantly overrepresented in the list of differentially expressed genes (the target set) compared to the totality of genes contained on the Affymetrix chip (background set) using the standard Hyper Geometric statistics. Analysis of gene ontology terms by processes show biological processes that were mainly affected by electrostimulation. S3. Transcriptomic analyses of differentially expressed genes in "core matrisome" sub-panel. Annotation according to [START_REF] Naba | The extracellular matrix: Tools and insights for the "omics" era[END_REF]. The extracellular matrix: Tools and insights for the "omics" era. Matrix Biol. 49, 10-24 (2016)). Genes in bold are modulated after electrostimulation and described in columns "LP" and "SP": fold changes are indicated in brackets after gene symbols, down-regulated genes (green), up-regulated genes (red). Col = Collagens; PG = Proteoglycans. p-value < 0.05. n=4.

Figure 1 .

 1 Figure 1. Human tissue-engineered 3D dermal substitutes are rich in collagens and present a top/bottom polarisation. A. Top surface (cell culture medium side) of dermal cell sheet observed by scanning electron microscopy (SEM). B. Bottom surface (Petri side) of dermal cell sheet observed by SEM. C. Transversal section of dermal substitute observed by transmission electron microscopy (TEM). D. Striated collagens fibrils observed by TEM. E. Second Harmonic Generation (SHG, in cyan) signal detected in the top view of substitute by two-photon microscopy. F. Nucleic acids and collagens repartition between the medium side (top side) and Petri side (bottom side) measured by FTIR-ATR. n>6.

Figure 2 .

 2 Figure 2. RNA content is modulated in dermal substitutes after electrostimulation. A. RNA indicators in the top (enriched in cells) and bottom (enriched in ECM) sides of the dermal substitute measured by FTIR-ATR 24h and 48h after electrostimulation. n>6. Control condition (Ctrl •), long pulses (LP ■), and short pulses (SP ▲) protocols. B. Differentially

Figure 3 .

 3 Figure 3. Collagens content transiently decreased in dermal substitute after electrostimulation. A. Collagens indicator in the top (enriched in cells) and bottom (enriched in ECM) sides of the dermal substitute measured by FTIR-ATR 24h and 48h after electrostimulation. B. Hydroxyproline content in the whole dermal substitute measured over

Figure 4 .

 4 Figure 4. MMPs activity but not expression is increased after electrostimulation. A. Expression of MMP-1, -2, -3, and -9, quantified in the supernatant by ELISA. B. Global MMPs activity quantified by fluorimetry after chemical activation of all pro-MMPs present in the supernatant. C. Quantification by fluorimetry of endogenously active MMPs in the supernatant. D. Modulation of TIMPs family mRNA 4h after electrostimulation. E. TIMP-3 expression quantified in the supernatant by ELISA. Control condition (Ctrl •), long pulses (LP ■), and short pulses (SP ▲) protocols.

Figure 5 .

 5 Figure 5. Pharmacological MMPs inhibitor GM6001 partly restores dermal substitute collagens content altered by ROS-dependent MMPs over-activation. A. Quantification by fluorimetry of endogenously active MMPs in supernatant 48h after electrostimulation in the presence of GM6001. B. Hydroxyproline content in the whole dermal substitute 1 week after electrostimulation, cultured in the presence (blank symbols) or absence of GM6001 (full symbols). Statistical analysis between the two controls was analyzed with Unpaired t-test. C. Collagen indicator in the bottom side (enriched in ECM) of dermal substitutes measured by FTIR-ATR 24h and 48h after LP and SP electrostimulation in the presence (blank symbols) or absence of GM6001 (full symbols). D. Quantification by fluorimetry of endogenously active

Figure 6 .

 6 Figure 6. Pulsed electric fields down-regulate pro-fibrosis inducer TGF-β1. A. Modulation of TGF-β 1 mRNA 4h after electrostimulation. E. TGF-β 1 expression quantified in the supernatant by ELISA. C. IL-4 expression quantified in the supernatant by ELISA. D. IL-13 expression quantified in the supernatant by ELISA. Control condition (Ctrl •), long pulses (LP ■), and short pulses (SP ▲) protocols.

  Figure S2. Mean FTIR-ATR spectra in the [1450-950 cm -1 ] range. Top (rich in cells) and bottom (rich in ECM) sides of dermal substitutes in control (Ctrl) condition, and 24h and 48h after long pulses (LP) and short pulses (SP) protocols.

Figure S3 .

 S3 Figure S3. Modulation of (a) collagens and (b) matrix metalloproteinases (MMPs) family's mRNA 4h after electrostimulation. Transcriptomic analyse using Clariom S Affymetrix array. n=4. Long pulses (LP ■): 10 pulses lasting 5ms, 600V/cm at 1Hz frequency; Short pulses (SP ■): 8 pulses lasting 100 µs, 1000V/cm at 1Hz frequency.

Figure S5 .

 S5 Figure S5. Long pulses (LP) and short pulses (SP) electric parameters induce transient and reversible fibroblasts electropermeabilization in human dermal substitute. a. Macroscopic aspect of dermal substitute (arrow), electropulsator and electrodes stimulating the whole substitute. b. Applied electric parameters profiles, extracted with an oscilloscope. Scale bar = 0.8 cm. c. Plasma membrane integrity assessed with propidium iodide (red) penetration, observed by two-photon microscopy. Scale bar = 50µm. d. Optical transversal section of SP electrostimulated dermal substitute observed by two-photon microscopy. Nuclei

  We want to acknowledge the GeT Genotoul technological platform that realized the transcriptomic analyses, especially Nathalie Marsaud for her help in optimizing nucleic acids extraction from the substitutes, and Dr Elisabeth Bellard for her valuable help for the SHG imaging on TRI-Genotoul platform. We would like to warmly thanks Jean-Michel Martin (ICT) and Loïck Royant (Leroy Biotech) for data extractions from the electropulsator,

	and are accessible through number GSE180644 ORCID Gouarderes S (0000-0001-9080-7876); Doumard L (0000-0001-9407-7989); GEO Series accession Dandurand J (0000-0001-7287-2973); Vicendo P (0000-0001-5874-1547); Golberg A (0000-0001-8782-8879); Samouillan V (0000-0003-0571-3985); Gibot L (0000-0001-7255-4123) CONFLICT OF INTEREST. The authors state no conflict of interest. ACKNOWLEDGEMENTS. This research was funded by the French Agence Nationale de la recherche (ANR-17-CE19-0013-01) and performed in the scope of HAPPYBIO GDR. Multiplex ELISAs were read with the technological support of Genoskin company English proofreading and editing. (Toulouse). and Dr Mingotaud for her helpful scientific discussions. Lab2screen is acknowledged for AUTHOR'S CONTRIBUTIONS STATEMENT. Investigation: SG, CO, LD, JD, IF;

(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE180644). Formal Analysis: SG, CO, VS; Visualization: SG, CO, LG; Validation: SG; Writingoriginal draft: SG, CO, AG, VS, LG; Writingreview and editing: SG, AG, VS, LG; Conceptualization: PV, AG, VS, LG; Methodology: LG; Funding acquisition: LG; Supervision:

  The 10 most affected processes are presented, ranked by statistical significance.

	Table
	GO Term	Description	P-value	FDR q-value Enrichment
		LP protocol
	GO:0031323	regulation of cellular metabolic process	6.67E-29	1.02E-24	1.68
	GO:0019222	regulation of metabolic process	3.16E-28	2.42E-24	1.63
	GO:0080090	regulation of primary metabolic process	1.97E-25	1.01E-21	1.65
	GO:0051171	regulation of nitrogen compound metabolic process	3.81E-25	1.46E-21	1.66
	GO:0060255	regulation of macromolecule metabolic process	2.71E-24	8.31E-21	1.63
	GO:0048523	negative regulation of cellular process	2.14E-22	5.47E-19	1.74
	GO:0048519	negative regulation of biological process	2.55E-21	5.58E-18	1.66
	GO:0009889	regulation of biosynthetic process	3.99E-21	7.63E-18	1.78
	GO:0031326	regulation of cellular biosynthetic process	6.39E-20	1.09E-16	1.76
	GO:0031324	negative regulation of cellular metabolic process	1.36E-19	2.09E-16	2.05
		SP protocol
	GO:0019222	regulation of metabolic process	6.36E-14	9.74E-10	1.56
	GO:0065009	regulation of molecular function	1.18E-12	9,00E-09	1.95
	GO:0031323	regulation of cellular metabolic process	1.94E-12	9.89E-9	1.56
	GO:0010033	response to organic substance	2.33E-12	8.92E-9	2.21
	GO:0048522	positive regulation of cellular process	4.38E-12	1.34E-8	1.62
	GO:0048523	negative regulation of cellular process	5.41E-12	1.38E-8	1.68
	GO:0048519	negative regulation of biological process	1.82E-11	3.98E-8	1.61
	GO:0060255	regulation of macromolecule metabolic process	2.07E-11	3.96E-8	1.54
	GO:0048518	positive regulation of biological process	2.23E-11	3.8E-8	1.54
	GO:0042221	response to chemical	2.36E-11	3.61E-8	1.99

Supplementary text S1: Vibrational signature of human dermal substitutes

The FTIR-ATR spectra of the top and bottom sides of dermal substitute in control condition, in the [3800-1300 cm -1 ] range is shown in Fig S1a . Main absorption bands detected from the spectrum of both the bottom and/or top sides, and their respective assignment are summarised in Table S1. Those were interpreted according to values reported in literature. In the [3000-2800 cm -1 ] range, asymmetric and symmetric stretching absorption modes of methylene group as(CH2) and s(CH2) located at 2927 cm -1 and 2850 cm -1 respectively, are mainly associated with lipids and widely used to quantify them. In the [1800-1500 cm -1 spectral region, protein absorption is represented by Amide I and Amide II bands. The amide I band, mainly arising from C=O stretching, is very sensitive to secondary structures in proteins. It must be underlined that O-H bending vibration of water is superimposed to the Amide I vibration in hydrated samples. In contrast with Amide I, Amide II, that mostly combines N-H bending coupled to C-N stretching, is not sensitive to water and largely used as a normalisation peak to compare whole spectra. Another absorption mainly attributed to proteins is the methyl bending δas(CH3) visible at 1452 cm -1 , whose area is used in further ratio indicators. The enlargement of the [1500-900 cm -1 ] range (Fig S1b), generally known as the fingerprint region, can reveal subtler molecular differences in biological tissues.

The 1338 cm -1 band (wagging of proline side chain) is thus referenced in literature as a specific absorption of collagen and can be used to quantify this major ECM protein [START_REF] Tang | Identification of ageing biomarkers in human dermis biopsies by thermal analysis (DSC) combined with Fourier transform infrared spectroscopy (FTIR/ATR)[END_REF][START_REF] Belbachir | Collagen types analysis and differentiation by FTIR spectroscopy[END_REF]. The area ratio A(1338 cm -1 )/A(1452 cm -1 ) is thus computed to determine collagen content from tissue side.

As referenced in Table S1, the 1082 cm -1 band absorption is mainly assigned to the s(PO2 -) of DNA and phospholipids of cell membranes [START_REF] Wood | The importance of hydration and DNA conformation in interpreting infrared spectra of cells and tissues[END_REF], with the contribution of the C-O of collagen and glycosaminoglycans. In addition, the 1060 and 1032 cm -1 are mainly assigned to the C-O stretching vibration of ribose and other carbohydrates residues [START_REF] Sanden | The use of Fourier-transform infrared spectroscopy to characterize connective tissue components in skeletal muscle of Atlantic cod (Gadus morhua L.)[END_REF]. This area ratio A(1140-1000 cm -1 )/A(1452 cm -1 ) zone is thus assigned to a "mixed" zone, peculiar sensitive to cellular material and GAGs.

Finally, the 990 band is specifically assigned to uracil ring motions of RNA while the 970 cm - 1 is assigned to DNA (mainly) and RNA ribose-phosphate skeletal motions (Wood et al, Table S4. Transcriptomic analyses of differentially expressed genes in "matrix-associated proteins" sub-panel. Annotation according to [START_REF] Naba | The extracellular matrix: Tools and insights for the "omics" era[END_REF]. Genes in bold are modulated after electrostimulation and described in columns "LP" and "SP": fold changes are indicated in brackets after gene symbols, down-regulated genes (green), up-regulated genes (red). p-value < 0.05. n=4.

Gene symbol LP SP

ECM-affiliated Proteins

Supplementary text S4: DSC characterization of whole dermal substitutes after electrostimulation Method. DSC measurements on hydrated dermal samples were performed with a DSC Pyris calorimeter (PERKIN ELMER, Waltham, MA) using an empty pan as reference. Samples (10-30 mg by mass) were sealed in hermetic aluminium pans and then submitted to a temperature program under helium atmosphere: a first cooling scan was performed from 20 to -100 °C at -10 °C min -1 followed by a heating scan from -100 to 85 °C at 10 °C min -1 . After completing the DSC measurements, pans were reweighted to check that they had been correctly sealed. The sample pans were pierced and dried to constant mass at 105 °C for 14 h to determine the sample dry mass and by difference the total amount of water in defrosted samples. are labelled with Hoechst (blue), electropermeabilized cells with propidium iodide (pink) and collagens are observed by second harmonic generation (cyan). e. Cell viability determined with PrestoBlue assays 24h after electric field applications (n=12) indicates that these electrical parameters induce a reversible electroporation not leading to cell death. Data are represented as the mean value ± SEM. ns: non-significant. Long pulses (LP): 10 pulses 5 ms long, 600 V/cm at 1 Hz frequency; Short pulses (SP): 8 pulses 100 µs long, 1000 V/cm at 1 Hz frequency.

Supplementary references

Badea, E., Della Gatta, G. & Budrugeac, P. Characterisation and evaluation of the environmental impact on historical parchments by differential scanning calorimetry. J. Therm. Anal. Calorim. 104, 495-506 (2011).


Zaheer Ahmed Ujjan

Aqeel Muhammad Ali Bhatti

Ahmed Shah

Aneela Tahira

Nek Muhammad Shaikh

Shusheel Kumar

Abdul Qayoom Mugheri

Shymaa S Medany

Ayman Nafady
email: anafady@ksu.edu.sa

Fahad Alnjiman

Aqeel Ahmed Shah

Aymannafady

Mélanie Emo

Brigitte Vigolo

Zafar Hussain

Dr M A Kazi

Hussain Zafar

Ayman Ibupoto
email: zaffar.ibhupoto@usindh.edu.pk

Nafady

Simultaneous doping of sulfur and chloride ions into ZnO nanorods for improved photocatalytic properties towards degradation of methylene blue

Keywords: Nonmetal dopant, ZnO, Methylene blue, photodegradation 1. Introduction

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

Over the recent years, the textile industry is one of the most pollutants releasing industry into the environment. These pollutants contain a heavy load of chemicals including dyes and they have been cause of harming our environment. These dyes are found at large quantities into the waster of textile industry which not only affect the human life but as well as aquatic life. Furthermore, these dyes exist in various forms such as an acidic, disperse, fabric, mordant, reactive, vat, and ingrain azo. These dyes are found as colored in the wastewater. The structural information of dyes tells that they contain an aromatic ring in their structure, and they can be dissolved either into organics or inorganic solvents and they can be found as suspended solids which are supposed to be cause of toxicity. These colored substances cause serious health problems including skin disease, allergy, cancer and mutation. These substances are not only affecting the human organs such as kidney, liver, central nervous system and brain, but also at the same to the aquatic life by acting as shield for the sunlight and thereby resisting the access of light to aquatic life. In addition, the survival of human and aquatic life in their ecosystem is probably impossible without the availability of pure water to them. The inappropriate disposal of by-products and an excessive use of chemicals into the industry have degraded the quality of natural water [1][2][3]. The methylene blue (MB) discharged by thedying industries is one of the most prominent dye among the different dyes. For the removal of MB various methods such as reverse osmosis, adsorption on activated carbon, coagulation, poly-electrolyte promoted, nano-filtration, electrochemical treatment, biological treatment in particularly enzyme and photo-catalytic degradation have been employed [START_REF] Viswanathan | Photocatalytic Degradation of Dyes: An Overview[END_REF].Photocatalytic degradation is one of the most competitive methods whicheffectively solve the degradation issue of dyes from wastewater and it has several advantages including simplicity, efficient, and do not produce secondary pollutants [5]. The photocatalytic method requires a high energy light irradiation source and an oxidant to generate hydroxylradicals which are involved degradation process. All the renewable technologies have attracted huge attention because they offer promising results and can be used as potential alternative for the energy generation and wastewater treatments. Photocatalysis is supposed to be an efficient technique to degradethe recalcitrantin wastewater [6]. Many researchers reported the use of semiconducting materials (TiO2, CdS, ZnO, WO3, Fe2O3, SnO2 etc) as photocatalyst or the wastewater treatments [7][8][9][START_REF] Saravanakkumar | Synthesis and characterization of CuO/ZnO/CNTs thin films on copper substrate and its photocatalytic applications[END_REF][START_REF] Zyoud | Alternative natural dyes in water purification: Anthocyanin as TiO2-sensitizer in methyl orange photo-degradation[END_REF][START_REF] Elango | Efficacy of SnO2 nanoparticles toward photocatalytic degradation of methylene blue dye[END_REF].

Among the various semiconductor materials, ZnO is generally considered as a good photocatalyst for the degradation oforganic dyes due to its stability in chemicals properties, non-toxicity and inexpensiveness [START_REF] Ameen | Solution-processed CeO2/TiO2 nanocomposite as potent visible light photocatalyst for the degradation of bromophenol dye[END_REF]. However, some major drawbacks including its wide band gap 3.37 eV (bulk) which may limit the absorption of light in the ultraviolet region and fast rate of electron-hole recombination rate which severely affects the performance of ZnO as photocatalyst. Therefore, the development of efficient and stable photocatalysts based on ZnO for the practical applications are highly needed. We need to utilize the large region of solar spectrum and to overcome the recombination rate of electron and hole pairs. These are the main challenges in the current state of art of photocatalysts based on ZnO [START_REF] Das | Engineering of ZnO nanostructures for efficient solar photocatalysis[END_REF]. The photo-response of ZnO may be enhanced in the visible spectrum of solar light by employing various alternative strategies including doing into ZnO, co-doping, coupling with lower band semiconductors, surface plasmon response, quantum dots, sensitization with natural and synthetic dyes [START_REF] Schumann | Promoting Strong Metal Support Interaction: Doping ZnO for Enhanced Activity of Cu/ZnO:M (M = Al, Ga, Mg) Catalysts[END_REF][START_REF] Kumari | N-and C-doped ZnO as semiconductor photocatalysts: A review[END_REF][START_REF] Bharat | Synthesis of Doped Zinc Oxide Nanoparticles: A Review[END_REF][START_REF] Poornaprakash | Effects of Ce incorporation on the structural, morphological, optical, magnetic, and photocatalytic characteristics of ZnO nanoparticles[END_REF][START_REF] Poornaprakash | Achieving enhanced ferromagnetism in ZnTbO nanoparticles through Cu co-doping[END_REF][START_REF] Poornaprakash | Co-doped ZnS nanoparticles: Structural, chemical, optical, and photocatalytic properties[END_REF][START_REF] Kaur | Rapid Solar-Light Driven Superior Photocatalytic Degradation of Methylene Blue Using MoS2-ZnO Heterostructure Nanorods Photocatalyst[END_REF][START_REF] Vallejo | Methylene Blue Photocatalytic Degradation under Visible Irradiation on In2S3 Synthesized by Chemical Bath Deposition[END_REF][START_REF] Subash | Highly Efficient, Solar Active, and Reusable Photocatalyst: Zr-Loaded Ag-ZnO for Reactive Red 120 Dye Degradation with Synergistic effect and Dye-Sensitized Mechanism[END_REF][START_REF] Aby | Plasmon Mediated Photocatalysis by Solar Active Ag/ZnO Nanostructures: Degradation of Organic Pollutants in Aqueous Conditions[END_REF][START_REF] Díaz-Uribe | Photocatalytic Activity of Ag-TiO2 Composites Deposited by Photoreduction under UV Irradiation[END_REF][START_REF] Chen | Synthesis and photocatalytic application of Au/Ag nanoparticle-sensitized ZnO films[END_REF][START_REF] Poornaprakash | Co-Doped ZnS Quantum Dots: Structural, Optical, Photoluminescence, Magnetic, and Photocatalytic Properties[END_REF][START_REF] Youssef | Dye-sensitized nanoparticles for heterogeneous photocatalysis: Cases studies with TiO2, ZnO, fullerene and graphene for water purification[END_REF][START_REF] Vallejo | Methylene blue photocatalytic degradation under visible irradiation on TiO2 thin films sensitized with Cu and Zn tetracarboxyphthalocyanines[END_REF][START_REF] Vallejo | Photocatalytic activity of graphene oxide-TiO2 thin films sensitized by natural dyes extracted from Bactris guineensis[END_REF][START_REF] Diaz-Uribe | Potential use of an anthocyanin-rich extract from berries of Vaccinium meridionale Swartz as sensitizer for TiO2 thin films-An experimental and theoretical study[END_REF]. Doping is known as the suitable approach to increase the optical spectrum and catalytic properties [START_REF] Hamid | PhotocatalyticWater Oxidation on ZnO: A Review[END_REF][START_REF] Türkyılmaz | Photocatalytic efficiencies of Ni, Mn, Fe and Ag doped ZnOnanostructures synthesized by hydrothermal method: The synergistic/antagonistic e_ect between ZnO andmetals[END_REF]. The literature survey shows that some nonmetal dopants like (e.g. carbon (C), nitrogen (N), and sulfur (S)) into ZnO cause the creation of intermediate or lower energy levels between the band gap, enabling ZnO to absorb visible light and minimize the recombination rate of electron-hole pairs due to photoexcited electrons to nanosized nonmetal atoms on the surface of the catalyst, therefore efficiently degraded the dyes in the wastewater [START_REF] Chen | Characterization and photoreactivity of N-, S-, and C-doped ZnO under UV and visible light illumination[END_REF][START_REF] Liu | Improved visible-light photocatalytic activity of porous carbon self-doped ZnO nanosheetassembled flowers[END_REF][START_REF] Lavand | Synthesis, Characterization, and Visible Light Photocatalytic Activity of Nanosized Carbon Doped Zinc Oxide[END_REF]. The use of S and Cl separately as nonmetal dopant into ZnO has been carried out, however the simultaneous doping of S and Cl into ZnO is not reported elsewhere.

In this study, the doping of S and Cl into ZnO simultaneously has been studied for the photodegradation of MB. The doping of S and Cl into ZnO has been carried out by hydrothermal method. The structural studies of doped samples were carried out by SEM, XRD, HRTEM and EDS techniques. The optical characterization on the nonmetal doped ZnO was studied by UVvisible spectroscopy. The performance of doped samples was found very efficient for the degradation of MB and efficiency of 99.66 % is reported herein.

Experimental Methodology

Materials

The chemicals use din this study include ammonium sulphide (NH4)2S or N2H8S, ammonium chloride (NH4Cl), sodium hydroxide (NaOH), zinc acetate-dihydrate(ZnC4H6O4), 25% aqueous ammonia solution Merck), methylene blue (C16H18N3SCl) organic dye, and ethanol (C2H5OH, 99.5%) were purchase from Sigma-Aldrich Karachi, Pakistan and used without further purification. The deionized water (DI) was used in all the experiments.

Synthesis of simultaneously S and Cl doped ZnO nanorods by hydrothermal method

The hydrothermal method was employed for the synthesis of ZnO and S as well as Cl doping ZnO nanostructures. We used zinc acetate di-hydrate (ZnC4H6O4) and 25 % ammonia for the fabrication of ZnO. The 0.01 M concentration of ammonium chloride and 0.01 M concentration of ammonium sulphide, 0.1 M of sodium hydroxide and 0.1 M of zinc acetate di-hydrate were mixed in equal volume of ethanol and deionized water and their well dispersion was obtained by employing ultrasonic bath for 30 min.The pristine ZnO was prepared by same method by dissolving 0.1 M zinc acetate di-hydrate and 0.1M NaOH. The aluminum foil was employed to seal the beakers in order to avoid the evaporation of aqueous solution during hydrothermal process. The growth process was carried out in preheated electric oven at 90 °C for 5 h. Finally, filtration and drying processes were employed to collect the white product of ZnO nanostructures. X-ray diffraction was performed with powder diffractometer (D8 Advance, Bruker) employing the experimental conditions of CuKα radiation (λ= 1.54050 Å), 45 mA, 45 kV and at a scan rate of 1 o min -1 .Scanning electron microscopy was carried out with a ZEISS Gemini SEM 500 equipped with field emission gun. The elemental analysis was determined by energy dispersive X-ray spectroscopy (EDS) embedded in the scanning electron microscope. The scanning transmission electron microscopy (STEM) observations were carried out with a JEOLARM 200-Cold FEG at 80 kValong with energy dispersive X-ray (EDX). For the observations, the powder was dispersed in ethanol and a drop of the dispersion was deposit on a holey carbon grid (200 mesh size). The degradation rate and the quantitative decrease in the cocnetratin of MB was quantified by a UV-visible spectrophotometer (PerkinElmer, Lambda 1050).

Photocatalytic Activity of prepared Zno nanostructures

The photocatalytic activity of as synthesized ZnO nanostructures was investigated with the degradation of methylene blue (MB) dye using different dose concentrations0.62×10 -6 , 1.87×10 - 6 , 2.50×10 -6 M in deionized water. Pristine and S-Cl doped ZnO nanostructures of 10 mg were placed horizontally in the two 100 mL beakers containing 1.87×10 -6 M of MB dye and exposed to ultraviolet (UV) light. Typically the reaction suspension was prepared by adding different dose concentration of (5, 10, 15) mg of the as-prepared S-Cl-doped ZnO powders into 100 mL of 10 mg methyl blue aqueous solution.Before applying UV light, the aqueous suspension of stock solution containing different catalyst and MB (0.62×10 -6 , 1.87×10 -6 , 2.50×10 -6 M) dye dose concentration placed in sonicator for 30 min for achieving adsorption-desorption equilibrium.

Later on, the MB stock solution was irradiated with UV-Visible light under the room temperature.

The UV-Visible light was employed for carrying out the degradation activity through locally made photoreactor using a compact UV lamp of (365 nm) and a power of 18 watt were applied. The UV-visible spectrophotometer (PE Lamda35) was employed to record the main absorbance peak to quantify the MB dyes which detected at (664) nm. The UV-visible spectrophotometer recorded the absorption spectrum after every 30 min intervals to monitor the MB concentration in the stock solution. The intensity of absorption spectrum at characteristic wavelength of (664) nm was observed to determine the concentration of MB by recording UV visible spectra in the wavelength range of 200-800nm. The mathematical formula used for the calculation of decolorization efficiency for as synthesized intrinsic ZnO and S-Cl doped ZnO nanostructures was as follows:

Degradation Efficiency (%) = ( ) × 100 (1)
Where Co(gm/ ) is the initial dye concentration and Ct (gm/ ) shows MB dye (mg/L) after certain interval of time. EDX is a very reliable technique to quantitatively analyze the presence of zinc, oxygen, sulfur and chlorine in the S-Cl-doped ZnO sample as shown in Figure 2(a &b). The EDX patterns reveal only peaks at the energies of zinc and oxygen for pristine ZnO (Figure 4a), indicating clearly the purity of synthesized nanostructures as shown in Figure 2(a), while for S-Cl doped ZnO, the spectrum shows peaks corresponding to sulfur, chlorine, zinc and oxygen atoms (Figure 2b), confirming the successful doping of S-Cl atoms in the lattice of ZnO. STEM High Angle Annular Dark Field (HAADF) images of hydrothermally synthesized pristine and S-Cl doped ZnO nanostructures are presented in Figures 3a and4a respectively. Figure 3a-shows that pristine ZnO possesses a rod-like structure with a length of several microns, as shown previously by SEM. The product obtained by addition of S and Cl as a dopant into ZnO is heterogeneous and consists of a mixture of nanorods and nano-platelets (Figure 4a). The length of nanorods decreases with the addition of S and Cl. X-maps of detected elements are shown on Figures 3b-c and4b-d. These observations highlighted that sulfur is incorporated mainly in the nano-platelets. The chlorine is mainly shown by EDS via SEM analysis and the percent distribution of chlorine was not uniform throughout the sample.

Results and Discussions

Structural characterization of various ZnO nanostructures

The pristine ZnO and S-Cl doped ZnO nanostructures were characterized by powder XRD. Fig. 5(a,b) reveals that the powder X-xay diffraction detected the patterns within the range of 20-79 o for pristine ZnO and S-Cl doped ZnO nanostructures with a scanning step of 0.01 o .The sharpness as well as absence of impurity indicates the high purity of as-synthesized nanomaterial.

Furthermore, the sharp peaks may be related to the planes which measured at 2 theta angle of [START_REF] Kang | A facile gelatin-assisted preparation and photocatalytic activity of zinc oxide nanosheets[END_REF]. The XRD results are according to the reported reference JCPDS card No. 01-075-1533, and could be assigned to the hexagonal wurtzite structure of ZnO [START_REF] Gautam | Role of Cl Doping on the Growth and Relaxation Dynamics of ZnO Nanorods Synthesized by Hydrothermal Method[END_REF]. However,the XRD results for the doped sample clearly indicates the increasing order in the red shift. The red shift of dominant peaks is observed at 2ϴ angle of 31.24, 33. The ratio of c/a was found 1.732 which is very close value with ideal value of 1.6.This increase in lattice parameters of S-Cl doped ZnO may be caused by addition of dopant whose ionic radii such as S(r =1.09 Å),Cl -(r =1.81Å) into pristine ZnO with atomic radii such as Zn(1.38 Å ) and O(1.40), resulting in expansion of the S-Cl-ZnO lattice structures [START_REF] Jiamprasertboon | Low-Cost One-Step Fabrication of highly Conductive ZnO:Cl Transparent Thin Films with Tunable Photocatalytic Properties via Aerosol-Assisted Chemical Vapor Deposition[END_REF]. In addition the doping of S-Cl into ZnO can further alter the distance in crystal structure as well as in diffraction angle. Moreover, the crystallite size of the as-prepared samples was evaluated from Scherer's relation [START_REF] Xu | Quality-Improved GaN Epitaxial Layers Grown on Striped Patterned Sapphire Substrates Ablated by Femtosecond Laser[END_REF].

( ) = ( ) ( ) (2)
Whereas D represents crystalline size (nm), K denotes shape factor K= 0.94, λdepicts wavelength al has also reported the decrease in crystalline size of La-doped ZnO nanostructures [START_REF] Nguyen | Preparation, Characterization and Photocatalytic Activity of La-Doped Zinc Oxide Nanoparticles[END_REF]. These size characteristic is an essential parameter for possibly improving the photocatalytic properties of S-Cl-doped ZnO nanostructures. These crystal structure parameters values are summed up in Table 1.

Optical properties of synthesized nanostructures

The exploration of optical properties of pristine ZnO and S-Cl doped ZnO nanostructures were carried out using UV-visible absorption spectroscopy. The UV-vis absorption spectrum whose wavelength range between (200-800 nm) was recorded as shown in Fig. 6(a)

The effect of incorporation of S-Cl as dopant into ZnO was carefully analyzed which depicted the change appears in absorption wavelength and it shifted to longer wavelength for S-Cl doped ZnO, which may be assigned to agglomeration of the nanostructures. The literature survey shows that the shifting of absorbance wavelength to larger wavelength is in line with reported work [START_REF] Nguyen | Preparation, Characterization and Photocatalytic Activity of La-Doped Zinc Oxide Nanoparticles[END_REF].It is worth noting that the band gap energy is considered to be main indicator for the determining the potential and for assigning the difference between conduction band (CB) and valence band (VB.Tauc-equation was used to evaluate the band gap energy (Eg) (* -+! ,-./,0 1) + ℎ3 → / 5 + ℎ 65 7 (4)

(αhν) % = k(hν -E ) ) (3)

Photodegradation activity towards methylene blue

/ 5 + 1 8(9: ;<=:) → 1 8 • ⇒ 21 8 • + A 7 → A 8 1 • + 1 8 • (5) ℎ 65 7 + A 8 1 → A 7 + A1 • (6)
/ 5 + ℎ 65 7 → (* -+! ,-./,0 1) + A/BC (7)

A 8 1 8 + / 5 → A1 • + 1A (8) A 8 1 8 + 1 8 • → A1 • + 1A + 1 8 (9) A 8 1 8 + ℎ3 → 2A1 • (10)
Finally the hydroxyl radicals are used as the scavengers to decompose MB dye pollutant through following reactions [START_REF] Jiamprasertboon | Low-Cost One-Step Fabrication of Highly Conductive ZnO:Cl Transparent Thin Films with Tunable Photocatalytic Properties via Aerosol-Assisted Chemical Vapor Deposition[END_REF] The degradation of MB was carried out at various initial concentrations such as (0.62, 1.87 and 2.50) ×10 -6 M using pristine ZnO and S-Cl doped ZnO with dosage of 10 mg under UV irradiation.

D/ + (1 8 • → A1 • + A1 8 • ) → E C/F /,GBC/ → HF-,IJC (11)
The obtained results revealed that the degradation efficiency and rate constant of MB with constant dosage of photocatalyst 10 mg was found in inversely relation with the initial concentration dye as shown in Fig 12 . The decreased performance is shown the effect of varying MB dye concentration on photocatalytic performance may be assigned to increasing the adsorption of MB dye on the surface of S-Cl doped ZnO, resulting in reduction of -OH concentration on sites of catalyst [START_REF] Lavand | Synthesis, Characterization, and Visible Light Photocatalytic Activity of Nanosized Carbon Doped Zinc Oxide[END_REF][START_REF] Malik | Visible light-driven mesoporous Au-TiO2/SiO2 photocatalysts for advanced oxidation process[END_REF]. Beer-Lambert's law also supports the reverse relation between initial dye concentration and photocatalytic activity because the intensity of photons as well as the adsorption of photons on the surface of catalyst is limited which leading to the lowering the rate of photodegradation process. The degradation efficiency of 0.62×10 -6 M MB concentration could reach up to the 93.46% with photocatalyst dosage of 10 mg over the same time of 150 min. ZnO caused the reduction in band gap from 3.25 eV to 2.9 eV which attributed incorporation of S and Cl in lattice of ZnO [START_REF] Bhatti | Efficient photo catalysts based on silver doped ZnO nanorods for the photo degradation of methyl orange[END_REF]. The band gap findings are summarized in table 2, whereas the degradation efficiency and rate coefficient findings are tabulated in Table 3. Moreover, the recent literature survey reveals that numerous oxidants such as peroxymonosulfate, hydrogen peroxide (H2O2), peroxydisulfate, ClO 3-etc. in heterogeneous photocatalytic system may lead to the increase in the degradation efficiency following either by limiting e -/h + recombination rate with the generation of superoxide radical ion (O2• -) by the capturing of conduction band electrons at the surface of ZnO photocatalyst or by providing additional oxygen atoms acting as an agent to capture electron [START_REF] Subhan | Photocatalytic and Antibacterial activities of Ag/ZnO Nanocomposities Fabricated by Co-Precipitation Method[END_REF]. Herein, reports of previous study on the effect of varying dose of oxidant H2O2 (5 -30 m mol) revealed that addition of H2O2 into thin film N-doped ZnO/CNT

  The morphological investigations of pristine ZnO and S-Cl doped ZnO were evaluated by employing the scanning electron microscope. It is well observed through the SEM study that pristine ZnO and S-Cl-doped ZnO nanostructures have almost homogeneous morphology, however some randomly oriented particles are associated with the S and Cl doped sample as shown in Figure1. The purse ZnO exhibits well defined nanorod like morphology with random alignment.The length of ZnO nanorods could be few microns and the average diameter around 200 nm as shown in Figure1a-b. The S and Cl doped ZnO sample possesses a heterogenous morphology consisting nanorods and cluster of particles as shown in Figure1c-d. The size of particles could be larger than 100 nm and the length of nanorods is decreased with the addition of S and Cl source due to etching effect.

  78 and 35.84 degree lying at plane (100), (002) and (101) respectively as depicted in Fig 5(b) .The shift in S-Cl doped ZnO sample might effect on the optical and morphological properties. Furthermore, the increasing order was observed in lattice parameters a and c and volumes V in the doped ZnO nanostructures. The lattice parameters 'a' and 'c' value were observed larger in S-Cl doped ZnO as compared to pristine ZnO.

  photogenerated charge carriers react with absorbed molecules to form hydroxyl and superoxide radicals. These values were obtained in decreasing order with incorporation of S-Cl as a dopant concentration into ZnO. The observed decreasing behavior of crystalline size could be attributed to the diffusion and growth impairment of ZnO crystal grains, which is caused by the replacement of oxygen (r =1.40 Å) in ZnO with either of S(r=1.09 Å) orCl -(r= 1.81Å). Loan T. T. Nguyen et.

  photon energy respectively and the optical band gap is denoted by Eg (e.V) and n=2.Tauc's plots of ZnO and S-Cl doped ZnO are represented in Fig6(a-b). The insets in Fig3(a-b) reveals the linear relation between (αhν) 2 and hν and this linear portion of Tauc's plot was extrapolated to the ordinate as to be equal to 3.24 eV. The addition of S-Cl as a dopant slightly reduces this value to 2.9 eV as shown in inset of Fig 3b,which reveals the ability of the synthesized nanostructures to harvest visible light. This band gap Eg value agrees with most of the reported studies about the nonmetal doped ZnO[START_REF] Chen | Characterization and photoreactivity of N-, S-, and C-doped ZnO under UV and visible light illumination[END_REF][START_REF] Liu | Improved visible-light photocatalytic activity of porous carbon self-doped ZnO nanosheetassembled flowers[END_REF][START_REF] Lau | Zheng Eco-Friendly Photocatalysts for Degradation of Dyes[END_REF]. The decreased value of band gap was assigned to defect states between the conduction band and valence band of S-Cl-ZnO, which may possibly arise due to the interaction of S-Cl-doping into ZnO. Thus, the S-Cl doped ZnO results in decline in the optical band gap energy which might enhance the photocatalytic properties of S-Cl-ZnO nanostructures to perform efficient degradation of organic pollutants.

Fig. 7

 7 Fig. 7 shows the chemical structure of Methylene Blue (MB).

Fig: 6

 6 Fig:6(a)schematically reveals that the MB dye decreases slowly over the time of 150 min at the photocatalyst dose of 10 mg. Whereas in the case of S-Cl-doped ZnO the decreases the height of absorbance peak in concentration of the MB over the irradiation time 150 min as shown in Fig (9, 10). The photocatalytic degradation activity may be affected by various factors such as amount of catalysts, concentration of MB and radiation of energy and irradiation time. For the optimization of photocatalysis process, it is essential to study these parametersto observe their effect on photocatalytic degradation.

Fig 12 (

 12 photocatalyst which is almost 13.74 times of pristine ZnO. The degradation efficiency of S-Cl doped ZnO was obtained almost 100%, which is almost 2.36 times of pristine ZnO. The enhanced photocatalytic efficiency was attributed to multi approaches reported in the previous studies[START_REF] Senthilvelan | 484 TiO2,ZnO and nanobimetallic silica catalyzed photodegradation of methylgreen[END_REF][START_REF] Xu | J Liu Photo-degradation of Acid Yellow 11 in aqueous on nano ZnO/Bentonite under ultraviolet and visible light irradiation[END_REF][START_REF] Chakrabarti | Dutta Photocatalytic degradation of model textile dyes in wastewater using ZnO as semiconductor catalyst[END_REF][START_REF] Soltani | Visible light-induced degradation of methylene blue in the presence of photocatalytic ZnS and CdS nanoparticles[END_REF]. In addition, the band gap of pristine ZnO and S-Cl doped ZnO play influential role in enhancing photocatalytic activity for the MB. The previous research reveals that lowering the band gap is proportional to the mineralization of MB. In our case the nonmetal doping such as S-Cl into

Figure 1 :

 1 Figure 1: Distinctive SEM images (a-b) Pristine ZnO, (c-d) S-Cl doped ZnO nanostructures

Figure 4 :

 4 Figure 4: STEM HAADF (a) image and corresponding X-maps of Zn (b), O (c) and S (d) elements for the S-Cl doped ZnO sample.

Figure 5 .

 5 Figure 5. (a) showsXRD patterns for pure and S-Cl-doped ZnO nanostructures, which can be marked as a single phase of wurtzite ZnO structure (b)shows increasing shift in peak at 2Theta angle of 31.17, 33.58, 35.68 degree.

Figure: 6 .

 6 Figure: 6. (a-b) shows findings of the band gap of Pristine ZnO and S-Cl doped ZnO deduced from recording the UV-vis spectrum.

Figure: 7 .

 7 Figure: 7. Chemical Structure of Methyl Blue

Figure: 9 .

 9 Figure: 9. (a-c) UV-Vis absorption spectrum of MB = 1.87x10 -6 M at different concentration of photocatalyst 5, 10 and 15mg.

Figure: 10 .

 10 Figure: 10. (a-c) showing UV-Vis absorption spectrum for the different concentration of MB Dye (0.62, 1.87 and 2.50) x10 -6 M at photocatalyst loading of 10 mg.

Figure: 11 .

 11 Figure: 11. (a) Depicts graph Ct/Co versus time indicating decreasing trend for (5, 10 and 15 mg) of S-Cl-ZO at 1.87 x10 -6 M of MB (b) linearly fitted graph Ln (Co/Ct ) versus time showing increasing degradation rate for (5, 10 and 15 mg) of S-Cl-ZnO (c) Photodegradation Efficiency of S-Cl-ZnO nanostructures at (5, 10 and 15 mg)

Figure: 12 .

 12 Figure: 12. (a) Depicts graph Ct/Co versus time indicating decreasing trend for (0.62, 1.87 and 2.50) x10 -6 M for 10 mg of S-Cl-ZnO (b) linearly fitted graph Ln (Co/Ct ) versus time showing increasing

Figure: 13 .

 13 Figure: 13. (a) Degradation rate constant for different catalyst loadings (b) Degradation rate constant for varying MB dye concentration

  31.86 • (100), 34.54 • (002), 36.32 • (101), 47.62 • (102), 56.66 • (110), 62.96 • (103), 66.44 • (200), 68.04 • (112), 69.18 • (201), 72.34 • (004), 76.76 • (202) andshowing the clear randomly oriented ZnO nanostructures

Table 1 :

 1 Crystal Parameters of Pristine ZnO as well as S and Cl doped ZnO Nanostructures.

Table 2 :

 2 Band gap of Pristine ZnO and S-Cl doped ZnO Nanostructures.

	Samples	Band Gap (eV)
	Pristine ZnO	3.25
	S-Cl-ZnO	2.90

Table 3 :

 3 Photocatalytic degradation rate constant (k) and Degradation efficiency, RegressionCoefficient for different catalyst loading and different methyl blue under UV radiation.

	Index	Parameters		Pristine ZnO			S-Cl-ZnO
	1	Crystalline Plane	(100)	(101)	(110)	(100)	(101)	(110)
	2	2ϴ (Degree)	31.0869 35.6364 55.9486 31.7745 36.0246	56.3820
	3	FWHM (degree) 0.45938	0.6200	0.3971	0.6201	0.5377	0.6830
	4	Maximum Height 191.328	94.304	121.930 210.469 102.774	157.540
	5	Crystalline Size (nm)	35.292	50.2117	42.1591 26.2092	44.4225	23.4961
	6	d-spacig ( Å)	4.7324	0.9032	1.8822	4.3458	1.0433	6.6916

Acknowledgments

We would like to thank the platform "Microscopies, Microprobes and Metallography (3M)" (Institut Jean Lamour, IJL, Nancy, France) for TEM and SEM facilities and J. Ghanbaja for his valuable help for analysis TEM. A.N and S.S.M extend sincere appreciation to the Researchers Supporting Project (RSP-2021/79) at King Saud University, Riyadh, Saudi Arabia.

nanocomposite photocatalyst increase the degradation efficiency for the removal of MB dye in the presence of UV light irradiation [START_REF] Lv | Enhanced photocatalytic degradation of methylene blue by ZnO-reduced graphene oxide composite synthesized via microwaveassisted reaction[END_REF][START_REF] Elias | A Highly Efficient and Stable Photocatalyst; N-Doped ZnO/CNT Composite Thin Film Synthesized via Simple Sol-Gel Drop Coating Method[END_REF]. The oxidant H2O2 may be considered the responsible for the formation of highly reactive radical intermediates and the electron capturing agents.

Consequently, the concentration of •OH radicals may be increased by the addition of H2O2 to the heterogeneous system.

Conclusions

In summary, photocatalytic performance of the hydrothermally synthesized non-metal S-Cl doped ZnO and pristine ZnO nanostructures have been reported. The characterizing techniques such as SEM mapping, XRD and EDS, TEM were used to elucidate the structure, morphology, composition and crystal information. The optical band of ZnO was dropped from 3.25 eV to 2.9 eV with simultaneous doping of S and Cl. The maximum degradation rate constant was obtained 0.03188 min -1 for 0.15 mg/L loading of photocatalyst which is almost 15 times of pristine ZnO.

The degradation efficiency of 100% for the S-Cl doped ZnO over the time of 150 min was obtained which is almost 2.36 times of pristine ZnO. Thus, S-Cl doped ZnO nanostructures may be considered as the potential photocatalysts for purifying the waste water of textile industry and could be sued for other environmental applications.

Conflict of interest statement

Authors declare no conflict of interest in this study.

References:

Catalyst

Different Catalyst Loading (mg) at 1.87x10