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“Safe” Chitosan Zinc Oxide Nanocomposite Has Minimal OrganSpecific Toxicity on Early Stages of Zebrafish Development Nadin Younes, Gianfranco Pintus, Maha Al-Asmakh, Kashif Rasool, Salma Younes, Simone Calzolari, Khaled A. Mahmoud, and Gheyath Nasrallah ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01144 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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ACS Biomaterials Science & Engineering

“Safe” Chitosan/Zinc Oxide Nanocomposite Has Minimal Organ-Specific Toxicity on Early Stages of Zebrafish Development Nadin Younes1,2, ǂ, Gianfranco Pintus1,2 ǂ, Maha Al-Asmakh1,2, Kashif Rasool3, Salma Younes1,2, Simone Calzolari4, Khaled A. Mahmoud3,5, Gheyath K. Nasrallah1,2* 1Department

of Biomedical Science, College of Health Sciences, Qatar University, Doha,

Qatar 2Biomedical 3

Research Center, Qatar University, Doha, Qatar

Qatar Environment and Energy Research Institute (QEERI), Hamad Bin Khalifa University

(HBKU), Qatar Foundation, P.O. Box 34110, Doha, Qatar 4 ZeClinics 5

SL, PRBB (Barcelona Biomedical Research Park), 08003 Barcelona, Spain

Department of Physics & Mathematical Engineering, Faculty of Engineering, Port Said

University, 42523 Port Said, Egypt ǂ Equal contribution * Correspond

to: Gheyath K. Nasrallah

Department of Biomedical Science, College of Health Sciences, Qatar University, Doha, Qatar. Women’s Science building, C01, Tel: +974 4403 4817, Fax: +974-4403-1351, P.O Box: 2713, email: [email protected]

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Abstract Marine biofouling is considered one of the most challenging issues affecting maritime industries worldwide. In this regard, traditional biocides, being used to combat biofouling, have high toxicity toward the aquatic system. Recently, a new chitosan/zinc oxide nanoparticle (CZNCs) composite has been used as promising “green” biocides. It is thought that due to the eco-friendly nature of chitosan , CZNCs may pave the way to develop less toxic surfaces for combating marine fouling. Zebrafish has become one of the most employed models for ecotoxicology studies. Therefore, this study aims to comprehensively evaluate any potential acute, cardio, neuro, or hepatotoxic effect of CZNCs using zebrafish embryos. As evidenced by the acute toxicity assays, exposure of zebrafish embryos to CZNCs (25-200 mg/L) did not elicit any signs of acute toxicity or mortality, suggesting a hypothetical LC50 higher than the maximum dose employed. CZNCs, at a concentration of 250 mg/L also showed no cardiotoxic or neurotoxic effects. At the same dosage, a minor hepatotoxic effect was observed in zebrafish embryos exposed to CZNCs. However, the observed hepatotoxicity had no effect on embryos survival even after long-term (10-days) exposure to CZNCs. We believe our results add valuable information to the potential toxicity of chitosan/metal oxide nanocomposites, which may provide new insights for the synthesis of ecofriendly coatings with improved antifouling performance and a low adverse impact on marine environment. Keywords: chitosan zinc oxide, cardiotoxicity, neurotoxicity, hepatotoxicity, zebrafish


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Introduction: Water flooding or water injection is a process commonly used by oil-producing companies to increase the oil reservoir pressure, and therefore stimulate oil production1. Due to its size, the complexity of the injection system and the high salinity of water (~55,000 mg/L), this process faces several challenges including microbial growth, fouling and corrosion of the pipelines. One of the major problems associated with the water flooding process is the formation of biofilms, which accelerates the corrosion in the biologically conditioned metalsolution interface2. This microbiologically-influenced corrosion (MIC) processes represents about 10% of metal corrosion as a whole, which alone costs the American oil and gas industry about 10 billion dollars per year3. Samples from the seawater of the Arabian Gulf area indicated the presence of different microbial communities including sulfate-reducing bacteria (SRBs)4. Although present in small concentration, the level of these bacteria is enough to pose a pressing threat to the oil recovery process5. To overcome this problem, antimicrobial agents and corrosion inhibitors are widely used as biocides in the oil and gas industry6-7. However, traditional biocides may induce bacterial resistance and/or be detrimental to the environment by forming harmful disinfection byproducts8. Chitosan is a natural biopolymer obtained from chitin with a wide range of promising applications.9 In addition to its ecofriendly nature,10 chitosan has hydroxyl amino groups providing an excellent adsorbing capacity towards a wide range of contaminants.11 The abundant number of reactive hydroxyl and amino groups in chitosan can act as a natural capping agent during nanoparticles synthesis.12 However, the major drawback of chitosan is the low solubility in neutral and alkaline solution.9, 13-15 To overcome this issue and to increase its solubility and stability, several types of cross-linkers have been introduced to chitosan in order to make it more useful as a biopolymer. Cross-linkers can be present in several forms including polymers16, oxides17, metals18, and amino acids19 among other chemical entities. Due



to their photocatalytic and antimicrobial activities, chitosan-metal oxide nanocomposites have attracted much interest in several fields of the applied research. Due to its unique properties such as wide band gap energy (3.37 eV)20 , high surface area, high catalytic efficiency, chemical stability,20 and UV protection ability,21 zinc oxide is one of the most used crosslinkers for the synthesis of chitosan nanocomposite. In our previous study, we have primarily focused on evaluating the antimicrobial activity of CZNCs nanocomposite and assessing its stability, biocompatibility and environmental impact towards mixed SRB biofilm in seawater.22 Our previous findings showed a highly stable behavior of CZNCs when exposed to high salt concentrations of injecting water and a dose-dependent inhibition (0-250 mg/L) of the SRB growth.22 Although in the same work, we demonstrated no general acute toxicity effects of CZNCs on zebrafish embryos, to our knowledge there are no data concerning potential organ-specific and long-term toxicity of CZNCs. Therefore, using zebrafish as a model system, this study aims to design and implement a number of organ-specific (heart, muscles, nervous system, and liver) and long-term (10-day of exposure) toxicity assays to comprehensively evaluate any potential in vivo adverse effects of CZNCs. Since no in vivo toxicity studies have been performed on CZNCs, we investigated a wider range of concentrations (25-250 mg/L) that are consistent with the acute toxicity rating scale provided by U.S. Fish and Wildlife Service (USFWS).23 Studies investigating ZnO nanoparticles preparations,24-27 characterizations,26-30 including XRD studies,26-28 and toxicity

29-41

are extensively described in the literature. However, we

reported here that only 10% of ZnO linked to chitosan could not induce the same extent of acute toxicity 29-34 and organ-specific30, 35-41 reported earlier for ZnO alone. Our previous results 22

along with the current data indicates the CZNCs has minimal organ specific toxicity, even

at high dose, and thus are potentially safe biocide for aquatic use.


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2. Materials and Methods 2.1. Materials Low molecular weight (LMW) chitosan with 85 % degree of deacetylation was purchased from Sigma Aldrich Co., Ltd (USA). Zinc nitrate (hexahydrate), trypolyphosphate (TPP) and sodium hydroxide (NaOH) were obtained from Fisher Scientific. Dimethyl sulfoxide (DMSO); diethylaminobenzaldehyde (DEAB), a competitive inhibitor of aldehyde dehydrogenases known to cause mortality and teratogenic phenotype in zebrafish embryos was used as positive control (PC) in acute toxicity assays;42-44 haloperidol, an antipsychotic drug was used as a PC in cardiotoxicity assays;44-46 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), a neurotoxic drug causing permanent symptoms of Parkinson's disease, was used as PC in neurotoxicity assays;47 and APAP or paracetamol, was used as a PC in hepatotoxicity assays,48-49 were all purchased from sigma Aldrich. Stock solution of 60X E3 egg media was prepared from 8.765 g NaCl, 380 mg KCl, 1.185g MgSO4, and 1.765G CaCl2 dissolved 0.5 liters MilliQ water. All the drugs employed as PC where used within their reported biologically active concentration, as indicated in the appropriate references cited above. 2.2. Zebrafish embryos culture Fertilized embryos of wild type and transgenic green fluorescent proteins (GFP) AB zebrafish strains (Danio rerio) were collected in Petri dishes containing prepared E3 egg media. All experiments were performed on zebrafish embryos within the first four days post fertilization (4-dpf) with the exception of the long-term toxicity assays (10 days). All animal experiments were performed in accordance to national and international guidelines for the use of zebrafish in experimental settings50 and according to the animal protocol guidelines required by the laboratory animal and Policy on Zebrafish Research established by Zeclinic (Spain) and Department of Research in the Ministry of Public Health, Qatar.51 For more details about our fish husbandry and embryo culture, the readers may refer to our previous articles.22, 43, 52-53



2.3. Preparation of CZNCs CZNCs was synthesized by mixing the ChNPs and ZnONPs22. The detailed description of CZNCs composite synthesis is described in the supporting information (SI-1). Briefly, ChNPs was synthesized by cross-linking of chitosan solution (0.1% at 5.5 pH) with trypolyphosphate (TPP). The reaction was carried out by adding TPP solution drop wise to the chitosan solution at mass ratio of 2:1 for 10 min, washed three times and collected the pellets by centrifugation as ChNPs. ZnONPs were synthesized by adding 0.5% soluble starch to zinc nitrate (0.1 M) and mixed by magnetic stirrer. The solution was then titrated by adding NaOH (0.2 M) solution and allowed to stir for 2 h to get white suspension. The obtained suspension was washed, centrifuged and then finally dried to get ZnONPs. CZNCs were synthesized by adding ZnONPs (10% w/w) to ChNPs and dispersed uniformly by ultrasonication. The obtained suspension was centrifuged, thoroughly washed with DI Water, and finally freeze-dried. The freeze-dried powder was used as CZNCs for the further characterization and toxicity evaluation studies.

2.4. CZNCs characterization Synthesized CZNCs was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Zetasizer, and X-ray diffraction (XRD). SEM images of the samples were acquired after gold sputter coatings by a FEI Quanta 650 FEG SEM. For TEM, samples were prepared by dispersing in ethanol and mounted on a Cu grid and images were acquired by FEI Talos F200X TEM. The zeta potential of CZNCs was measured by analyzing electrophoretic mobilities (EPMs) using ZetaPALS analyzer (Malvern Instruments, Zetasizer Nano ZS). The hydrodynamic diameter was measured by dynamic light scattering using ZetaPALS analyzer (Malvern Instruments, Zetasizer Nano ZS). XRD analysis was done using a Bruker D8 Advance with a step scan of 0.02° per step and a scanning speed of 1°/min (Bruker AXS, Germany).


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2.5. Acute Toxicity Assays Mortality and developmental toxicity of the CZNCs nanomaterial was investigated with an acute toxicity assay adapted by the Organization of Economic Co-operation and Development (OECD) guideline for testing chemical toxicity (Nº 203 and 236). The purified (acetic acid-free) lyophilized CZNCs nanomaterial was weighted and dissolved in MilliQ water, then sonicated in a water bath for 15 min for several cycles until a complete homogeneous solution was achieved. The early life stages of zebrafish, embryos in particular, are generally more sensitive to chemical toxicants than adults.54-55 Therefore, embryos at 24– 96-hour post fertilization (96-hpf) were selected to test the potential toxicity of CZNCs. At 3hpf, healthy embryos were selected, then moved to a clean petri dish, where abnormal embryos were discarded. At 24-hpf, the fertilized embryos were dechorionated with pronase 1.0 mg/mL (Sigma Aldrich) and retained in 6 well-plates containing E3 egg medium. Next, zebrafish E3 egg medium was removed and replaced with the following treatment dissolved in E3 egg medium (i) CZNCs (25, 50,100, 250 mg/L) (ii) the positive control DEAB (0.1, 1, 10, 100, and 1.0 M); and (iii) the negative control 0.1% DMSO. Then, embryos were incubated at 28.5 ºC for additional 72 h until their age reached 96-hpf. Cumulative mortality was recorded at 3-time point intervals (48, 72, and 96-hpf), which are the recommended observational time54. In addition to the acute toxicity, all embryos were observed daily for the presence of teratogenic abnormalities such as body deformity, scoliosis, pigmentation, yolk size, heart edema, heartbeat, movement. The mortality rate was expressed as the number of dead embryos compared to the control groups after 96-hpf. After that, we attempt to identify the no observed effect concentration (NOEC) and the low observed effect concentration (LOEC) for both DEAB and CZNCs.56 Finally, the median lethal dose (LC50) was calculated by fitting sigmoidal curve to mortality data with a 95% confidence interval using GraphPad Prism 7. A



total of 100 embryos were used for each tested dose condition of the CZNCs, and 20 embryos for DEAB and DMSO concentrations. 2.6. Cardiotoxicity evaluation The cardiotoxicity assay were performed using the Tg[cmlc:GFP] transgenic AB strain of zebrafish. This strain expresses the GFP in the cardiac myocytes, thus allowing a good quality of cardiac imaging. Briefly, at 24-hpf, healthy embryos were selected and allowed to develop until 96-hpf. At this time, 10 embryos were incubated at 28.5ºC with the following treatment dissolved in E3 medium: (i) 250 mg/L CZNCs; (ii) 0.1% DMSO (negative control); and (iii) 10 µM haloperidol (positive control). After 4 h of treatment, the treated embryos were then anesthetized by immersion in 0.7 µM Tricaine methanesulfonate/E3 solution (A4050, Sigma-Aldrich, Saint Louis, MO, USA). For imaging, every embryo was positioned under the microscope using agarose based mold and the fluorescent heart was recorded for 60 sec57. Videos were acquired by high speed cameras and analyzed with the ZeCardio® software for the presence of any heart dysfunctions. In particular, the following cardiac parameters were assessed: heart rate, QTc corrected interval: [Framingham formula: QTc = QT + 0.154 (1 – RR)]58, cardiac arrest and ejection fraction [(Ef %) = ((DD-SD)/DD) x100. DD, ventricle diastolic diameter (max dilatation); SD, ventricle systolic diameter (max contraction)]. 2.7. Neurobehavioral toxicity evaluation At 24-hpf, healthy zebrafish embryos were selected and allowed to develop normally until 96-hpf. the embryos were then transferred to a 96-well plate (one embryo per well) and incubated for additional 24 h with the following treatments dissolved in E3 egg medium: (i) 250 mg/L CZNCs; (ii) 0.1% DMSO (negative control); and (iii) 100 µM of the neurotoxin 1methyl-4-phenyl-1 2 3 6-tetrahydropyridine (MPTP) (positive control). At 120-hpf, the neurotoxicity was analyzed by locomotion assessment using the EthoVision XT 11.5 tracking software and the DanioVision device (Noldus Information Technologies, Wageningen,


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Netherlands). DanioVision allows the tracking of individual zebrafish embryos during alternating dark and light cycles. The 96-wells plate was placed in the DanioVision chamber and illuminated for 20 min to allow embryos acclimation. Next, embryo movements were measured for 50 min under the following light/dark conditions: 10-min alternative light/dark trial (5 cycles) as shown in Figure 3B. Neurotoxicity was evaluated by comparing the above parameters between treated and control groups. Due to circadian rhythms, all locomotion assays were performed from 13:00 pm onwards to ensure steady activity of zebra fish larvae.59 2.8. Hepatotoxicity evaluation The hepatotoxicity assays were performed using the Tg[cmlc:GRP] transgenic AB strain of zebrafish. This strain expresses the RFP in the hepatocytes thus allowing a good quality of liver imaging. To evaluate the toxic effect of the nanomaterial on zebrafish liver, the following parameters were assessed: liver size (hepatomegaly or liver necrosis), yolk retention and steatosis. At 24-hpf, healthy embryos were selected and allowed to develop normally until 120hpf., which is the time at which the liver usually fully develops.60 The 120-hpf embryos were then incubated for additional 32h at 28.5 ºC with the following treatments: (i) 250 mg/L CZNCs; (ii) 0.1% DMSO, and (iii) 2% EtOH (positive control for steatosis assessment), 2640 µM Acetaminophen (APAP) and/or 2% EtOH (positive control for yolk retention and necrosis assessment). After that, the treated embryos were fixed in 4% paraformaldehyde (SigmaAldrich, Saint Louis, MO, USA) for 2-4 h at room temperature (RT) and then washed 3 times with 1X PBS. 2.8.1. Liver area analysis The fluorescent fixed embryos were observed under a fluorescence stereo microscope (Olympus MVX10) using a digital camera (Olympus DP71). RFP filtered images were obtained using cell’D and FIJI image processing package software for hepatomegaly or liver necrosis detection.



2.8.2. Detection of steatosis and yolk retention Steatosis and yolk retention were evaluated by Oil Red O staining (Sigma-Aldrich, USA). Zebrafish embryos were stained as previously described61. Briefly, the skin pigment of the fixed embryos was removed by an incubation with 0.1 ml of 5% sodium hypochlorite bleaching solution for 20 min followed by 5 washes with PBS at RT. Then, the bleached embryos were submerged in 85% propylene glycol (PG) (Sigma-Aldrich, USA) for 10 min, and then in 100% PG for another 10 min, before staining them with Oil Red 0.5% in 100% PG (overnight, at RT and with a gentle rocking). Oil Red O stained embryos were then washed in 100% PG for 30 min, 85% PG in PBS for 50 min and finally 85% PG in PBS for 40 min. Next, the embryos were washed with 1X PBS before adding 80% glycerol. Finally, bright field images were taken to detect both steatosis and yolk retention. For the analysis of steatosis, embryos were considered to be positive when 3 or more round lipid droplets were visible within the hepatic parenchyma44. Then, the percentage of steatosis was calculated by dividing the number of embryos showing steatosis with the total number of embryos observed. For the yolk retention, embryos showing red strong signal in the yolk area were considered positive. The percentage was calculated by dividing the number of positive embryos with the total number of embryos. 2.9. Statistical analysis Results were expressed as average ± SEM (standard error of mean). Statistical evaluation of differences between experimental group means was performed using one-way analysis of variance (ANOVA), followed by the Dunnet test. The Chi-square test was used for the hepatotoxicity assays (steatosis and yolk retention) to compare the significance between the percentages. Outliers were eliminated by using the Graph Pad software. Significance (*) = p

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