In Vitro Assessment of Anthelmintic Activities of AgO Nanoparticle in Comparison to Closantel Against Liver Fluke Dicrocoelium Dendriticum

Background: Dicrocoeliasis is a rare Food-Born parasitic disease of the grazing herbivores as well humans, caused by Dicrocoelium dendriticum making severe pathological changes of the liver and bile systems, and therapeutic options for treatment are limited. With the appearance of drug resistance in liver ukes, there is a need to focus on alternative approaches to control helminth parasites of veterinary importance. Because of low-performance medications; drug delivery poses a great challenge for better treatment of Dicrocoeliasis. The current study aims to determine the anthelmintic properties of silver oxide nanoparticles (AgO) as a new method in dicrocoeliasis treatment, in vitro assay. Methods: The impacts of various concentrations of AgO nanoparticles (50-200 µg/ml) for 12-24 hours were compared with the Closantel, as the chemical drug. The anthelmintic ecacy was measured by scanning electron microscopy (SEM) technique. Results: SEM images of treated worms by AgO (200 µg/ml) showed severe damage, including complete loss of sensory papillae and destruction of prominent network structures and tegument vesicles. The mortality rates how the anthelmintic properties of AgO were highly relied on time and concentration, as far as increasing the time and concentration cause increasing the mortality rate. According to the MTT assay, the toxicity of AgO, at concentrations, 800 µg/ml is 8.7 %. Conclusions: Hence, it could be concluded that AgO NPs performed anthelmintic properties effects. To the best of our knowledge, no previous reports have assessed the effect of AgO NP on liver uke D.dendriticum. Therefore, the present study provides a basis for future research on the control of this common trematode.

There are narrow therapeutic choices for the treatment of dicrocoeliasis in animals and drugs need to be used as an unapproved indication. It is troublesome to regulate whether anthelmintic drugs applied at dose rates and routes endorsed for grazing herbivores cause able to eradicate liver uke in a de nitive host as well. The possible hazard of either ine cient levels and the danger of expansion of anthelmintic resistance or leading to toxic levels is accordingly high [9,10].
Currently, there is no vaccine available for the prevention, and hence chemotherapy is one of the most widely used strategies to control dicrocoeliasis. Nevertheless, due to the emergence of resistance and the cost of treating small ruminants, alternative treatments have been seeking [4]. At present, chemical anthelmintic drugs, including Benzimidazole, pro-benzimidazole families, and Albendazole have been widely used. However, these drugs are not easily available in distant rural areas and also have some serious disadvantages such as the development of drug resistance, adverse drug reactions, residual effects, toxicity problems, and high veterinary costs. Albendazole, which can be used to treat dicrocoeliasis, has been reported to be toxic in camelids [9]. For other (pro) benzimidazoles further studies are required concerning the safety in animals since higher dose rates need to be used against D. dendriticum than those used against tapeworms, lungworms, and gastrointestinal nematodes [10]. Therefore, it is vital to design an easily operated and non-invasive compound. In the recent decade, nanoparticles (NPs) due to their de ned properties have gained lots of attention and considerable interest which makes them a favorable candidate for anthelmintic application since present antiparasitic drugs have some side effects and their e cacy is not fully proved. Among a wide variety of nanoparticles, Silver nanoparticles (AgNPs) are one of the most vital and fascinating nanomaterials among several metallic nanoparticles that are involved in the biomedical application and created the mass reports in the last years [4,11]. Notably, dramatic consideration was toward the biomedicine-related appraisal of AgNPs that rst invited worldwide consideration as eccentric antimicrobial, antifungal, and antiviral agents. Ag NPs have shown excellent properties against a wide range of microorganisms. One of the most vital and unique applications of AgNPs make them ideal in the eld of medicine is using them as antimicrobial agents, as well as for use in nanotoxicology studies [12].
During the past years, AgNPs became one of the most investigated nanostructures that proved to have promised, and interesting characteristics suitable for various unconventional and enhanced biomedical applications [13]. AgNPs are convinced to have legitimate dramatic and countenances capability for the improvement of ction antimicrobial agents, drug-delivery formulations, identi cation and diagnosis platforms, biomaterial and medical apparatus blankets, and performance-enhanced therapeutic alternatives [14]. However, their mechanism of action is not completely clari ed. Several mechanisms have been assumed for the effect of silver NPs on the cell. First of all, direct contact with microorganisms, the ability to penetrate to cell walls, changing the structure of cell membranes, and nally resulting in cell death. Second of all, by binding to the proteins, in the cell membrane, which is involved in the generation and transportation of APP [15]. Thus, the current study aimed to investigate the in vitro anthelmintic activities of AgONPs, against adult D. dendriticum in comparison with the chemical drug, Closantel, and the negative control, RPMI culture media. Surface alterations to the tegument were evaluated by SEM technique and were applied to investigate the concentration effect of AgONPs and with the impact of different incubation times on the death rate of D.dendriticum in comparison with Closantel. However, to the best of the author's knowledge, this is the rst report on the impact of AgO on the structure and motility of adult D. dendriticum.

Materials and physical measurements
All synthesized nanoparticles used in this method were of analytical grade and used as received without any further puri cation. X-ray diffraction (XRD) patterns were recorded by a Philips-X'PertPro, X-ray Diffractometer using Ni-ltered Cu Kα radiation at scan range of 10 < 2θ < 80. The size and shape of metal nanoparticles are measured by scanning electron microscopy (SEM). SEM images were obtained on LEO-1455VP equipped with energy-dispersive X-ray spectroscopy. Spectroscopy analysis (UV-Vis) was carried out using Shimadzu UV-Vis scanning UV-Vis diffuse re ectance spectrometer. The energy dispersive spectrometry (EDS) analysis was studied by XL30, Philips microscope.

Synthesis of AgO nanoparticle
To synthesize AgONPs, 1 g of silver nitrate was dissolved in 20 mL of deionized water under ultrasonic irradiation (180 W) and stirring to make a clear solution. Afterward, 1.5 g of potassium persulfate (K 2 S 2 O 8 ) as the powerful oxidant was added to the above solution under ultrasonic irradiation (180 W) and stirring for 15 min. Finally, the gray precipitate was ltered and washed three times with distilled water and then was dried at 60°C for 24 hours.

Collection of D. dendriticum
Adult fresh D. dendriticum were collected from the liver of slaughtered goats and sheep from the Kashan slaughterhouse. The samples after transported to the laboratory were washed three times with PBS buffer (pH:7-7.3) for future processing. Finally, they were immediately transferred into the 24 well plates containing RPMI1640 (50 IU/ml of penicillin, 50 IU/ml of streptomycin, 50 % V/V of Fetal bovine serum (FBS), and 2% of Sheep red blood cells for different treatment. Only worms with normal tegument and exhibit motility by visual inspection were selected.

MTT viability assay
The MTT assay was used to measure the viability of the Hela cells to nd the optimum concentrations of AgO. Some 10 5 Hela cells per well were placed in 96-well plates along with introducing different concentrations of AgO to each well and keeping at 37°C, 5% CO 2 for 24 h. Afterward, the MTT solution (20 µl, 5 mg/ml in PBS) was added to each well and further incubated for 3 h. After 3 hours, the supernatant of each well was removed and 100 µl of DMSO was added to each well. After 15 min incubation with DMSO, the ELISA plate reader was used for reading the absorbance of each well at 570 nm [16]. The percentage of cell viability was calculated based on Eq. 1 [17].

Structural study
Crystal structure and phase purity of the as-synthesized AgONPs were measured by X-ray powder diffraction (XRD). Based on the XRD pattern of Silver Oxide NPs (Fig. 1), the observed diffraction peaks can be indexed to the pure Monoclinic phase of AgO with space group P21/c (JCPDS no. 80-1269). The crystallite diameter (Dc) of AgONPs based on the Scherrer equation (D c = Kλ/ βcosθ) was calculated to be 19.59 nm. Where β is the breadth of the observed diffraction line at its half intensity maximum, K is the so-called shape factor, which usually takes a value of about 0.9, and λ is the wavelength of the X-ray source used in XRD. Further prove the composition of AgO was performed by energy dispersive spectroscopy (EDS), as shown in Fig.2 a. EDS spectrum reveals that NPs composed of only Ag and O atoms. Fig.2  was also carried out to check the presence of certain functional groups in AgONPs, as depicted in Fig. 3 a. FT-IR spectrum shows three absorption bands at 3182, 1059, 705 cm -1 which can be attributed to the stretching and bending vibrations of H 2 O molecules (3182 and 1059 cm -1 ) and the Ag-O bond (705 cm -1 ).
The optical property of as-synthesized AgONPs was determined using UV-vis spectroscopy. Fig.3 b depicts (αhν) 1/2 vshν curve of AgONPs which was calculated based on the Wood and Taucequation [2]. Worm motility assays The number of viable adult worms was measured by the eosin staining method after 12, 18, and 24h incubation with AgO, and Closantel respectively. The results clearly showed that after 12 h incubation at concentration 150µg/ml of AgONPs as well as the highest concentration to 200 µg/ml all worms were dead. Furthermore, after 12 h incubation at 200µg/ml of Closantel, all adult D.dendriticum died. Results indicated that less motile with the increasing concentration of the AgONPs. On the other hand, the decrease in the motility rate of ukes treated with experimental drugs was both time and concentrationdependent (Table 2).

Scanning electron microscopy (SEM) of adult D. dendriticum
The tegumental topographical changes of D.dendriticum liver ukes were investigated by the SEM technique to assess the effects of AgO on their surface structures. The control worms seem normal with unchanged tegument around suckers and their oral and ventral suckers are round and smooth. Besides, sensory papillae at the edges and inside the oral sucker, tegumental ridges network, and vesicles look unaltered. Besides, ridge walls and valley oors cover densely by tegumental vesicles in the entire body, which seem to be like spherical structures (Fig. 5, a-d). SEM images of treated worms with AgO NPs (100 µg/ml) demonstrated that their tegumental region endured a variety of changes, including appearing severe swelling, swollen and blister (Fig. 6a), destroying sensory papillae (Fig. 6b), and also destroying the network structure and tegument vesicles (Fig. 6, c, d). SEM images of treated D. dendriticum with Closantel (200 µg/ml) are shown in Fig. 7a-f. Based on Fig. 7a, swelling, erosions, and blebs appeared on the surface tegumental. Besides, cirri were damaged and lost their natural appearance (Fig. 7b). Also, sensory papillae were disappeared completely and oral and ventral suckers are aky completely insofar as a little recognizable structure was remained (Fig. 7c-f).

Discussion
Dicrocoeliasis, caused by Dicrocoelium dendriticum, is a common disease among ruminates that has important economic and veterinary aspects [8]. Although anti-parasitic medicine, chemicals are readily available, they have irreversible side effects. Among the different groups of metallic nanoparticles, silver nanoparticles (Ag-NPs) have become one of the common in-demand nanoparticles in debt to their exponential number of operations in various districts. The increased use of Ag-NPs-enhanced products may result in an increased level of toxicity affecting living organisms such as parasites [17,19,20]. Given the widespread use of AgNPs, risk assessment of these nanoparticles is of great importance. Numerous studies have demonstrated the potency of AgNPs to induce deleterious biological and cellular effects [21]. These nanoparticles adhere to the cell walls and membranes of microorganisms and may reach the cell interior. They injury the cellular organelles, engender the production of reactive oxygen species, and alter the mechanisms of signal transduction [22]. Several studies report utilizations in which good results have been achieved using silver nanoparticles for the control of pathogenic microorganisms in the elds of public health and medicine [14,23]. Despite the widespread use of silver nanoparticles, few studies have been performed on AgNPs against platyhelminth parasitic infections [18, [24][25][26]. Besides, many investigations have been performed on the anti-parasitic impact of the AgNPs on the Gigantocotyle explanatum, Haemonchus contortus, Ancylostoma caninum, and Fasciola hepatica [26][27][28][29]. It has been suggested that metal oxide NPs such as AgO have a high attitude to create reactive oxygen species (ROS) and free radicals, which are responsible for causing oxidative stress and apoptosis leading to cell death, which ends up with acceptable antibacterial, antifungal, antioxidants and anti-parasite [30,31]. Indeed, the enormous creation of ROS in cells by direct interaction with particles is at present accepted as one of the major mechanisms of cellular toxicity of nanoparticles [1,[32][33][34]. ROS have many signaling and information functions; however, excessive ROS can collapse the antioxidant defense system, leading to the damage of DNAs, lipids, and proteins [34,35]. Ag NPs with larger surface areas provide better contact with microorganisms. Since Ag NPs are smaller than microorganisms, they easily diffuse into cells and rupture the cell wall and pathogens. It has also been shown that smaller nanoparticles are more toxic than bigger ones. The toxicity of Ag NPs is dependent on the concentration, pH of the medium, and exposure time to pathogens [36]. Also, their e cacy is due not only to their nanoscale size but also to their large ratio of surface area to volume. They can increase the permeability of cell membranes, produce reactive oxygen species, and interrupt the replication of deoxyribonucleic acid by releasing silver ions [37]. Also, antioxidant enzymes have been recognized as important modulators in AgNP induced oxidative stress. Two of them, catalase (CAT) and superoxide dismutase (SOD), are prominent for maintaining the level of ROS in organisms and are used as bio-indicators of increased ROS production [17]. Previous research demonstrated that AgNPs induce oxidative stress by altering the activity of both enzymes in vivo and in vitro assays [38]. It is currently accepted that the alteration of the enzyme activity might be due to either regulation of genes or to direct surface interaction of the enzymes with AgNPs [39]. The molecular mechanisms of the interaction between enzymes and nanoparticles were also explored in some in vitro studies [40][41][42]. The organisms treated by Ag NPs are under oxidative stress and induced autophagic cell death and mitochondrial damage via reactive oxygen species generation along with the inhibition of the major antioxidant enzyme, SOD [43,44].
In the current study, the effect of AgO NPs on adult D. dendriticum was investigated using in vitro model. Anti-parasitic results of AgO NPs demonstrated that 100 µg/ml of AgO at 24 h shows a high destruction effect on the tegument surface of D. dendriticum. Future research is needed to realize the mechanism of action of AgNPs in the tegument of this liver uke as well as to detect internal injure to the parasitic ultrastructure and molecular changes dominant to parasite death.

Conclusion
In summary, scanning electron microscopy (SEM) was applied to the anthelmintic e ciency of AgO NPs. Besides, the impacts of various concentrations and reaction time on the anthelmintic e ciency of AgO NPs were also investigated. Also, the e ciency, mortality rate, which is based on the number of live and dead adult D. dendriticum, of NPs was investigated after 12, 18, and 24 h expose time. SEM results demonstrated that NPs showed dose-dependent anthelmintic e ciency. Nonetheless, additional research is necessary to evaluate the in vivo the e cacy of this treatment as well as its toxicity on a de nitive host. Oxidative stress is accepted as a key mechanism of AgNPs toxicity in living organisms. Therefore, mechanistic studies related to the impact of AgNPs on the structure and function of antioxidant enzymes at the molecular level are essential for a comprehensive evaluation of their toxicity.