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

doi:10.21203/rs.3.rs-827258/v1

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

https://doi.org/10.21203/rs.3.rs-827258/v1

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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 flukes, 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 efficacy 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 fluke D.dendriticum. Therefore, the present study provides a basis for future research on the control of this common trematode.

Pathology

Dicrocoelium dendriticum

AgO nanoparticle

Scanning Electron Microscopy

in vitro

Dicrocoeliasis is a widespread hepatic bile duct trematode disease, which parasite both humans and a wide range of grazing herbivores and counts as one of the major threats to livestock production in endemic areas [1, 2]. Dicrocoelium spp. as well as Fasciola spp. designed a group as the food-borne trematodes [3, 4]. The lancet liver fluke, Dicrocoelium dendriticum has been distributed throughout Europe, Asia, North Africa, and North America, known as the major cause of the disease. Its parasite is now recognized as Neglected Tropical Diseases, causing major public health problems as well as significant economic impact. Most D.dendriticum infections cause no symptoms or only minor ones, hence remain undetected. The clinical infection of dicrocoeliasis is normally resulting in mild symptoms, but heavy infections can lead to serious animal health problems [5, 6]. Dicrocoeliasis causes severe pathological changes of the liver and bile system such as abscesses, granulomas, and fibrosis. Cholangitis with the thickened bile ducts appearing as white spots on cut surfaces of the liver was diagnosed. Chronic disease can develop into cirrhosis [7]. There have been only rare documented cases of re-infection. The parasitizing infectious diseases result in abdominal pain; flatulence, dyspepsia, and watery diarrhea have been reported. Besides, infected animals show a slight increase in their bilirubin (7%) and albumin (3%), not related to the worm burden. However, the clinical symptoms are not pathognomonic in harsh infectious and animal scan causes edemas, anemia, icterus, and a reduction in their production [8].

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 fluke in a definitive host as well. The possible hazard of either inefficient 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 defined 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 efficacy 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 first 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 field 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 fiction antimicrobial agents, drug-delivery formulations, identification and diagnosis platforms, biomaterial and medical apparatus blankets, and performance-enhanced therapeutic alternatives [14]. However, their mechanism of action is not completely clarified. 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 finally 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 first 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 purification. X-ray diffraction (XRD) patterns were recorded by a Philips-X’PertPro, X-ray Diffractometer using Ni-filtered 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 reflectance 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 (K₂S₂O₈) 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 filtered 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 find the optimum concentrations of AgO. Some 10⁵Hela 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% CO₂ 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].

Cell Viability (%) = 100 * OD sample/OD control [1]

In vitro assays

The investigation was carried out in four groups. Every treatment considered of different concentrations of experimental groups NPs: 50, 100, 150, and 200 µg/ml), positive control treatment (Closantel: 50, 100, 150, and 200 µg/ml) and negative control treatment (only RPMI1640 media culture). For this purpose, under sterile conditions in a laminar flow cabinet, adult liver flukes D. dendriticum were transferred into the 24 well, each well contains 1ml of RPMI1640 (50IU/ml of penicillin, 50IU/ml of streptomycin, 50% V/V of FBS, and 2% of sheep red blood cells). Afterward, 1ml of AgONPs were added individually to each well and incubated for 12, 18, and 24h at 37^°C in an atmosphere of 5% CO₂. The number of live adult D.dendriticum was checked during these times.

Motility examination

The motility time (12, 18, and 24 h) of worms after incubation in different concentrations of the treatments and control groups was calculated and the viability of experiments was measured based on the motility criteria (the whole body moving high, parts of the body moving, less movement of the whole body and complete loss of motility). The trematodes revealed no movement visually were examined by a light microscope for movement conformation. The motility assay in control groups was noted, according to the absence of the experimental. compounds. All worms in each experiment were observed individually at an hour interval according to the motility criteria established.

Scanning electron microscopy (SEM) sample preparation

For the determination of the ultrastructural alteration in the tegumental of worms in the test and control groups, we used electron microscopy (SEM). In the first step, the treated and control flukes were fixed with sodium cacodylate buffer (pH: 7.4, 0.2 M) and glutaraldehyde in phosphate buffer (2.5 % v/v) for 4 h at 4^°C, and then the parasites were washed 3 times in phosphate buffer (pH: 7.4). In the second step, they were dehydrated in ascending ethanol concentrations (70%, 80%, 95%, and 100%) for 30 min, final dehydration was achieved in hexamethyldisilazane and consequently dried in the vacuum oven [18]. Finally, mounted on stubs, sputter-coated with gold, and they were photographed by SEM (ZEISS-DSM 960A, Germany) at the central laboratory of the Institute for Color Science and Technology, Tehran, Iran.

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 b demonstrates the SEM images of Silver Oxide NPs. It showed AgO mainly consists of rice shape NPs with an average size of 20-40 nm. Besides, Furrier transform infrared spectroscopy (FT-IR) 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^-1which can be attributed to the stretching and bending vibrations of H₂O 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)^1/2vshν curve of AgONPs which was calculated based on the Wood and Taucequation [2].

αhʋ = (hѵ-Eg)ⁿ

where α is the absorbance, h the Planck constant, ν the photon frequency, Eg the energy gap, and n the pure numbers associated with the different types of electronic transitions. For n=1/2, 2, 3/2, and 3, the transitions are directly allowed, indirectly allowed, directly forbidden, and indirectly forbidden, respectively. Each energy gap was determined by the extrapolation of each linear portion of the curves to a= 0. Hence, the energy gap of AgONPs was calculated to be2.9 eV.

Anthelmintic treatment study

In vitro toxicity assay

Toxicity values (TC50) of HeLa cells were measured after 24 h incubation and were compared with positive and negative control groups. The toxicity results of AgO were shown in Table1and Fig. 4. The toxicities of AgO at 100 µg/ml were 8.7 %. Results indicated the toxicity of AgO NPs has low.

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 flukes treated with experimental drugs was both time and concentration-dependent (Table 2).

Scanning electron microscopy (SEM) of adult D. dendriticum

The tegumental topographical changes of D.dendriticum liver flukes 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 floors 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 flaky completely insofar as a little recognizable structure was remained (Fig. 7c-f).

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 fields 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, 24–26]. 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 [26–29]. 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, 32–34]. 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 efficacy 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 [40–42]. 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 fluke as well as to detect internal injure to the parasitic ultrastructure and molecular changes dominant to parasite death.

In summary, scanning electron microscopy (SEM) was applied to the anthelmintic efficiency of AgO NPs. Besides, the impacts of various concentrations and reaction time on the anthelmintic efficiency of AgO NPs were also investigated. Also, the efficiency, 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 efficiency. Nonetheless, additional research is necessary to evaluate the in vivo the efficacy of this treatment as well as its toxicity on a definitive 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.

Acknowledgments

The authors are grateful to Deputy of Research, Kashan University of Medical Sciences, Kashan, Iran, for its support.

Authors' contributions

Designing the study: MA. Collecting data and statistical analysis: MA and AH. Literature review: MA, AH, SMHM, HH. Data interpretation: MA, SMHM. Drafting the manuscript: MA. Critical revision of manuscript: MA, AH. All authors read and approved the final manuscript.

Funding

The present study was supported by the Deputy of Research, Kashan University of Medical Sciences, Kashan, Iran, through grant no. 9740.

Availability of data and materials

The datasets used analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This project was found to be in accordance with the ethical principles and the national norms and standard for conducting Medical Research in Iran. The study protocol was approved by the Ethics Committee of Kashan University of Medical Sciences, Iran, (Approval ID is IR.KAUMS.MEDNT.REC.2018.23).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors' information (optional)

¹Department of Medical Parasitology and Mycology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran. ²Department of Medical Parasitology and Mycology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran. ³Institute for Biomedical Materials and Devices, School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, NSW 2007, Australia.⁴Department of Medical Parasitology and Mycology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran.

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Table.1. Toxic values (TC50) of Hela cell exposed to the AgO nanoparticles

Viability (%)	Concentration (µg/ml)
100	50
99.3	100
80.2	150
79.3	200

Table 2. Comparison Lethal concentration (LC) values of AgO with control group (Closantel) during 12, 18, and 24 h.

Groups

LC (Hour)

LC10

LC25

LC50

LC75

LC90

AgO nanoparticle

87.9

39.7

39.1

95.03

60.6

45.8

102.9

83.8

52.5

110.9

101.2

120.1

115.1

62.5

Closantel

89.5

87.8

106.3

125

102.9

143.6

110.9

160.4

118

Comparison between groups

P<o.oo1

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

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Abstract

Figures

Background

Methods

Materials and physical measurements

Synthesis of AgO nanoparticle

MTT viability assay

In vitro assays

Motility examination

Scanning electron microscopy (SEM) sample preparation

Results

Discussion

Conclusion

Declarations

References

Tables

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