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Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices
Accepted Manuscript Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices Mohammad Taghi Rahimi, Ehsan Ahmadpour, Bahman Rahimi Esboei, Adel Spotin, Mohammad Hasan Kohansal Koshki, Ahad Alizadeh, Soheila Honary, Hamed Barabadi, Milad Ali Mohammadi PII:
S1743-9191(15)00257-5
DOI:
10.1016/j.ijsu.2015.05.043
Reference:
IJSU 1903
To appear in:
International Journal of Surgery
Received Date: 8 May 2015 Accepted Date: 15 May 2015
Please cite this article as: Rahimi MT, Ahmadpour E, Esboei BR, Spotin A, Kohansal Koshki MH, Alizadeh A, Honary S, Barabadi H, Mohammadi MA, Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices, International Journal of Surgery (2015), doi: 10.1016/j.ijsu.2015.05.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices Mohammad Taghi Rahimia, Ehsan Ahmadpourb,*, Bahman Rahimi Esboeic, Adel Spotind,
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Mohammad Hasan Kohansal Koshkie, Ahad Alizadehf, Soheila Honaryg, Hamed Barabadig, Milad Ali Mohammadig
a
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Department of Parasitology and Mycology, Sari Medical School, Mazandaran University of Medical Sciences, Sari, Iran b Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of parasitology and mycology, school of public health, Tehran University of Medical, Tehran, Iran d Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Medical Parasitology and Mycology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran f Department of Epidemiology and Biostatistics, School of Health, Tehran University of Medical Sciences, Tehran, Iran. g Mazandaran University of Medical Sciences, School of pharmacy, Sari, Iran.
*
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Running title: silver nanoparticles scolicidal activity
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Corresponding Author: Ehsan Ahmadpour Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Email address: [email protected]
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Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus
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granulosus protoscolices
Abstract
Cystic hydatid disease (CHD), a helminth infection with various clinical complications caused
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by the larval stage of the dog tapeworm Echinococcus granulosus is considered as a public health problem in different regions of the world. To date, scolicidal agents have been broadly
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applied for inactivation of the fertile cysts and these scolicidal agents have several side effects on patients. Therefore, the objective of this study was to evaluate the scolicidal efficacies of synthesized silver nanoparticles (AgNPs) derived from the aqueous aerial extract of Penicillium aculeatum against protoscolices of CHD in-vitro. Protoscolices were aseptically aspirated from
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infected livers of sheep with CHD. Various concentrations (0.025, 0.05, 0.1 and 0.15 mg/mL) of green synthesis of Ag-NPs and different exposure times (10, 30, 60 and 120 min) were used against protoscolices of CHD Viability of protoscolices was confirmed by 0.1% eosin staining.
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The findings showed that the Ag-NPs at all concentrations have high scolicidal effects. The concentrations 0.1 and 0.15 mg/mL after 120 min of exposure times showed 83% and 90%
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mortality rate, respectively. The least scolicidal activity of biosynthesized Ag-NPs was 40% (0.025 mg/mL and 10 min). Current investigation indicated that applying biogenic Ag-NPs may be considered as a potential scolicidal agent for CHD surgery due to being economical, safer and non-toxic compared to the used chemical materials. However, further studies are required to evaluate the efficacy of Ag-NPs in vivo. Keywords: Cystic hydatid disease, Hydatid cyst, Green Silver Nanoparticles, Scolicidal, Echinococcus granulosus.
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1. Introduction Cystic hydatid disease (CHD) is a major neglected cyclo-zoonotic disease with various clinical complications caused by the larval stage (metacestode) of Echinococcus granulosus in many
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countries of the world [1-3]. The annual incidence rate of CHD can vary from 1 to 200 per 100,000 inhabitants in various endemic regions. Iran is considered as an endemic (southern parts) and hyperendemic (northern parts) area of CHD [3]. CHD is highly important in both
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medical and veterinary fields due to considerable economic damages and losses of animal proteins [1, 4-6]. In addition, CHD can cause 10% decline of life performance of infected
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animals including the decrease in meat quality, milk production, fiber and the number of surviving offspring [7].
Canids are the definitive hosts of E. granulosus whereas human, sheep and other mammalian species are intermediate hosts. Humans and herbivores acquire infection by accidental ingestion
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of eggs of E. granulosus. Oncospheres (embryo) of eggs are able to penetrate the intestinal mucosa and disseminate mainly, through the portal system in the liver and lungs [8].
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Surgery, PAIR (puncture-aspiration-injection-respiration) technique, and chemotherapy are commonly available cure for CHD. A lot of efforts have been conducted to prepare an efficient
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vaccine. However, there is not an effective vaccine against CHD for human [9]. The standard approach for CHD treatment is still remains surgery but we cannot ignore some unexpected repercussions such as anaphylactic shock, disease recurrence and mortality of this invasive method [10, 11]. Besides, the surgery becomes impractical when cysts are formed either in multiple organs or in risky locations including brain and spinal tissues. In these cases, the alternative options for treatment are chemotherapy and/or PAIR [2]. Removal of the cysts combined with chemotherapy either using Albendazole and/or Mebendazole before and after
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surgery are the best and selective methods. However, these drugs have demonstrated some side effects including thrombocytopenia, leucopenia and hepatotoxicity [12-15].
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To date, the routine scolicidal solutions such as, Ag-nitrate, cetrimide, hypertonic saline and ethanol, are broadly employed for infertility of the protoscolices whilst, they have different adverse effects such as liver necrosis, methaemoglobinaemiate and sclerosane colangititis
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(biliary tract fibrosis). Therefore, it is indispensable to develop new scolicidal materials with more efficacies and low adverse effects, which can be implemented during surgery [15-17].
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Synthesized green nanoparticles of biological sources like plants are widely accomplished because they are less toxic to human compared with the chemical medicines. Several metal nanoparticles such as copper (Cu), alginate magnesium (Mg), gold (Au), zinc (Zn), titanium (Ti), and silver (Ag) have been evaluated that among them, the synthesized silver nanoparticles
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(AgNps) have been introduced as the most effective one owing to having a large capacity in direct contact with their environment and acceptable antimicrobial efficacy against parasites, viruses and bacteria [18-22].
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There is no investigation concerning the impacts of green silver nanoparticles on protoscolices of CHD in-vitro. The objective of current study was to evaluate the scolicidal efficacies of cost
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effective and environmentlly friendly synthesized silver nanoparticles (AgNPs) derived from the aqueous aerial extract of Penicillium aculeatum against protoscolices of CHD in-vitro. 2. Materials and methods 2.1. Nanoparticle preparation Yeast extract was purchased from Liofilchem, Italy and silver nitrate and other chemical reagents were purchased from Merck, Germany. The pure colony of P. aculeatum was isolated from soil
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and approved by Department of Mycology and Plant Pathology of Sari Agriculture and Natural Resources University, Mazandaran Province, Iran. The fungus was cultured on Czapek dox broth, including 21g sucrose and 3g yeast extract in 1000mL distilled water, and then incubated
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at 28°C at 200 rpm for seven days (IKA KS 4000). The culture was centrifuged at 10,000 rpm for 5min, and the supernatant was used for the synthesis of AgNPs. A solution of silver nitrate (3mM) was prepared by dissolving 0.5096g of the compound in 100mL of double distilled
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water. Thereafter, 100mL of silver nitrate solution at a concentration of 3mM was added to 100 ml of the supernatant and incubated again for 24 hours at 28°C (total concentration of silver
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nitrate = 1.5mM). The colors of the solutions changed to dark buff, indicating the formation of AgNPs in the solution. The solutions were centrifuged at 20,000 rpm for 5min; the particles were separated from the supernatant. Then, the nanoparticles re-dispersed in double distilled water and centrifuged again. This action was repeated for three times to improve purification of AgNps.
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The solution was filtered through a 0.22µm millipore syringe filter to remove any impurity before characterization tests. Finally the AgNPs was freeze-dried and weighted by a digital scale to prepare concentrations of 0.025, 0.05, 0.1 and 0.15 mg/mL for in-vitro studies [23]. Evaluation of nanoparticles
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2.2.
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The formation of silver nitrate to AgNPs was confirmed by ultraviolet (UV) spectroscopy (Genesys 2 spectrophotometer, USA) and scanning electron microscope (SEM) model 2360 (Leo Oxford England). The size and polydispersity of particles were determined using photon correlation spectroscopy (PCS) by Zetasizer Nano Particle Analyzer (model 3600, Malvern instruments, UK) at 25°C with a scattering angle of 90°. PCS is a non-invasive technique that measures the size and size distribution of nanoparticles dispersed in a liquid. 2.3.
Protoscolices collection
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Hydatid cysts of E. granulosus were obtained from livers of naturally infected sheep in Mazandaran slaughterhouse, Mazandaran Province, North of Iran. The hydatid fluid was aseptically removed and transferred into a container and left to set for 30 min. The protoscolices
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settled down at the bottom of the container and then centrifuged at 800 rpm for 5 minutes. The supernatant was removed and the yielded protoscolices were left and the live protoscolices were
2.4.
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stored at 4°C for further use [24]. Scolicidal assay
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In the current study, different concentrations of Ag-NPs including 0.025, 0.05, 0.1 and 0.15 mg/mL were used for different exposure times including 10, 30, 60 and 120 min. To prepare the mentioned dilutions 0.025, 0.05, 0.1 and 0.15 µL of Ag-NPs were dissolved in 9.7 ml of normal saline in a test tube. Next, the obtained solution was gently mixed. Thereafter, in each
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experiment, 100 µl of protoscolices-rich sediment was added to 2.5 ml of the solution. After mixing the contents, the test tube was incubated at 37 ⁰C for 10, 30, 60 and 120 min. At the end of incubation periods in order to assess the viability of protoscolices 10µl of 0.1%
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eosin was added to the 20µl remaining pellet of protoscolices and mixed gently. The stained protoscolices was smeared on a manually scaled glass slide, which was covered with a cover
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glass (24 × 50 mm), and examined using light microscope. The percentages of dead protoscolices were estimated by counting a minimum of 200 protoscolices. Hydatid cyst fluid was considered as negative control group. Besides, NaCl 5% (5 g/1000 ml) were used as positive control group [25]. The experiments were performed in duplicate. 2.5.
Viability test
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Eosin stain with the concentration of 0.1% (1 g of eosin powder in 1000 ml distilled water) was utilized to check the viability of the protoscolices. Five min after exposure times to the eosin stain the protoscolices which did not absorbed dye with the movement of the flame cells were
2.6.
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potentially recorded as viable, otherwise, they were considered as dead protoscolices. Statistical analysis
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Differences between test and control groups were analyzed using the chi-square test. P values
GraphPad InStat software. 3. Results 3.1. Nanoparticle preparation
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<0.05 were considered significant. In addition, statistical analysis was performed using
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Addition of P. aculeatum supernatant to 1mM aqueous AgNO3 solution led to a color change to yellowish brown in the solution after 24 h of reaction, indicating the formation of AgNPs. The UV-Vis spectrum of AgNps produced by P. aculeatum exhibited an absorption band at around
(Figure 1).
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420 nm, which is a typical plasmon band, suggesting the formation of silver nanoparticles
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Figure 2 shows that the average size of the AgNps was 80.42 nm with a polydispersity index (PDI) of 0.238. The PCS spectra demonstrated that the AgNps formed had fairly well-defined dimensions and suitable monodispersity. PDI measures the second moment of the size distribution of the nanoparticle population. PDI ranges were from a value of 0.01 to 0.5 – 0.7 for monodispersed particles. Samples with very broad size distribution had polydispersity index >
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0.7. The SEM micrograph of nanoparticles showed that the AgNPs produced by P. aculeatum had a spherical shape and well distributed in solution (Figure 3).
Scolicidal effects of Ag-NPs
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3.2.
Figure 4 depicts the scolicidal effects of different concentrations of biosynthesized Ag-NPs (0.025, 0.05, 0.1 and .015 mg/mL) with different time expousre (10, 30, 60, and 120 min) against
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protoscolices of Hydatid cysts. Statistically the difference between the scolicidal effects of biosynthesized Ag-NPs was significant for all examined concentrations and exposure times in
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comparison to the control group (P<0.0001). It is noteworthy that with a rise in all concentrations of biosynthesized Ag-NPs the viability of protoscolices was decreased significantly (P<0.0001). At concentration of 0.1 and 0.15 mg/ml for 60 min treatment, the observed mortality rate was 80 % and 79%, respectively; while, up to 83% and 90% mortality
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was observed with 0.1 and 0.15 mg/ml for 120 min. In addition, the most scolicidal activity of biosynthesized Ag-NPs was 90% (0.15 mg/mL and 120 min) while the least scolicidal activity of
4. Discussion
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biosynthesized Ag-NPs was 40% (0.025 mg/mL and 10 min) (Table 1).
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In this study various dilutions (0.025, 0.05, 0.1 and .015 mg/mL) of biosynthesized Ag-NPs with different exposure times (10, 30, 60 and 120 min) were employed for scolicidal assessment. The most scolicidal activity of biosynthesized Ag-NPs were observed 90% (0.15 mg/mL and 120 min). Scolicidal effect of biosynthesized Ag-NPs may be attributed to electrostatic interaction between Ag-NPs (electrically positive) and the parasite (electrically negative) associated to organic complexes on its surface. A recently published study, showed fungal chitosan could kill 100% of protoscolices at concentration of 400 µg/ ml after 180 min [26].
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Surgery is current treatment of choice for complicated cases of CHD. Nevertheless, its success depends on formation of new cysts, relapse or secondary dissemination of CHD after surgery which can result in death due to spillage of the cyst contents [10]. Indeed, the infertilization and
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inactivation of protoscolices via scolicidal agents accompanied by minimum side effects and high efficacy instead of opening or removing cyst is highly recommended [12].
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Up to now, many protoscolicidal agents such as hypertonic saline, mannitol, chlorhexidine gluconate, huaier aqueous, Allium sativum, Sambucus ebulus, fungal chitosan and Berberis
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vulgaris have been used for inactivation of hydatid cyst content [26-29]. Unfortunately, using aforementioned protoscolicidal agents were limited because of their low efficacy, toxicity and undesirable side effects. Hypertonic saline (20 %) has been found to be 100% effective on protoscolices of hydatid cyst [25], but acute hypernatremia can cause severe nerve system symptoms such as necrosis, myelinolysis, convulsions, and intracranial bleeding. Silver nitrate
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and cetrimide have been shown to be 100% effective against protoscolices of the hydatid cyst; however, toxic reactions may also be caused by the absorption of these ingredients [30]. Although albendazole sulfoxide is the first choice for protoscolices, it increases the level of liver
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enzymes [16]. Besides, demanding preparation, and high cost are the principle disadvantages of
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the mentioned protoscolicidal agents. For these reasons, it cannot be considered as a perfect scolicidal remedy [12].
A similar study, showed that biogenic Selenium NPs at all concentrations have potent scolicidal effects against protoscolices of hydatid cyst especially at concentrations 500 and 250 mg/ml the scolicidal activity was 100% after 10 and 20 min of application, respectively [31].
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Presently, nanoparticles play crucial role in drug delivery, gene therapy, diagnostics, and many areas of research. The plant mediated biosynthesis of nanoparticles is cost effective and environmentally green method that does not require employing energy, pressure, toxic chemicals
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and temperature. Indeed, these characteristics are considered advantageous over chemical and physical methods [32, 33].
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This investigation for the first time presents the scolicidal effects of biosynthesized Ag-NPs against protoscolices of hydatid cysts in vitro model. Our results showed that Ag-NPs have an
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effective scolicidal activity in all concentrations especially at concentration 0.15 mg/ml (90% mortality rate) after 120 min exposure times. Consequently, these findings indicated that scolicidal effects of Ag-NPs at concentration 0.15 mg/ml was comparable with scolicidal effects of 20% silver nitrate (20 min) , 0.5-1% cetrimide (10 min), 20% hypertonic saline (15 min), 95%
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ethyl alcohol (15 min) and H2O2 3% (15 min), as previously described [12, 13, 29, 34-37] The permeation behavior through the hydatid cyst membrane was evaluated by albendazoleloaded nanoparticles (about 300 nm) which were prepared by the emulsion solvent evaporation
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method revealed a sufficient entrapment efficacy (36.4±6.4%) to increase the apparent solubility of albendazole. In fact, benzimidazole-loaded nanoparticles appear to be promising formula
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regarding treatment of hydatid cyst. Xie et al., 2010 evaluated the efficacy of praziquantel loaded hydrogenated castor oil solid lipid nanoparticle (PZQ-HCO-SLN) suspension in dogs naturally infected with E. granulosus [38]. The therapeutic effect of the novel formulation was promising to elevate the therapeutic efficiency of PZQ. At the dose of 5 mg/kg, stool ova declined and negative conversion rates and tapeworm removal rate of the suspension were recorded 100%, whereas the native PZQ were recorded 91.55%, 87.5%, and 66.7%. Once the dose decreased to 0.5 mg/kg, no effect was observed using the native drug, while the novel suspension still
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remained the same therapeutically effective as the 5 mg/kg native PZQ. An ideal scolicidal agent should be effective in low concentration in a shortest possible time, stable in hydatid cyst
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fluid, able to kill the protoscolices, nontoxic and easily available with low cost. Currently, the need to effective methods to control parasites has increased the use of nanomaterial products in biomedical sciences. Various researches conducted on parasite showed
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promising results. A study conducted by Said et al on antiparasitic effects of silver, chitosan, and curcumin nanoparticles as anti Giardia agent shows that the number of the parasites in stool
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and small intestinal sectors decreased in treated rats compared with non-treated ones [39]. larvicidal effects of Green synthesized silver nanoparticles against Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti suggest that they can be used as a rapid and safe biopesticide [40].
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In conclusion, this is noteworthy to mention that this is the first report on the biosynthesized AgNPs as scolicidal agent on hydatid cysts protoscolices in vitro. The evaluation of Ag-NPs against hydatid cyst protoscolices showed an effective and promising scolicidal activity. The prepared
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Ag-NPs using biotechnological methods can be considered as an effective scolicidal agent that could be applied for pharmaceutical applications, because the mentioned eco-friendly process is
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likely to be cost effective, safe in addition, it requires non-toxic materials compared to existing chemical methods.
Acknowledgments
This work was financially supported by Traditional Medicine & Materia Medica Research Center (TMRC) Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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Table 1. Protoscolicidal effects of biosynthesized Ag-NPs on protoscolices of hydatid cyst using different exposure time and concentrations of nanosilver. Agents
Repeat
Time
Control Positive
1 2 (Mean±SD)
0.54 0.45 (0.50±0.06)
0.64 0.55 (0.60±0.06)
0.62 0.65 (0.64±0.02)
0.77 0.78 (0.78±0.01)
Group 1
1 2 (Mean±SD)
0.4 0.35 (0.38±0.04)
0.56 0.49 (0.53±0.05)
0.68 0.65 (0.67±0.02)
0.68 0.61 (0.65±0.05)
Group 2
1 2 (Mean±SD)
0.43 0.47 (0.45±0.03)
0.59 0.59 (0.59±0.00)
0.69 0.64 (0.67±0.04)
0.75 0.73 (0.74±0.01)
Group 3
1 2 (Mean±SD)
0.49 0.51 (0.50±0.01)
0.56 0.53 (0.55±0.02)
0.75 0.85 (0.80±0.07)
0.85 0.81 (0.83±0.03)
Group 4
1 2 (Mean±SD)
0.45 0.47 (0.46±0.01)
0.59 0.54 (0.57±0.04)
0.79 0.78 (0.79±0.01)
0.9 0.9 (0.90±0.00)
Control Negative
1 2 (Mean±SD)
0.19 0.2 (0.20±0.01)
0.2 0.2 (0.20±0.00)
0.2 0.2 (0.20±0.00)
0.2 0.2 (0.20±0.00)
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Figure 1: UV–visible spectrum of the AgNps
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Figure 2: Photon correlation spectroscopy (PCS) of the AgNps; Average size: 80.42 nm, PDI: 0.238
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Figure 3: SEM image of the silver nanoparticles (Ag-NPs) synthesized by P. aculeatum.
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Figure 4. Scolicidal effects of biosynthesized Ag-NPs against protoscolices of Echinococcus
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granulosus at the different concentrations following various exposure times. Control positive (NaCl 5%); Group1 (0.025mg/mL of nanosilver); Group2 (0.05mg/mL of nanosilver); Group3
fluid).
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(0.1mg/mL of nanosilver); Group4 (0.15mg/mL of nanosilver); Negative control (Hydatid cyst
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Highlights:
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Green synthesis of silver nanoparticles using aqueous aerial extract of Penicillium aculeatum.
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Ag-NPs showed potent in vitro scolicidal activity.
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Assessment of silver nanoparticles against protoscolices of hydatid cysts, in vitro.
S1743-9191(15)00257-5
DOI:
10.1016/j.ijsu.2015.05.043
Reference:
IJSU 1903
To appear in:
International Journal of Surgery
Received Date: 8 May 2015 Accepted Date: 15 May 2015
Please cite this article as: Rahimi MT, Ahmadpour E, Esboei BR, Spotin A, Kohansal Koshki MH, Alizadeh A, Honary S, Barabadi H, Mohammadi MA, Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices, International Journal of Surgery (2015), doi: 10.1016/j.ijsu.2015.05.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices Mohammad Taghi Rahimia, Ehsan Ahmadpourb,*, Bahman Rahimi Esboeic, Adel Spotind,
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Mohammad Hasan Kohansal Koshkie, Ahad Alizadehf, Soheila Honaryg, Hamed Barabadig, Milad Ali Mohammadig
a
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Department of Parasitology and Mycology, Sari Medical School, Mazandaran University of Medical Sciences, Sari, Iran b Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of parasitology and mycology, school of public health, Tehran University of Medical, Tehran, Iran d Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Medical Parasitology and Mycology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran f Department of Epidemiology and Biostatistics, School of Health, Tehran University of Medical Sciences, Tehran, Iran. g Mazandaran University of Medical Sciences, School of pharmacy, Sari, Iran.
*
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Running title: silver nanoparticles scolicidal activity
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Corresponding Author: Ehsan Ahmadpour Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Email address: [email protected]
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Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus
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granulosus protoscolices
Abstract
Cystic hydatid disease (CHD), a helminth infection with various clinical complications caused
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by the larval stage of the dog tapeworm Echinococcus granulosus is considered as a public health problem in different regions of the world. To date, scolicidal agents have been broadly
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applied for inactivation of the fertile cysts and these scolicidal agents have several side effects on patients. Therefore, the objective of this study was to evaluate the scolicidal efficacies of synthesized silver nanoparticles (AgNPs) derived from the aqueous aerial extract of Penicillium aculeatum against protoscolices of CHD in-vitro. Protoscolices were aseptically aspirated from
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infected livers of sheep with CHD. Various concentrations (0.025, 0.05, 0.1 and 0.15 mg/mL) of green synthesis of Ag-NPs and different exposure times (10, 30, 60 and 120 min) were used against protoscolices of CHD Viability of protoscolices was confirmed by 0.1% eosin staining.
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The findings showed that the Ag-NPs at all concentrations have high scolicidal effects. The concentrations 0.1 and 0.15 mg/mL after 120 min of exposure times showed 83% and 90%
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mortality rate, respectively. The least scolicidal activity of biosynthesized Ag-NPs was 40% (0.025 mg/mL and 10 min). Current investigation indicated that applying biogenic Ag-NPs may be considered as a potential scolicidal agent for CHD surgery due to being economical, safer and non-toxic compared to the used chemical materials. However, further studies are required to evaluate the efficacy of Ag-NPs in vivo. Keywords: Cystic hydatid disease, Hydatid cyst, Green Silver Nanoparticles, Scolicidal, Echinococcus granulosus.
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1. Introduction Cystic hydatid disease (CHD) is a major neglected cyclo-zoonotic disease with various clinical complications caused by the larval stage (metacestode) of Echinococcus granulosus in many
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countries of the world [1-3]. The annual incidence rate of CHD can vary from 1 to 200 per 100,000 inhabitants in various endemic regions. Iran is considered as an endemic (southern parts) and hyperendemic (northern parts) area of CHD [3]. CHD is highly important in both
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medical and veterinary fields due to considerable economic damages and losses of animal proteins [1, 4-6]. In addition, CHD can cause 10% decline of life performance of infected
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animals including the decrease in meat quality, milk production, fiber and the number of surviving offspring [7].
Canids are the definitive hosts of E. granulosus whereas human, sheep and other mammalian species are intermediate hosts. Humans and herbivores acquire infection by accidental ingestion
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of eggs of E. granulosus. Oncospheres (embryo) of eggs are able to penetrate the intestinal mucosa and disseminate mainly, through the portal system in the liver and lungs [8].
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Surgery, PAIR (puncture-aspiration-injection-respiration) technique, and chemotherapy are commonly available cure for CHD. A lot of efforts have been conducted to prepare an efficient
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vaccine. However, there is not an effective vaccine against CHD for human [9]. The standard approach for CHD treatment is still remains surgery but we cannot ignore some unexpected repercussions such as anaphylactic shock, disease recurrence and mortality of this invasive method [10, 11]. Besides, the surgery becomes impractical when cysts are formed either in multiple organs or in risky locations including brain and spinal tissues. In these cases, the alternative options for treatment are chemotherapy and/or PAIR [2]. Removal of the cysts combined with chemotherapy either using Albendazole and/or Mebendazole before and after
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surgery are the best and selective methods. However, these drugs have demonstrated some side effects including thrombocytopenia, leucopenia and hepatotoxicity [12-15].
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To date, the routine scolicidal solutions such as, Ag-nitrate, cetrimide, hypertonic saline and ethanol, are broadly employed for infertility of the protoscolices whilst, they have different adverse effects such as liver necrosis, methaemoglobinaemiate and sclerosane colangititis
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(biliary tract fibrosis). Therefore, it is indispensable to develop new scolicidal materials with more efficacies and low adverse effects, which can be implemented during surgery [15-17].
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Synthesized green nanoparticles of biological sources like plants are widely accomplished because they are less toxic to human compared with the chemical medicines. Several metal nanoparticles such as copper (Cu), alginate magnesium (Mg), gold (Au), zinc (Zn), titanium (Ti), and silver (Ag) have been evaluated that among them, the synthesized silver nanoparticles
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(AgNps) have been introduced as the most effective one owing to having a large capacity in direct contact with their environment and acceptable antimicrobial efficacy against parasites, viruses and bacteria [18-22].
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There is no investigation concerning the impacts of green silver nanoparticles on protoscolices of CHD in-vitro. The objective of current study was to evaluate the scolicidal efficacies of cost
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effective and environmentlly friendly synthesized silver nanoparticles (AgNPs) derived from the aqueous aerial extract of Penicillium aculeatum against protoscolices of CHD in-vitro. 2. Materials and methods 2.1. Nanoparticle preparation Yeast extract was purchased from Liofilchem, Italy and silver nitrate and other chemical reagents were purchased from Merck, Germany. The pure colony of P. aculeatum was isolated from soil
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and approved by Department of Mycology and Plant Pathology of Sari Agriculture and Natural Resources University, Mazandaran Province, Iran. The fungus was cultured on Czapek dox broth, including 21g sucrose and 3g yeast extract in 1000mL distilled water, and then incubated
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at 28°C at 200 rpm for seven days (IKA KS 4000). The culture was centrifuged at 10,000 rpm for 5min, and the supernatant was used for the synthesis of AgNPs. A solution of silver nitrate (3mM) was prepared by dissolving 0.5096g of the compound in 100mL of double distilled
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water. Thereafter, 100mL of silver nitrate solution at a concentration of 3mM was added to 100 ml of the supernatant and incubated again for 24 hours at 28°C (total concentration of silver
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nitrate = 1.5mM). The colors of the solutions changed to dark buff, indicating the formation of AgNPs in the solution. The solutions were centrifuged at 20,000 rpm for 5min; the particles were separated from the supernatant. Then, the nanoparticles re-dispersed in double distilled water and centrifuged again. This action was repeated for three times to improve purification of AgNps.
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The solution was filtered through a 0.22µm millipore syringe filter to remove any impurity before characterization tests. Finally the AgNPs was freeze-dried and weighted by a digital scale to prepare concentrations of 0.025, 0.05, 0.1 and 0.15 mg/mL for in-vitro studies [23]. Evaluation of nanoparticles
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2.2.
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The formation of silver nitrate to AgNPs was confirmed by ultraviolet (UV) spectroscopy (Genesys 2 spectrophotometer, USA) and scanning electron microscope (SEM) model 2360 (Leo Oxford England). The size and polydispersity of particles were determined using photon correlation spectroscopy (PCS) by Zetasizer Nano Particle Analyzer (model 3600, Malvern instruments, UK) at 25°C with a scattering angle of 90°. PCS is a non-invasive technique that measures the size and size distribution of nanoparticles dispersed in a liquid. 2.3.
Protoscolices collection
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Hydatid cysts of E. granulosus were obtained from livers of naturally infected sheep in Mazandaran slaughterhouse, Mazandaran Province, North of Iran. The hydatid fluid was aseptically removed and transferred into a container and left to set for 30 min. The protoscolices
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settled down at the bottom of the container and then centrifuged at 800 rpm for 5 minutes. The supernatant was removed and the yielded protoscolices were left and the live protoscolices were
2.4.
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stored at 4°C for further use [24]. Scolicidal assay
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In the current study, different concentrations of Ag-NPs including 0.025, 0.05, 0.1 and 0.15 mg/mL were used for different exposure times including 10, 30, 60 and 120 min. To prepare the mentioned dilutions 0.025, 0.05, 0.1 and 0.15 µL of Ag-NPs were dissolved in 9.7 ml of normal saline in a test tube. Next, the obtained solution was gently mixed. Thereafter, in each
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experiment, 100 µl of protoscolices-rich sediment was added to 2.5 ml of the solution. After mixing the contents, the test tube was incubated at 37 ⁰C for 10, 30, 60 and 120 min. At the end of incubation periods in order to assess the viability of protoscolices 10µl of 0.1%
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eosin was added to the 20µl remaining pellet of protoscolices and mixed gently. The stained protoscolices was smeared on a manually scaled glass slide, which was covered with a cover
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glass (24 × 50 mm), and examined using light microscope. The percentages of dead protoscolices were estimated by counting a minimum of 200 protoscolices. Hydatid cyst fluid was considered as negative control group. Besides, NaCl 5% (5 g/1000 ml) were used as positive control group [25]. The experiments were performed in duplicate. 2.5.
Viability test
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Eosin stain with the concentration of 0.1% (1 g of eosin powder in 1000 ml distilled water) was utilized to check the viability of the protoscolices. Five min after exposure times to the eosin stain the protoscolices which did not absorbed dye with the movement of the flame cells were
2.6.
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potentially recorded as viable, otherwise, they were considered as dead protoscolices. Statistical analysis
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Differences between test and control groups were analyzed using the chi-square test. P values
GraphPad InStat software. 3. Results 3.1. Nanoparticle preparation
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<0.05 were considered significant. In addition, statistical analysis was performed using
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Addition of P. aculeatum supernatant to 1mM aqueous AgNO3 solution led to a color change to yellowish brown in the solution after 24 h of reaction, indicating the formation of AgNPs. The UV-Vis spectrum of AgNps produced by P. aculeatum exhibited an absorption band at around
(Figure 1).
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420 nm, which is a typical plasmon band, suggesting the formation of silver nanoparticles
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Figure 2 shows that the average size of the AgNps was 80.42 nm with a polydispersity index (PDI) of 0.238. The PCS spectra demonstrated that the AgNps formed had fairly well-defined dimensions and suitable monodispersity. PDI measures the second moment of the size distribution of the nanoparticle population. PDI ranges were from a value of 0.01 to 0.5 – 0.7 for monodispersed particles. Samples with very broad size distribution had polydispersity index >
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0.7. The SEM micrograph of nanoparticles showed that the AgNPs produced by P. aculeatum had a spherical shape and well distributed in solution (Figure 3).
Scolicidal effects of Ag-NPs
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3.2.
Figure 4 depicts the scolicidal effects of different concentrations of biosynthesized Ag-NPs (0.025, 0.05, 0.1 and .015 mg/mL) with different time expousre (10, 30, 60, and 120 min) against
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protoscolices of Hydatid cysts. Statistically the difference between the scolicidal effects of biosynthesized Ag-NPs was significant for all examined concentrations and exposure times in
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comparison to the control group (P<0.0001). It is noteworthy that with a rise in all concentrations of biosynthesized Ag-NPs the viability of protoscolices was decreased significantly (P<0.0001). At concentration of 0.1 and 0.15 mg/ml for 60 min treatment, the observed mortality rate was 80 % and 79%, respectively; while, up to 83% and 90% mortality
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was observed with 0.1 and 0.15 mg/ml for 120 min. In addition, the most scolicidal activity of biosynthesized Ag-NPs was 90% (0.15 mg/mL and 120 min) while the least scolicidal activity of
4. Discussion
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biosynthesized Ag-NPs was 40% (0.025 mg/mL and 10 min) (Table 1).
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In this study various dilutions (0.025, 0.05, 0.1 and .015 mg/mL) of biosynthesized Ag-NPs with different exposure times (10, 30, 60 and 120 min) were employed for scolicidal assessment. The most scolicidal activity of biosynthesized Ag-NPs were observed 90% (0.15 mg/mL and 120 min). Scolicidal effect of biosynthesized Ag-NPs may be attributed to electrostatic interaction between Ag-NPs (electrically positive) and the parasite (electrically negative) associated to organic complexes on its surface. A recently published study, showed fungal chitosan could kill 100% of protoscolices at concentration of 400 µg/ ml after 180 min [26].
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Surgery is current treatment of choice for complicated cases of CHD. Nevertheless, its success depends on formation of new cysts, relapse or secondary dissemination of CHD after surgery which can result in death due to spillage of the cyst contents [10]. Indeed, the infertilization and
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inactivation of protoscolices via scolicidal agents accompanied by minimum side effects and high efficacy instead of opening or removing cyst is highly recommended [12].
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Up to now, many protoscolicidal agents such as hypertonic saline, mannitol, chlorhexidine gluconate, huaier aqueous, Allium sativum, Sambucus ebulus, fungal chitosan and Berberis
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vulgaris have been used for inactivation of hydatid cyst content [26-29]. Unfortunately, using aforementioned protoscolicidal agents were limited because of their low efficacy, toxicity and undesirable side effects. Hypertonic saline (20 %) has been found to be 100% effective on protoscolices of hydatid cyst [25], but acute hypernatremia can cause severe nerve system symptoms such as necrosis, myelinolysis, convulsions, and intracranial bleeding. Silver nitrate
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and cetrimide have been shown to be 100% effective against protoscolices of the hydatid cyst; however, toxic reactions may also be caused by the absorption of these ingredients [30]. Although albendazole sulfoxide is the first choice for protoscolices, it increases the level of liver
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enzymes [16]. Besides, demanding preparation, and high cost are the principle disadvantages of
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the mentioned protoscolicidal agents. For these reasons, it cannot be considered as a perfect scolicidal remedy [12].
A similar study, showed that biogenic Selenium NPs at all concentrations have potent scolicidal effects against protoscolices of hydatid cyst especially at concentrations 500 and 250 mg/ml the scolicidal activity was 100% after 10 and 20 min of application, respectively [31].
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Presently, nanoparticles play crucial role in drug delivery, gene therapy, diagnostics, and many areas of research. The plant mediated biosynthesis of nanoparticles is cost effective and environmentally green method that does not require employing energy, pressure, toxic chemicals
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and temperature. Indeed, these characteristics are considered advantageous over chemical and physical methods [32, 33].
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This investigation for the first time presents the scolicidal effects of biosynthesized Ag-NPs against protoscolices of hydatid cysts in vitro model. Our results showed that Ag-NPs have an
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effective scolicidal activity in all concentrations especially at concentration 0.15 mg/ml (90% mortality rate) after 120 min exposure times. Consequently, these findings indicated that scolicidal effects of Ag-NPs at concentration 0.15 mg/ml was comparable with scolicidal effects of 20% silver nitrate (20 min) , 0.5-1% cetrimide (10 min), 20% hypertonic saline (15 min), 95%
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ethyl alcohol (15 min) and H2O2 3% (15 min), as previously described [12, 13, 29, 34-37] The permeation behavior through the hydatid cyst membrane was evaluated by albendazoleloaded nanoparticles (about 300 nm) which were prepared by the emulsion solvent evaporation
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method revealed a sufficient entrapment efficacy (36.4±6.4%) to increase the apparent solubility of albendazole. In fact, benzimidazole-loaded nanoparticles appear to be promising formula
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regarding treatment of hydatid cyst. Xie et al., 2010 evaluated the efficacy of praziquantel loaded hydrogenated castor oil solid lipid nanoparticle (PZQ-HCO-SLN) suspension in dogs naturally infected with E. granulosus [38]. The therapeutic effect of the novel formulation was promising to elevate the therapeutic efficiency of PZQ. At the dose of 5 mg/kg, stool ova declined and negative conversion rates and tapeworm removal rate of the suspension were recorded 100%, whereas the native PZQ were recorded 91.55%, 87.5%, and 66.7%. Once the dose decreased to 0.5 mg/kg, no effect was observed using the native drug, while the novel suspension still
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remained the same therapeutically effective as the 5 mg/kg native PZQ. An ideal scolicidal agent should be effective in low concentration in a shortest possible time, stable in hydatid cyst
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fluid, able to kill the protoscolices, nontoxic and easily available with low cost. Currently, the need to effective methods to control parasites has increased the use of nanomaterial products in biomedical sciences. Various researches conducted on parasite showed
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promising results. A study conducted by Said et al on antiparasitic effects of silver, chitosan, and curcumin nanoparticles as anti Giardia agent shows that the number of the parasites in stool
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and small intestinal sectors decreased in treated rats compared with non-treated ones [39]. larvicidal effects of Green synthesized silver nanoparticles against Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti suggest that they can be used as a rapid and safe biopesticide [40].
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In conclusion, this is noteworthy to mention that this is the first report on the biosynthesized AgNPs as scolicidal agent on hydatid cysts protoscolices in vitro. The evaluation of Ag-NPs against hydatid cyst protoscolices showed an effective and promising scolicidal activity. The prepared
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Ag-NPs using biotechnological methods can be considered as an effective scolicidal agent that could be applied for pharmaceutical applications, because the mentioned eco-friendly process is
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likely to be cost effective, safe in addition, it requires non-toxic materials compared to existing chemical methods.
Acknowledgments
This work was financially supported by Traditional Medicine & Materia Medica Research Center (TMRC) Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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Table 1. Protoscolicidal effects of biosynthesized Ag-NPs on protoscolices of hydatid cyst using different exposure time and concentrations of nanosilver. Agents
Repeat
Time
Control Positive
1 2 (Mean±SD)
0.54 0.45 (0.50±0.06)
0.64 0.55 (0.60±0.06)
0.62 0.65 (0.64±0.02)
0.77 0.78 (0.78±0.01)
Group 1
1 2 (Mean±SD)
0.4 0.35 (0.38±0.04)
0.56 0.49 (0.53±0.05)
0.68 0.65 (0.67±0.02)
0.68 0.61 (0.65±0.05)
Group 2
1 2 (Mean±SD)
0.43 0.47 (0.45±0.03)
0.59 0.59 (0.59±0.00)
0.69 0.64 (0.67±0.04)
0.75 0.73 (0.74±0.01)
Group 3
1 2 (Mean±SD)
0.49 0.51 (0.50±0.01)
0.56 0.53 (0.55±0.02)
0.75 0.85 (0.80±0.07)
0.85 0.81 (0.83±0.03)
Group 4
1 2 (Mean±SD)
0.45 0.47 (0.46±0.01)
0.59 0.54 (0.57±0.04)
0.79 0.78 (0.79±0.01)
0.9 0.9 (0.90±0.00)
Control Negative
1 2 (Mean±SD)
0.19 0.2 (0.20±0.01)
0.2 0.2 (0.20±0.00)
0.2 0.2 (0.20±0.00)
0.2 0.2 (0.20±0.00)
TE D EP AC C
120 min
RI PT
60 min
SC
30 min
M AN U
10 min
SC
RI PT
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Figure 1: UV–visible spectrum of the AgNps
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Figure 2: Photon correlation spectroscopy (PCS) of the AgNps; Average size: 80.42 nm, PDI: 0.238
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Figure 3: SEM image of the silver nanoparticles (Ag-NPs) synthesized by P. aculeatum.
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Figure 4. Scolicidal effects of biosynthesized Ag-NPs against protoscolices of Echinococcus
EP
granulosus at the different concentrations following various exposure times. Control positive (NaCl 5%); Group1 (0.025mg/mL of nanosilver); Group2 (0.05mg/mL of nanosilver); Group3
fluid).
AC C
(0.1mg/mL of nanosilver); Group4 (0.15mg/mL of nanosilver); Negative control (Hydatid cyst
ACCEPTED MANUSCRIPT
Highlights:
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Green synthesis of silver nanoparticles using aqueous aerial extract of Penicillium aculeatum.
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Ag-NPs showed potent in vitro scolicidal activity.
SC
Assessment of silver nanoparticles against protoscolices of hydatid cysts, in vitro.