Scolicidal Effects of Silver – Copper (core-shell) Nanoparticles Against Echinococcus granulosus Protoscolices in vitro
Alaa Adnan Aljanabi¹Kawther A. M. Al-mussawi²Abbas Matrood Bashi³ 1. AlSafwa University College _ Iraq
2. Karbala University - College of Education for Pure Sciences _ Iraq 3. College of Altuff _ Iraq .
The current study included the preparation and diagnosis of the Silver - Copper (core - shell) nanoparticlesand the nanoparticles on which the commercial drug Albendazole is loaded and the study of their effect on the Protoscolices of hydatid cyst disease in vitro , this is a comparison with the effect of the commercial drug alone with the presence of the control treatment that includes the addition of distilled water only , The results showed that the Silver-Copper (shell- core) nanoparticles had a very high effect on the vitality of the Protoscolices and this effect increased with increasing concentration and duration of exposurewith high significant differences but less than the intensity of the effect of the nanoparticles alone, the two compounds had a high and significant effect compared to the commercial drug when used alone.
Keywords : Protoscolices , Nanoparticles , Echinococcosis , Hydatid cyst.
Echinococcosis is a zoonotic disease caused by the tapeworm of the genus Echinococcus . This parasite lives in the intestine of the final hosts in an adult form . In the intermediate host, the larval form is present as cysts within the internal organs and this disease is one of the biggest health problems common in some countries and has different forms, as cystic Echinococcosis is the most common and caused by E. granulosusbecause its larvae develop as single and separate cysts and it is less dangerous and more treatable than other forms such as E. multilocularis causing the disease. alveolar echinococcosis and E.vogeli that cause polycystic echinococcosis (CFSPH, 2020) .
According to Higuitaet al. (2016) the squamous cyst parasite infects more than one million people around the world annually and is the cause of economic losses estimated at about three billion dollars each year.Hydatid cyst disease in developing countries such as countries in the Middle East remains a major health obstacle, and its highest prevalence rates are concentrated in rural areas where there are many sheep pastures, which represent the most important intermediate host Abdulhameedet al., 2019; Thys et al., 2019).
In Iraq, in particular, it is one of the main health problems (CDC, 2012), It is one of the endemic diseases in Iraq country and there are no organized national programs for disease control and surveillance (Athmar& Ban-Abbas, 2014). The most cases of human infection were recorded in the central and southern regions, including the governorates of Basra, DhiQar and Muthanna
an effective drug against it and the absence of pathological symptoms even after the disease reaches an advanced state, especially in the important organs such as the brain and heart, so it is difficult to control therapeutic and surgical in those organs, because of this risk generated attempts Solutions were used to reduce the disease and reduce its spread (Kharebov, 1997).
Some studies have shown that nanoparticles of silver AgNps, gold AuNps, chitosan and other metal oxides have an inhibitory or toxic effect for many parasites such as Giardia, Leishmania, Toxoplasma, Plasmodium and some insect larvae (Elmiet al., 2013). The nanoparticles of silver made from nanoparticles are the most effective due to their direct contact with the environment and their anti-parasite, bacterial and viral effects (Mahmoudet al., 2014). Therefore, it is recommended to use nanoparticles to get rid of parasites as they are effective and less harmful drugs as well as an effective vaccine for controlling parasites (Elmiet al., 2013).
MATERIAL AND METHODS
Collection of hydatid Cysts and Protoscolices
Sheep livers naturally infected with hydatid cysts were collected from locally stores , Karbala city , Iraq placed in clean containers and transferred to the Parasitology Lab.- Department of Medical Laboratory Techniques / Al-Safwa University College, the livers were washed with running water to get rid of blood and impurities, then their surfaces were sterilized with dilute ethyl alcohol at a concentration of 70% and at laboratory temperature the fluid of the cysts was withdrawn by sterile medical syringes 5 ml and by means of clean and sterile scissors the cyst was opened.Phosphate Buffer Saline Solution(PBS) was added to the washing bottle and antibiotics were added to the buffer (Crystalline penicillin at 2000 units / liter and Streptomycin at 1 g / L).Where the germ layer was washed to remove the suspended protoscolices in it, the liquid was left for a period of time for all the protoscolices to be deposited , after then were collected and deposited and the float was removed and a little of what was left of it was placed in plastic test tubes for sedimentation in the Centrifuge machine, where it was deposited for 15 minutes 3000 rpm/m.(Smyth, 1985) .
Preparation of Nanoparticles
The Silver-Copper (shell-core) nanoparticles was preparedaccording to (Nadagouda&Varma, 2007) . 10 mL (0.1 N) of ascorbic acid (vitamin C) was allowed to interact with 2 mL (0.1) N of calcium chloride CuCl2 which act as the core , then added to the shell which is about 2 ml of (0.01 N) AgNo3and left to interact with shaking at room temperature for an hour to form a gray precipitatethat was separated from the filtrate and washed with distilled water and spread it until drythen grinded and collected in tubes glass and kept at room temperature until use.
Preparation of commercial drug
The drug was obtained from a commercial pharmacy and it is Albendazole 200 mg tablets, manufactured by Julphar Company - United Arab Emirates. The tablets were crushed and the drug was used as a control treatment for comparison with the nanoparticles .
Loading the commercial drug on NPs
250 mg of the nanoparticles which preparedPreviously was mixed with 250 mg of the commercial drug in 25 ml of distilled water, the mixture was left for 12 hours on the magnetic stirrer and then left to precipitate and the filtrate was separated from the sediment and the sediment was washed with distilled water and left to dry, after complete drying, the sediment was collected , grinding and kept in a glass container at room temperature until use.
Diagnosis of NPs
The prepared nanoparticles were diagnosed to ensure that they carry the nanoscale properties by Fourier-transform infrared spectroscopy (FTIR), Atomic force microscopy (AFM) and X-ray diffraction (XRD), and the shell-core characteristic between the two compounds was confirmed by Scanning electron microscope (SEM) and examined at Al-Fadhelfoundation / Babel - Al- Hillah branch, SEM and XRD examination were carried out in Iran.
The efficacy of Nanoparticles and Albendazole drug in the viability of Protoscolicesin vitro Concentrations of 50, 125, 250 and 500 micrograms / ml were used. 0.5 ml of the protoscolices suspension was placed in 10 ml test tubes and then the Nanoparticles was added after the stock solution was prepared. Three replications were made for each of the four concentrations of the drug and the Nanoparticles with The presence of a control treatment added to it with distilled water only. Protoscolices vitality were examined after 10, 30 and 60 minutes of addition at 37 °C.
Protoscolices viability was examined by taking 10 μL of the suspension and adding the dye and examining under a microscope where the living heads were stained green and dead heads were stained red (Nematollahiet al., 2018( .
RESULTS AND DISCUSSION
Effect of Albendazole drug in the viability of Protoscolices
The results showed according to Table 1, that the effect of treatment with a concentration of 50 mg / ml had no significant effect after 10 minutes of treatment, and the situation continued after 30 minutes and after 60 minutes of treatment , compared with the control treatment .The significant differences began to appear with increasing concentration, but these significant differences were insignificant compared with the control treatment when using concentrate 125mg / ml and after 10 minutes, and these slight differences continued with increasing time up to 60 minutes.
Significant differences increased at concentration 250 mg / ml compared to control treatment, but the increase in the time period of exposure to treatment did not affect in the vitality of protoscolices .The greatest value of the significant differences appeared with a concentration of 500 mg / ml compared with the control treatment, but there was no clear significant difference with the time increase when the protoscoliceswere exposed to this concentration.
When comparing the concentrations, we will find that the effect of the treatment increased with the increase in concentration compared to the control treatment, as the concentration 50 mg / ml
had no effect on the vitality of the substrates, while this effect increased with the concentration of 125 mg / ml, reaching a maximum of 500 mg / ml. With high significant differences.As for time, it did not have a clear effect on vitality, and there were no significant differences . These results were in agreement with Al-hamiary(2010) and Al-Nakeeb (2004) who indicated that the treatment of Albendazole had an effect on primordial viability that increased with increasing concentration.
As this drug is considered one of the best drugs used in parasite resistance, the principle of its action depends on binding with the B-tubulin protein, causing the polymerization process to be canceled in the microtubules of this protein causing a group of damage to the parasite cells from the transfer, growth and obstruction to the absorption of glucose in worms and parasitic larvae ,this leads to the depletion of the stock of animal starch (Glycogen), thus reducing energy production ATP until the parasite dies (Karakay , 2007) .
Table (1) : Effect of Albendazole drug in the viability of Protoscolices
10 M. 30M. 60M.
Mean SD Mean SD Mean SD
37.67 2.08 36.00 14.93 35.00 5.00
50 31.00 12.12 26.67 0.58 25.67 1.53
125 24.00 1.73 21.67 2.89 21.00 1.73
250 18.33 1.53 17.00 1.00 15.00 1.73
500 12.67 0.58 12.00 1.73 10.33 0.58
LSD 10.194 12.490 4.721
Sig. 0.002 0.014 0.0001
Effect of Silver – Copper (core –shell) Nps in the viability of Protoscolices
The results showed that the silver-copper nanoparticles (core-shell) had a significant effect on the vitality of the substrates compared to the treatment of Albendazole, as this compound had a significant effect even with the lowest concentration of 50 mg / ml, where significant differences appeared compared to the control treatment, and these significant differences increased. By increasing the concentration significantly and clearly, it reached its peak concentration at 500 mg / ml, where the colic killing rate was 100%.The increase in the exposure period also had an effect on the vitality of the Protoscolices as well with very large significant differenceseven with the lowest concentration of 50 mg / ml, reaching a peak at 60 minutes, and with the concentration of 125 mg / ml, the nanoparticles had an effect at 10 minutes which increased dramatically with increasing time and significant differences.As for the concentration of 250 and 500 mg / ml, their effect was the greatest and clear since the first ten minutes of exposure, with very large significant differences.
As shown in Table 2, the two concentrations 250 and 500 mg / ml are the most effective in the vitality of the protoscolices , compared with the lowest concentrations of 50 and 125 mg / ml .The results are in agreement with Norouziet al. (2020) who showed that silver nanoparticles have a scolicidaleffect, and this effect increases with increasing concentration and increasing the exposure time, as is the case with copper nanoparticles, but with less effect than silver .
The nanoparticle oxides, including the silver nanoparticles have heterogeneity in surface propertiesbandgap causing a high increase in detonation that affects the surface adsorption process and thus their penetration into the surfaces (Immanuelet al., 2019) .
Table (2) : Effect of Silver – Copper (core –shell) Nps in the viability of Protoscolices
10 M. 30 M. 60 M.
Mean SD Mean SD Mean SD
Silver – Copper (core –shell) Nps
37.67 2.08 36.00 14.93 35.00 5.00
50 27.33 2.31 20.33 5.51 0.00 0.00
125 14.00 7.00 0.00 0.00 0.00 0.00
250 0.00 0.00 0.00 0.00 0.00 0.00
500 0.00 0.00 0.00 0.00 0.00 0.00
LSD 6.232 12.950 4.068
Sig. 0.0001 0.0001 0.0001
Effect of Drug loading on Silver - Copper (shell – core) nanoparticles in the viability of Protoscolices
The drug loading on the silver-copper nanoparticles (core-shell) had a clear effect on the vitality of the protoscolicescompared to the control treatment and even with the lowest concentration usedwhich was 50 mg / ml, where there were clear significant differences in the effect and this effect continued to increase with increasing concentrations until the significant differences became very high with a concentration of 500 mg / ml, as show in table 3 .The duration of exposure also had an effect on the vitality of the protoscolices as well as the results showed that the viability of the protoscolices decreases with the increase in the duration of exposure compared to the control treatment. Results were similar with higher concentrations 125, 250 and 500 mg / ml and with greater significant differences, the viability reached zero after 30 minutes of exposure time with concentrations of 250 and 500 mg / ml.These results agreed withTorabiet al. (2018) who used the drugs Albendazole and Praziquantel loaded with chitosan nanoparticles in the treatment of microcysts of E.granulosusin vitroand found it very effective .
It also agreed with Bakhtiaret al. (2019) that showed that Albendazole loaded on selenium nanoparticles at concentrations of 250 and 500 mg / ml has an effect of 100% in killing protoscolices after exposure for 5 days .
Table (3) : Effect of Drug loading on Silver - Copper (shell – core) nanoparticles in the viability of Protoscolices
M.10 M. 30 M. 60
Mean SD Mean SD Mean SD
Drugloading on Silver - Copper (shell – core) nanoparticles
control 37.67 2.08 36.00 14.93 35.00 5.00
50 15.33 6.43 14.33 5.13 0.00 0.00
125 3.00 1.00 0.67 0.58 0.00 0.00
250 2.67 0.58 0.00 0.00 0.00 0.00
500 1.33 1.15 0.00 0.00 0.00 0.00
LSD 5.656 12.856 4.068
Sig. 0.0001 0.0001 0.0001
According to the results, the commercial drug loaded on the nanoparticles is more effective in reducing the vitality of the protoscolices which was agreed with Bakhtiaret al. (2019) who indicated that Albendazole loaded on the selenium nanoparticles kills 100% of the protoscolices in 5 days compared to the drug alone, which kills 100% after 7 days .
The current research has found that the commercial drug becomes effective in eliminating the protoscolicesof hydatid cystin vitro when it is loaded onto the Silver-Copper nanoparticles (core- shell) compared to the drug alone, and the nanoparticles alone is better than the commercial drug and is also betterfrom the drug loaded on the nanoparticles .
1. Abdulhameed, M. F., Robertson, I. D., Al-Azizz, S. A., & Habib, I. (2019). Neglected Zoonoses and the Missing Opportunities for One Health Education: The Case of Cystic Echinococcosis among Surgically Operated Patients in Basrah, Southern Iraq. Diseases, 7(1), 4.
2. Athmar, K. A. A., & Ban-Abbas, A. M. (2014). Immunization mice with DNA from protoscolices of human hydatid cyst. Immunological study. Int. J. Adv. Bio. Res., 4(1): 89- 95.
3. Al-hamiaryA. Kh. A. (2010) . Evaluation of the Activity Daturastramonium Seeds Extracts on Growth and Development of Hydatid Cysts for Echinococcus granulosus in White Mice Balb/c (Therapeutic , Histologic and Immunologic Study ) . doctorate thesis.
4. Al-Nakeeb, S. A. R. (2004). Seroepidemiological and therapeutic study on hydatid cyst infection in Kirkuk and Tikrit provinces. M. Sc. Thesis , coll. Med. , Univ. Tikrit: 125 pp.
5. BakhtiarN. M. ;Akbarzadeh, A.;Casulli, A.;Mahami-Oskouei M. ; Ahmadpour, E.&Nami, S.&Rostami, A.&SpotinA. (2019) Therapeutic efficacy of nanocompounds in the treatment of cysticand alveolar echinococcoses: challenges and future prospects . Parasitology Research . 118:2455–2466 .
6. CFSPH . (2020) Echinococcosis . page 1-14 . www.cfsph.iastate.edu
7. Elmi, T, Gholami, S, Fakhar, M, Azizi, F. (2013) . A Review on the se of nanoparticles in the treatment of parasitic infections. J. Mazand. Univ. Med. Sci. 23, 102:126-33.
8. Higuita N. I. A., Brunetti E., McCloskey C.(2016) . Cystic Echinococcosis . Journal of Clinical Microbiology Volume 54 Number 3 . 518-523 .
9. Immanuel S, Aparna TK, Sivasubramanian R. Graphene-based electrochemical sensors for biomolecules.(2019) . Micro Nano Technol. , 113–138.
10. Karakay, K. (2007). Spontaneous rupture of hepatic hydatid cyst into the peritoneum causing causing only mild abdominal pain: A case report. World, J. Gastroenterol. , 13(5):806-808.
11. Kharebov, A. ; Nahmias, J. and El-On ,J. ( 1997 ) . Cellular and humoral immune responses of hydatidosis patients to E. granulosus purified antigens
12. Mahmoud , H., Fasihi-Harandi, M., Shakibaie, M., Aflatooni, M.R., Ziaali, N., Makki, M.S. and Jahanbakhsh, S. (2014) . Scolicidal effects of biogenic selenium nanoparticles against protoscolices of hydatid cysts. Int. J. Surg. 12, 399-403.
13. Mohanad F. Abdulhameed 1,2, Ian D. Robertson 1,3 , Suzan A. Al-Azizz 2 and Ihab Habib .(2019) . Neglected Zoonoses and the Missing Opportunities for One Health Education: The Case of Cystic Echinococcosis among Surgically Operated Patients in Basrah, Southern Iraq Diseases, 7, 4;
14. Nadagouda, M.N.; Varma, R.S. A (2007) .Greener Synthesis of Core (Fe, Cu)-Shell (Au,
Pt, Pd, and Ag) Nanocrystals Using Aqueous Vitamin C. Cryst. Growth Des., 7, 2582–
15. Nematollahi A. ; Shahbazi P. , Rafat A. and Ghanbarlu M. ( 2018) . Comparative survey on scolicidal effects of selenium and silver nanoparticles on protoscolices of hydatid cyst . Open Veterinary Journal, Vol. 8(4): 374-377 .
16. Norouzi R. ; Ataei A. ; Hejazy M. ; Noreddin A. and El Zowalaty M. E. (2020) . Scolicidal Effects of Nanoparticles Against Hydatid Cyst Protoscolices in vitro . International Journal of Nanomedicine . 15 1095–1100 .
17. Smyth, J. D. (1985). In vitro culture of Echinococcus spp. Proc. 13th Int. Cong. Hydatid.
Madrid : 84-95.
18. Torabi, N.;Dobakhti, F.;Faghihzadeh, S. &Haniloo, A. (2018) . In vitro and in vivo effects of chitosan-praziquanteland chitosan-albendazole nanoparticles on EchinococcusgranulosusMetacestodes . Parasitology Research . 1- 9 .
19. Thys, S., Sahibi, H., Gabriël, S., Rahali, T., Lefèvre, P., Rhalem, A.,&Dorny, P. (2019).
Community perception and knowledge ofcystic echinococcosis in the High Atlas Mountains, Morocco. BMCpublic health., 19(1): 118.