Effect of Gold Nanoparticles on Bilharziasis

Abstract

Gold nanoparticles (AuNPs) gained a great attention in biomedical researches and become more applicable in nanomedicine in recent years because they have distinctive physicochemical properties. The current study was planned to assess the effect of the AuNPs with three doses levels on genes expressions, histology and oxidative stress status of Schistosoma mansoni infected mice liver. Inoculation of mice with 100 μl AuNPs at different doses (0.25, 0.5 and 1 mg/kg mice body weight) twice per week on day 46 and day 49 post infection reduced the total worm burden. Meanwhile, it reduced egg load in the liver and reduce the granuloma size. Also, AuNPs were able to significantly decrease the activities of malondialdhyde and nitric oxide as compared to infected untreated group. However, they increased the level of glutathione as compared toinfected untreated group. Concomitantly, AuNPs ameliorate the inflammatory response through decreasing the mRNA expression of IL-1ß, IL-6, TNFα, IFNγ, and iNOS. In consistence with molecular, histopathological and biochemical data, AuNPs could ameliorate the infection induced damage in the liver of mice .Our results indicated that, AuNPs are effective anti-schistosomal and anti-oxidantagent.to confirm the role of nanogold as an antischistosomal agent and its mechanism of action, more studies are required to be done in the future.

Keywords: Nanogold, Schistosoma mansoni, liver, gene expressions, histopathology, oxidative stress, mice.

Introduction

Bilharziasis is one of the most common parasitic diseases, which mostly affect the liver causing granuloma formation and hepatic fibrosis. Since, severe morbidity can result of schistosomal infection; the disease is still an important helminthic infection. Schistosomasis excessively affects people who have limited access to potable water and sanitation lived in the tropics and subtropics, approximately 240 million people infected with over 700 million people at risk of getting infected.¹

Praziquantel (PZQ) is the known effective anti-schistosomal drug but the reinfection occurs rapidly after massive drug administration, thus, an efficient therapy used is the optimal way, especially in Schistosoma mansoni endemic areas. From a long time, gold nanoparticles (AuNPs) was have been used for drug delivery into cells.^2,3,4Moreover, AuNPs have a strong potential role in cancer treatment and apoptosisinduction.⁵Accumulation of nanosystems at the targeted site I soften higher than normal drugs and usually leads to reduced systemic toxicity. However, chrysotherapy; gold was used in some diseases treatment “smallpox, skin ulcers, syphilis and measles” in ancient cultures in Egypt, India and China.²

Gold complexes showed potential antileishmanial and antimalarial activity, which have interesting role against Leishmania promastigotes in culture medium, becoming promising for using as “band-aids” to treat skin lesions. In addition, the effects of AuNPs as larvicidal for a mosquito vector of malaria have been reported. Thus, the impact of researches on gold for human tropical diseases therapy is considerable.⁶ The recent interest in using AuNPs in medicine has altered the methods of diagnosis and treatment.²For example, employing AuNPs in PCR has optimized the specificity of this diagnostic method.⁷Also some researchers have took advantage of AuNPsin transferring drugs into the biological cells, which provided a good basic for nuclear targeted delivery.⁸ Additional in vivo investigations are wished for the antihelminthic efficacy of AuNPs.⁹ Therefore, the present work aims to determine the cure rate of three doses of AuNPs against hepatic injury induced by schistosomasis in CD-1 mice.

Materials and methods

Gold nanoparticles (AuNPs)

AuNPs have been prepared by chemical reduction method as reported by Turkevich¹⁰. A solution of HAuCl₄ has been used as Au³⁺ ions precursor. Sodium citrate has been used as both of mild reducing and stabilizing agent. The color of the solution slowly turned into faint pink color, indicating the reduction of the Au³⁺ ions to Au nanoparticles. The fabrication of AuNPs were performed with the colloidal reduction process of chloroauric acid (HAuCl₄.3H₂O) with salt of trisodium citrate (N₃C₆H₅O₇) purchased from Aldrich (99% pure) Chemical Co. Ltd and used without further purification. In a typical experiment: 2 mM of HAuCl₄.3H₂O was dissolved in 100 mL of double distilled water. To this solution, 1% N₃C₆H₅O₇ (~3 mM) was mixed. The pH of this solution was measured via pH meter (Cole parmer U.SA.), which was reached to 7.88. The obtained pinkish colored solution was stirred vigorously and refluxed the solution at boiling temperature for 15-20 min. pinkish color was changed to deep red colored solution of AuNPs. The obtained colloidal solution was stored for the further morphological and other elemental analysis.

Characterization

Size and morphology of AuNPs were done by using transmission electron microscopy (TEM) equipped with high resolution transmission electron microscopy (HR-TEM). Samples for TEM were prepared using the colloidal solution of nanoparticles. The colloidal sample solution was sonicated for 10 min in a bath sonicator before the observation and dipped in carbon coated copper grid (400 mesh) and dried at room temperature for the morphological analysis.A TEM picture was taken by a JOEL JEM 2000 EX 200 microscope at 200kv.

Animals

Sixty male CD-1 mice weighing 18-20 g were used in all experiments. The animals were obtained from a closed random bred colony at the Schistosome Biological Supply Center (SBSC) at Theodor Bilharz Research Institute, Giza, Egypt. Animals were housed in polycarbonate boxes with steel-wire tops (not more than six animals per cage) and bedded with wood shavings. Ambient temperature was controlled at 22 ± 3 °C with a relative humidity of 50± 15% and a 12-h light/dark photoperiod. Food and water were provided ad libitum. This study was conducted in accordance with legal ethical guidelines of the Medical Ethics Committee of the Theodor Bilharz Research Institute (TBRI), Giza, Egypt (Approval No. 4018/2011).

Mice Infection

S. mansoni cercariae (Egyptian strain) were obtained from infected intermediate host snails (Biomphalaria alexandrina) maintained at the SBSC. Mice were infected subcutaneously with freshly shed 100± 10 cercariae/mouse according to Liang et al.¹¹method.

Experimental design

Animals were divided into six groups of ten mice each. Group I served as a control (non-infected); the animals were received saline (100 μl saline water/mouse) by intraperitoneal (ip) injection for 10 days. Group II and Groups III, IV, V and VI were infected with 100±10 S. mansoni cercariae. The animals of groups III, IV and V were intrapretonially inoculated with 100 μl AuNPs at different doses (0.25, 0.5 and 1 mg/kg mice body weight) twice per week on day 46 and day 49 post infection respectively. Finally, infected animals of Group VI were orally administered 100 µl of PZQ (600 mg/kg mice body weight) on day 46 post infection at an interval of 24 h for 2 days.¹²

Study of parasitological criteria

Immediately after mice killed by cervical decapitation, hepatic and portomesenteric vessels were perfuse for worms ‘recovery and subsequent counting.¹³ After perfusion, a piece of liver was used for the determination of the number of ova in liver and the percentage change in egg density was determined. The percentage of eggs at various developmental stages was examined in three samples from each mouse and the mean number of eggs at each stage/animal was determined.¹⁴

Sample preparation

After dissection, the liver of all groups were immediately removed and divided into three parts, the first part for RNA extraction, the second one for histopathological studies and the third part was homogenized (10% w/v) in ice-cold 0.1 M Tris-HCl buffer (pH 7.4). The homogenate was centrifuged at 2000 Ã-g for 15 min. at 4 °C and the resultant supernatant was used for biochemical analysis.

Histopathological investigations and granuloma size

Tissue samples of the liver of all groups were immediately fixed after animal dissection in 10% neutral buffered formalin dehydrated and processed for paraffin sectioning. Sections were then deparaffinized, stained with hematoxylin and eosin stains. To assess the size of tissue granuloma, the mean diameter (μm) was measured. For each group, 30 granulomas were chosen from different hematoxylin-eosin stained liver sections from different mice.

Assessment of oxidative stress markers

Estimation of the reduced glutathione (GSH) level

GSH level in liver was determined by the methods of Ellman.¹⁵ The method based on the reduction of Ellman’s reagent with GSH to produce a yellow compound; the reduced chromogen directly proportional to GSH concentration and its absorbance can be measured at 405 nm.

Determination of thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS) were assayed through colorimetric tests of the liver homogenates according to the method of Ohkawa et al.¹⁶ In this method, TBARS was determined by using 1 ml of trichloroacetic acid 10% and 1 ml of thiobarbituric acid 0.67% which were then heated together in a boiling water bath for 30 min. TBARS which react with the amount of malondialdehyde found in liver homogenate to give a red color were then measured at 535 nm.

Determination of nitric oxide level

Nitric oxide (NO) level was assayed colorimetrically in liver homogenate according to the method of Green et al.¹⁷ The nitrite/nitrate level was determined where in an acid medium and in the presence of nitrite the formed nitrous acid diazotisesulphanilamide is coupled with N-(1-naphthyl) ethylenediamine. The resulting azo dye has a bright reddish-purple color which can be measured at 540 nm.

Quantitative PCR

Tissues frozen at -80^oC were thoroughly grounded with a mortar under liquid nitrogen and total RNA was isolated with Trizol (Sigma-Aldrich). Quality and integrity of RNA were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A260nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).¹⁸ Real time PCR was performed as detailed previously.^{19, 20} In brief, total RNA freed from DNA using the DNA free kit (Applied Biosystem, Darmstadt, Germany) was used to synthesize cDNA using QuantiTectTM Reverse QuantiTectTM SYBR® Green PCR kit (Qiagen) was applied for amplifications in the ABI Prism® 7500HT Sequence Detection System (AppliedBiosystems, Darmstadt, Germany) with gene-specific QuantiTectTM primers delivered by Qiagen (Hilden, Germany). We investigated the genes encoding the mRNAs for the following proteins: interleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα), interferone- (IFN), and inducible nitric oxide synthase (iNOS). PCR reactions were performed and evaluated as detailed elsewhere.¹⁸

Statistical analysis

The obtained data were presented as means ± standard error. One-way ANOVA was carried out, and the statistical comparisons among the groups were performed with Duncan’s test using a statistical package program (SPSS version 17.0). P≤0.05 was considered as significant for all statistical analysis in this study.

Results

Morphological analysis of colloidal AuNPs

The structural morphology and crystalline character of AuNPs were examined via transmission electron microscopy (TEM) equipped with high resolution transmission electron microscopy (HR-TEM). The obtained images shows the corresponding TEM results, which are shown in figure (1), the low magnified image (Fig. 1A) shows that, AuNPs are spherical in shape within the range of 10-15 nm in diameter. From the observation, it’s depicted that all the NPs are in definite spherical shape with rough surface and free from agglomeration behavior. Another obtained image represents the high resolution TEM (HR-TEM) image of AuNPs (figure 1B), which shows the lattice difference fringes between two adjacent planes are about 0.235 nm. The obtained lattice difference clearly corresponds to the lattice constant of face centered cubic (FCC) of AuNPs and are analogues with the previously reported information.^{21, 22} The crystal lattice fringes of HRTEM observation (Fig. 1B), again shows a confirmation of good crystalline nature of synthesized AuNPs and it is consistent with the low magnified image of AuNPs. ^{21, 22}

AuNPs treatment induced a significant reduction in hepatic worm burden at all examined doses (0.25, 0.5 and1 mg/kg) as compared to infected group. The worm burden reduction rate was approximately, 32%, 49% and 64%, respectively (Table 1). Similarly, figure 2 shows that, the three dose levels of AuNPs caused a highly significant reduction on egg density in liver tissues of infected mice, and the highest reduction (69.8 %) was recorded at 1mg AuNPs dose level .

Table 2 and figure 3 show the alternation on the liver histology from S. mansoni infected animals, compared with that from control animals. Figure 3A displays a histological section of liver from a control mouse. The center-lobe vein has normal morphological characteristics while figure 3B shows a histological section of liver after 56 days of S. mansoni infection in mice. Cellular alteration was verified on liver. There are leukocyte aggregations near blood vessels and evident vascular congestion. Histological investigation of hepatic tissue sections reveals that S. mansoni infection caused a severe inflammatory response of the liver, as indicated by inflammatory cellular infiltration as well as cytoplasmic vacuolation and degeneration of hepatocytes. In addition, the hepatic sinusoids were dilated and apparently contained more Kupffer cells. Treated livers of S. mansoni infected mice with the three dose levels (0.25, 0.5 and 1 mg/kg) of AuNPs as shown in figures 3C, 3D and 3E, respectively appeared with moderate inflammatory cellular infiltration.

Figure4 showed that, the granuloma size in hepatic tissue showed a marked and a significant reduction in the granuloma diameter at P<0.05as a result of AuNPs treatment to schistosome infected mice at all investigated doses (0.25, 0.5 and 1 mg/kg) as compared to untreated schistosome infected mice. Likewise, PZQ gavage induced a significant decrease in hepatic granuloma size infected of S. mansoni versus infected group.

Schistosomiasis induced a significant elevation in hepatic levels of MDA and NO (table 3). In the same manner, injection of 3 different doses of AuNPs and PZQ to infected mice increased the levels of MDA and NO significantly as compared to non-infected group. Oppositely, a significant reduction was observed in hepatic MDA and NO levels as a result of AuNPs (0.25, 0.5 and 1 mg/kg) and PZQ injection versus infected group. Finally, GSH which involved in the down-regulation of substances formed during oxidative stress has been determined (Table 3). It was striking that GSH was significantly down-regulated by S. mansoni infection but that this effect was largely ameliorated by AuNPs treatment.

Moreover, the S. mansoni infected mice revealed a significant up-regulation in mRNA of IL-1β, IL-6, TNFα, IFNγ and iNOs in hepatic tissue; likewise, injection of different doses of AuNPs and PZQ induced significant up-regulation versus control group. On the other hand, treatment with AuNPs as well as, PZQ to infected mice decreased the expression of IL-1β, IL-6, TNFα, IFNγ and iNOs-mRNA significantly as compared to infected group (Fig. 5).

 

4. Discussion

Newly in several fields of nanomedicine; AuNPs have been actively used for diagnostic and therapeutic applications. It has been debated that nanoparticles of gold could be used in nearly all medical purposes.²³ Abraham and Himmel²⁴proved the successful usage of colloidal gold in rheumatoid arthritispatients. In addition, AuNPs caused cestode paralysis, which is followed by death; the authors attributed to alterations in cestode enzymatic activity of the parasite.⁹

Our results revealed that schistosomiasis caused marked and significant histopathological impairments in liver sections, and granulomatous inflammation was recorded. Ameret al.²⁵ reported that S. mansoni induced granulomas which were characterized by concentric fibrosis هى the trapped eggs surrounded by many fibroblasts. In addition, Toussonet al.²⁶observed main histopathological injuries in schistosomiasis mansoni such as granulomas, diffuse infiltration of inflammatory cells “eosinophils and small mononuclear cells” and fibrosis of portal areas and interlobular septa. In the same manner, El- Banhawey et al. ²⁷ cleared that schistosomiasis causes necrotic changes in the liver tissues.

On contrary, our treatment with different doses of AuNPs appeared moderate inflammatory cellular infiltration, decreased the granuloma diameter. Moreover, AuNPs reduced the hepatic worm burden as compared the infected group. Dkhilet al.²⁸ deduced that gold nanoparticles treatment to infected schistosome mice improved the histological disturbances of infected brain mice.

Schistosomiasis mansoni imbalanced the hepatocellular antioxidant system and liberated the free radicals which are evidenced by decrement in GSH level and increased levels of both nitrite/nitrate and MDA in hepatic tissue. Meanwhile, AuNPs increased hepatic GSH level and decreased the levels of nitrite/nitrate and MDA. It was reported that, schistosomiasis disturbed the levels of enzymatic and non-enzymatic antioxidants which impaired liver GSH content of mice and decreased hepatic antioxidant capacity inducing lipid peroxides generation which may act a main role in the pathology associated with bilharziasis.^{25, 29} Furthermore, S. mansoni caused oxidative stress in different mice organs such as brain. Neuroschistosomiasis induced reduction of GSH level and increased nitrite/nitrate and MDA levels. Otherwise, gold nanoparticles (0.25, 0.5 and 1 mg/kg) injection to schistosome infected mice ameliorated GSH level and reduced levels of nitrite/nitrate and MDA in brain.²⁸

In the present study, AuNPs injection (0.25, 0.5 and 1 mg/kg) to schistosome infected mice showed a significant down-regulation of IL-1β, IL-6, TNFα, IFNγand iNOs-mRNA expressions in hepatic tissue versus infected mice.

IL-1 and TNF-α are the major pro-inflammatory cytokine, they considered as ‘alarm cytokines’ which secreted by macrophages. IL-1 plays a role in initiation and propagation of the inflammation by stimulating the expression of adhesion molecules on endothelial cells and leukocytes. In addition, TNF-α may aggravate fibrosis and ameliorate the granulomatous reaction results from presence of schistosome eggs. So, in mice lacking of IL-1β gene, characterized by delayed disease development and declined systemic inflammatory responses.^{30, 31} Moreover, lower expression of IL-6 and IL-1β (pro-inflammatory cytokines) cause a down modulation of granulomatous inflammation and hepatocyte necrosis.³²Also, Macrophages could be activate to produce NO and other inflammatory mediators by IFNγ is which considered as an important inducer of iNOs. In addition, Abdallahi et al.³³detectediNOs mRNA in the liver at the onset of parasite egg laying; the authors cleared that the levels then increased as the eggs accumulated liver.³⁴ However, Mwatha et al.³⁵ reported that increased TNFγ is correlated with the development of severe hepatosplenic disease.

Khan et al.³⁶ concluded that gold nanoparticles (50 nm) showed a normal level of IL-6 gene expression in hepatic tissue of rat on day 5 of injection meanwhile, IL-1β, TNFα m-RNA expression was down regulated significantly on day 5. Moreover, nanoparticles of gold have no cytotoxic effect as they decrease the production of reactive oxygen species and do not stimulate secretion of proinflammatory cytokines (TNF𝛼and IL1-𝛽) making them suitable candidates for nanomedicine.^{37, 38}Gold nanoparticles are not induced apoptosis, moreover, not activated gene expression related to oxidative stress and inflammatory response (TNF𝛼) while their low reactivity with biomolecules and cells provides a promising medical platform.³⁹

Conclusion

Collectively, our investigations suggest that the way in which AuNPs exert their ameliorating effects on Schistosomiasis mansoni promoted oxidative stress may be attributed to its ability to scavenging free radicals , and that this action could find a clinical use in the treatment of hepatic dysfunction in schistosomiasis. Additional studies are still necessary, however, with a view to clarify the exact mechanism of this modulatory effect, and to examine its potential therapeutic effects in more detail.