Newly fabricated zinc oxide nanoparticles loaded materials for therapeutic nano delivery in experimental cryptosporidiosis

Cryptosporidiosis is a global health problem that threatens the lives of immunocompromised patients. This study targets to fabricate and investigate the efficiency of zinc oxide nanoparticles (ZnO-NPs), nitazoxanide (NTZ)-loaded ZnO-NPs, and Allium sativum (A. sativum)-loaded ZnO-NPs in treating cryptosporidiosis. Further FTIR, SEM, XRD, and zeta analysis were used for the characterization of ZnO-NPs and loaded materials. The morphology of loaded materials for ZnO-NPs changed into wrapped layers and well-distributed homogenous particles, which had a direct effect on the oocyst wall. The charge surface of all particles had a negative sign, which indicated well distribution into the parasite matrix. For anti-cryptosporidiosis efficiency, thirty immunosuppressed Cryptosporidium parvum-infected mice, classified into six groups, were sacrificed on the 21st day after infection with an evaluation of parasitological, histopathological, and oxidative markers. It was detected that the highest reduction percent of Cryptosporidium oocyst shedding was (81.5%) in NTZ, followed by (71.1%) in A. sativum-loaded ZnO-NPs-treated groups. Also, treatment with A. sativum and NTZ-loaded ZnO-NPs revealed remarkable amelioration of the intestinal, hepatic, and pulmonary histopathological lesions. Furthermore, they significantly produced an increase in GSH values and improved the changes in NO and MDA levels. In conclusion, this study is the first to report ZnO-NPs as an effective therapy for treating cryptosporidiosis, especially when combined with other treatments that enhance their antioxidant activity. It provides an economical and environment-friendly approach to novel delivery synthesis for antiparasitic applications.


Animals and experimental design
The current study was implemented on 30 laboratory-outbred Swiss albino mice of the most widely cost-effective and fully genetically variable known strain (CD1 strain), Each mouse was about 3-5 weeks old and weighing 20-25 gm each.All experimental procedures were carried out in accordance with the internationally valid guidelines for animal experimentation and were approved by the Institutional Animal Care and Use Committee of Beni-Suef University (BSU-IACUC).The study was carried out in accordance with the ARRIVE guidelines.They were used and divided into six groups; each group consists of five mice.The provided animals were from the Schistosoma Biological Supply Programme (SBSP) at Theodor Bilharz Research Institute (TBRI).Mice were housed in the animal house of the Faculty of Science at Beni-Suef University with clean wood-chip bedding, well-aerated cages, with food pellets, water, and libitum at a temperature of 24 °C.Their stools were examined by iodine wet mount and modified Ziehl-Neelsen stain 39 before and after concentration techniques 40 to exclude the existence of parasites.
(i) All included mice were immunosuppressed and classified into 2 main groups, and each group was subdivided into 3 sub-groups: (ii) Control groups: G1.Negative control: non-infected non-treated group.G2.Positive control: (C.parvum) infected non-treated group.G3.C.parvum infected mice receiving NTZ (drug control group).

Immunosuppression
Immunosuppression was carried out by synthetic dexamethasone (Dexazone) given orally using an esophageal tube at a dose of 0.25 µg/g/day for 15 days before inoculation with Cryptosporidium oocysts 41 .Dexazone (dexamethazone) (0.5 mg) was commercially produced and presented by Kahira Pharmaceuticals and Chemical Industries Company, Shoubra-Cairo, Egypt.

Preparation of Cryptosporidium oocysts and mouse inoculation
Cryptosporidium Parvum oocysts used for infecting mice were obtained from diarrheal young calves coming to veterinary clinics in the Faculty of Veterinary Medicine at Beni-Suef University.Faecal specimens were collected in clean sterile cups, provided that samples were not mixed with urine or water, and then transferred to the Zoology Department at the Faculty of Science at Beni-Suef University.
Collected stool specimens were exposed to macroscopic examination and then examined microscopically by Modified Ziehl-Neelsen stain (MZN) (cold method) to identify Cryptosporidium oocysts 42 .The same volume of 2.5% potassium dichromate (K 2 Cr 2 O 7 ) was used to preserve the proved positive samples for Cryptosporidium and stored at 4 °C) until used for mouse inoculation 43,44 .Distilled water was used to wash Cryptosporidium oocysts three successful times to get rid of potassium dichromate and centrifuged at 1500×g for 10 min just before use 45 .
The calculated infecting dose was done by taking the mean of three counts of MZN-stained oocysts and examining them under the oil immersion lens.Each count was done by taking 50 µl from the stool specimen and counting the oocysts in it, repeating this three times, and then taking their mean.Multiplication by 20 was done to get the result in 1 ml of the specimen 46 .Animals other than the negative control group were infected with Cryptosporidium oocysts orally by esophageal intubation; traumatic injury to animal throats was avoided.Nearly each mouse was infected by a dose of 104 Cryptosporidium oocysts 45 .
To make sure of mice infection, stool pellets were brought from different mice groups from the 2nd day postinfection (PI) up to 1 week later, then examined parasitologically by MZN stain (cold method) for detection of Cryptosporidium oocysts 42 .

Drug dosage and administration
All doses of used drugs in this study were calculated in grams according to mice weight (each mouse is approximately 20-25 gm), starting 7 days PI, once daily, and lasting for 5 days later.NTZ (Nanazoxid) was administered orally using an esophageal tube in suspension form at a dose of 100 mg/ kg body weight 47,48 .Powders of all the drugs loaded on ZnO-NPs were added to distilled water and then sonicated to form a suspension that was administered orally using an esophageal tube.NTZ-loaded ZnO-NPs powder was given in a dose of 100 mg/kg 48 .A. sativum-loaded ZnO-NPs were administered at a dose of 500 mg/kg 49 .ZnO-NPs were given at a dose of 10 mg/kg 50 .Experimental animals were scarified on the 21st day of PI.Scarification was done by intraperitoneal anesthesia.An anticoagulant anaesthetic solution (100 units/ml heparin and 500 mg/ kg thiopental) was injected intraperitoneally into the mice 51 .

Preparation of ZnO-NPs
Zinc sulphate Zn (So4) 2H2O, sodium hydroxide (NaOH), and trisodium citrate solution were used for the preparation of ZnO-NPs.All chemicals were provided by Sigma Aldrich, ready to use, with no further purification.Both solutions contain 0.2 molar NaOH and 0.1 molar Zn sulfate.2H2O was dissolved in deionized water.After that, both solutions were poured into one beaker and stirred at 1000 rpm for up to 4 h (at a temperature of 60 ºC).Then, the transparent original solution became milky white.The solution was centrifuged at 4500 rpm for 2 min., resulting in the precipitation of a white product.Later, it was washed with deionized water and then with methanol.Powdered ZnO-NPs were calcinated at 350 ºC for 3 h 52 .

Preparation of NTZ and A. sativum-loaded ZnO-NPs
ZnO-NPs (0.05 g) were added to 100 ml of trisodium citrate solution 5.4 mM (Merck, Germany) and stirred for 1 h.When 2 gm of grinded NTZ powdered in an aqueous solution and 2 gm of A. sativum powdered in another aqueous solution, ZnO-NPs were added to each solution separately under sonication, stirred overnight at room temperature, and filtrated.The powder was washed twice with distilled water and methanol, then dried 53,54 .

Characterization of fabricated samples
The fabricated samples were characterised by XRD (PANalytical Empyean, Sweden) at 40 kV, 30 mA current, ranging from 5 to 60o scan angle, accelerating voltage, and a scan step of 0.05.To determine the vibration of chemical bonds, Bruker (vertex 70 FTIR Raman) Germany spectrophotometry (serial number 1341) covering a frequency range of 400-4000 cm1 was applied using a potassium bromide disc.The morphology of materials had been assessed through a scanning electron microscope (SEM) (JEOL (JSM-5200), Japan).
For evaluating zeta potential and particle size distribution, the stability of the suspensions was evaluated in water.A Malvern Instruments Ltd., Worcestershire, UK, Zetasizer Nano ZS device with an MPT-2 automatic titrator was used to assess zeta potential in 10 mM NaCl.Due to its ideal ionic strength, this solution is useful for determining zeta potential.The hydrodynamic diameter of the particles in ultrapure water was measured by dynamic light scattering (PCS-Photon Correlation Spectroscopy).A 173° angle was measured between the scattering and the incident He/Ne laser beam.The Contin algorithm's autocorrelation functions were used to generate particle size distributions that were intensity-weighed.The intensity-weighted average hydrodynamic diameter was obtained by doing a second-order cumulant analysis of the correlation function and using the Stokes-Einstein relation while taking the suspension's viscosity into consideration.

Parasitological assessment
On day 21, PI, faecal pellets were weighted, dissolved in formol saline at 10%, and filtered by sterile gauze to obtain a clear film.Then, by micropipette, 50 µl were taken from each specimen and examined parasitologically www.nature.com/scientificreports/using MZN stain to count the number of Cryptosporidium oocysts 36 .Then, OPG (oocyst per gram of faeces) was calculated 55 .The % reduction in the OPG count was calculated referring to this equation: %R = 100(C − E)/C.%R: reductions; C: control group; and E: experimental groups of mice 56 .

Histopathological assessment
The small intestinal (ileum) segments, liver, and lung specimens excised from scarified mice were subjected to histopathological preparation.The excised specimens were cut both transversely and longitudinally, fixed in 10% formalin, and oriented on filter paper.Tissues were processed for paraffin embedding after fixation.Histopathological sections of 5 μm thickness were processed from each block and stained with Haematoxylin and Eosin stain (Hx&E) according to Drury and Wallington 57 .Histopathological examination was performed for detection of Cryptosporidium oocysts, associated pathological changes, and assessment of drug efficacy after treatment regimen.

Oxidative stress marker assessment
From different groups, parts of the ileum were gathered and homogenised in cold buffer (i.e., 100 mM potassium phosphate, pH 7.0, containing 2 mM EDTA) to yield a 50% (w/v) homogenate.Next, the homogenate was centrifuged for 15 min at 4000 rpm.Determination of Glutathione Reduced (GSH): The principle was producing a yellow compound from the reduction of 5,5′ dithiobis (2-nitrobenzoic acid) (DTNB) with GSH.The GSH concentration is directly proportional to the reduced chromogen.Its absorbance was measured at 405 nm and expressed as mmol/gm tissue following the manufacturer's instructions (Biodiagnostics, Co.) 58 .
Determination of Nitric Oxide (NO): The principle was the formation of nitrous acid diazotise sulphanilamide in the presence of nitrite and in an acidic medium.N-(1-naphthyl)-ethylenediamine was coupled to the product.A bright reddish-purple colour appeared to the resulting azo dye that was measured at 540 nm and expressed as μmol/gm tissue following the manufacturer's instructions (Biodiagnostics, Co.) 59 .
Determination of Lipid Peroxide (Malondialdehyde [MDA]): The principle was the formation of a thiobarbituric acid reactive product by the reaction of thiobarbituric acid (TBA) with MDA in an acidic medium at a temperature of 95 °C for 30 min.The resultant pink product absorbance was measured at 534 nm and expressed as nmol/gm tissue following the manufacturer's instructions (Biodiagnostics, Co.) 60 .

Ethical consideration
All experimental procedure and animal maintenance were approved by the Institutional Animal Care and Use Committee of Beni-Suef University (BSU-IACUC) with approval number (021-127).

Statistical analysis
All data were quantitatively analyzed by one-way ANOVA.Subsequently, multiple comparisons were done between groups.Results were enumerated as mean ± SEM, and the value of significance was set at P ≤ 0.05.GraphPad Prism v.6.0 (GraphPad Software, San Diego, CA, USA) was used to accomplish all calculations.

Characterization of the prepared samples
The existence of diverse functional groups accountable for the production of ZnO-NPs in bacterial biomass was explored using FTIR spectroscopy.FTIR analysis was looked into to determine the chemical functional group of the obtained ZnO, A. sativum-loaded ZnO-NPs (Zn/All), and NTZ-loaded ZnO-NPs (Zn/NTZ).Figure 1 displays FTIR spectra of the loaded samples and Zn-ONPs.The analysis of the FTIR spectrum gave insight into the bimolecular structure carrying various functions that are present in the underlying system.In the case of ZnO, a large absorption peak that was attributable to the adsorbed water molecules emerged at about 3920 cm1.At 1600 cm1, a short, sharp absorption peak was seen, which was attributed to the aromatic bending of the alkene group (C=C).The sharps peak at 1130 cm1 is ascribed to the C-H band's deformation vibration. 61,62 ile after loading with A. sativum (Zn/All), the FTIR spectrum was represented in Fig. 1, there was a red shift after loading.Absorption peaks at 3405 cm −1 were corresponding to the presence of flavonoids, nonflavonoids, and saponins in illum 63,64 .
In addition, peaks appeared at 2920 cm1, which corresponded to the asymmetric stretching of C-H groups in aromatic compounds.While organosulfide compounds such as alliin, allicin, and diallyl disulfide were detected at the peak at 1130 cm1, which was attributed to the S=O group.
Furthermore, in the loading of NTZ on ZnO-NPs (Zn/NTZ), some absorption peaks appeared; a sharp peak appeared at 3357 cm1, which was attributed to N-H stretching vibration; and some new peaks were created as a result of the formation of a complex between the drug and ZnO-NPs.In addition, some absorption peaks shifted to a higher frequency 65 .
The structural properties of these materials were examined through XRD diffraction, as shown in Fig. 2. The XRD pattern of pure ZnO exhibited high crystallinity with no secondary phases, where the presence of ( 100), (002), and (101) planes matched the hexagonal wurtzite crystal structure of ZnO .The structural properties were modified after loading.The position of the diffraction peaks of (Zn/All) changed and shifted due to the formation of a complex between ZnO and A. sativum.
To know about the phase identification and nano-crystalline structure of the ZnO and loaded nanoparticles,XRD diffraction must be investigated 66 .The XRD of pure NTZ, physical mixture and solid dispersion is shown in Fig. 2. The XRD spectra of NTZ showed many distinct peaks, indicating that the drug was in www.nature.com/scientificreports/highly crystalline form, and the same was also depicted in the case of physical mixtures.But the intensity of these peaks decreased in solid dispersions, exhibiting a decrease in the crystallinity of NTZ [67][68][69][70][71] .Pure ZnO-NPs were confirmed by SEM to be aggregated into a layered structure, as shown in Fig. 3a.According to Fig. 3b, Zno-NPs emerged as uniform, spherical particles stacked on top of one another.These particles ranged in size from 50 to 65 nm on average.SEM images of (ZN/All) were displayed in Fig. 4 a, b after loading.There seemed to be a lot of granules evenly dispersed across the surface of A. sativum.ZnO is visible as strewn particles covering the entire surface of A. sativum.In the SEM of Zn/NTZ, the composite morphology between the drug and ZnO was fully altered and transformed at Fig. 5 a, b, and the surface showed wrapped layers 72,73 .
Zeta potential measurements in Figs. 6, 7, and 8 validated the stability and charge of all samples based on ZnO-NPs.For pure ZnO, Zn/All, and Zn/NTZ, respectively, it was seen that all samples had negative charges (− 12.4 mV, − 25.1 mV, and − 0.11 mV).The stability and charge of the nanoparticles were assessed using zeta potential assays based on their electrophoretic mobility.Zeta potential value was considerably reduced after being loaded with A. sativum and NTZ.As-made ZnO-NPs and samples that had been loaded with A. sativum and NTZ both had hydrodynamic diameters of 535, 1268, and 2726 nm, respectively, as shown in Figs. 6, 7, and 8b 74 .

Morphology of immunosuppressed murine models
Signs of immunosuppression started to appear on mice after two weeks from drug administrative doses by existence of subcutaneous edema, cutaneous ulcerations and hair loss.

Effect of NTZ and different NPs treatment regimens on OPG count
On day 21 PI, oocyst shedding was quantified and estimated per gram of faeces.The data was analyzed by oneway analysis of variance (ANOVA) with Duncan post-hoc test to show the difference between the means of either infected versus non-infected or versus treated groups.In compared to the infected non-treated control group (G2), oocyst output was significantly reduced in the groups treated with NTZ and A. sativum loaded ZnO-NPs and ZnO-NPs alone (P ≤ 0.001).G3 treated with NTZ had the largest reduction in Cryptosporidium oocyst shedding (81.5%), followed by G6 (71.1%) treated with A. sativum loaded ZnO-NPs, G5 (69.6%)NTZ loaded ZnO-NPs, and G4 (64.3%) treated with ZnO-NPs.However, there was no statistically significant difference between the NTZ loaded ZnO-NPs, NTZ, and ZnO-NPs treated groups (P ˃ 0.05).When compared to ZnO-NPs, A. sativum loaded ZnO-NPs significantly reduced the oocyst count (P ≤ 0.05) (Fig. 9).

Effect of NTZ and different NPs treatment regimens on oxidative parameters from ilea tissue
Cryptosporidium infection revealed a significant decline in the level of GSH (P ≤ 0.001) compared to negative control groups.In versus to positive control, all treated groups showed a significant increase in GSH levels (P ≤ 0.001).Loading NTZ into ZnO-NPs increased NTZ effectiveness and resulted in a considerable increase in GSH level (P ≤ 0.001).Furthermore, A. sativum loaded ZnO-NPs demonstrated a substantial increase in GSH values when compared to the ZnO-NPs treated group (P ≤ 0.001).Moreover, the infection provoked a significant increase (P ≤ 0.001, P ≤ 0.01, respectively) in the levels of MDA and NO.All treated groups highly ameliorated these changes in MDA and NO compared with negative and positive control groups (P ≤ 0.001) with more efficacy to A. sativum loaded ZnO-NPs and NTZ loaded ZnO-NPs when compared to ZnO-NPs alone (P ≤ 0.001) (Fig. 13).

Discussion
Cryptosporidium spp. is one of the major zoonotic health problems in humans, domesticated animals, and immunosuppressed individuals as well.Despite the rapid development of variable aspects of this protozoan, there has been no effective treatment available until now [75][76][77] .
Only NTZ is currently approved by the FDA for cryptosporidiosis treatment.Its main mechanism of action depends on inhibition of the basic enzyme in the anaerobic metabolism pathway, pyruvate ferredoxin www.nature.com/scientificreports/oxidoreductase 78 .However, it has limited cure rates, with the marked disadvantage of being unbeneficial to immunosuppressed patients and malnourished children 79 .
Targeting and designing new nanoparticle dependent drug carriers achieves enhancement of treatment efficacy 80 .Concerning the characterization results, the SEM images showed the morphological studies for all samples to explain the performance of these materials for biological activity.Their morphology had an impact on the distribution in the body and diffusion rate, hence affecting the pharmacokinetics of its payload 81 .In case of (ZN /All) ZnO appeared as scattered particles filled the whole A. sativum surface and that is favored in penetration the wall of parasite.For SEM of Zn/NTZ, the surface appeared as wrapped layers which enhanced its surface area and improved its activity.Significant reduction was observed in the zeta potential value after loading with A. sativum, and NTZ.This revealed that interactions between ZnO-NPs with A. sativum and NTZ affected the structure and charge of these nanocarriers 82 .
The distribution of loaded nanoparticles inside the parasite matrix can be improved with an increase in the negative surface charge in the intestine.Hence, Zn/All has higher biological activity against parasites than Zn/ NTZ and pure ZnO-NPS.The distribution and transport characteristics of a nanoparticle in vivo are significantly influenced by its size.Different-sized nanoparticles in this study might be transported differently within the body and, as a result, suppress parasite growth in different ways.
The most popular scaling method used in pharmaceutical technology since the advent of NEP is dynamic light scattering (DLS).Controlling the size of nanoparticles is a promising technique to target the distribution where the parasites dwell and improve antiparasitic drug residence 83 .
The hydrodynamic diameter of the synthesized ZnO-NPs and the loaded sample with A. sativum and NTZ was measured to be 304 nm and 535, 1268, 2726 nm, respectively.When the particle size of loaded samples increases, it is considered evidence of loading-driven particle agglomeration.
In the preceding literature, induction of immunosuppression in mice was done by using dexamethasone, as it was previously used in different studies as a well-known immunosuppressive drug, particularly in mice 41,84 .It has an inhibitory mechanism on the pathway of interferon-gamma and hence the immune response due to the high activity of glucocorticoids 84 .
In this study, immunosuppressed groups developed skin manifestations after 2 weeks of experimentation.Multiple dermatological lesions were related to the administration of glucocorticoids 41,85 .Uner et al. noticed petechial haemorrhage in the skin on the mice ears also after 14 days of immunosuppression 86 .
As regards the obtained parasitological results in the present study, Cryptosporidium oocyst shedding revealed higher significance in the feces of the infected immunosuppressed non-treated mice group than other infected treated groups all through the period of the experiment.Cryptosporidium oocysts occurred in the stool of immunosuppressed mice on the 2nd day of PI with a high percentage, and this is in line with other results 41,87 .Abdou et al. reported that oocyst shedding continued until the 30th day of PI in experimental C. parvum-infected mice 88 .
In the preceding literature, the highest reduction percent of Cryptosporidium oocyst shedding per gram feces (81.5%) was detected in G3 (NTZ treated group), followed by ZNO-NPS, A. sativum-, loaded ZnO-NPS-treated groups, and the least was found in the NTZ-loaded ZnO-NPS-treated group (G5) (69.6%).The obtained results were in line with some authors 89 whose reduction percentage of oocyst for the group treated by NTZ was 92.05%, revealing the highest result as well.This was in contrast to Moawad et al. 41 , who found that treatment with NTZ-loaded Chitosan NPs produced the best oocyst counts reduction, followed by the NTZ and Chitosan NPs treatment groups.Mohi-Eldin et al. had pointed out that the reduction rate in oocyst per gram of feces in Eimeria stiedae infected rabbits treated with A.sativum loaded ZnO-NPs was less than group treated by A.sativum extract alone 49 .
On the other hand, Dkhil et al. documented that oral administration of 10 mg/kg/day ZNO-NPs for continuous 5 days reduced oocyst shedding and jejunal inflammatory injury significantly in Eimeria papillate-infected mice 50 .
In regard to our histopathological examination results of the intestinal sections, the intestinal lumen in the positive control group showed heavy infestations of oocysts associated with moderate oedema and mild inflammatory cell infiltrate, as well as congestion of submucosal capillaries.Previous studies were in agreement with these results 5,41,[90][91][92] .They illustrated that infection with Cryptosporidium in mice caused some alterations in the intestinal mucosa, including variable degrees of villous atrophy and crypt hyperplasia.Marked oedema in lamina propria was exhibited, and moderate chronic inflammatory cell infiltrates accompanied by congestion with dysplastic changes of the intestinal tract were noticed as well.
Infected mice treated with NTZ alone revealed partial enhancement in the intestinal pathological lesions after being infected with Cryptosporidium.This obtained result was in line with Moawad et al. and Abd El Wahab et al. who reported that NTZ-treated rats showed moderate changes when compared to a positive control group that showed severe pathological alterations 5,41 .Also, the ZnO-NPs-treated group showed moderate improvement in the intestinal lesions.
In the current study, mice received A. sativum-loaded ZnO-NPs, followed by NTZ-loaded ZnO-NPs, revealed the highest enhancement in histopathological intestinal lesions.That was indicated by normal intestinal villi and broadening of villi with moderate inflammatory oedema and few inflammatory cells, respectively, with the absence of Cryptosporidium oocysts in the intestinal villi in both groups.These obtained findings were in parallel with others who recorded amelioration of the intestinal histopathological sections by detecting minimal Cryptosporidium oocysts at the intestinal epithelium with mild inflammatory cellular infiltration in mice receiving combined NTZ-artesunate loaded on polymeric nano-fibre and NTZ-loaded Chitosan NPs, respectively 41,91 .
According to histopathological changes in liver tissue sections, the positive control group showed marked diffuse hydropic degeneration of hepatocytes that had vacuolated cytoplasm and was associated with moderate congestion.Scattered dysplastic cells with hyperchromatic large nuclei and the absence of inflammation had been noticed.Certad et al. and Abdou et al. demonstrated a high grade of hepatic and bile dysplasia in severe immunodeficiency and immunosuppressed mice 88,93 .Meanwhile, Elmahallawy et al. reported the hepatic injury in the form of high levels of serum liver enzymes 94 .Also, C. parvum oocyst induction led to pathological liver tissue changes in the form of hepatocyte degeneration and focal infiltration with mononuclear cells 95 .
Zno-NPs-treated mice group showed nearly no improvement, and in group treated by NTZ, the pathological alterations ranged from marked to moderate.Treated mice receiving A. sativum-loaded ZnO-NPs and NTZloaded ZnO-NPs revealed the highest amelioration of the liver picture by finding minimal and mild degrees of hydropic degeneration.Moderate congestion and the absence of dysplastic hepatocytes were noticed in both groups.These results went hand in hand with Moawad et al. 41 , who used NTZ loaded Chitosan NPs in Cryptosporidium infected immunosuppressed mice, and Mohi-Eldin et al. who found that garlic-loaded ZnO-NPs had anti-parasitic action on hepatic coccidiosis in rabbits 49 .
The pathogenicity of respiratory cryptosporidiosis has not been fully pointed out.Previous research reported that pulmonary cryptosporidiosis may complicate immunosuppressed subjects and even occur in immunocompetent ones 96 .A similar study about Cryptosporidium oocysts establishment within the lung tissue was reported recently 97 .www.nature.com/scientificreports/ In the present study, lung tissue sections of the immunosuppressed positive control group showed markedly severe inflammation and thick alveolar interstitial spaces with rupture of the alveolar septa.This finding was illustrated by other recent studies 41,95 .In immunosuppressed mice groups receiving NTZ, Zno-NPs, and NTZloaded ZnO-NPs, the pathological changes ranged from mild to moderate inflammation with partial improvement.Similar results were recorded previously 41,95 .Moreover, Madbouly et al. found interstitial inflammation and pulmonary haemorrhage in the lung tissue of immunosuppressed infected mice, and they got better after combined therapy with NTZ and atorvastatin 98 .For the first time, treated immunosuppressed mice receiving A. sativum-loaded ZnO-NPs showed the highest improvement in the lung picture by finding mild inflammation with minimal destruction of the alveolar septa.
A.sativum has the ability to treat experimental cryptosporidiosis through disruption of the normal physiological bioactivities of the parasite, such as food absorption, reproduction, and mobility.Immune system enhancement by stimulating the natural killer cells' activity and phagocytosis may be another mechanism 99 .
The antioxidant activity of all garlic compounds is well documented, with allicin and S-allylcysteine showing the highest activity through enhancement of endogenous radical scavenging mechanisms, inhibition of free radical formation, and elevation of the antioxidant cellular enzymes 100 .The antioxidant activity of A. sativum and its compounds becomes of great interest as regards their anti-hepatotoxic, anti-atherogenic, and anti-cancer properties 13 .
Furthermore, ZnO-NPs have the ability to prevent the wasting of GSH through oxidative damage prompted by infections, as previously demonstrated by Dkhil et al. 101 .It is supposed that the antibacterial activity of these metal oxide particles is attributed to the generation of active oxygen species 102 .Joe et al. illustrated that ZnO-NPs have the ability to disrupt the oocyst wall glycoproteins, facilitating penetration and consequently increasing harmful Zn2 + ions 103 .In addition, Siddiqi et al. and Prasad et al. observed that when ZnO-NPs were exposed to light, their photosensitive capabilities were activated, resulting in the formation of reactive oxygen radicals that affect the parasite virulence-associated surface glycoprotein molecules with decreased oocyst infectivity 104,105 .Moreover, the tiny size of the NPs allowed for easy passage through cell membrane pores to exert a direct, harmful effect 106 .Furthermore, it binds to DNA molecules, disrupting critical metabolic processes in C. parvum 107 .
In the current study, treatment with A. sativum and NTZ-loaded ZnO-NPs groups produced a significant increase in GSH levels compared to non-infected, infected control groups, and NTZ-and ZnO-NPs-treated groups.All treated groups improved changes in NO and MDA values compared with control groups, with more efficacies for A. sativum-loaded ZnO-NPs and NTZ-loaded ZnO-NPs when compared to ZnO-NPs alone.These obtained results match with Abdelhamed et al. who stated that the highest free radical scavenging capacity in serum and different tissue homogenates was observed in mice receiving combined NTZ and artesunate-loaded polymeric nanofiber 91 , and Dkhil et al. who concluded that administration of ZnO-NPs helped in protection against the oxidative stress negative effects in E. papillata-infected mice 50 .Moreover, ZnO-based nano-formulations inhibited Leishmania tropica promastigotes growth because of the production of reactive oxygen species by these NPs 108 .
The antioxidant property of A. sativum is owed to many bioactive compounds that act as antioxidants either directly through modifications in the pre-apoptotic oxidative mechanism or indirectly via stimulation of the endogenous synthesis of antioxidant enzymes or molecules.The capability of the antioxidants to scavenge generated free radicals during cell respiration protects the human body against infections 109 .
ZnO-NPs almost show their antimicrobial characteristics through two main mechanisms: their direct effect through inhibition of cell growth, impairment of cell permeability, and apoptosis induction.The second is their indirect effect by H 2 O 2 production that induces oxidative stress and the release of zinc ions in the environment, which enables their cell wall penetration and their antimicrobial effects 110,111 .
Our results demonstrated that ZnO nanomaterials can be used further in a wide range of biological applications, and this is equally important, as reported by Khan et al. 112 .The obtained results support our hypothesis that ZnO-NPs could have significant medical importance in pharmaceutical and medical science for their biocatalytic activities and improve their utilisation in different biological antiparasitic applications.The therapeutic properties of garlic were empowered by being loaded with ZnO-NPs and acting synergistically, causing potent anti-inflammatory and anti-oxidative effects.

Conclusion
In the current study, the synthesis of low-cost ZnO-NPs through co-precipitation was investigated.These nanoparticles were modified to be nanocarriers for A. sativum and NTZ.These newly materials were well characterized and for the first time ZnO-NPs were used as a successful, effective alternative therapy in treating experimental cryptosporidiosis especially when combined with other treatments that enhanced their antioxidant activity.The study also provides an economical and environment-friendly approach towards the synthesis of novel delivery for antiparasitic applications.Unlikely, this study didn't investigate the effect of ZnO-NPs loaded materials in vitro or evaluate their prophylactic effect on C. parvum infection.To obtain a dosage response curve for the used drugs, more doses should be further evaluated.Additional metal oxides or new therapeutic measures could be used to treat parasitic infections and manage a dose-effective regimen that protects against any hazardous side effects that might occur over an extended period of time. https://doi.org/10.1038/s41598-023-46260-3 https://doi.org/10.1038/s41598-023-46260-3

Figure 3 .
Figure 3. SEM of ZnO-NPs illustrating the surface morphology of sample.

Figure 4 .
Figure 4. SEM of Zn/All illustrating the surface morphology of sample.

Figure 5 .
Figure 5. SEM of Zn/NTZ NPs illustrating the surface morphology of sample.

Figure 10 .Figure 11 .
Figure 10.Histopathological ilea sections of different treatment groups stained with hematoxylin and eosin (×100).(a) Light microscopy of small intestinal section from negative control group showed normal mucosa; (b) Positive control group in which the intestinal lumen showed heavy infestations of oocysts (red cycle) associated with moderate edema (red arrow); (c) NTZ treated group showed broadening of villi by moderate inflammatory edema (red arrows) & intestinal villi with absence of Cryptosporidium oocysts; (d) ZnO-NPs treated group showed minimal residual intraluminal Cryptosporidium oocysts (red cycles) associated with marked few inflammatory cells & edema (red arrow), with mild congestion (black arrow); (e) NTZ loaded ZnO-NPs treated group showed broadening of villi with moderate inflammatory edema and few inflammatory cells (red arrows), intestinal villi with absence of Cryptosporidium oocysts; (e) A.sativum loaded ZnO-NPs treated group showed almost normal intestinal villi with absence of Cryptosporidium oocysts.

Figure 12 .
Figure 12.Light microscopy of lung tissue sections from different mice groups included in the study stained with hematoxylin and eosin (× 100).(a) Negative control group showed delicate and thin alveolar walls (black arrows) and emphysematous changes with severe destruction of alveolar septa (red arrows); (b) Positive control group showed destructed and dilated airspaces and rupture of alveolar septa(yellow arrow), with severe inflammation (red arrows) associated with thickening of some alveolar interstitial spaces (black arrows); (c) NTZ treated group showed rupture of many alveolar septa (red arrows) and mild inflammation beside the alveolar spaces (black arrows); (d) ZnO-NPs treated group showed moderate inflammation (black arrows) associated with destruction of some alveolar septa (red arrows); (e, f) NTZ loaded ZnO-NPs and A.sativum loaded ZnO-NPs groups showed mild to moderate inflammation between nearly normal alveolar spaces with minimal destruction of alveolar septa in group f.

Figure 13 .
Figure 13.Effect of variable treatment regimens used in the study on GSH, MDA, and NO levels in ileum of infected mice with Cryptosporidium.Values were enumerated as mean ± SEM (n = 5).

Table 1 .
Variable degrees of hepatic injury in different treatment groups included in the study.