Trichinella spiralis is a parasitic nematode associated with food-borne disease, known as trichinellosis, which is frequently acquired through the consumption of undercooked meat, predominantly pork and its derived goods harboring the encysted larvae1. Due to its diverse host range, encompassing 150 distinct species of animals as well as humans, trichinellosis presents an urgent public health challenge; with an estimated 11 million individuals infected worldwide2. Out of 24 parasites associated with international trade, T. spiralis has been deemed the most noteworthy by the Food and Agriculture Organization (FAO) of the United Nations (UN) and the World Health Organization (WHO)3,4. T. spiralis undergoes three distinct stages in its life cycle: the adult form, newborn larvae (NBL), and infectious muscle larvae (ML)5. Throughout these phases, cysteine proteases are released, serving a pivotal function in pathogenesis by aiding in metabolic processes related to nutrition, molting, tissue infiltration, evasion of immune responses, and/or alterations6. An interesting finding about a cysteine protease from T. spiralis (Ts-CF1) is its expression throughout life cycles, as well as its localization within the stichosome and cuticle. This finding lends credence to the idea that Ts-CF1 could be a prospective therapeutic target or vaccine candidate for T. spiralis infection7,8. Moreover, this parasite exhibits a diverse array of strategies for immune evasion, skillfully circumventing both the host’s adaptive and innate defense mechanisms9. Calreticulin (Ts-CRT) represents a sophisticated mechanism that enables survival within a challenging host environment by engaging in overlapping interactions, and disrupting the complement immune system within the host10.

The parasite’s β-tubulin monomer is selectively bound to the benzimidazole-2-carbamate derivatives (BzC) as the approved treatment, thereby inhibiting the polymerization of microtubules11. Nevertheless, the efficacy of the treatment is significantly diminished owing to the mammalian host’s tubulin binding. Although they are not recommended during pregnancy and discouraged for children under the age of two, they are also not entirely effective against newborn or encysted larvae due to the emergence of drug resistance and low bioavailability11. The ongoing evolution of helminth resistance to synthetic anthelmintic medications, however, calls for the investigation of natural compounds as a potential replacement for these drugs12. On top of that, natural pigments tend to be more accessible and economically feasible13.

A multitude of studies has recently explored the potential uses of natural pigments, such as carotenoids, in the fight against both non-parasitic and parasitic infections. The findings of these studies have been shaped by their ethnopharmacological relevance in addressing coughs respiratory ailments, dysentery, diarrhea, gastrointestinal parasite infections, and rheumatic diseases14,15,16. Staphyloxanthin (STX) is a notable pigment derived from Staphylococcus aureus, classified within a distinct category of apocarotenoid triterpenoid pigments17. In microorganisms and plants, oxidative cleavage of C40 isoprenoids produces apocarotenoids. Carotenoid cleavage dioxygenases (CCDs) play a major role in the enzymatic production of apocarotenoids, whereas additional processes are used in the non-enzymatic process17. The biological potential of carotenoids is chiefly ascribed to the production of reactive oxygen species (ROS), leading to oxidative stress and suppression of ion-membrane interactions and active solute transport, thereby altering the cellular permeability18. Regarding to the aforementioned scenario, this study relies on the evaluation of STX anthelmintic activity against T. spiralis.

The intrinsic physicochemical characteristics, such as low solubility in water and insufficient permeability, present a significant challenge and restrict the oral administration of certain drugs due to their suboptimal bioavailability19. Nano-carriers such as niosomes have been widely employed to facilitate drug delivery and enhance their oral bioavailability. Niosomes are bilayered structures consisting of biocompatible materials, specifically non-ionic surfactants and cholesterol, which serve as lipids20. These nanovesicles are water-soluble, exhibiting exceptional biocompatibility and possessing the capability to carry both hydrophobic and hydrophilic agents. This approach of drug delivery significantly improves the physical stability of the pharmaceutical compound, preventing chemical deterioration and the detrimental effects of unfavorable environmental conditions21. Therefore, STX was formulated as a niosomal dispersion to improve oral bioavailability and enhance the therapeutic efficacy against T. spiralis.

To the best of our knowledge, this is the first report to examine the impact of STX on the treatment of trichinellosis. The objective of this research was to assess the novel antiparasitic activity of STX against T. spiralis both in vitro and in vivo as a drug solution and niosomal dispersion. Moreover, homology modeling and molecular docking analysis were performed to investigate the potential affinity of STX to block T. spiralis-target proteins.

Materials

The STX pigment was extracted from Staphylococcus aureus A2 (accession number PP197164). The laboratory extraction, purification, chemical validation, and safety profile on normal Vero cells of STX were covered in our previous research22. Cholesterol and sorbitan monostearate (Span 60) (Sigma Chemical Co., St. Louis, MO, USA) were obtained from the Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University, Egypt. Albendazole (ABZ) served as the reference anthelmintic drug, supplied by Pharma Cure Pharmaceutical Industries, Egypt, as a 400 mg/10 mL oral suspension (Alzental).

In silico pharmacokinetics, drug‑likeness, and toxicity predictions

STX and ABZ were transformed into their canonical simplified molecular-input line-entry system (SMILE) representations. Subsequently, these models were analyzed using the ADMETlab 3.0 server to determine their pharmacokinetic parameters. This included assessments of A (absorption, specifically MDCK, skin permeability, human intestinal absorption (HIA), and Caco-2), D (distribution, encompassing Blood-Brain barrier penetration (BBB), P-glycoprotein substrate, inhibitor, and plasma protein binding (PPB)), M (metabolism, focusing on CYP family substrate/inhibitor), E (excretion, particularly elimination half-life (t1/2)), and T (toxicity, including carcinogenesis and ames mutagenesis), in addition to the evaluation of the drug-likeness score.

Preparation of Staphyloxanthin-loaded Niosomal formulation

The composition of the niosomal formulation listed in Table 1 was utilized following the previously reported investigation23. Niosomes were formulated using hydration method as previously described21,23. In brief, span 60 (surfactant) and cholesterol were melted on a water bath at 55 °C. STX was dissolved in 5 mL ethanol and the solution was then added to the melted components with mixing to form clear liquid. A homogeneous mixture was formed by gradually incorporating 5 mL of water upon mixing. Proniosomal creamy gel was formed with continuous mixing upon cooling. Hydration of proniosomes was used to prepare the surfactant nanovesicles. This was accomplished through the incremental incorporation of the remaining water, with continuous mixing to develop homogenous niosomal dispersion. The niosomal dispersion was allowed to undergo hydration and swelling by overnight incubation at room temperate. For size reduction, the prepared swollen vesicles were bath sonicated for 45 min. Ice cubes were used to prevent heating of the sample during sonication. This technique produced STX-loaded niosome at a final concentation of 12 mg/mL.

Morphology and vesicle size determination

The morphological attributes and size of the prepared niosomal formulation were examined utilizing a transmission electron microscope (TEM) (JEOL – JSM1400 PLUS, Tokyo, Japan)21. Niosomes were suitably diluted (1:200) with HPLC filtered distilled water. One droplet of niosomal dispersion was loaded on a carbon grid and left to dry. Afterwards, the grid endured a sequential staining process, first with saturated uranyl acetate dissolved in 70% ethanol for 5 min, followed by lead citrate for 2 min. The sample was delicately affixed in the holder for examination under the electron microscope. Photomicrographs were obtained through meticulous calibration of the magnification power.

Dynamic light scattering spectroscopy (DLS)

The determination of niosome size was further validated using DLS, which yields the average vesicle size (Z-average)24. This analysis was conducted utilizing the Zetasizer (Zetasizer Nano ZS, Malvern Instruments Ltd, Malvern, Worcestershire, United Kingdom). The uniformty of vesicle dispersion were also assessed using DLS, as indicated by the polydispersity index (PDI) value. The same technique was employed to record the zeta potential. HPLC filtered distilled water was used to appropriately dilute (1:200) the formulation before loading into the cell. The measurements were taken with the diffraction angle set to 90° at 25 °C. The mean of 14 measurements was used to calculate the Z-average. The system software was used to calculate the PDI value. The value of zeta potential was ascertained from a comprehensive dataset comprising 100 determinations.

Entrapment efficiency and drug loading

The evaluation of the encapsulation efficiency of the niosomes was conducted after the separation of the free STX pigment, as previously described20,25. This was accomplished by centrifuging the prepared nanovesicles for 90 min at 12,000x g, and then carefully separating the liquid phase supernatant from the solid residue pellet. The unentrapped STX content in supernatant was determined spectrophotometrically by measuring the absorbance at 456 nm. The entrapment efficiency EE% was calculated by subtracting the free STX content from the total amount of STX initially used in vesical formulation, according to the provided equation:

A: The total amount of STX initially incorporated in vesical formulation. B: The unentrapped STX content in supernatant.

The loading efficiency (LE) was shown as the amount of STX encapsulated per lipid. This gave a more accurate picture of the niosomes’ ability to load STX compared to the total amount of lipid in the formulation, as shown in the equation:

LE (mg/g) = actual amount of encapsulated drug/total lipid in the formulation.

Stability of niosomes

The stability of niosomes can be confirmed by measuring particular characteristics over a specified period25. To put it briefly, 1 mL of STX-nisomes’ size and EE% were evaluated. For a month, the requirements were observed at two distinct temperatures: 4 and 25 °C.

Parasite and animals

Male Swiss albino mice used in the study were parasite-free, laboratory-bred, and weighed an average of 25 g. The mice were purchased from the animal house at the College of Veterinary Medicine of Cairo University (Cairo, Egypt). The ethics committee of Faculty of Pharmacy, Tanta University approved the care and manipulation of the animals, which were carried out in accordance with the National Institutes of Health’s guide for the use and care of laboratory animals (TP/RE/3/25 p-01). Mice were fed a standard pellet diet and had unrestricted access to water. The T. spiralis (code: ISS6158) utilized in this investigation was supplied by the Medical Parasitology Department, Faculty of Medicine, Tanta University, Egypt. The initial isolate of T. spiralis was procured from infected pork sourced from a slaughterhouse in Cairo. This was upheld through continuous transmission to mice and rats. This Trichinella isolate was genotyped as T. spiralis at the Superior Institute of Health in Rome, Italy, which is the European Union Reference Laboratory for Parasites. Each mouse was infected with 200–300 larvae of T. spiralis through oral administration26,27.

Isolation of T. spiralis adult worms and muscle larvae

Muscle larvae and adults of T. spiralis were collected from the infected animals, as previously described27. After 30 days of infection with T. spiralis, the Swiss albino mice were sacrificed, their muscles were removed and ground, and the larvae of the infected muscles were digested by submerging them in a solution of acid pepsin. The mixture was stirred continuously for 2 h at 37 °C before the digest was filtered. The recovered larvae were rinsed with tap water many times and then let to settle in a conical flask for 30 min. In order to isolate adult T. spiralis worms, the small intestines of infected untreated mice were washed and then cut longitudinally along their entire length. The resulting pieces were 2 cm in diameter, and they were then placed in normal saline at 37 °C for 3 or 4 h to facilitate the worms’ migration out of the tissue.

In vitro experimental design

A sterile 24-well tissue culture plate from SoCal BioMed in the USA was used for all investigations. A 2 ml of RPMI-1640 medium (Lonza, Belgium) supplemented with 10% fetal bovine serum, 200 µg/ml streptomycin, and 200 U/ml penicillin (Omega Scientifc, USA) was used to incubate muscle larvae (ML) of T. spiralis (60 ML per well). This study established five distinct groups as follows: Group I consists of muscle larvae cultured solely in RPMI, while Group II comprises muscle larvae cultured in RPMI that includes purified STX pigment, dissolved in 1% dimethyl sulfoxide (DMSO) at a concentration of 100 µg/mL. Group III: muscle larvae cultured in RPMI with ABZ at a concentration of 100 µg/mL. Group IV: muscle larvae cultured in RPMI with the niosomal formulation of STX at a concentration of 100 µg/mL. Group V: muscle larvae cultivated in an incubation medium comprising a combination of STX niosomes (50 µg/mL) and ABZ (50 µg/mL). The experiment was conducted in four replicates. The plates were meticulously sealed and incubated at a controlled temperature of 37 °C within an environment enriched with 5% CO2 for durations of 1, 6, 24, and 48 h28. The assessment of parasite viability was conducted through manual counting. To evaluate the viability rates of parasites, only definitively dead parasites were counted. Despite exhibiting minimal motility, the parasites were classified as living organisms. The survival rate in each well was determined by considering that non-motile worms were deceased. The criteria for a deceased body were as follows: the parasite’s body was either linear, comma-shaped, or immotile. Parasite mortality % =(the number of dead ML/the total number of ML)×100%27.

Ultrastructural examination using scanning electron microscopy

The effect of STX on the ultrastructural features of T. spiralis muscle larvae compared to ABZ is examined by scanning electron microscopy (SEM)28,29. The isolated larvae were incubated with different treatments of STX and ABZ as described in the aforementioned in vitro experiment. After 24 h of inoculation, T. spiralis muscle larvae were harvested and transferred to a freshly prepared fixative solution containing 2.5% glutaraldehyde (w/v) in 0.1 M sodium cacodylate. The mixture was maintained at a pH of 7.2 at 37 °C and allowed for overnight incubation. Before being post-fixed in a solution of 2% (w/v) osmium tetroxide in sodium cacodylate buffer for 1 h, larvae were rinsed in 0.1 M sodium cacodylate buffer at pH 7.2 for 5 min. Liquid carbon dioxide was used to dry the specimens after dehydrating in an ethanol succession. The process of applying and affixing the desiccated larvae onto stubs equipped with a dual-sided carbon adhesion tape was executed prior to the gold coating using a sputtering coater (EIKO Engineering CO, Japan). The samples were then analyzed using a SEM (Jeol GSM-IT200, Japan) operating at 5 to 20 kV, Electronic Microscope Unit, Institute of Nanoscience and Nanotechnology, Kafrelsheikh University, Egypt.

Drug administration

A 100 mg/kg/day dose of purified STX, ABZ, and STX niosomes was given. A 50 mg/kg/day dose of both STX niosomes and ABZ was given as a combination treatment. The required dose (250 µL) was administered orally for 3 consecutive days.

Experimental design

Animals were divided into seven distinct groups (n = 10 per subgroup), as summarized in Table 2. Group I served as a negative normal control: uninfected and untreated. Group II was employed to evaluate the safety of STX. Group III served as a positive control, receiving infection without treatment. Group IV: infected and treated with purified STX pigment dissolved in 1% DMSO. Group V: infected and treated with ABZ. Group VI: infected and treated with STX niosomal formulation. Group VII: infected and treated with a combination therapy. Groups were further subdivided into two subgroups (I and M) to evaluate the impact of the administered drugs for three consecutive days during the intestinal phase (I) (3–5 days post-infection), and the muscular phase (M) (30–32 days post-infection).

Assessment of parasite burden

On the specified termination day, all animals experienced an overnight fasting period and were subsequently sacrificed via intraperitoneal anesthesia using urethane (1.3–1.5 g/kg in approximately 1.5 g/5 mL of 0.9% normal saline solution; Sigma-Aldrich, St. Louis, MO). Subgroup I (intestinal phase) involved scarifying mice on day 6 post-infection, processing the small intestine according to previously instructions, obtaining adults of T. spiralis, and calculating the worm reduction rate. In addition, the mice in subgroup M (muscular phase) were sacrificed on 35 post infection, and the pepsin digestion technique was used to retrieve the muscle larvae. The McMaster counting chamber, Faust-Germany, was used for the microscopic counting of the larvae4,24,30.

Histopathological examination

The studied groups’ intestinal and skeletal muscle segments were fixed in 10% formalin for 24 h, rinsed in water for 12 h, dehydrated in increasing alcohol grades, and then cleaned in xylene. For two h at 55 °C, impregnation was carried out in pure soft paraffin. A microtome was used to cut ten hard paraffin sections that were 5 μm thick. Haematoxylin and eosin stain was applied to the sections. The presence or absence of Trichinella larvae, any pathological abnormalities, and the degree of inflammatory cellular response were assessed histopathologically in intestinal sections. Skeletal muscle sections were inspected for Trichinella cysts, and comments regarding the capsule’s integrity, content, and inflammatory cellular response were recorded for each group27.

Immunohistochemical examination

Skeletal muscle specimens’ paraffin sections were deparaffinized and rehydrated. Microwaving the slices in a citrate buffer (pH 6.0) allowed the antigen to be recovered. The endogenous peroxidase was inhibited using methanol that contained 3% hydrogen peroxide. Sections were incubated with the primary antibodies, Anti-CD34 Antibody (Supplier: DAKO, Catalog Number: M7165, Clone: QBEnd-10, Mouse anti-Human), in an ideal dilution of 1:50–1:100 for an overnight at 4 °C in a humid chamber. The secondary antibody, Biotin streptavidin link, DAKO, was then applied. To further pinpoint the antigen’s location, a chromogen solution of 3,3’-diaminobenzidine tetrahydro chloride (DAB) substrate was added (Universal Detection Kit, DAKO Envision, Denmark). Slides were then mounted, dehydrated in alcohol, and counterstained with hematoxylin4.

Histopathological and immunohistochemical scoring

Small intestine sections were histologically scored using a semiquantitative scoring approach that assigned grades to the following characteristics: goblet cell hyperplasia (0, normal; 1, mild-only in crypts; 2, moderate-in crypts and villi); the presence of mast cells (0, absent or 1, present); eosinophils in epithelium and muscularis mucosae (0, absent or 1, present); plasma cells in lamina propria (0, absent or 1, present); crypt hyperplasia (0, absent or 1, present); villous atrophy (0, normal; 1, mild; 2 moderate and 3 severe)31. The inflammatory reaction encompassing the capsule was assessed and scored for the skeletal muscle specimens as follows: +1 denotes mild, + 2 moderate, and + 3 an extreme reaction32. The percentage of positive stained cells (quantity score) and the staining intensity score were combined to determine the immunohistochemistry score (IHS). Scores were assigned to the intensity of the staining: 0 (negative), 1 (mild), 2 (moderate), and 3 (strong). The quantity score was calculated on a scale of 0 to 4, as indicated below: 0 indicates that there is no immunostaining; 1 indicates that 1–10% of the cells are positive; 2 indicates that 11–50% of the cells are positive; 3 indicates that 51–80% of the cells are positive; and 4 indicates that 81–100% of the cells are positive. To calculate the IHS, the quantity score (0–4) was multiplied by the staining intensity score (0–3), which ranged from 0 to 12. Strong immunoreactivity was defined as a score of 9–12 (+ 3), moderate immunoreactivity as 5–8 (+ 2), feeble immunoreactivity as 1–4 (+ 1), and negative immunoreactivity as 032.

Computational inspection of STX binding affinity for the parasite’s essential proteins

The binding efficiency of STX for blocking essential proteins involved in different stages of T. spiralis life cycle, including T. spiralis β-tubulin, calreticulin (Ts-CRT), and cathepsin F (Ts-CF1), was examined in silico4,29. From the RCSB protein data repository, the model of β-tubulin protein’s X-ray crystallographic structure bound to albendazole sulphate (PDB ID: 1OJ0) was obtained (https://www.rcsb.org/). Due to the lack of crystallographic structures for T. spiralis calreticulin and cathepsin F, homology modeling was generated for the prediction of the 3D protein structures.

Structure prediction and validation of target proteins

The NCBI GenBank database provided the T. spiralis calreticulin and cathepsin F amino acid sequences in the FASTA format. Their respective GenBank accession numbers are KAL1242782.1 and XP_003378245.1, respectively. The “Easy Modeller 4.0” tool in the “Threading Assembly Refinement (I-TASSER)” software was used to implement homology modeling iteratively. Templates and binding sites for these sequences were identified using the I-TASSER service (http://zhanglab.ccmb.med.umich.edu/I- TASSER/). Utilizing the LOMETS tool for template-based protein structure prediction within the I-TASSER, homology modeling of the mature domain of Ts-CF1 was conducted, employing the wild-type human procathepsin K crystal structure (PDB ID: 7PCK) as a template, with a Z-score of 4.39. Furthermore, the human calreticulin arm domains crystal structure (PDB ID: 3RG0), with a Z-score of 6.40, was used as a template for constructing the 3D T. spiralis calreticulin structure. The best model with a confidence score (C-score), which gauges the caliber of the anticipated model and prospective energy, was chosen for more research out of the five models of each protein that were generated by I-TASSER. The parameters produced during structure assembly simulations, the template that was employed, and the effects of threaded structure alignment were the basis for the computed C-score. The C-score typically ranges from − 5 to 233. Structure analysis and verification server version 6 (SAVES v6.0) was used to authenticate the target proteins’ best validation structures. The 3D sequence profile for protein models was tested using the scores of the VERIFY-3D and ERRAT tools using SAVES v6.0 outputs, and the PROCHECK tool was utilized to confirm structure using the Ramachandran plot. PROCHECK analyzes the overall structural geometry and residue-by-residue geometry to determine the stereochemical nature of a protein structure34.

Protein interaction and molecular Docking analysis

Molecular Operating Environment (MOE, 2020.09) was used to conduct the molecular docking investigations. The partial charges were automatically computed using the MMFF94x force field after all minimizations were carried out using MOE up to a Root Mean Square Deviation (RMSD) gradient of 0.05 kcal mol − 1 Å−1. Using the protonate 3D protocol in MOE with its default settings, the protein chains under investigation were prepared for molecular docking simulations by adding polar hydrogens and deleting all residues, creating protonation states that were advantageous for the simulations. Docking pose building was accomplished using the London-dG scoring function and the Triangle Matcher placement approach4,29.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5. The results were presented as the mean ± standard deviation and statistical significance was set at P-value < 0.05. The significance of the differences between the experimental and control groups was analyzed by the student’s t-test. The in vitro experiments were analyzed using repeated measures ANOVA with post hoc Bonferroni test. One-way ANOVA was used to analyze the statistical significance between the in vivo groups with Tukey’s test as post hoc for pair wise comparison. Histopathological and immunohistochemical scoring were analyzed using Kruskal-Wallis test and Dunn’s post-test.

Drug-likeness and ADMET predictions

The drug-likeness of STX was assessed by ADMETlab 3.0 predictions of physicochemical properties, as shown in Fig. 1. Lipinski’s rule is utilized to determine the permeability and solubility using five parameters. Compounds that contravene Lipinski’s Rule of Five (RO5) are more prone to demonstrate inadequate absorption or penetration. The analysis revealed that STX exhibited two violations, whereas ABZ did not violate Lipinski’s rule. Number of hydrogen bond acceptors (nHA), hydrogen bond donors (nHD), and topological polar surface area (TPSA), of STX are predicted to be in the optimal range. However, the molecular weight and octanol/water partition coefficient (Log Po/w) of STX exceed the optimal values, suggesting poor absorption and oral drug-likeness administration.

It is vital for drug discovery efforts to target both pharmacological action and pharmacokinetics features. Human intestinal absorption (HIA), madin-darby canine kidney (MDCK) cells permeability, cancer coli-2 (Caco-2) cells permeability, and parallel artificial membrane permeability assay (PAMPA) were among the various criteria used to evaluate STX’s permeation capacity. The results displayed in Table 3 revealed that the Caco-2 permeability model of STX was found to be close to the acceptable range, while MDCK and PAMPA models showed low passive permeability (< 2 × 10−6 cm/s). STX showed HIA value of < 30% lower than HIA value of ABZ (> 30%). The distribution and partitioning in body tissues were also predicted. The plasma protein binding (PPB) affinity of STX is the optimal range (87.908%), even more than ABZ (85.36%). STX exhibited minimal BBB permeability (0.001), which elucidates its limited capacity to traverse the blood-brain barrier (BBB) and induce neurotoxicity. Moreover, the findings indicate that STX exhibits a negative predictive capacity concerning the inhibition of the CYP family (CYP1A2, CYP2C9, CYP2D6, and CYP3A4), as shown in Table 3. The anticipated excretion parameters, characterized by half-life (T½) and plasma clearance (CL), indicated that STX demonstrated a short half-life of 1.342 h and a low clearance rate of 2.33 ml/min/kg. The toxicological properties presented in Table 4 are crucial pharmaceutical considerations, encompassing mutagenicity as assessed by the ames test and carcinogenicity. The information gathered points to STX as non-mutagenic and suggests a negative prediction of genotoxicity. The acquired data indicate a negligible probability of detrimental impacts from the studied STX on cardiac rhythm, as evidenced by the minimal inhibition of the human ether-a-go-go related gene in comparison to ABZ. The ADMET predictions further suggest that STX exhibits a lower index of rat oral acute toxicity (ROA), neurotoxicity, hepatotoxicity, respiratory toxicity, and immunotoxicity, juxtaposed with ABZ.

Radar charts of physicochemical properties predictions of STX (A) and ABZ (B). logP: logarithm of the n-octanol/water distribution coefficient at pH = 7.4; logS: logarithm of the aqueous solubility value; logD: logarithm of the n-octanol/water distribution coefficient; nHA: number of hydrogen bond acceptors; nHD: number of hydrogen bond donors; TPSA; topological polar surface area; nRot: number of rotatable bonds; nRing: number of rings; MaxRing: number of atoms in the biggest ring; nHet: number of heteroatoms; fChar: formal charge; nRig: number of rigid bonds; MW: molecular weight.

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Characterization of the formulated niosomes

The niosomal formulation’s morphology, zeta potential, and particle size were assessed. Representative TEM micrographs of the formulated niosomes are displayed in Fig. 2. The micrographs depict spherical particles exhibiting diameters within the nanoscale range. DLS measurement provided further verification of the vesicle size, showing that it was in the nanoscale range with an average size of 177.8 nm. Noteworthy, according to the recorded polydispersity index value (0.383), the produced niosomes exhibited a consistent vesicle size. Our findings revealed that the zeta potential of the prepared niosomes is a high negative value of −35.23 ± 1.26, indicating the strength and stability among the niosomal vesicles. The encapsulation efficiency of STX into niosomal vesicles is 92.7%. The estimation of drug loading involved calculating the amount of drug encapsulated per gram of lipid. The results revealed that 185.4 mg of STX was loaded per gram of lipid in the formulation. Moreover, the stability of niosomes was assessed over a 30-day period under storage conditions of ambient temperature and refrigeration (4 °C). The results of our study indicated that niosomes stored in a refrigerator at 4 °C exhibited superior stability, as no discernible sedimentation or creaming was observed during the storage period. Additionally, the niosomal suspension maintained its initial particle size and EE%. Conversely, the niosomes that were stored at a temperature of 25 °C exhibited comparable changes in their initial particle size (836.2 nm; supplementary material Fig. S1) and EE% (85.3%).

In vitro anthelmintic activity

The anthelmintic activity against T. spiralis muscle larvae using different treatments of purified STX pigment, STX niosomal formulation, and combination therapy was studied in vitro compared to the reference drug ABZ. Survival numbers and mortality rates of T. spiralis muscle larvae after treatment are demonstrated in Table 5. The assessment of viability was conducted by observing the movement and coiling behaviors of muscle larvae, as the presence of immotile, straight, or comma-shaped larvae is a characteristic indicator of mortality, as shown in Fig. 3. The results displayed in Table 5 indicated that the effect of different treatments on muscle larvae survival is contingent upon exposure time. The statistically significant (P < 0.05) impact of all treatments was apparent from the first hour of incubation. Complete eradication of muscle larvae was observed after 48 h of incubation for niosomes and combinational therapy, while STX showed a mortality rate of 93.75%. Incubation of muscle larvae with STX pigment for 1 h showed higher killing activity and mortality rate (10.8%) compared to delayed-in-vitro-release niosomes (8%). Nevertheless, after 6 h of inoculation, niosomes demonstrated higher activity and mortality rate (30.83%) compared to mortality rates of STX (16.66%) and ABZ (22.5%). After 24 h of inoculation, all the tested agents showed significant activity (P < 0.05) and remarkable larvicidal control. Regarding to combination therapy, the synergistic effect between niosomes and ABZ was observed, as attested by higher mortality rates at all the duration of exposure compared to other groups.

Ultrastructural alterations of the cultured T. spiralis larval stage

SEM findings revealed that the muscle larvae incubated in RPMI medium without treatment showed an array of bacillary apertures, normal cuticle appearance, and a typical coiled structure, featuring fine transverse patterns and clearly defined annulation, as shown in Fig. 4. Concerning the impact of STX on cultured larvae, it was observed that there were excessive cuticular deformities, characterized by the appearance of a large mass with a cauliflower shape, numerous notches, blebs, and massive transverse and longitudinal fissures. ABZ demonstrated a comparatively less impact on the cuticle when juxtaposed with STX. The results indicated a damaged cuticle, characterized by notches, blebs, shallow longitudinal fissures, and a loss of its customary annulations.

In vivo evaluation of anthelmintic activity

The anti-parasitic activity of purified STX pigment, STX niosomal formulation, and combination therapy was further evaluated in an in vivo study. The experiment was compared to the reference drug ABZ, in order to validate the prospective in vitro findings. During the intestinal phase, all treatment groups exhibited a significantly lower mean number of adult worms in the small intestine (P < 0.001) compared to the infected untreated group (85.33 ± 6.02). The results presented in Table 6 demonstrated that the efficacy of STX treatment on the infected mice (G IVI) was 65.62%, resulting in a significant reduction (P = 0.0002) in the mean adult worm count (29.33 ± 4.04) in comparison to the untreated infected group (G IIII). Administration of ABZ suspension (G VI) significantly decreased the intestinal adult worm count (4.00 ± 2.00) with eliminating efficacy of 95.53% compared to groups G IIII and G IVI. Enhanced drug delivery of STX niosomal nanocarrier (G VII) augmented the killing efficacy of STX up to 98.05%, and reduced the mean adult worms count (1.66 ± 1.52). Notably, the combination therapy administered to G VIII successfully eradicated (100%) T. spiralis adult worms in infected mice, with no observable adult worms (0.00 ± 0.00).

In the muscular phase, the average count of muscle larvae per mouse was recorded as a measure of treatment efficacy. The results displayed in Table 7 revealed that all treated groups demonstrated a significant decrease in mean larvae count (P < 0.001) compared to the infected untreated group (32900 ± 1708.801). The combination therapy administered to group G VIIM showed the highest efficacy (90.73%) of eliminating the muscle larvae and a significant reduction of mean count (3047.66 ± 145.55), followed by the niosomes-treated group (G VIM) (83.08%) and ABZ-treated group (G VM) (74.96%), with mean counts of 5563.66 ± 545.03 and 8235.66 ± 670.04, respectively. In contrast, the STX-treated group (G IVM) showed the lowest efficacy (40.02%) and capacity of drug delivery, with a mean count of muscle larvae of 19733.33 ± 1159.02 compared to other treated groups.

Histopathological examination

First, the toxicity of the tested STX pigment was evaluated in mice to validate the safety profile obtained by ADMET predictions and cytotoxicity on normal Vero cells. The results displayed in Fig. 5. revealed that STX administration to GII showed normal tissue findings with no pathological abnormalities compared to normal control (GI).

Photomicrographs of H&E-stained small intestine were examined to evaluate the efficacy of STX, niosomes, and combination therapy on T. spiralis infection during the intestinal phase compared to ABZ, as shown in Fig. 6. After 6 days of muscle larvae inoculation, histopathological findings of the infected untreated group (GIII) showed a dense infiltration of inflammatory cells, primarily in the villi’s core and extending into the submucosa with shortened villi and increased villous width. The villous inflammation is observed with dense leukocytic cell infiltration consisting mainly of eosinophils, neutrophils, plasma cells, and lymphocytes extending into the lamina propria. A further decline in the villous height to crypt depth ratio, along with obvious goblet cell hyperplasia in epithelial lining villi and crypts, further demonstrated the flattening of the villi and hyperplasia of the Lieberkühn crypts. The intestinal wall of the STX-treated group (GIV) showed a slight reduction of leukocytic cell infiltration in villi and lamina propria with shortened villi, increased villous width, and crypt hyperplasia. The ABZ-treated group (GV) sections exhibited a notable reduction in the intensity of the leukocytic cellular infiltration in the villous core with normal crypts and normal lamina propria. Photomicrographs of the niosomes-treated group (GVI) showed fewer leukocytic cell infiltrations in the lamina propria with elongated villi and goblet cell hyperplasia in epithelial lining crypts. Regarding to the examined sections of the combination-treated group (GVII), the intestinal wall demonstrated a significant decline of inflammatory cellular infiltration, alongside a restoration of the normal villous architecture, crypts, and lamina propria. Moreover, the semi-quantitative histopathological scoring revealed that the treated groups demonstrated improved scores compared to GIII, as presented in Fig. 6M.

Skeletal muscle sections were also examined to evaluate the efficacy of the tested drugs on the encysted larvae during the muscular phase, as shown in Fig. 7. In the infected untreated group, histopathological examination of muscle sections revealed various chronic inflammatory cells, including eosinophils, plasma cells, lymphocytes, and histiocytes penetrating muscle bundles and encompassing the encysted larvae. Additionally, there was a notable dissipate degeneration of the infected muscle and a numerous number of encysted T. spiralis larvae, each encased with the collagen capsule, dispersed throughout the muscles’ sarcoplasm. Following STX treatment, the infected animals’ muscles displayed entire larvae enclosed in clearly defined, intact capsules as well as pericapsular histio-lymphocytic inflammatory cells infiltrate. Muscle sections of ABZ-treated group revealed a notable presence of pericapsular inflammatory cellular infiltration, alongside a reduced number of trichina capsules exhibiting focal deterioration. STX delivery via noisome nanovesicles resulted in a small number of larval deposits within the modified nursing muscle fibers, accompanied by a less pronounced inflammatory infiltration. The most significant enhancement, characterized by a reduced presence of larval cysts, localized degeneration of capsules, and a milder inflammatory infiltrate, was observed in the context of combination therapy.

Histopathological findings of H&E-stained intestinal sections. (A; 100x), (B; 400x), and (C; 400x) infected untreated group (GIII) showing dense leukocytic cells infiltration (arrowhead), goblet cells hyperplasia (blue arrow) besides hyperplasia of the crypts (thick black arrow). (D; 100x) and (E; 400x) STX-treated group (GIV) showing shortened villi (arrowhead), crypts hyperplasia (thick black arrow), leukocytic cells infiltration (arrowhead). (F; 100x) and (G; 400x) ABZ-treated group (GV) showing fewer leukocytic cells infiltration (arrowhead). (H; 100x), (I; 400x), and (J; 400x) niosomes-treated group (GVI) showing few leukocytic cells infiltration (arrowhead). (K; 100x) and (L; 400x) combination treated group (GVII) showing normal crypts and normal lamina propria. (M) Histopathological lesion scores in small intestine (n = 6). * Means significant p < 0.05 when compared to normal control group G1. # means significant p < 0.05 when compared to GIII & GIV by Kruskal-Wallis test and Dunn’s post-test.

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Histopathological findings of H&E-stained skeletal muscle sections. (A; 100x), (B; 400x), and (C; 400x) infected untreated group (GIII) showing many encysted trichnoid larvae (thin black arrow) and intense inflammatory infiltration (thick black arrow). (D; 100x) and (E; 400x) STX-treated group (GIV) showing numerous larval deposits (thin black arrow) and dense inflammatory infiltration. (F; 100x) and (G; 400x) ABZ-treated group (GV) showing reduced larval deposits (thin black arrow) and moderate inflammatory infiltration. (H; 100x) and (I; 400x) niosomes-treated group (GVI) showing few larval deposits (thin black arrow) and mild inflammatory infiltration. (J; 100x) and (K; 400x) combination-treated group (GVII) showing least number larval deposits (thin black arrow) and mild inflammatory infiltration. (L) Histopathological lesion scores of inflammatory reactions (n = 6). * Means significant p < 0.05 when compared to normal control group G1. # means significant p < 0.05 when compared to GIII & GIV by Kruskal-Wallis test and Dunn’s post-test.

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Immunohistochemical examination

The immunostaining of the skeletal muscles to detect the expression of proinflammatory cytokine (TNF-α) was also employed to assess the efficacy of the tested treatments on T. spiralis infection during the encysted larvae stage. As illustrated in Fig. 8, T. spiralis infection revealed trichina capsules characterized by localized degeneration, encompassed by inflammatory cellular infiltration demonstrating strong expression of TNF-α, with a score of 11.00 ± 1.549 in the untreated control group. Skeletal muscle sections of the STX-treated group revealed trichina capsules encircled with marked infiltration of inflammatory cells demonstrating strong expression of TNF-α, with a score of 10.00 ± 1. 459. Treatment with ABZ greatly decreased the number of inflammatory cells in the trichina capsule of skeletal muscle compared to the infected control group, with a score of 7.833 ± 0.9832 showing moderate TNF-α expression. Loading STX in a niosomal nanocarrier significantly decreased the number of inflammatory cells infiltrating the trichina capsule of skeletal muscle compared to treatment with STX pigment, which showed moderate levels of TNF-α (with a score of 5.000 ± 1.095). The synergistic activity of the combination treatment was verified by immunostaining of skeletal muscle showing the highest alleviation of inflammatory cellular infiltration and mild expression of TNF-α, with a score of 2.667 ± 1.033.

Homology modeling and validation of modeled proteins

The cluster density of each protein was used to construct five models using I-TASSER. The generated models exhibited varying confidence scores for each protein, and the models with the highest C-scores were selected for further analysis. For T. spiralis cathepsin F protein, the selected model, with the highest C-score of −1.52, displayed an estimated RMSD of 10.1 ± 4.6 Å and an estimated TM-score of 0.53 ± 0.15. The selected model for the T. spiralis calreticulin protein showed a C-score of 0.15, an estimated RMSD of 6.5 ± 3.9 Å, and an estimated TM-score of 0.73 ± 0.11. The selected models displayed the 3D-modeled protein structures, as shown in Fig. 9. Final validation of the refined structure: The SAVES v6.0 server was used to validate the refined I-TASSER structure. The Ramachandran plot statistics of T. spiralis cathepsin F model indicated that 87.8% of its residues are located in the most favored regions, 11.1% in additional allowed regions, 0.7% in generously allowed regions, and 0.4% in disallowed regions. The 3D-modelled T. spiralis calreticulin protein exhibited 85.2% of residues positioned within the most favored regions of the Ramachandran plot, alongside 13.5% in the additional allowed regions, 0.6% in the generously allowed regions, and 0.6% in the disallowed regions, as shown in Fig. 9. Lastly, Ts-CF1 and Ts-CRT were both modeled to a high standard, as evidenced by their overall quality factors of 94.412 and 98.196, respectively.

Molecular Docking analysis

Employing the “molecular operating environment (MOE) version 2019.0102,” a molecular docking study was carried out to enhance the comprehension of the binding interactions of the STX residue and the T. spiralis target proteins crucial to the parasite’s life cycle, as shown in Fig. 10. The docking results of STX into the binding pocket of the co-crystallized β-tubulin structure demonstrated that STX exhibited a binding score of −8.7283 kcal/mol, compared to ABZ (score= −6.9504 kcal/mol). This interaction is characterized by hydrogen bonding with Ser138, alongside two hydrophobic interactions with Tyr222. The interactions between the STX and the homology-modeled 3D protein structures, Ts-CRT and Ts-CF1, were further examined through docking analysis. The results of Ts-CF1 docking revealed that STX displayed a hydrogen bond interaction with Glu232, with a binding score of −10.603 kcal/mol compared to ABZ (score= −6.1692 kcal/mol). In relation to Ts-CRT, the docking results for STX indicated a binding score of −8.1168 kcal/mol in in contrast to ABZ (score= −5.7630 kcal/mol), demonstrating a hydrogen bonding interaction with Ser39, alongside a hydrophobic interaction with Phe8. Consequently, it can be inferred that STX may impede the activity of critical proteins in T. spiralis.

The advanced development of therapeutic drugs necessitates an in-depth comprehension of pharmacokinetic and toxic properties. The prediction of this data greatly improves the possibility of success while simultaneously decreasing the time needed for drug development35. ADMET predictions are commonly employed to assess the characteristics and suitability of a compound for pharmaceutical use19,36. In this study, the computed models of STX showed positive pharmacokinetic features of STX, including a negative effect on metabolic rate, biodegradability in the environment, and a low probability of being toxic or mutagenic, compared to ABZ. However, inadequate absorption and permeability decrease the oral bioavailability of STX. Therefore, a promising drug delivery carrier was formulated to overcome this drawback and enhance the therapeutic ability of STX as a promising antiparasitic agent.

This was accomplished through the encapsulation of STX within niosomes, which are cholesterol-based vesicular carriers created using non-ionic surfactant. The encapsulation of hydrophilic and lipophilic medicines is possible with niosomes37. When compared to other vesicular carriers, they offer numerous benefits. These include a higher degree of physicochemical stability and a lower cost in comparison to liposomes, the original vesicular carrier prototype38. Niosomes demonstrated encouraging potential for increased biological activity in the defense against pathogens21. It was hypothesized that these carriers could increase the drug’s permeability through the parasite membrane, have a greater inhibitory effect on the parasite, and have fewer harmful effects on healthy cells39,40. Our decision to use niosomes as the delivery mechanism was supported by these data. In this investigation, cholesterol was principally administered using niosomes manufactured with span 60 as the primary surfactant. The incorporation of cholesterol as a membrane stabilizing agent has the potential to improve the drug retention and entrapment41. As anticipated, the prepared vesicles exhibited a spherical form. The observed dimensions of the vesicles ranged from 100 to 200 nm in the nanoscale spectrum. There is a consensus with the morphology and size values of the reported data42,43. It is critical to highlight that the characteristics of the drug and the specific type of lipid are pivotal determinants of the efficiency of drug entrapment, particularly for lipophilic drugs24. Considering the lipophilic nature of STX, it is clear that the STX predominantly localized in the vesicle’s lipid core, thereby emphasizing the recorded drug loading and entrapment efficiency. In light of the values reported by other researchers, the observed entrapment efficiency falls within the appropriate range20,44,45.

To the best of our knowledge, this is the inaugural report on the influence of STX in the context of combating trichinellosis. This entailed an examination of the impact of free and niosomes-encapsulated STX on various stages of T. spiralis. First, the therapeutic efficacy of STX as an antiparasitic agent was determined in vitro. The findings of this investigation demonstrated that niosomal encapsulation of STX significantly increased the death rate of muscle larvae compared to free STX. This observed result can be ascribed to the vesicles’ instigation of ultra morphological changes. It is conceivable that the enhanced membrane permeability of the noisome is responsible for the changes in parasite morphology46. In addition to mortality, muscle larvae experienced notable alterations in their cell walls, including membrane destructions and surface blebs, as evidenced by electron microscopy. Cuticle integrity, a crucial component of body wall of Trichinella, along with the somatic musculature and hypodermis, is required for osmoregulation and determines the parasite’s shape, sustenance, and defense30,47. Thompson and Geary discovered that transcuticular passive diffusion serves as the primary mechanism for drug entry into helminths, leading to the development of anti-anthelmintics designed to compromise the integrity of the worm’s surface48. Blebbing transpires as the parasite endeavors to restore the compromised surface membrane in reaction to pharmacological intervention. Consequently, tegumental alterations may serve as a reliable signal of a drug’s potential anthelmintic efficacy49.

Anthelmintic agents can inflict damage upon their cuticle or cytoskeleton or impair parasite metabolism, ultimately resulting in paralysis and subsequent mortality50. Hence, the effective antiparasitic activity of STX can primarily be ascribed to its chemical nature of carotenoids. Briefly, the biological potential of carotenoids is primarily attributed to the generation of reactive oxygen species, which in turn causes oxidative stress and alterations in cell permeability18. Garcia-Bustos et al. postulated that the pro-oxidative traits enhance the anthelmintic potential by interfering with the helminth parasites’ decoupling oxidative phosphorylation, energy generation, and attaching to the glycoprotein on the parasite’s cuticle, ultimately leading to their demise51. Furthermore, Coronel et al., demonstrated that carotenoids have the potential to uncouple specific reductase-mediated mechanisms that impede helminth parasites’ energy production capabilities52. A plethora of research studies have proven the encouraging potential of carotenoids in the management of parasitic infections14,53,54. Furthermore, the antiparasitic activity was enhanced through the niosomal encapsulation of STX, as demonstrated by the eradication of larval counts. Numerous research studies clarifying the mechanism behind increased efficacy after encapsulation within niosomes as vesicular carriers have focused on the propensity of vesicular systems to adsorb to the pathogen cell membrane21,55. Other reports have proposed the fusion of vesicles with the cellular surface. An increased influx of medication into the microorganisms can be facilitated by the adsorption or fusion process, which can render the cell membrane more permeable56. The extent of the antiparasitic activity enhancement that is consistent with nano-based formulation is supported by the documented findings observed across an array of parasites57,58. Hassan et al. reported that the nano-formulation of hydrazones, compared to the synthesized hydrazone, exhibited exceptional activity against Toxoplasma infection, resulting in a substantial reduction in the burden of brain cysts57. Another example is the chitosan-coated nanocarrier, which improved the efficacy of miltefosine in combating T. spiralis infection in mice across different life cycle stages, including the intestinal, migratory, and muscular phases, in contrast to its free form58. However, to date, there is no published data reporting the antiparasitic potential of STX in drug solution or niosomal dispersion.

In light of the enhanced impact of niosomal encapsulation of STX, the study expanded to assess its in vivo efficacy. The in vivo evaluation entailed the oral delivery of the drug, either in solution form or as niosomes, thereby simulating the authentic administration process in humans. In the current study, the obtained results revealed the superior activity of STX-niosomes in the eradication of adult worms and diminishing the larval count compared to the drug solution and reference drugs. It is posited that vesicular systems may either adhere to or merge with the organism’s surface, leading to the direct conveyance of a substantial concentration of the drug into the organism, emphasizing the augmented activity of niosomal encapsulation46. Niosomes have proven this impact in their anti-trichinellosis and other anti-parasitic trials39,58. Moreover, the histological deterioration observed in the small intestine and skeletal muscles of infected mice was mitigated after treatment. An important barrier against gastrointestinal nematode infections is the mucus layer, a dynamically produced matrix that is carefully controlled31. Hyperplasia of goblet cells and increased mucin synthesis, which is associated with parasite expulsion, are symptoms of a T. spiralis infection, as many authors have before described31,59. Groups receiving combination therapy demonstrated the most significant enhancement in reducing goblet hyperplasia, focal peri-capsular plasma-lymphocytic inflammatory cellular infiltration, exhibiting the fewest trichina capsules with degeneration, and reinstating normal architecture. These findings corroborate those of a prior study that found that niosomes reduced T. spiralis infection burden and inflammatory expression surrounding trichina capsules60,61.

Monitoring the life cycle of T. spiralis demonstrated that β-tubulin, which is implicated in microtubule polymerization, and cathepsin F specific (Ts-CF1), a cysteine protease found in the stichosome and cuticle, are crucial for its life stages, thereby supporting their potential as therapeutic targets for T. spiralis infection8,62. Moreover, T. spiralis secretes calreticulin protein (Ts-CRT) at various stages of muscle larvae and adult worm development; this protein disrupts, interacts with, and overlaps with the host immune system, allowing T. spiralis to evade both the innate and adaptive immune systems of the host. Therefore, targeting this protein receptor could be an effective strategy to combat T. spiralis infection63. To reveal the manner in which STX inhibits T. spiralis infection, the 3D structures of Ts-CF1 and Ts-CRT were prepared using a homology modeling protocol. Based on our molecular docking results, STX may have the capacity to influence the target enzyme systems in a manner that is comparable to or superior to that of the reference drug, indicating their possible antiparasitic efficacy against T. spiralis at various stages of its life cycle.

The oral administration of STX via niosomal nanocarriers presents a promising delivery approach for the treatment of trichinellosis. These vesicles have the potential to augment the efficacy of STX in both in vitro and in vivo settings against T. spiralis. The enhanced oral bioavailability, along with the capacity of niosomal dispersion to induce ultrastuctural tegumental alterations resulting in paralysis and subsequent mortality, contributes significantly to the heightened activity observed. Moreover, the results of our molecular docking analysis indicate that STX may induce larval mortality through its capacity to disrupt enzymatic pathways essential to the life cycles of T. spiralis. The observed efficacy of STX-loaded niosomes paves the path for potential application and translation into clinical practice. However, further studies are required to assess the pharmacokinetic properties of STX.