Introduction

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the larvae of Echinococcus granulosus (Egs) parasitizing the human body [1]. After ingestion, Egs locate and parasitize the liver and other organs through the digestive and blood circulation systems and cause serious damage to organs [2, 3]. China is one of the regions with a high incidence of CE, accounting for 91% of the 18,000 cases in the world each year, and is still a serious health risk factor, especially in western China [4]. Surgery is the main treatment for echinococcosis. However, spillage of cyst fluid caused by surgery leads to postoperative recurrence or secondary infection [5]. Therefore, the use of anti-echinococcosis drugs is particularly necessary.

Albendazole (ABZ) is a kind of benzimidazolyl drug, and serves as a no alternative drug in treating echinococcosis clinically [6, 7]. The anti-echinococcosis efficacy of the drug has been demonstrated in vitro experiments, in vivo animal experiments and clinical use [8, 9]. However, ABZ has the disadvantages of low intestinal solubility, poor absorption and low bioavailability, which cannot achieve complete antiparasitic efficacy [10]. Some new agents, such as emulsions, liposomes and chitosan microspheres, have been used to improve the efficacy of ABZ [7, 11, 12], but they still have the disadvantage of insufficient targeting. A previous study has proved that nanoparticles (NPs) of ABZ can improve the liver targeting of drugs [13]. The possible mechanism is that NPs can be absorbed by liver Kupffer cells through the lymphatic reticular endodermal system as foreign substances, and then produce a specific passive targeting effect [14]. The characteristics of improving the bioavailability of poorly soluble drugs and liver targeting make NPs a great prospect in clinical application.

The mechanism of ABZ is believed to interfere with the formation of microtubules by polymerizing with free β-microtubules of the parasite, thereby hindering glucose uptake, leading to the blockage of some important physiological processes of parasite growth and development, and leading to the death of the parasite due to energy failure [15, 16]. ABZ also inhibits the anaerobic glycolysis pathway and partially reverses the tricarboxylic acid cycle by inhibiting the fumarate reductase system in mitochondria, thus hindering the production of ATP and making the parasite unable to survive and reproduce [16]. Atovaquone (ATO) is a mitochondrial complex III inhibitor originally developed to block mitochondrial respiration in Plasmodium falciparum and other protozoa [17] for the prevention of malaria, as well as for the prevention and treatment of pneumocystis pneumonia and toxoplasmosis in acquired immune deficiency syndrome patients [18]. Recent studies have shown that ATO or in combination with other drugs (ABZ, 3-bromopyruvic acid, etc.) kills Echinococcus multilocularis by inhibiting respiratory pathways [19,20,21]. However, whether ATO combined with ABZ can enhance the efficacy of anti-CE of ABZ remains unclear.

This study compared the anti-CE efficacy of ABZ-ATO free and NPs, and evaluated the synergistic effect of the two drugs on the energy metabolism of protoscolexes. This study lays a theoretical foundation for the clinical compound treatment of echinococcosis and also provides an effective new drug treatment strategy for CE treatment.

Materials and methods

Determination of drug loading of drug NPs

ABZ NPs, ATO NPs and ATO-ABZ NPs were provided by Professor Jiang Hulin’s team from China Pharmaceutical University (Nanjing, China). ABZ (Sigma, USA) stock solution (50 µg/mL) and ATO (Macklin, Shanghai, China) stock solution (50 µg/mL) were prepared and diluted according to an isometric gradient to be used as the standard solution. The absorbance of two drug solutions was measured at 295 nm and 494 nm by ultraviolet spectrophotometer, respectively, and the standard curve was established to obtain the regression equation. The ABZ NPs, ATO NPs, and ATO-ABZ NPs were demulsified. In brief, 100 µL of NPs were centrifuged at 4000 g for 15 min at 4 °C. 20 µL of the supernatant was added with 1980 µL of methanol, and the mixture tested at 295 nm for ABZ NPs, at 494 nm for ATO NPs, and at 295 nm and 494 nm for ATO-ABZ NPs by ultraviolet spectrophotometer, respectively. Absorbance values were substituted into the corresponding regression equation to obtain the concentration of the corresponding NPs. NPs were diluted with ultrapure water (1:100), and examined by Malvern Zetasizer NanoZS90 (Malvern Instruments Ltd., UK) to detect particle size and zeta potential.

Collection and culture of protoscolexes

Egs were collected from liver cysts of sheep infected with Egs at Hualing abattoir in Urumqi, Xinjiang. In brief, under sterile conditions, the cyst fluid from the liver cyst was collected with a syringe and transferred to a sterile centrifuge tube, left to rest, and the sediment was washed repeatedly with sterile saline. The liver cyst was open, and the internal capsule was placed in sterile saline. The internal capsule was cut into pieces, filtered through a screen several times, and washed with sterile saline until the color of the internal capsule was milky white. Then, the cyst fluid combined with the protoscolexes in the inner capsule was stranded, and sediments were the protoscolexes to be harvested. The protoscolex was washed several times with normal saline supplemented with penicillium streptomycin (double antibody), and digested with 1% pepsin in a 37 °C thermostatic water bath for 30 min. The obtained protoscolexes were kept in the medium (RPMI 1640: FBS: Double antibody = 89:10:1) at 37℃ in a 5% CO2 incubator. The protoscolexes with an activity ≥ 98% detected by the 1% methylene blue dye exclusion test could be used for the following test. This study was in accordance with the ARRIVE guidelines, and approved by the Animal Ethics Committee of the Animal Center of Xinjiang Medical University (approval no. 123–123).

Determination of the in vitro survival rate of protoscolexes with different drug interventions by methylene blue dye exclusion test

To assess the effect of a single agent, protoscolexes were evenly seeded in 96-well plates according to approximately 200 protoscolexes per well, and randomly divided into groups with three replicates per group. Protoscolexes were intervened with different concentrations of NPs (10, 20, 30, 40, 50, 100, 200 µg/mL) or free of ATO or ABZ for 1–5 days. Protoscolexes were stained with methylene blue for 3 min at room temperature, and then observed under an inverted microscope (Nikon, Japan) to evaluate its survival.

According to the effect of a single drug on the activity of protoscolexes, the synergistic ratio of the two drugs NPs was screened. To assess the effect of ATO-ABZ, vesicles with 2–3 mm diameter cultured from protoscolexes for more than 2 months were seeded in a 48-well plate with 20 ~ 30 vesicles per well with three replicates per group. Vesicles received different concentrations of NPs or free of ATO-ABZ (10, 20, 30, 40, 50, 100, 200 µg/mL) for 5 days, and then were collected to evaluate protoscolex activity.

Hematoxylin-eosin (HE) staining

The protoscolexes were fixed in 4% paraformaldehyde solution for 48 h, embedded in paraffin, and sectioned. The sections were deparaffinized, and then subjected to HE staining. In brief, the sections were dehydrated with alcohol solution and washed by distillation, placed in hematoxylin staining solution (ZSBB-Bio, Beijing, China) for 55 s and washed with distilled water to remove the excess staining solution. After 2 s of differentiation with ethanol hydrochloride, the protoscolexes were placed in an eosin staining solution (ZSGB-BIO, Beijing, China) for 55 s, and the excess staining solution was washed away with distilled water. The sections were dehydrated with gradient alcohol before being processed for transparency, and then sealed with neutral gum. Protoscolexes were observed under a microscope to observe the pathological damage.

Scanning electron microscope (SEM) observation

Protoscolex after 3 days of drug intervention was placed in centrifuge tubes and washed three times with PBS buffer (Hyclone, USA) for 10 min each. The protoscolexes were fixed with 2.5% glutaraldehyde for 24 h, and washed 3 times with 0.1 mol PBS buffer (pH = 7.2) for 15 min each time. After fixation with 1% osmium tetroxide for 1 h, the protoscolexes were washed three times with PBS buffer and received gradient dehydration by 50%, 70%, 80%, 90%, and 100% tert-butyl alcohol. After drying by vacuum spray, the samples were coated with metal, and the surface ultrastructure of protoscolexes were observed under a SEM (1430VP; LEO, Germany).

Cell culture, and determination of cell viability and mitochondrial membrane potential of LO2 cells

Human normal liver cells (LO2 cells) were donated by the Collaborative Central Laboratory of Xinjiang Medical University and cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (HyClone, USA) and 0.1% penicillin-streptomycin (HyClone, USA) at 37℃ and 5% CO2. The viability of LO2 cells with different treatments was detected by methyl thiazolyl tetrazolium (MTT) assay. Briefly, LO2 cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 1 × 105/well, and cultured overnight. Then, drugs (blank NPs or ABZ NPs or ATONPs or ATO-ABZ NPs) at different concentration (0.01, 0.1, 1, 10, 30 and 50 µg/mL) were used to treat the cells for 24 h. After that, 10 µL MTT solution was added to each well, and after incubated for 4 h, DMSO (100 µL) was added to dissolve crystallization. The absorbance (OD) value of each well was measured at 490nmthe cell viability was calculated as follows: cell viability (%) =(ODtest-ODblank)/(ODcontrol-ODblank)×100%.

The mitochondrial membrane potential of LO2 cells was detected with the Mitochondrial Membrane Potential Assay Kit with JC-1 (Solarbio, Beijing, China) based on the protocols of manufacturer. In brief, LO2 cells were seeded in a 24-well plate at a density of 1 × 104/well and cultured overnight. Drug intervention was performed for 24 h, and then stained with JC-1 staining working solution for 20 min at 37 ℃ in the dark. After removing the staining solution, the cell culture medium was added, and the cells were observed and photographed under a microscope.

Determination of reactive oxygen species (ROS) in protoscolexes

ROS of protoscolexes was detected by the Reactive Oxygen Species Assay Kit (S003M; Beyotime, China). In brief, protoscolexes with viability ≥ 98% were randomly divided into four groups: control, ABZ NPs, ATO NPs, and ATO-ABZ NPs groups. The protoscolexes in the ABZ NPs, ATO NPs, and ATO-ABZ NPs groups were treated with 30 µg/mL ABZ NPs, 10 µg/mL ATO NPs, and 10 µg/mL ATO-ABZ NPs, repectively. After that, the Egs in all the groups were added with 1 mL of 10 µmol/L 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA), and cultured at 37 ℃ in an incubator with 5% CO2 for 20 min. After washing with serum-free cell culture medium three times, the protoscolexes were observed and photographed by an Ti-S inverted fluorescent microscope (Nikon, Japan).

Western blot

The total protein of protoscolexes with different treatments was extracted with RIPA buffer (Thermo, USA) supplemented with serine proteases inhibitor Phenylmethylsulfonyl fluoride (PMSF) (Solarbio, Beijing, China). After centrifugation at 12,000 g for 10 min, the supernatant was collected to determine the protein concentration using a Bicinchoninic acid assay (BCA) kit (Thermo, USA), and then the protein concentration was uniformly adjusted to 2500 µg/mL. Subseqeunt, the protein samples were subjected to 12.5% SDS-PAGE for 2 h, wet transferred to PVDF membranes (Millipore, USA) for 1 h, and blocked with 5% bovine serum albumin (BSA) solution at room temperature for 1 h. Then, the membranes were incubated with primary antibodies against dihydroorotate dehydrogenase (DHODH) (1: 1000; ab246901, Abcam, USA) and GAPDH (1: 1 000) at 4℃ overnight. After washing with TBST solution (0.05% Tween20) three times, the membranes were incubated with the secondary antibody of goat anti-rabbit IgG/HRP (1: 5 000; Bioss, Beijing, China) for 1 h at room temperature. After washing with TBST solution three times, the protein bands were visualized by ECL solution (Thermo, USA) and photographed in a gel imager (BioRad, USA). The grayscale values of the bands were analyzed using Image J software with GAPDH as an internal reference.

Enzyme-linked immunosorbent assay (ELISA)

The contents of lactic acid (LD), lactic dehydrogenase (LDH), pyruvic acid (PA), and ATP levels in protoscolexeswith different treatments were measured using the corresponding ELISA assay kits, including a LD kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute, China), a LDH activity detection kit (BC0680, Solarbio, China), a PA activity assay kit (BC0680; Solarbio, China) and an enhanced ATP detection kit (S0027; Beyotime, China), respectively. Each experiment was repeated three times.

In vivo experiments

Kunming mice (female, weight 18 ~ 22 g) were purchased from Laboratory Animal Research Center, Xinjiang Medical University (license NO. SYXK (New) 2018-0003) and kept at the temperature of 20 °C to 22 °C, relative humidity of 50–55%, and 12 h of light/ darkness. The CE mouse model was established by intraperitoneal injection of 50 vesicles of Egs, and validated hydatid foci after 3 months of infection by B-ultrasonography. The mice with successful modeling were randomly grouped in control, model, ABZ NPs, ATO NPs, and ATO-ABZ NPs, and received a dose of 50 mg/kg intragastric administration for 28 days once a day. Mice were weighed once a week. After 28 days of drug intervention, mice were anesthetized by 1% sodium pentobarbital and vesicles in the abdominal cavity were isolated. The size, shape and wet weight of vesicles were recorded to calculate the inhibition rate of vesicle wet weight as an equation: (mean value of vesicle wet weight in the model group value of vesicle wet weight in the drug intervention group)/C× 100%. The other organs of mice were collected and weighed to calculate the organ index as an equation: organ index (mg/g) = organ weight (mg)/body weight (g) of mice. The pathological changes of the organs were observed by HE staining.

Statistical analysis

SPSS 17.0 software (USA) was used for statistical analysis. Data were represented as mean ± SD. One-way analysis of variance was used for comparison between groups. P < 0.05 was considered statistically significant. GraphPad Prism6.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to visualize the data.

Results

Characterization of ATO NPs, ABZ NPs, and ATO-ABZ NPs

After linear regression, the standard curve of ABZ was y = 0.0470 × x + 0.0045 (R2 = 0.9992), which showed a good linear correlation in the concentration range of 4–16 µg/mL (Figure S1A). The standard curve for ATO was y = 0.0033 × x − 0.0071 (R2 = 0.9990), which showed a good linear correlation in the concentration range of 40 ~ 160 µg/mL (Figure S1B). Figures S1C-E showed the particle size and zeta potential of ABZ, ATO, and ATO-ABZ of the NPs. The drug loading of ABZ NPs was 0.995 mg/mL ABZ NPs; the drug loading of ATO NPs was 1.079 mg/mL ATO NPs; as well as the drug loading of ATO-ABZ NPs (1: 1) was 0.458 mg/mL ATO NPs and 0.436 mg/mL ABZ NPs (Table 1). The particle size of ATO-ABZ NPs was 267.5 nm, which was higher than that of ABZ NPs (161.6 nm) and ATO NPs (201.2 nm). Additionally, the zeta potential of ABZ NPs, ATO NPs, and ATO-ABZ NPs was − 20.7 mV, -42.3 mV, and − 21.6 mV, respectively (Table 1). These results showed the three drug NPs had uniform size, stability, and high drug loading, among which zeta potential was negative, indicating that the drug nanoparticles had a weak negative charge on the surface, which was not easy to cause hemolysis, and had high biocompatibility and good stability. These results indicated that the ABZ NPs, ATO NPs, and ATO-ABZ NPs could be used for further experiments.

Table 1 Physical and chemical properties of nanoparticles of different drugs
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Effects of ABZ free/NPs, ATO free/NPs and ATO-ABZ free/NPs on the viability of protoscolexes

During the 1–5 days of intervention, ABZ, ABZ NPs, ATO and ATO NPs inhibited the viability of protoscolexes, and the inhibitory effect was enhanced with the increase of the drug concentrations (Table 2and Table 3). On the 1st, 2nd, 3rd, 4th and 5th day of intervention, the IC50 values of ABZ free were 17740.209, 7869.119, 1678.318, 1510.222, and 426.169 µg/mL, respectively, that of ABZ NPs 367126.442, 110526.086, 73295.703, 3118.444, 2959.905 µg/mL, respectively; that of ATO free 86.150, 64.659, 17.459, 7.497, 5.914 µg/mL, respectively, and that of ATO NPs 697.392, 193.522, 24.593, 3.727, 3.101 µg/mL, respectively.

Table 2 Survival rate of protoscolexes after albendazole (ABZ) free and nanoparticles (NPs) intervention (%)
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Table 3 Survival rate of protoscolexes with atovaquone (ATO) free and nanoparticles (NPs) intervention (%)
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According to the in vitro pharmacodynamic results of a single drug, ATO was set at 10 µg/mL, and mixed with ABZ in different ratios (ATO: ABZ = 1:0.25, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:1:1:1) for intervention of protoscolexes. The results showed that during the 1–5 days of intervention, ATO-ABZ free and NPs at different ratios inhibited the activity of protoscolexes (Table 4). The optimal ratio of ATO and ABZ to inhibit the protoscolex activity was 1:1. At this drug ratio, the activity of protoscolexes was not only strongly inhibited but also gradually decreased with the increase of drug exposure time (Table 4).

Table 4 Survival rate of protoscolexes with treatment of different ratios of atovaquone (ATO): albendazole (ABZ) with the 10 µg/mL of ATO under free and nanoparticles (NPs) conditions
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Further we examined the effect of the optimal ratio (ATO-ABZ 1:1) on protoscolex survival. The results showed that the activity of protoscolexes was inhibited by ATO-ABZ free and NPs at a concentration of 1:1 at 1–5 days of intervention (Table 5). The inhibition effect of high concentration groups (50, 100 µg/mL) of ATO-ABZ NPs 1:1 and ATO-ABZ free 1: 1 was stronger than that of the low concentration groups (5–40 µg/mL), and by the 5th day of the intervention, all the protoscolexes in the 50 and 100 µg/mL ATO-ABZ NPs groups died (Table 5). The IC50 values of ATO-ABZ free 1:1 group on 1d, 2d, 3d, 4d and 5d were 499.405, 83.313, 16.902, 2.585, and 0.753 µg/mL, respectively and that of of ATO-ABZ NPs 1:1 group were 586.297, 74.745, 11.763, 1.154 and 0.569 µg/mL, respectively.

Table 5 Survival rate of protoscolexes with treatment of different concentrations of atovaquone (ATO)-albendazole (ABZ) free and nanoparticles (NPs) at the ratio of ATO: ABZ = 1:1
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We also examined the effect of the optimal ratio (ATO-ABZ 1:1) on vesicle survival. The results showed that different concentrations of ATO-ABZ free and NPs 1:1 inhibited the activity of vesicles, and the effect was enhanced with the increase of time and concentration of intervention. The viability of 12.5, 25, 50, 100 and 200 µg/mL ATO-ABZ NPs 1:1 group was reduced to 66.67%, 63.33%, 60.00%, 53.33% and 45.00%, respectively, while the corresponding ATO-ABZ free 1: In group 1, 63.33%, 60.00%, 50.00%, 48.33%, 48.33% vesicles survived. The inhibition effect of the ATO-ABZ NPs 1:1 and ATO-ABZ free 1: 1 at high concentration groups(100, 200 µg/mL) on the activity of vesicles was stronger than that of the low concentration groups (12.5–50 µg/mL). On the 5th day, the activity of vesicles in 100 and 200 µg/mL ATO-ABZ NPs groups was 26.67% and 13.33%, respectively. The viability of 100 µg/mL ATO-ABZ free 1:1 group and 200 µg/ mL ATO-ABZ free 1:1 group were 28.33% and 25.00% (Table 6). The IC50 values of ATO-ABZ free 1:1 group on 1d, 2d, 3d, 4d and 5d were 391.879, 178.884, 97.344, 43.270 and 6.656 µg/mL, respectively and that of ATO-ABZNPs 1:1 group were 296.469, 272.264, 134.618, 30.989 and 10.389 µg/mL, respectively.

Table 6 Survival rate of vesicles of protoscolexes with treatment of different concentrations of atovaquone (ATO)-albendazole (ABZ) free and nanoparticles (NPs) at the ratio of ATO: ABZ = 1:1
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Effects of ABZ, ATO free/ NPs and ATO-ABZ free/ NPs on the morphology of protoscolexes

With the increase of the two drugs free intervention time (from 1 day to 5 days), the protoscolexes showed increased methylene blue staining and obvious morphological changes and ATO free had a stronger inhibitory effect on protoscolexes than ABZ free (Fig. 1A). Morphologically, the protoscolexes showed abnormal morphology, shrunken size, unclear internal structure, absence of calcium particles, and abscission of the hook (Fig. 1A). Particularly attention was that the two drug interventions resulted in different degrees of drug crystal precipitation. (Fig. 1A). In the 50 µg/mL ABZ NPs group and ATO NPs group, the protoscolexes showed weakened activity or even death, smaller blue-stained volume, shrinkage, increased eversion type, and destroyed structure. At the concentration of 50 µg/mL, ATO NPs had a stronger inhibitory effect on protoscolexes than ABZ NPs, and no crystal precipitation was observed in either group (Fig. 1B). We also examined the effect of the optimal ratio: ATO-ABZ 1:1 on protoscolex viability. The results showed that the ATO: ABZ free and ATO: ABZ NPs interventions reduced protoscolex viability, and the viability became lower with the increase of intervention time (Fig. 1C and D). Particularly, the morphological changes of protoscolexes in NPs groups were more significant than those in the drug free groups at the same concentration, and no crystal precipitation was observed (Fig. 1C and D). These data suggest that NPs of the single drug (ATO or ABZ) or the combined drug (ATO-ABZ) were more stable and had stronger inhibitory effect on protoscolex than the free drugs.

Fig. 1
figure 1

Effect of different drugs on the morphology of protoscolexes. (A-C) The morphology of protoscolexes on the 1st, 3rd, and 5th days of free drug intervention (A), NPs (B), and ATO-ABZ synergistic ratio with fixed ATO 10 µg/mL (C) were observed by inverted microscope after methylene blue staining. 40× magnification. (D) Validation of ATO-ABZ NPs 1:1 drug effect in each experimental group on 1, 3, and 5 days of protoscolex morphology (40× magnification)

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Effects of ATO-ABZ free/NPs on the morphology of vesicles and histopathology of protoscolexes

During the observation period of 5 days, the volume and size of vesicles in the control group were unchanged and that in the drug intervention groups showed different degrees of damage (Fig. 2A). With the increase of drug intervention time, vesicle damage more serious, mainly manifested in reduced volume, thickened stratum corneum, content collapse into black mass, unclear surface texture is not clear (Fig. 2A). The pathological examination of protoscolexes with 3 days of drug intervention showed that the protoscolexes in the ABZ NPS group showed shrinkage, partial tissue shedding, abnormal parasite morphology, and deep coloring and that in the ATO NPs group showed shrinkage, disintegrated edge structure, abnormal morphology, disordered internal structure, and dissolved matrix and reduced size (Fig. 2B). The surface ultrastructure of protoscolexes with 3 days of in vitro drug intervention showed that in the ABZ NPs intervention group, the protoscolexes lost their normal morphology and collapsed; in the ATO NPs treatment group, the protoscolexes was severely damaged, showing rough surface, collapse and shrinkage of the worms; in the ATO-ABZ NPs intervention group, the protoscolexes was severely damaged, showing structure collapse, volume reduction, and eroded edge in the epidermis (Fig. 2C).

Fig. 2
figure 2

Effect of different drugs on vesicle morphology and structure. (A) The morphology of echinococcus granulosa vesicles with different drug interventions (40× magnification). (B) Histological characteristics of protoscolexes after different drug interventions (40× magnification). (C) Ultrastructural changes on the surface of protoscolexes were detected by scanning electron microscope (40× magnification)

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Effects of different drugs on the toxicity, morphology, and mitochondrial membrane potential of LO2 cells

Compared with the control group, the concentrations of ABZ NPs and ATO NPs in the range of 0.01 µg/mL to 30 µg/ ml did not change the cell viability of LO2 cells (P > 0.05), representing the safe concentration range, and the safe administration concentration range of ATO-ABZ NPs was 0.01–50 µg/ mL (Fig. 3A). The IC50 values of ABZ NPs, ATO NPs, ATO-ABZ NPs and blank NPs were 573.045 µg/mL, 467.314 µg/mL, 271.439 µg/mL, and 310757.710 µg/mL, respectively. Within the safe concentration range of the three drugs, we chose ABZ NPs 30 µg/mL, ATO NPs 10 µg/mL, and ATO-ABZ NPs 10 µg/mL to observe their effects on cell morphology. The results showed that the cells in the control group adhered to the wall and grew well, while the cells in each drug treatment group showed varying degrees of reduction in the number of cells, unclear cell boundaries, and cell shedding. However, the cell state of the blank NPs group was close to that of the control group, indicating that the blank NPs had no obvious damage to LO2 cells, and the liver damage of each administration group was small at the corresponding concentration (Fig. 3B).

Fig. 3
figure 3

Effect of drug intervention on LO2 cells. (A) Cell viability was evaluated by the MTT method. (B) cellular morphology was observed by an optical microscope. (C) mitochondrial membrane potential of LO2 cells was evaluated by fluorescence micrograph

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The intensity of green fluorescence and red fluorescence in the control group was similar, indicating that JC-1 was accumulated in the mitochondrial matrix and the mitochondrial membrane potential was high. After treatment with ABZ NPs, ATO NPs and ATO-ABZ NPs, the red fluorescence intensity was significantly decreased, and the green fluorescence intensity was slightly increased, indicating that JC-1 could not aggregate into the mitochondrial matrix and the mitochondrial membrane potential was slightly lower than that of the blank control group indicating that each drug group had little effect on the mitochondrial membrane potential of LO2 cells at the corresponding concentration (Fig. 3C).

Effects of different drug NPs on oxidative stress of protoscolexes

The ROS level of normal protoscolexes was very low, but was significantly increased by ABZ NPs, ATO NPs, and ATO-ABZ NPs, indicating that drug-induced ROS accumulation may induce some initiating mechanism of protoscolex death (Fig. 4A). The results of DHODH protein expression analysis showed that ABZ NPs, ATO NPs and ATO-ABZ NPs interventions significantly reduced DHODH expression, respectively, and ATO-ABZ NPs inhibiting DHODH expression effect was stronger than the ATO or ABZ NPs (Fig. 4B, Figure S2). The intervention of ABZ NPs, ATO NPs and ATO-ABZ NPs reduced the contents ofLD and LDH, increased PA content, and reduced ATP production in protoscolex, and the effect of ATO-ABZ NPs was stronger than that of ABZ NPs and ATO NPs (Fig. 4C and F). Regarding the effects on the contents of LD, LDH and ATP, the effects of ATO NPs were stronger than those of ABZ NPs (Fig. 4D and F).

Fig. 4
figure 4

Pharmacological interventions affect oxidative stress of protoscolexes. (A) Stained fluorescence micrographs (200× magnification) indicated ROS in protoscolexes with different drugs. (B) Western blot detected DHODH protein expression. (C) ELISA assay analyzed energy metabolism indicators. *P < 0.05 vs. control, #P < 0.05 vs. ABZ NPs, $P < 0.05 vs. ATO NPs.

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Establishment of a hydatid mouse model, and physiological indexes of mice after drug intervention

Egs microcapsules were injected intraperitoneally to establish a mouse model of secondary infection with Egs. Three months after infection, multiple univentricular cysts were grown in the abdominal cavity and liver of the mice (Fig. 5A). After 28 days of treatment with each drug, the mice were dissected and the vesicles in the body were removed to observe the vesicles in each group. In the model group, the surface of the vesicles was smooth, round and transparent, while all drug treatments caused vesicles to shrink and smaller. Among them, the volume of vesicles in ABZ free and ABZ NPs groups was significantly lower than that in the model group. The vesicles in ATO free and ATO NPs groups showed obvious yellow staining, which may be related to the original color of ATO. In the ATO-ABZ free group, the yellow stained vesicles were observed, and in the ATO-ABZ NPs group, the yellow stained vesicles were significantly damaged, showing reduced size (Fig. 5B). ABZ NPs, ATO NPs and ATO-ABZ NPs significantly reduced cyst weight in mice and the effect of ATO-ABZ NPs was stronger than that of ABZ NPs and ATO NPs (Fig. 5C). The results of weekly body weight measurement showed that the weight change was a normal physiological growth state in the control group, increased rapidly in the model group and showed the same trend of “increase-decrease” change in drug treatment groups (Fig. 5D).

Fig. 5
figure 5

Verification of the inhibiting effect of the drug on cyst formation in mice. (A-D) Mice were infected with Egs by intraperitoneal injection for 3 months and received B-ultrasound to confirm the success model (A). *P < 0.05 vs. model, #P < 0.05 vs. ABZ NPs, $P < 0.05 vs. ATO NPs. Mice were then received drug intervention for 28 days and sacrificed to test vesicles (B), weight of cyst (C), organ weight (D), and organ index (E). *P < 0.05 vs. blank, #P < 0.05 vs. model

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To observe whether the drug treatment had any effect on the weight of each organ, the organ index was calculated and compared with the model group. The results showed that the liver index, spleen index and kidney index of each drug group were significantly different from those of the model group (P < 0.05), while the changes in heart index, brain index and lung index were not statistically significant between the model group and the drug treatment groups (P > 0.05, Fig. 5E). These outcomes indicated that the NPs treatment groups could have fewer toxic effects on the main organs of mice, such as heart, brain, and lung.

3Effects of different drug NPs on the pathological changes of organs in mice

Further, the pathological changes of various organs after drug treatment were detected. The results of the liver pathological examination showed that the model group had abnormal liver structure, with a large number of inflammatory cells infiltration in the hepatic portal area and necrosis of the lesion. After treatment with each drug NPs, the degree of inflammatory response in liver tissue showed a decreasing trend. In the ATO NPs treatment group, the nuclei of most hepatocytes were pyknotic, and the “empty halo” formed around the nucleus, the venules in the portal area appeared congested, a large number of pathological inflammatory cells infiltrated, and a large number of focal necrosis were seen in the liver tissue. The liver tissue structure of the ATO NPs treatment group was closer to that of the control liver than that of the ABZ and ATO-ABZ NPs treatment groups (Fig. 6A).

Fig. 6
figure 6

Pathological changes of organs in mice with ATO-ABZ NPs intervention. (A-G) Pathological changes of (A) liver, (B) spleen (C) lung (D) renal (E) heart (F) brain and (G) small intestinal of mice after 28 days of treatment with various drugs by HE staining

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Pathological examination of renal tissue showed that the model group had abnormal renal structure, showing obvious congestion of glomeruli and renal interstitium, and a large number of pathological inflammatory cell infiltration. In the ABZ NPs treatment group, the renal tubular epithelial cells were swollen, the renal interstitial cells were hyperemia and inflammatory cell infiltration, and some renal tubular necrosis was observed. In the ATO NPs treatment group, the renal tissue structure was abnormal, the renal tubular epithelial cells were partially swollen, and the renal interstitial inflammatory cells were infiltrated. Renal in the ATO-ABZ NPs treatment group showed no significant changes in renal glomeruli and tubules and alleviated interstitial hyperemia and hemorrhage (Fig. 6B).

Pathological examination of the spleen showed that the model control group had local lymphopenia, and increased spleen cell spacing, accompanied by a large number of red blood cell infiltration and increased neutrophils. The cell spacing decreased and the cytoplasm tended to be uniform in each drug NPs treatment group (Fig. 6C).

Pathological examination of heart tissue showed that the myocardial fibers were blurred and some myocardial fibers fused with myocardial cells in this area were degenerated, necrotic and local congestion in the model group. In drug NPs treatment groups, no significant pathological changes were observed (Fig. 6D).

Pathological examination of lung tissue showed congestion of the alveolar septum, thickening of the alveolar wall, and infiltration of a large number of neutrophils in the alveolar space in the model group. Inflammatory cell infiltration and hemorrhage emerged in the alveolar cavity of the ATO NPs treatment group. Lung in the ABZ NPs treatment group, showed slight congestion in the alveolar septum and alveolar cavity, slight hyperplasia of fibrocytes and a small amount of inflammatory cell infiltration around the bronchus. Lungs in the ATO-ABZ NPs treatment group showed slight congestion and normal pulmonary tubules at all levels (Fig. 6E).

Pathological examination of brain tissue showed that the structure of each layer of brain tissue was clear, and no hemorrhage and inflammatory cell infiltration were found in the model group. The brain tissue structure of the drug treatment groups showed no obvious pathological changes (Fig. 6F).

Discussion

To date, the search for effective drug strategies with lower side effects is still the direction of efforts for the treatment and prevention of CE. Although ABZ and ATO can kill parasites by inhibiting energy metabolism [10, 22], their low water solubility limits their bioavailability. The advantages of NPs, such as targeting, controlled release, biocompatibility, increasing the solubility of water-soluble drugs, and improving drug stability and bioavailability, have the potential to enhance the efficacy of ABZ and ATO [23, 24]. In the present study, ABZ and ATO were prepared into NPs, respectively, and their combination into “energy uptake suppression” and “energy metabolism inhibition” NPs, to explore the feasibility of energy dual resistance for the treatment of CE. The optimal ratio of ATO-ABZ NPs was 1:1, which could effectively inhibit the activities of protoscolexes and vesicles. The activity of the protoscolexes was inhibited and the morphology of the protoscolexes was impaired by NPs more than that of the free form. In terms of pathological mechanism, ATO-ABZ NPs had little effect on the viability and mitochondrial membrane potential of liver cells, inhibited the energy metabolism of protoscolexes, and had little toxic effect on the organs of mice.

ABZ is a drug inhibiting hydatid cyst, which has been proven in clinical and animal experiments [10, 25]. Similarly, we observed that the viability of protoscolexes was inhibited by ABZ in a dose - and time-dependent manner, which was consistent with the published literature [12, 26]. A special phenomenon was that the survival rate of the 200 µg/mL ABZ free intervention group was higher than that of the 100 µg/mL ABZ free intervention group in the first 3 days. The possible reason was that drug crystallization occurred at the concentration of 200 µg/mL of ABZ, and the protoscolexes avoided the toxic effects of drugs through incomplete intake of medication. Pharmaceutical NPs play a convenient role in increasing ABZ bioavailability. Shohreh Aminpour et al. [27] and Kobra Kohansal et al. [24] intervened protoscolexes with ABZ NPs or in vivo treatment of mouse protoscolex-infected hydatid disease models and found that ABZ-loaded NPs were more lethal to protoscolexes than free drug. Consistent with these studies, our results showed that ABZ NPs were more stable than ABZ-free in inhibiting protoscolex viability.

The significant finding of this study was that ABZ combined with ATO-loaded NPs had an inhibitory effect on protoscolex activity. Multiple drugs have been used in combination with ABZ for the inhibition of protoscolexes of Egs in mice [27, 28]. ABZ combined with other drugs has a higher degree of damage to Egs [29]. In the combined treatment of ABZ and ATO for alveolar echinococcosis, the use of ATO can enhance the therapeutic effect of ABZ on experimental alveolar echinococcosis in mice [19]. ATO alone has been shown to reduce the development of alveolar echinococcosis in mice; however, ATO has limited efficacy in treating Egs infection in mice compared with ABZ [21]. In our study, NPs of ATO combined with ABZ are stronger that ABZ NPs alone in inhibiting protoscolex activity and vesicle growth, and promoting morphological damage in a mouse model of CE.

We evaluated the histopathological effects of ATO-ABZ NPs on mice. Organ weight and organ indexes are often used to measure the functional status of an animal to evaluate the comprehensive toxicity of chemical drugs to this organ [30, 31]. Previous studies on the toxicity of ABZ NPs to the kidney of the hydatid disease models found that NPs, like ABZ, had no pathological damage to the kidney [24]. Our results also showed that ATO-ABZ NPs were superior to ABZ NPs and ATO NPs in comparison with the model group, with no pathological changes in glomeruli and tubules and slight interstitial congestion in tight. In addition to kidney, we also found that ATO-ABZ NPs increased liver and spleen indexes, especially in treated mice with hydatid liver similar to healthy controls, indicating that ATO-ABZ NPs kill parasites and have high safety on host organ function. In particular, mice treated with 50 mg/kg ATO NPs still had significant liver tissue damage (e.g., inflammatory cell infiltration, focal necrosis), while the liver tissue of mice treated with the same dose of ATO-ABZ NPs and ABZ NPs was close to normal liver indicating that ATO-ABZ NPs and ABZ NPs were safer than ATO at doses that inhibited hydatid vesicles.

Parasites obtain nutrients and energy from the host to maintain their growth and development and mainly rely on anaerobic glycolysis to achieve energy metabolism [32]. LDH, as a terminal enzyme in the glycolytic pathway catalyzing the catabolism of PA to LD, is very important for the survival of parasites [33]. LDH expression is increased in parasitic disease, which is often considered to be a good target for anti-parasite drugs [34, 35]. When LDH activity is inhibited, glycolytic metabolism will be blocked, thereby affecting energy generation, and can lead to the accumulation of pyruvic acid in the parasite, or even the death of the parasite [34]. Our results showed that LDH and LD content decreased while PA accumulated suggesting the inhibiting efficacy of ATO-ABZ NPs on energy metabolism of protoscolexes. The inhibition of energy metabolism by ATO-ABZ NPs was also reflected in the reduction of ATP production. Previous studies have confirmed that ATO combined with other drugs can synergistically inhibit the respiratory pathway of Echinococcus multilocularis [20]. Our study is the first to demonstrate the inhibitory effect of ATO combined with ABZ loading NPs on energy metabolism of protoscolex via inhibiting LDH to catalyze PA catabolism.

DHODH is the only rate-limiting enzyme in the biosynthesis pathway of the de novo pyrimidine synthesis pathway that functions within mitochondria [36, 37], and rapidly growing and dividing cells require large amounts of de novo pyrimidine synthesis to maintain their growth. DHODH belongs to the mitochondrial antioxidant system and also plays a key role in organism metabolism. Studies have shown that (1) inhibition of DHODH expression could slow down the cell proliferation and arrest G2/M cell cycle [38]; (2) DHODH deletion could cause mitochondrial dysfunction such as reduced mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and reduced the quantity of mitochondrial DNA [39]. In cancer cells, DHODH inhibition leads to pyrimidine depletion, induces abnormal metabolism and inhibition of proliferation, which is manifested as ROS overproduction and ATP consumption [39, 40]. In the parasite Plasmodium, inhibition of DHODH activity prevents the dihydroorotic acid from being converted to orotic acid, resulting in the inability to synthesize pyrimidines, resulting in DNA and RNA synthesis disorders, and ultimately preventing metabolism of Plasmodium falciparum to achieve the purpose of treating malaria [41]. Additionally, lack of coenzyme Q10 and its subgroup could lead to the increased intracellular ROS, abnormal mitochondrial morphology, and reduced mitochondrial membrane potential, which may contribute to intracellular conversion, low energy mode of mitochondria, and eventually cell death [42]. Our results show that ATO-ABZ NPs suppressed DHODH expression, increased ROS, and decreased ATP production. We, therefore, concluded that ATO-ABZ NPs may promote the death of protoscolexes by inhibiting DHODH to promote oxidative stress (ROS) and inhibiting metabolism (ATP, energy metabolism and anabolism). However, the role of DHODH in the protoscolexes killing-effect of ATO-ABZ NPs still needs to be confirmed by further studies in animal models.

In conclusion, ATO-ABZ NPs are safe drugs for CE due to their inhibition of activity and vesicle growth of protoscolexes and non-toxicity to host organs. In term of the pharmacological mechanism, ATO-ABZ NPs inhibited oxidative stress and energy metabolism of protoscolexes via inhibiting DHODH. Our finding provides a theoretical basis for the research of new drugs for CE treatment, and finds a new drug delivery method and a new treatment mode such as the application of nano-preparations to improve the therapeutic efficacy of CE.