Nematocidal activity of chitosan nanoparticles conjugated with albendazole against the enteral and parenteral phases of trichinosis in experimentally infected mice

Abstract

Trichinosis is a serious parasitic zoonotic disease caused mainly by Trichinella spiralis. The used drugs for treatment of trichinosis showed limited bioavailability and high degree of resistance. Moreover, they have a very poor effect in treatment of encysted larvae. Therefore, there is a need for development of new agents which help in improving the bioavailability of the used drugs and enable them to reach different tissues. This study was designed to assess the use of chitosan nanoparticles (CSNPs) in conjugation with full and half dose albendazole (ABZ) in treatment of intestinal and muscular trichinosis. Albino mice (84 mice) were used to evaluate the efficacy of drugs and divided into seven groups; I: control, II: ABZ (50 mg/kg) treated, III: ABZ (25 mg/kg) treated, IV: ABZ (50 mg/kg) conjugated CSNPs treated, V: ABZ (25 mg/kg) conjugated CSNPs treated, VI: CS treated and VII: CSNPs treated. Parasitological and histopathological examinations were used to evaluate the therapeutic efficacy of the used drugs. Results showed significant reduction of adult Trichinella extracted from intestine of all ABZ treated groups either conjugated or not with the highest reduction rate in group IV followed by group V with percentage of reduction of 99.33% and 98.11%, respectively and marked improvement of histopathological examination. Also, results showed significant reduction of Trichinella larvae extracted from muscles of group IV, V and VII with the highest reduction rate in group IV with percentage of reduction of 100% in muscle larvae and marked improvement of histopathological examination. It was concluded that albendazole full dose conjugated chitosan nanoparticles can be a good candidate drug for treating both intestinal and muscular trichinosis.

Introduction

Trichinosis is a widely prevalent foodborne parasitic zoonotic disease. It shows a cosmopolitan distribution in temperate and equatorial climate zone (Pozio 2007a). Infection with genus Trichinella has been recorded in all continents except Antarctica (Pozio 2007b). There are about 10,000 cases recorded per year. Moreover, acute trichinosis may also occur in outbreaks affecting thousands of people (Kumar et al. 2017).

Infection occurs by consumption of undercooked or raw pork meat that harbors the nurse-cell first stage larva complex (Nöckler et al. 2005). The life cycle of T. spiralis is divided into two phases; an intestinal phase and a muscle phase. The female lays about 500–1500 newly born immature larvae (L1) during their entire life. The larvae enter through the lamina propria and penetrate into the blood stream to reach different organs (Bruschi and Dupouy-Camet 2014). They predominately localize in striated muscles rich with blood supply such as diaphragm, intercostal, extra-ocular muscles, masseter, tongue and deltoid. The changes that occur in the skeletal muscle cells result in its transformation into new cells known as the nurse cells that is unique to Trichinella infection and thus forming a nurse-cell larva complex (Wu et al. 2005; Bruschi and Murrell 2020).

Various factors including complex life cycle and different structural forms throughout the cycle share in the persistence of the disease. The usual treatment of T. spiralis infection depends on albendazole or mebendazole. Unfortunately, these drugs have low water solubility and high degree of resistance (Arundel et al. 2012). Moreover, both drugs are active against adult stage and non-encysted larvae only, with no significant effect against encysted larvae in muscles. Subsequently, there is great need for development of safer and more effective antiparasitic agent against this infection (Kukhaleva et al. 2015; Codina et al. 2015; Mukaratirwa et al. 2016).

Nanotechnology is found to improve the drug delivery system of the drugs including the stability, solubility area and the dissolution rate (Ranghar et al. 2014; Norouzi 2017). In addition to the idea of merging nanotechnology with plant-derived extracts which will open a cost-effective safe route for ethnomedicinal anthelmintics (Majumdar and Kar 2023).

Chitosan has been widely used as antimicrobial agent (Kong et al. 2010; Aziz et al. 2012). Whereas, Chitosan nanoparticles is considered a natural carrier and shows biodegradable and non-toxic characters which minimizes any possible side effects of conventional NPs (Liu et al. 2013; Ibrahim et al. 2015). The aim of this study is to investigate the effect of conjugation of chitosan nanoparticles with full and half dose of albendazole in treatment of intestinal and muscular phases of T. spiralis infection in experimentally infected mice.

Material and methods

Preparation of Trichinella spiralis larvae used for infection

Trichinella spiralis- infected mice (free from other parasites) used in this study was supplied from Medical Parasitology Department, Faculty of Medicine, Tanta University. Infected mice were sacrificed five weeks post infection, skinned and shredded. The larvae were isolated by incubating minced pieces in artificial gastric juice at 37°C for two hours according to Ozkoc et al. (2009). The excysted larvae were collected using (50 mesh/inch) then (200 mesh/inch) copper sieve and suspended in distilled water for half an hour. The living larvae in the sediment (coiled and mobile) were counted.

Experimental animals and infection

The present study was carried out on 84 laboratory bred parasite-free male Swiss albino mice (3–4 weeks old weighing 25–30 gm at the time of the experiment). The animals were provided by the European Country Farms in Egypt and were housed in the biological unit of Theodor Bilharz Research Institute (TBRI) on a standard diet containing 24% protein, 4% fat and about 4–5% fiber and water ad-libitum. Each mouse was infected orally by 200 Trichinella spiralis larvae (Dunn and Wright 1985) in 0.25 ml suspension using intraesophageal inoculation. The protocol of this study was authorized by ethical committee of Kasr-Alainy school of Medicine, TBRI and the Cairo University of Institutional Animal Care and Use Committee (CU/III/F/22/18).

Treatment preparations

Chitosan nanoparticles

Chitosan was purchased from Acros Organics Company, USA, M. Wt. 100,000–300,000, degree of deacetylation 79%. Chitosan was dissolved in 1% (v/v) acetic acid and stirred for 24 h. The solution was well-mixed with Penta sodium tri polyphosphate (TPP) solution and left under ultra-sonication for 45 min (Ibrahim et al. 2015).

Albendazole preparations

Non modified purified albendazole powder was purchased from Sigma Pharmaceutical Industries, Egypt (Batch No. AEA001087A, QC No. 0022710). It was dissolved in distilled water to reach a concentration of 5 mg/ml or 2.5 mg/ml.

Part of the Albendazole solution was added to nano-chitosan solution in the same molar ratio with stirring for 20 min, then left under ultra-sonication for 45 min, to obtain the final albendazole conjugated chitosan nanoparticles (Ibrahim et al. 2015).

Characterization of chitosan nanoparticle and its loaded

The Fourier Transform Infra-red (FTIR) spectra of the solution were recorded by using an FT- IR spectrophotometer (Nexus 670, Nicolet, USA) in the region of 4000–400cm⁻¹ with spectra resolution of 4 cm⁻¹.

Shape and size of chitosan nanoparticle was investigated using JEOL TEM. TEM measurements were done through placing a drop of colloidal solution on 400 mesh copper grids coated by an amorphous carbon film and evaporating the solvent in air at room temperature. The average diameter of chitosan nanoparticle was determined from diameter of 100 nanoparticle found in several arbitrarily chosen are shown in enlarged microphotographs (Fig. 1) (Ibrahim et al. 2015).

Fig. 1.

TEM images of nano chitosan and its nano size range

Experimental design

The 84 male albino mice included in this study were halved to be used in investigating effect of intestinal (experiment-I) and muscular phase (experiment-II). Each phase comprised 7 groups; of 6 mice each (Table 1). The treatment protocol of groups (II-VII) started on the second day post-infection and continued for 3 consecutive days. The mice of enteral phase (Experiment-I) were sacrificed on the sixth day to detect adults in the small intestine (Ashour et al. 2016).

Table 1.

Experimental groups designed according to treatment plan

Group	Description
I	Infected non-treated control group
Infected and treated groups
II	albendazole full-dose (50 mg/kg)
III	albendazole half-dose (25 mg/kg)
IV	albendazole full-dose conjugated chitosan nanoparticles (50 mg/kg)
V	albendazole half-dose conjugated chitosan nanoparticles (25 mg/kg)
VI	Chitosan (50 mg/kg)
VII	chitosan nanoparticles (50 mg/kg)

Parenteral phase (Experiment-II) was designed to evaluate the effect of drugs on Trichinella larvae in the muscular phase (Table 1). The treatment protocol of groups (II-VII) started on day 30 post-infection and continued for 7 consecutive days. The mice were sacrificed on day 45 post infection to detect encysted larvae in muscles (Ashour et al. 2016).

Evaluation of treatment

Animal scarification was done by intraperitoneal anesthetic–anticoagulant injection. Then, efficacy of treatment in both experiments was evaluated through the following equation:

$$\frac{\frac{Meannumberofadults}{Laraverecoveredfromcontrols}-\frac{Meannumberofadults}{Larvaerecoveredfromtreatedgroup}}{\frac{Meannumberofadults}{Laraverecoveredfromcontrol}}\times 100$$

Parasitological assessment

In intestinal (Experiment-I)

The mice were euthanized, and the intestinal pieces were rinsed with saline, and the fluid was collected and centrifuged at 1500 rpm for 5 min. The sediment was examined at a magnification of × 10 to count the adult worms (Basyoni and El-Sabaa 2013).

In muscular phase (Experiment-II)

The mice were euthanized, and the diaphragm of each mouse was carefully dissected, compressed between 2 slides and examined at a magnification of × 4 to count the larvae in the whole muscle (Basyoni and El-Sabaa 2013 and Muñoz-Carrillo et al. 2017).

Histopathological assessment

In intestinal (Experiment-I)

According to Nassef et al. (2018) a segment of one cm long from the small intestine of each animal was fixed and embedded in paraffin blocks. Sections of 5 microns thickness were stained by haematoxylin and eosin and examined microscopically for histopathological changes (Shalaby et al. 2010).

In muscular phase (Experiment-II)

Muscle specimens (parts of the thigh muscles) were taken from each mouse, fixed in 10% formalin, dehydrated and stained by haematoxylin and eosin and examined microscopically for histopathological changes (Shalaby et al. 2010).

Histopathological examination was done at the Pathology department at Kasr-Alainy School of Medicine. Hematoxylin and Eosin (H&E) stained intestinal and muscular specimens from sacrificed mice were assessed for presence of adults /larvae. Degree of inflammation was evaluated and classified into + 1 = mild, + 2 = moderate and + 3 = marked reaction (Drury and Wallington 1980).

Statistical analysis of data

Data were analyzed using the statistical package for the Social Sciences (SPSS) version 25 (IBM Corp., Armonk, NY, USA). Data was summarized using mean ± standard deviation in quantitative data. Comparisons between quantitative variables were done using the non-parametric Kruskal–Wallis and Mann–Whitney tests. For comparing categorical data, Chi square (χ²) test or Exact test were performed, accordingly. P values less than 0.05 were considered as statistically significant.

Results

Parasitological results

Examined sections showed significant reduction of adult Trichinella extracted from intestine of all Albendazole-treated groups either conjugated or not with the highest reduction rate in group IV (Albendazole full dose loaded chitosan nanoparticles) followed by group V (Albendazole half dose loaded chitosan nanoparticles) with percentage of reduction of 99.33% and 98.11%, respectively (Table 2). Also, results showed significant reduction of Trichinella larvae extracted from muscles of group IV (Albendazole full dose loaded chitosan nanoparticles), V (Albendazole half dose loaded chitosan nanoparticles) and VII (Chitosan nanoparticles) with the highest reduction rate in group IV with percentage of reduction of 100% in muscle larvae (Table 3).

Table 2.

Comparison between adult count in different treated groups and their percentage of reduction compared to control group

Groups	Adult count (Mean ± SD)	Percentage of reduction %
Group I	105.75 ± 16.50
Group II	2.17 ± 5.31**	97.95
Group III	23.80 ± 40.29*	77.49
Group IV	0.71 ± 1.89**	99.33
Group V	2.00 ± 4.47**	98.11
Group VI	55.00 ± 12.49	47.99
Group VII	63.80 ± 22.40	39.67

^*Statistically significant difference compared to group I (Infected untreated group); P value < 0.05

^**Statistically significant difference compared to group I (Infected untreated group), group VI (Chitosan) and group VII (Chitosan nanoparticles); P-value < 0.05

Table 3.

Comparison between larval count in different treated groups and their percentage of reduction compared to control group

Groups	larvae count Mean ± SD	Percentage of reduction %
Group I	408.00 ± 22.80
Group II	182.50 ± 69.94	55.27
Group III	223.00 ± 104.62	42.89
Group IV	0.00 ± 0.00**	100.00
Group V	118.75 ± 10.31*	70.89
Group VI	168.00 ± 21.90	58.82
Group VII	146.00 ± 14.32*	64.22

^*Statistically significant difference compared to group I (Infected untreated group); P value < 0.05

^**Statistically significant difference compared to group I (Infected untreated group), group II (Albendazole full dose), group III (Albendazole half dose), group VI (Chitosan) and group VII (Chitosan nanoparticles); P value < 0.05

Histopathological results

Histopathological examination of the intestinal sections from group I (infected non-treated) showed marked inflammatory reaction in the form of mixed cellular infiltrate including plasma cells, lymphocytes and neutrophils, distortion of villus architecture and oedema of lamina propria (Fig. 2). Groups II and III showed moderate inflammatory reaction in the form of mixed cellular infiltrate with preserved villus architecture (Fig. 3A). Group IV showed normal intestinal mucosa in the form of normal villous architecture well defined brush border with no inflammatory reaction or oedema found (Fig. 3B). Groups V, VI and VII showed mild inflammatory reaction (Fig. 3C).

Fig. 2.

Two sections of small intestine of control group (H&E 200X) showing distorted villous architecture (Black arrow), marked lymphoplasmacytic cellular infiltrate in lamina propria (Red arrow) and scattered Trichinella adults (Yellow arrow)

Fig. 3.

Small intestinal sections of: (A) Groups II and III (H&E 200X) showing preserved villous architecture (Black arrow), moderate lymphoplasmacytic cellular infiltrate of lamina propria (Red arrow) and mild oedema (Yellow arrow). (B) Group IV (H&E, 100x) showing preserved villous architecture with intact brush borders and lamina propria with no lymphoplasmacytic cellular infiltrate. (C) Groups V, VI and VII (H&E 100X) showing preserved villous architecture with intact brush borders (Black Arrow) and lamina propria with mild lymphoplasmacytic cellular infiltrate (Red arrow)

Histopathological examination of the muscle sections from group I (infected untreated) showed marked inflammatory reaction in the form of mixed cellular infiltrate including plasma cells, lymphocytes and macrophage, many living larvae with well-defined capsules around them (Fig. 4). Groups II, V, VI and VII showed mild inflammatory reaction with deposition of living and degenerated larvae (Fig. 5A, B). Group III showed moderate inflammatory reaction with deposition of living and degenerated larvae (Fig. 5C). Group IV showed normal muscle bundles with no larvae or inflammatory cells (Fig. 5D).

Fig. 4.

Two sections from infected thigh muscle (H&E 200x) of control group showing skeletal muscle bundles (Black arrow) surrounded by severe lymphoplasmacytic cellular infiltrate (Blue arrow) and living Trichinella larvae (yellow arrow)

Fig. 5.

Different sections from infected skeletal muscles (H&E 200x) of: (A, B) Groups II, V, VI and VII showing skeletal muscle bundles (Red arrow) with living (A) and degenerated (B) Trichinella larvae (Black Arrows) surrounded by mild lymphoplasmacytic cellular infiltrate (Blue arrow), neutrophils (Yellow arrow). (C) Group III showing skeletal muscle bundles (Red arrow) with living and degenerated Trichinella larvae (Black arrows) surrounded by moderate lymphoplasmacytic cellular infiltrate (Blue arrow), neutrophils as well as scattered giant cells (Yellow arrow). (D) Group IV showing skeletal muscle bundles with no larvae or inflammatory cells

From the above-mentioned results, it was found that only group IV (infected and treated with albendazole full dose conjugated chitosan nanoparticles) had resolved all the pathological changes occurred either in intestine or muscles and tissue had returned to its normal histology again after treatment (Fig. 6).

Fig. 6.

Comparison between different groups according to the inflammatory reaction intensity by histopathological examination

Discussion

The usual treatment of trichinosis depends on albendazole (Gottstein et al. 2009). However, the main obstacles against these drugs are their low water solubility and low bioavailability. Thus, they show minimal effect against encapsulated larvae (Attia et al. 2015). In the same context, the nanoscopic size (10–100 nm) and chemical properties of nano particles (Norouzi 2017) make them effective drug carriers (Eid et al. 2020). The present study was designed to assess the effect of conjugated albendazole and chitosan nanoparticles with different concentrations on intestinal and muscular phases of experimental trichinosis.

In the intestinal phase experiment, albendazole treated groups revealed significant decrease in the number of Trichinella adults when compared to the control group.

This significant effect of albendazole is achieved by its selective binding to the β-tubulin monomer of the parasite, leading to inhibition of the parasite microtubule polymerization (Yu and Qi 2015). Also, albendazole produces several biochemical changes in the target nematode like inhibition of mitochondrial fumarate reductase, reduction of glucose transport and uncoupling of oxidative phosphorylation (Shoheib et al. 2006). These results were in agreement with Nassef et al. (2018) and Nada et al. (2018) that revealed significant high reduction rate of T. spiralis adults in intestine. In contrary, Chung et al. (2001) reported lower degrees of reduction of T. spiralis adults when they used albendazole.

In group IV & V, Albendazole conjugated with chitosan nanoparticles revealed statistically significant reduction rate of T. spiralis adults in the intestine. These results were explained by Priotti et al. (2017) who assessed the albendazole-chitosan/cellulose micro-crystal formulations in treatment of intestinal trichinosis in vitro. It was postulated that albendazole micro-crystal formulations increased the solubility and dissolution rate of pure albendazole, however, chitosan-based crystal was the best option to optimize oral absorption of albendazole.

Concerning the muscular phase, using albendazole conjugated chitosan nanoparticles yielded statistically significant reduction of T. spiralis encysted larvae in comparison to the fair results of unconjugated albendazole with the same doses.

It is well-known that albendazole has low water solubility and low bioavailability in tissues (El-Melegy et al. 2019). In addition to its selective inhibition of the parasite's microtubule assembly and polymerization, without effect on the hosting nurse cell which is derived from human origin (Yadav 2012).

Moreover, the remarkable results of albendazole-chitosan nanoparticles are attributed to the increased absorption of drugs and the enhanced ability to penetrate different tissues (Jahangiri and Barghi, 2018). Nassef et al. (2019) reported significant reduction of T. spiralis encysted larval count by using albendazole full dose conjugated with chitosan nanoparticles. While using albendazole half dose conjugated with chitosan nanoparticles, yielded greater percentage of reduction than our study revealed.

Eid et al. (2020) proved high efficacy of chitosan coated nanostructured lipid carriers (NLCs) in conjugation with albendazole against intestinal and muscular trichinosis. Similarly, El-Melegy et al. (2019) used silver nanoparticle in conjugation with mebendazole (50 mg/kg/day) for treatment of muscular trichinosis, which yielded significant percentage of reduction of encysted muscle larvae when compared to infected untreated group.

In addition, conjugated drugs with chitosan were found to be protective against other parasites such as; albendazole conjugated chitosan nanoparticles used against alveolar echinococcosis causing damage of hydatid cysts and the overall infection was highly suppressed (Abulaihaiti et al., 2015). Likewise, albendazole chitosan microspheres displayed significant effect against experimental Toxocara (Barrera et al. 2010).

Regarding Chitosan and Chitosan nanoparticles, minimal reduction rate of T. spiralis adults encysted larvae in intestinal and muscles samples we revealed, respectively. It was suggested that interaction between positively charged amino group of chitosan and negatively charged microbial cell membrane components can enhance the permeability of the cell membrane and subsequent leakage of cellular contents leading to cell death (Raafat and Sahl, 2009). In addition to its antimicrobial and anti-inflammatory effects by stimulating both humoral and cellular immune responses. Different studies reported effect of chitosan and chitosan nanoparticles against various parasites. The variability in chitosan efficacy against various microbes may be attributed to several factors including; chitin type, degree of polymerization, molecular weight, some physiochemical properties and pH of the solution (El-Sherbiny and El-Baz 2015).

Intestinal histopathological sections from unconjugated albendazole groups showed moderate inflammatory reaction with restoration of the villus architecture. Similar results were recorded by Rayia et al. (2017). On the contrary, Attia et al. (2015) used full-dose albendazole with marked improvement of inflammation.

Intestinal sections from mice treated with full dose albendazole conjugated chitosan nanoparticles showed normal villous architecture with well-defined brush border. Whereas, intestinal sections from mice treated with half dose albendazole conjugated chitosan nanoparticles showed mild inflammatory reaction. Nassef et al. (2018) found that both doses resulted in residual mild inflammatory reaction.

Intestinal sections from mice treated with chitosan or with chitosan nanoparticles showed mild inflammatory reaction. Different results yielded by Nassef et al. (2018) in which intestinal sections revealed marked inflammatory reaction.

Muscle sections from mice of infected untreated group showed marked inflammatory reaction with well-defined capsules around them. Similar results were recorded by Ashour et al. (2016) and Basyoni and El-Sabaa (2013). Muscle sections from mice treated with albendazole showed mild-moderate inflammatory reaction as well as Attia et al. (2015) and Shalaby et al. (2010).

Albendazole conjugated chitosan nanoparticles treated groups showed normal muscle bundles with no larvae or inflammatory cells. Examination of muscle sections from chitosan and chitosan nanoparticles groups showed mild inflammatory reaction with deposition of living and degenerated larvae with capsules around them. On the contrary to our study, chitosan nanoparticles used in treatment of muscular trichinosis, revealed no improvement in the histopathological results in Nassef et al. (2019).

Conclusion

It was concluded that half/ full dose albendazole conjugated chitosan nanoparticles could be a promising anti-trichinellal agent in both intestinal and muscular phases. This was obvious with the statistically significant effects especially full dose regimen. Extension of the research is recommended to using chitosan at different doses, duration, sources, molecular weight and degree of deacetylation to evaluate the efficacy of these agents.

Author contributions

All manuscript authors contributed to every activity of it; idea of paper, study design, collection of materials, methodology, writing the paper and revising it.

Funding

No financial support, funding or grants were received for this manuscript.

Declarations

Conflict of interest

The authors declare that they have no competing interests.