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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2021 Mar 25;45(4):921–929. doi: 10.1007/s12639-021-01380-3

In vitro anti-Acanthamoeba activity of the commercial chitosan and nano-chitosan against pathogenic Acanthamoeba genotype T4

Hajar Ziaei Hezarjaribi 1,2, Elahe Toluee 1,3, Reza Saberi 2,3, Yousef Dadi Moghadam 2,3, Mahdi Fakhar 2,4,✉, Javad Akhtari 5
PMCID: PMC8556430  PMID: 34789973

Abstract

Acanthamoeba keratitis (AK) is a rare but serious infection of the eye and can lead to blindness. The effective and safe medical therapy remains unclear for AK until present. Antimicrobial activity and biological characteristic of chitosan encourage screening of it against Acanthamoeba. Thus, in vitro anti-amoebic activities of commercial chitosan and nano-chitosan were tested on pathogenic Acanthamoeba genotype T4, a causative agent of human AK. The Acanthamoeba spp. was isolated from the keratitis patient. The Acanthamoeba genotype T4 was approved using PCR method followed by sequencing technique. Chitosan nanoparticles was prepared using ionic gelation method and characterized by their physicochemical properties. In the present study, the in vitro activity of serial dilutions (12.5, 25, 50, 100, and 200 µL/mL) of commercial chitosan and nano-chitosan were evaluated against Acanthamoeba trophozoites and cysts. The finding of nano-chitosan particle size by DLS was 118 nm with a PDI of about 0.134. Zeta potential value was found to be 42.7 mV. The obtained results showed that the tested chitosan and nano-chitosan presented anti-amoebic activities dependent to time and concentration. The inhibitory effect of the chitosan and nano-chitosan is enhanced by increasing the concentration and incubation time. The inhibitory effect of nano-chitosan on both trophozoites and cyst was more than chitosan. According to the results, nano-chitosan shows the potent activity against Acanthamoeba T4 and could be used for the development of novel and safe therapeutic approaches in the future.

Keywords: Chitosan, Nano-chitosan, Acanthamoeba, In vitro, Inhibitory effect

Introduction

Acanthamoeba spp. are ubiquitous and pathogenic microorganism widely distributed in various habitats, including water, air, soil, dust, rivers, or medical instruments like, dental treatment units, hemodialysis unit and ophthalmology yard (Saberi et al. 2019a, b). Acanthamoeba spp. present two main stages in its life cycle: vegetative and motile form or trophozoite stage, and the latent and non-motile form or cyst stage that both forms can enter the body through various routs (Siddiqui and Khan 2012). Acanthamoeba spp. is capable of causing granulomatous amoebic encephalitis (GAE), a severe cerebral infection affecting mostly immunocompromised patients (Kilvington and White 1994). They can either cause Acanthamoeba keratitis (AK), primarily in contact lens wearers and may also occur in association with skin lesions (Kilvington and White 1994; Panjwani 2010). Although AK is still considered as a rare disease and is usually diagnosed late, the incidence in soft contact lens wearers has increased to reach 1 in 21,000 in 2015 (Randag et al. 2019).

Diagnosis of AK is difficult and in most cases diagnosed as bacterial, fungal or viral keratitis. The cultivation of Acanthamoeba from the corneal biopsy or from contact lenses can be used to diagnose AK. Also, confocal microscopy, and Immunofluorescence assays and multiplex real-time PCR methods were used for diagnosis of AK (Siddiqui and Khan 2012).

Treatment of AK is difficult due to the lack of standardized diagnostic tests and awareness among clinicians, since diagnosis of the condition is frequently elusive (Dart et al. 2009). Failure to achieve the optimum dose of select drug and phenotypic switching in Acanthamoeba spp. could be the causes of failure to treat Acanthamoeba infections (Khan 2006). Treatment of AK includes a combination of antimicrobial agents such as biguanides and diamidines, while in some cases a topical azole and/or neomycin is added in combination (Maycock and Jayaswal 2016). On the other hand, effective treatment with a combination of topical biguanide and diamidine therapy has been reported mostly when initiated in early stages of the disease (Clarke et al. 2012). Polyhexamethylene biguanide (PHMB) and chlorhexidine are the two biguanides that leading to loss of cellular components and inhibition of enzymes necessary for cell respiration. PHMB and chlorhexidine are effective at low concentrations (0.02%) but PHMB is toxic to human corneal cells (Turner et al. 2004). Propamidine-isethionate (0.1%), hexamidine-diisethionate (0.1%), and dibromopropamidine are diamidines used for the treatment of AK that by altering the structure and permeability of the cell membrane, causing denaturation of cytoplasmic contents (Szentmáry et al. 2019). The effort for safe and effective drugs remains a challenge.

Chitosan (poly-b-(1e4)-D-glucosamine), is a non-toxic polymer, obtained by N-deacetylation of chitin, which is mainly made from crustacean shells and is very much similar to cellulose (Younes and Rinaudo 2015). Chitin and chitosan are widely used as health foods in several countries. The application of chitosan includes a variety of areas such as pharmaceutical and medical applications, paper production, textile wastewater treatment, biotechnology, cosmetics, food processing and agriculture (Elieh-Ali-Komi and Hamblin 2016).

Chitosan has numerous pharmacological actions including immunopotentiating, antihypertensive, hypocholesterolemic and antibacterial activities. It has antioxidant activity and as an antibacterial agent can be used to help deliver drugs through the skin (Tripathi and Singh 2018). Previous studies on antimicrobial activity of chitosan have been proved against many microorganisms (Goy et al. 2009).

Nano-chitosan was obtained from chitosan by crosslinking with sodium tripolyphosphate (TPP). The effectiveness of nano-chitosan films in the treatment of cutaneous leishmaniasis was confirmed in animal models (Goy et al. 2009). Due to its antimicrobial activity, biocompatibility and biodegradability, chitosan has attained increasing commercial interest as suitable resource (Goy et al. 2009).

Antimicrobial activity and biological characteristic of chitosan encourage screening of it against Acanthamoeba. Hence, based on above literature on chitosan, current study was designed to test the anti-acanthamoebic effects of the chitosan and nano-chitosan against trophozoites and cysts of pathogenic Acanthamoeba genotype T4 that was isolated from a patient with keratitis.

Materials and methods

Preparation of the chitosan and nano-chitosan

Chitosan nanoparticles were prepared using ionic gelation method (Chabra et al. 2019). Briefly, 0.1 g chitosan (Mw 165 kD) were melted in acetic acid (0.1%) and stirred overnight at 25 °C. Tripolyphosphate (TPP) and double-distilled water were mixed together to reach a concentration of 0.05% w/v and the solution was added to chitosan (1:5 ratio) using an insulin syringe and was stirred at 25 °C. Ultrasonication was applied for 5 min to diminish the size of nanoparticles. Photon correlation spectroscopy of the chitosan nanoparticles was carried out by a Dynamic Light Scattering (DLS) instrument (Nano-ZS; Malvern, UK) to determine the size of the chitosan nanoparticles and zeta potential and polydispersity index (PDI).

Acanthamoeba isolate

Our study was performed with the Acanthamoeba genotype T4 that was isolated from a keratitis patient with Accession Number (KU877552). Before the experiments, the identification of the Acanthamoeba genotype was checked using PCR and sequencing techniques, which results displayed an Acanthamoeba genotype T4 (Saberi et al. 2019). The main reasons for choosing this genotype are abundant cases and its global distribution and high transmission.

Acanthamoeba cultivation

Amoeba were cultivated in 1% non-nutrient agar (Difco, USA) enriched with TYM (Trypticase, Yeast and Maltose) and incubated at room temperature. Plates were examined daily for 7–14 days.

Trophozoites and cysts preparation

The non-nutrient agar surface was carefully scraped using a sterile cell scraper, trophozoites and cysts were collected by pouring 4–5 mL of sterile phosphate-buffered saline (PBS) into the culture plates; the suspension was centrifuged at 2000 rpm for 5 min, the supernatant was discharged, and the pellet was washed twice with PBS. Trophozoites and cysts in a suspension were counted in a hemocytometer slice and the suspension was adjusted to 2 × 105 amoeba/mL and was subjected to drug testing (Dodangeh et al. 2017).

Anti-Acanthamoeba assay of chitosan and nano-chitosan

In vitro evaluation was performed in sterile 96-well plates (Sigma-Aldrich). In this way 250 µL of the adjusted 2 × 105 amoeba/mL was added with the same volume of serial dilutions of the chitosan and nano-chitosan (12.5, 25, 50, 100, and 200 µL/mL) in microcentrifuge tubes were vortexed and incubated at 37 °C for 24, 48, and 72 h. After the incubation periods, 20 µL of the amoebic suspensions were mixed with the same volume of 1% Methylene blue (MB) to detect and count the viable amoeba. Unstained (viable) and stained (non-viable) cells were then counted and all tests were performed in duplicate and were repeated at least three times. Negative and positive controls always monitored the experiment: 0.02% chlorhexidine digluconate and acetic acid were used as positive and negative controls, respectively.

Cytotoxicity test

Mouse Macrophages cell line J774A.1 (ATCC® TIB-67™) were seeded into 96-well plates (Nunc, Roskilde, Denmark) in 100 µl RPMI-1640 medium (Gibco, Life Technologies GmbH, Germany) supplemented with 10% fetal bovine serum (Gibco, Germany) and 1% Gentamycine (Gibco, Germany). Cells then were incubated at 37°Cwith 5% CO2 for 24 h. Different concentrations (12.5, 25, 50, 100, and 200 µL/mL) of nano-chitosan were added to each well in triplicate. After 24, 48 and 72 h of incubation time, 10 µL of MTT solution (Sigma, UK) (5 mg/mL in PBS) was added to each well, including controls. After 3 h of incubation at 37 °C, the supernatant was removed and 100 µL of DMSO (Darmstadt, HE, Germany) was added. Lastly, the absorbance at 570 nm was measured by a microtiter plate reader (BioTek ELX800, Winooski, VT, USA) and the viability was calculated by the following formula: Viability (%) = OD test /OD control × 100.

Statistical analysis

In this study to analyze the data, we first examine the assumption of normality of values using the Kolmogorov–Smirnov test with Z approximation. Parametric statistical methods were used for data analysis. Repeated measurement test, analysis of variance, independent t-test and LSD test were used. Significance level was set at < 0.05.

Results

Particle size and zeta potentials

The particle size result of nano-chitosan by DLS was 118 nm with a PDI of about 0.134 and these data were shown in Fig. 1. Surface charge and thereby the stability of the prepared nanoparticle systems was determined by zeta potential measurements. Zeta potential value was found to be 42.7 mV (Fig. 2). This value lies in the stable range, indicating that this nanoparticle is stable during experiment period.

Fig. 1.

Fig. 1

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DLS showing the size distribution of nano-chitosan with narrow size distribution

Fig. 2.

Fig. 2

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Zeta potential of chitosan nanoparticles showed positive charge

Effects of chitosan on trophozoites and cysts

The concentrations of 12.5, 25, 50, 100 and 200 µl/mL of chitosan were used against trophozoites and cysts at three different times (24, 48 and 72 h). Trypan blue dye exclusion test revealed that the viable Acanthamoeba cyst and trophozoites contained clear cytoplasm whereas a nonviable amoeba will have a blue cytoplasm. After adding the lowest concentration of the chitosan (12.5 µl/mL), 72.31% of trophozoites and 91.88% cysts were viable after 24 h, respectively. At another concentration and time, 25 µl/mL of chitosan after 72 h, 44.65% trophozoites and 54.29% cysts survived. Anti-Acanthamoeba activity at 100 µl/mL of the chitosan reveled survival of 28.84% of trophozoites and 34.18% of cysts after 72 h. The mean of trophozoites and cysts survival rate when incubated (for 72 h) at concentrations of 200 µl/mL of the chitosan was 17.03 and 16.02, respectively. The results of the effect of chitosan in different concentrations and times and survival percentage of trophozoites and cysts are tabulated in Fig. 3 and Table 1.

Fig. 3.

Fig. 3

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Percentage viability of Acanthamoeba trophozoites and cysts after treating with the chitosan

Table 1.

Effects of the chitosan on the proliferation of Acanthamoeba trophozoites and cysts

Time Concentrations Trophozoites Mean ± SD Cyst Mean ± SD P-value
24 h 12.5 µL/mL 72.31 ± 2.906 91.88 ± 0.370 0.000
25 µL/mL 62.23 ± 1.211 84.18 ± 0.740 0.000
50 µL/mL 55.57 ± 2.414 76.28 ± 1.110 0.000
100 µL/mL 47.61 ± 5.639 70.39 ± 2.018 0.003
200 µL/mL 32.41 ± 2.855 63.24 ± 2.906 0.000
Control −  99.00 ± 1.732 99.33 ± .667 0.795
Control +  10.47 ± 4.268 40.99 ± 2.273 0.000
48 h 12.5 µL/mL 62.08 ± 3.265 80.69 ± 3.350 0.002
25 µL/mL 51.28 ± 7.480 72.22 ± 4.811 0.015
50 µL/mL 45.70 ± 4.172 62.23 ± 1.211 0.003
100 µL/mL 38.88 ± 9.632 54.07 ± 0.403 0.112
200 µL/mL 19.94 ± 4.438 45.94 ± 4.170 0.002
Control −  97.33 ± 2.516 99.00 ± 1.00 0.398
Control +  0.000 ± 0.000 15.44 ± 1.191 0.000
72 h 12.5 µL/mL 50.00 ± 4.545 67.67 ± 4.628 0.009
25 µL/mL 44.65 ± 2.590 54.29 ± 4.172 0.027
50 µL/mL 39.92 ± 2.537 47.22 ± 6.159 0.131
100 µL/mL 28.84 ± 3.330 34.18 ± 3.916 0.146
200 µL/mL 17.03 ± 3.846 16.02 ± 13.911 0.910
Control −  97.00 ± 2.645 97.67 ± 1.202 0.749
Control +  0.000 ± 0.000 0.000 ± 0.000 0.000
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Effects of nano-chitosan on trophozoites and cysts

The results of study showed that survival percentage of trophozoites and cysts in treated with nano-chitosan was decreased in compared with chitosan (Table 2). The viability rate of the trophozoites and cysts treated with nonochitosan, after 24 h of exposure at concentration of 12.5 μl/mL was 57.07% and 80.55%, at 25 μl/mL was 48.48% and 68.37%, at 50 μl/mL was 38.88%, 57.50% and at 100 μl/mL was 27.68% and 55.57%, respectively. This was followed by adding 200 µl/mL of nano-chitosan after 24 h, survival rate of trophozoites and cysts was 19.96% and 39.92%. Trophozoites survival rate decreased with increasing concentration of the nano-chitosan and it was statistically significant (see Table 2). In addition, the nano-chitosan is more effective than the chitosan. In total, in the presence of 100 µL/mL of the nonochitosan, eliminate the whole population of trophozoites after 72 h, whereas only 2.56% of cysts were viable when incubated at 200 µL/mL concentration after 72 h (see Fig. 4 and Table 2).

Table 2.

Effects of the nano-chitosan on the proliferation of Acanthamoeba trophozoites and cysts

Time Concentrations Trophozoite Mean ± SD Cyst Mean ± SD P-value
24 h 12.5 µL/mL 57.07 ± 2.186 80.55 ± 4.811 0.002
25 µL/mL 48.48 ± 4.656 68.37 ± 1.480 0.002
50 µL/mL 38.88 ± 4.811 57.50 ± 3.859 0.006
100 µL/mL 27.68 ± 2.906 55.57 ± 2.414 0.000
200 µL/mL 19.96 ± 4.050 39.92 ± 2.537 0.002
Control −  99.00 ± 1.732 99.33 ± .667 0.795
Control +  10.47 ± 4.268 40.99 ± 2.273 0.000
48 h 12.5 µL/mL 41.16 ± 4.566 61.16 ± 2.670 0.003
25 µL/mL 32.47 ± 1.480 52.56 ± 2.220 0.000
50 µL/mL 26.19 ± 4.123 40.99 ± 2.273 0.006
100 µL/mL 17.94 ± 4.441 32.32 ± 4.628 0.018
200 µL/mL 7.72 ± 0.595 19.30 ± 3.350 0.004
Control −  97.33 ± 2.516 99.00 ± 1.00 0.398
Control +  0.000 ± 0.000 15.44 ± 1.191 0.000
72 h 12.5 µL/mL 23.71 ± 1.110 38.38 ± 6.308 0.017
25 µL/mL 15.44 ± 1.191 31.36 ± 1.745 0.000
50 µL/mL 5.34 ± 4.637 25.75 ± 1.312 0.002
100 µL/mL 00.00 ± 0.000 18.80 ± 3.700 0.013
200 µL/mL 00.00 ± 0.000 2.56 ± 4.441 0.423
Control −  97.00 ± 2.645 97.67 ± 1.202 0.749
Control +  0.000 ± 0.000 0.000 ± 0.000 0.000
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Fig. 4.

Fig. 4

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Percentage viability of Acanthamoeba trophozoites and cysts after treating with the nano-chitosan

Cytotoxicity activity

The MTT assay indicated that chitosan and nano-chitosan in different concentrations had no toxicity effects on the J774A.1 viability after 24, 48 and 72 h.

Discussion

According to the results, the tested chitosan and nano-chitosan have been shown to possess anti-Acanthamoeba effects. Despite progress in anti-Acanthamoeba therapeutic agents, such as combination therapies and keratoplasty, cysts are still resistant to therapeutic agents. This is a challenge that has not yet been addressed. For example, the morbidity and mortality associated with AK and GAE have remained high (Lorenzo-Morales et al. 2015; Visvesvara et al. 2007). Many of the available drugs lead to ineffective and unsafe treatment, due to their unwanted side effects. Therefore, the need for drug compounds that are effective against Acanthamoeba and also do not cause side effects in host cells seems necessary (Roshni et al. 2020; Trabelsi et al. 2012).

In this study, chitosan and nano-chitosan were tested as therapeutic agents at concentrations of 12.5, 25, 50, 100 and 200 µl/mL, as well as positive (chlorhexidine 20 µl/mL) and negative (acetic acid) controls always used in this study. Comparative assessment of the viability of trophozoites and cysts of Acanthamoeba T4 after exposure to the chitosan and nano-chitosan showed changes in the number of trophozoites and cysts, that with increased concentrations and time, the trend of viable trophozoites and cysts was decreasing. Anti-Acanthamoeba effects of chitosan and nano-chitosan on trophozoites showed that there was a significant difference in all concentrations and three times (see Tables 1 and 2); it means that the effect of of nano-chitosan on trophozoites was more efficient than chitosan. The results of this study showed that chitosan and nano-chitosan on trophozoites are more effective than cysts. Rigid double-layered wall of cyst causes a difference in the sensitivity to drug in the trophozoites and cysts (Dodangeh and Fakhar 2016). Acanthamoeba cysts are highly resistant and according to switch phenotypes, amoeba into a dormant cyst form is a major barrier in the development of effective treatment. Results of previous studies showed that the addition of cellulose enzyme to disrupt cyst wall structure present amoeba cysts susceptible to the effects of anti- Acanthamoeba drugs (Abjani et al. 2016). In other hand, mammalian cells lack chitosan and cellulose synthesis, so there is a need to investigate on the role of cellulose-degrading molecules in contact lens disinfectants as well as in drug formulations (Abjani et al. 2016).

Severe AK often treated with a combination of drugs such as PHMB or chlorhexidine (Alkharashi et al. 2015). One of the limitations of this treatment with the mentioned drugs was the toxic effects biguanides have on human keratocytes. Moreover, the treatment has a very lengthy course of the disease and it has a low effect on the cyst (Eldin et al. 2019). Finding a natural compound with anti Acanthamoeba activity and non-toxic to human cells is essential to treatment of AK. There were viability of Acanthamoeba isolated from keratitis patient cultivated in vitro examined after exposure to chitosan and nonochitosan. Natural products such as chitosan and nano-chitosan can be evaluated as a new generation of antimicrobial agents against Acanthamoeba infections, especially, if investigated with cyst targeting (2007). Many studies accomplished to date on chitin and derivatives as a source of bioactive material (Benhabiles et al. 2012; Salah et al. 2013). Surprisingly, there are no previous reports in the literature about bacterial resistance to chitosan. In addition, chitosan and its derivatives have demonstrated their promising application for the treatment of cancer (Kim 2010). Chitosan is applauded for its nontoxic nature, antimicrobial activity, polycationic, antitumor activity, antioxidative activity, anticholesterolemic, and analgesic effect (Kim and Rajapakse 2005). Using the incorporation of polyanion like TPPchitosan nanoparticle (ChNP) can be prepared easily in chitosan solution (Divya and Jisha 2018). Studies have reported antibacterial activity of ChNP against different pathogen bacteria (Qi et al. 2004). The antibacterial activity of chitosan is dependent on various factors included disruption and altering the membrane permeability, inhibition of DNAreplication and subsequently cell death and chelating agent that selectively binds to trace metal elements causing toxin production. In addition, other factors such as molecular weight and degree of deacetylation of parent chitosan, size and concentration of nanoparticle and pH, temperature and time are important to mechanism of chitosan (Kong et al. 2010).

The results of chitosan and nano-chitosan effects on cysts showed that in 25, 50, 100 and 200 µl/mL concentrations of both drugs at time 24 h was significant difference (P < 0.05), but in 12.5 µl/mL in time was no significant difference (P > 0.05). All concentrations at time 48 h showed that there was a significant difference (P < 0.05) between the efficacies of the chitosan and nano-chitosan on cysts. In 12.5, 25, 50 and 100 µl/mL concentrations of both drugs at time 72 h was significant difference (P < 0.05) while in 200 µl/mL in time was no significant difference (P > 0.05) between the efficacies of the both drugs on cysts. However, the effectiveness of nano-chitosan on cysts was more prominent than chitosan. In Siddiqui et al., study, liposomal complexation of pentamidine isethionate and ergosterol is shown to enhance its antiacanthamoebic activity (Siddiqui et al. 2009). In another study, Willcox et al., reported that silver nanoparticles have been successfully used to inhibit microbial growth and colonization on contact lenses, which shows the potential of nanoparticles in the development of contact lens solutions (Willcox et al. 2010).

Chitosan could be introduced as a promising compound, as in Shon et al., study, finding showed that growth of Acanthamoeba castellanii was inhibited by chitosan oligosaccharide (up to 20 mg ml−1). In this study, chitosan oligosaccharide inhibited polyamine biosynthesis and ornithine decarboxylase activity (Shon and Nam 2003).

Conclusion

The inhibitory effect of nano-chitosan on both trophozoites and cyst was more than chitosan. According to the results, nano-chitosan show the potent activity against Acanthamoeba T4 and could be used for the development of novel and safe therapeutic approaches in the future. As a whole, further studies are urgently needed to determine and/or confirm its mechanism of action against Acanthamoeba.

Acknowledgements

This project was funded by Toxoplasmosis Research Center, Department of Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran (Grant No: 2892).

Authors’ contributions

HZ, and MF conceptualization and study design. ET, YM, RS samples processing and down experiments. RS manuscript writing. MF and JA manuscript revision and editions.

Data availability

All data generated or analyzed during this study are included in this manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The current study was approved by the Ethical Committee of the Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran (IR.MAZUMS.REC.1398.880).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Mahdi Fakhar, Email: mahdif53@yahoo.com.

Javad Akhtari, Email: javad.akhtari@gmail.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated or analyzed during this study are included in this manuscript.

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