Antiparasitic properties of miltefosine-based nanoformulations against protozoan pathogen, Acanthamoeba castellanii

Noor Akbar; Roberta Cagliani; Jibran Sualeh Muhammad; Mutasem Rawas-Qalaji; Balsam Qubais Saeed; Naveed Ahmed Khan; Ruqaiyyah Siddiqui

doi:10.4103/abhs.abhs_35_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 1 | Issue : 4 | Page : 219-227

Antiparasitic properties of miltefosine-based nanoformulations against protozoan pathogen, Acanthamoeba castellanii

Noor Akbar¹, Roberta Cagliani², Jibran Sualeh Muhammad³, Mutasem Rawas-Qalaji⁴, Balsam Qubais Saeed⁵, Naveed Ahmed Khan⁶, Ruqaiyyah Siddiqui⁷
¹ Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, Unites Arab Emirates; Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates; Department of Biology, Chemistry and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, Sharjah, United Arab Emirates
² Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates
³ Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, Unites Arab Emirates; Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates; Department of Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
⁴ Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates; Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates; Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, USA
⁵ Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, Unites Arab Emirates
⁶ Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, Unites Arab Emirates; Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates
⁷ Department of Biology, Chemistry and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, Sharjah, United Arab Emirates

Date of Submission	01-Jun-2022
Date of Decision	11-Sep-2022
Date of Acceptance	15-Sep-2022
Date of Web Publication	28-Oct-2022

Correspondence Address:
Dr. Jibran Sualeh Muhammad
Department of Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah 27272
United Arab Emirates

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abhs.abhs_35_22

Abstract

Background: Acanthamoeba castellanii genotype T4 is the causative agent of the progressively increasing sight-threatening Acanthamoeba keratitis and central nervous system infections. Because of the increased prevalence and the ineffectiveness of the current antiamoebic drugs, we synthesized miltefosine poly(lactic-co-glycolic acid) nanoparticles (miltefosine PLGA NP) as a potential potent and biocompatible antiamoebic drug. The advantage to use PLGA NP is to preserve the cells from the toxic effect of miltefosine drug. In particular, miltefosine PLGA nanoformulation offers a better cellular uptake and a sustained drug release compared with the free drug that presents potent cytotoxicity at high concentrations against human colon cancer cell lines. Methods: The miltefosine NP were synthesized using a double emulsion-solvent evaporation method, characterized, and then assessed for their antiamoebic activity against A. castellanii belonging to the T4 genotype. Blank PLGA NP and miltefosine were used as controls. Results: Amoebicidal assays revealed that at 25 and 50 µM, unmodified miltefosine eradicated 83% and 93% of amoebae, respectively. At these same concentrations of 25 and 50 µM, the amount of miltefosine released form PLGA NP formulation was limited to 22.6%. However, it killed 36% and 56% of the protozoa, respectively. Thus, the efficacy of PLGA NP formulation was similar to that of the unmodified miltefosine. Both miltefosine and its PLGA NP significantly inhibited the pretreated amoebae (minimum inhibitory concentration 50% = 37.23 and 55.26 µM, respectively, compared with 147.2 µM of the blank NP; P < 0.05) and reduced amoebae-mediated host cell death. The blank NP and miltefosine NP exhibited minimal cytotoxicity against colon epithelial cell lines. In contrast, the unmodified miltefosine caused 37%, 71%, and 88% of cytotoxicity at 10, 25, and 50 µM, respectively. Conclusion: Overall, these findings suggest that controlling the release of miltefosine from PLGA NP for a short time was almost as effective as miltefosine alone against A. castellanii genotype T4 while reducing host cell toxicity. Hence, this study demonstrates the feasibility of using PLGA NP for the treatment of Acanthamoebic infections.

Keywords: Acanthamoeba castellanii, amoebicidal activity, cytopathogenicity, cytotoxicity, miltefosine

How to cite this article:
Akbar N, Cagliani R, Muhammad JS, Rawas-Qalaji M, Saeed BQ, Khan NA, Siddiqui R. Antiparasitic properties of miltefosine-based nanoformulations against protozoan pathogen, Acanthamoeba castellanii. Adv Biomed Health Sci 2022;1:219-27

How to cite this URL:
Akbar N, Cagliani R, Muhammad JS, Rawas-Qalaji M, Saeed BQ, Khan NA, Siddiqui R. Antiparasitic properties of miltefosine-based nanoformulations against protozoan pathogen, Acanthamoeba castellanii. Adv Biomed Health Sci [serial online] 2022 [cited 2023 Jun 9];1:219-27. Available from: http://www.abhsjournal.net/text.asp?2022/1/4/219/359866

Background

Free-living amoebae are ubiquitously present in various environments, including fresh water, ponds, swimming pools, soil, spring water and air, etc. [1]. Acanthamoeba castellanii exits in two morphological states, that is, trophozoites and the double-walled cysts. The cysts can withstand harsh conditions such as nutrient deficiencies, changes in pH, and the use of antiamoebic agents [2]. A. castellanii is the causative agent of sight-threatening infection known as Acanthamoeba keratitis (AK) [3] and infection of the central nervous system (CNS) called granulomatous amebic encephalitis (GAE) [4]. In AK, the patient may experience several complications including pupil constriction, epithelial abnormality, ring-like stromal infiltration, and ocular congestion. If not treated immediately, it could lead to vision impairment [2,5-8]. The prognosis of AK is quite challenging, and the available treatment options are inefficient in countering amoebal infections [9]. Similarly, GAE is a rare, but fatal brain infection, especially in immunocompromised patients, with 95% mortality rate due to delayed diagnosis [10,11]. Acanthamoeba infection is difficult for both the patient and the physician because it might take months to eliminate [12,13]. Unfortunately, most of the amoebicidal drugs are non-Food and Drugs Authority (FDA) approved (due to high toxicity) [12,14].

Miltefosine (an alkylphosphocholine) is known to exhibit potent antiprotozoal activity. For instance, the drug has been found to be effective against Entamoeba histolytica, Trypanosoma spp., Leishmania species, and Trichomonas vaginalis [15],[16],[17],[18]. The drug has also been shown to have a good therapeutic ability against Acanthamoeba infections in vitro [19],[20],[21]. Miltefosine treatment completely cured individuals with GAE and Acanthamoeba skin lesions [22].

In the present study, we found that miltefosine exhibited marked host toxicity. Therefore, we attempted to reduce miltefosine host toxicity while sustaining its antiprotozoal activity. Miltefosine was synthesized as poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP). It was demonstrated that this formulation has similar amoebicidal activity to that of miltefosine, but with minimal host toxicity. Hence, the use of miltefosine PLGA NP may reduce miltefosine undesired host cells by the sustained drug release while maintaining its amoebicidal effect. It is encouraging to note that the use of nanocarrier for drug delivery seems to be the most promising FDA-approved candidate and has a tremendous prospective for usage as a drug nanocarrier [23,24]. In addition, PLGA has been used for very long time in healthcare settings as a recyclable therapeutic agent [25,26]. The fact that PLGA biodegrades entirely in an aqueous media is a significant advantage when used as a drug nanocarrier [27]. In this study, the main advantage to use PLGA NP is to preserve the cells from the toxic effect of miltefosine drug. Hence, the use of miltefosine PLGA NP may reduce undesired side effects in the cells by exploiting the sustained drug release, and at the same time, this formulation may have an amoebicidal effect. In the present study, miltefosine was synthesized as PLGA NP and tested for its antiparasitic activity against A. castellanii.

Materials and methods

Chemicals and reagents

Miltefosine, poly(D,L-lactide-co-glycolide) 50:50 (PLGA), Mw 38–54 kDa, dichloromethane, TWEEN 80 viscous liquid, Kolliphor, and phosphate buffered saline tablet were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Deionized water, liquid chromatography-mass spectrometry (LC-MS) Chromasolv, and acetonitrile were purchased from Honeywell (Wunstorfer Strasse, Seelze, Germany). Formic acid was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).

Miltefosine PLGA NP synthesis

Triplicate batches of miltefosine PLGA NP were synthesized by a modified double emulsion method [28]. Briefly, 35 mg of PLGA was dissolved in 5 mL of dichloromethane containing Tween-80 (5% v/v) as an emulsifier. A volume of 750 µL of miltefosine (7.5 mM) solubilized in water was added to the polymer solution. The solution was emulsified using a probe sonicator (Qsonica L.L.C, Newtown, USA) amplitude 20% for 2 min. The primary W/O emulsion was dropped in 20 mL of water containing Kolliphor (1% w/v) to form the double emulsion (W/O/W) and sonicated for 3 min. The solution was stirred overnight at 700 rpm at room temperature to evaporate the organic solvent. The formed NP were collected by ultracentrifugation at 20,000 rpm for 20 min at 15°C and then washed three times with distilled water. Blank PLGA NP without miltefosine were synthesized using the same procedure.

NP characterization and morphology evaluation

The NP size distribution, polydispersity index (PDI), and zeta potential of miltefosine PLGA NP and blank PLGA NP were determined by dynamic light scattering (DLS) using Malvern Zetasizer, ZS (Malvern Panalytical Ltd, Malvern, United Kingdom). Briefly, 1 mL of nanosuspension was used for the DLS measurements. The intensity-weighted mean value and surface charge value of three measurements were reported as mean ± SD.

The morphology, size, surface structure, and topography of miltefosine PLGA NP and PLGA NP formulation were examined using a scanning electron microscope (SEM). Briefly, one drop of the redispersed sample was spread on a clean slide cover and left to dry under a vacuum. The dried sample was then mounted on carbon tape and sputter-coated using a gold sputter module under a high vacuum. The gold-coated samples were scanned, and photomicrographs were taken at an acceleration voltage of 5 kV using a Thermo Scientific Apreo SEM (FEI Company, Hillsboro, Oregon, USA).

Miltefosine entrapment efficiency in NP

Entrapment efficiency (EE) of miltefosine PLGA NP was determined using Acquity ultra-performance liquid chromatography (UPLC) H-class LC-MS/MS system (Waters, Milford, USA) coupled to a Xevo TQD triple quadrupole mass spectrometer equipped with an electrospray ionization source (Waters, Milford, USA) with a detection voltage of 3 kV capillary voltage, 54 V cone voltage, and 34 V collision energy. Agilent Eclipse Plus C18 (2.1 × 50 mm, 1.8 μm) column with a security guard column (1.7 μm, Acquity UPLC C18) was used to separate miltefosine. Miltefosine was eluted at 25°C using a gradient mobile phases composed of ultrapure water acidified with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) using an injection volume of 10 µL and a flow rate of 0.45 mL min^-1 for 4.00 min with 60A:40B from 0.00 to 0.50 min, 60A:40B to 0.0A:100B from 0.50 to 0.51 min, 0.0A:100B from 0.51 to 1.50 min, 0A:100B to 60A:40B from 1.50 to 1.51 min, and 60A:40B from 1.51 to 4.00 min.

Samples were quantified using a standard curve prepared by serial dilution over 0–121 µM range (R2 = 0.999). The EE% (mean ± SD) of miltefosine in PLGA NP was calculated from three different NP batches using the following equation:

In vitro release of miltefosine from NP

A dialysis technique was performed in triplicates to evaluate drug release from PLGA NP [29]. The freshly synthesized miltefosine PLGA nanosuspension was concentrated by centrifugation at 5000 rpm for 30 min using Macrosep centrifugal filter devices (Pall Corporation, Portsmouth, UK). One milliliter of the concentrated miltefosine nanosuspension (n = 3) was filled into cellulose dialysis tubing membrane with a 14 kDa molecular weight cut-off (Sigma-Aldrich, St. Louis, Missouri, USA). The dialysis device was then immersed in 10 mL of phosphate buffered saline (PBS) as a diffusion medium to ensure sink condition. The medium was maintained uniform by stirring in a heated water bath at 37°C. Aliquot samples of 100 µL were withdrawn over the 24 h at 0, 5, 15, 30 min, and then at 1, 2, 4, 6, and 24 h. Withdrawn volumes were replenished with a fresh medium.

The absorbance of the released miltefosine in the withdrawn aliquots was quantified according to the previously described analytical method using LC-MS/MS system. Results were expressed as the mean (±SD) percentage of the cumulative amount of drug released as per the following equation:

A. castellanii cultures

A. castellanii genotype T4 was received from American Type Culture Collection (ATCC 50492) and cultivated in 10 mL of a protease-peptone-yeast-glucose broth medium with the medium 0.75% yeast extract, 0.75% protease-peptone, and 1.5% glucose. Amoebae cultures were incubated at 30°C until confluency is attained. Next, the adherent trophozoites were dislodged by placing the cultured flask on ice for 10 min followed by gentle tapping. The amoebae culture was transferred to centrifuge tubes, and the culture was centrifuged for 5 min at 2500× g. The supernatant was discarded, and the pellet was resuspended in 1 mL of a serum-free Roswell Park Memorial Institute (RPMI) medium [30]. Finally, the initial inoculum of A. castellanii (5 × 10⁵) was adjusted by counting with a hemocytometer and then used for different assays.

Amoebicidal assays

A. castellanii was treated with blank PLGA NP, miltefosine, and miltefosine-loaded PLGA NP using amoebicidal assays as defined earlier. Briefly, 5 × 10⁵ amoebae trophozoites were treated with three different concentrations (10 µM, 25 µM, and 50 µM) of miltefosine, blank PLGA NP, and miltefosine PLGA NP for 24 h at 30°C amoebae in a 24-well plate with a final volume of 0.5 mL. Next, amoebae viability was determined by adding 0.1% trypan blue to each well, and viable trophozoites were enumerated by hemocytometer. Amoebae cultivated in RPMI alone were taken as a negative, while treated with sodium dodecyl sulfate (SDS) (0.25%) was taken as a positive control, respectively.

Human colorectal epithelial (HCT-116) cell line

Human colon epithelial cell lines were received from the ATCC CCL-247 and grown in complete media (RPMI supplemented with 1% minimum essential medium amino acids, 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin) at 37°C with 95% humidity and 5% CO₂ [31]. After this incubation, the adherent cells were disengaged enzymatically by incubating cells with 2 mL of trypsin ethylenediaminetetra acetic acid (EDTA) at 37°C for 5 min. Next, cell culture containing trypsin EDTA was centrifuged at 2500 g for 5 min. The cells pellet was resuspended in the above-mentioned complete media followed by seeding equal number of cells into 96-well plates that employed in different tests.

Cell cytotoxicity assays

Lactate dehydrogenase (LDH) assays were performed to determine the host cell cytotoxicity of miltefosine, PLGA NP, and miltefosine PLGA NP using human colon epithelial cell lines [32,33]. HCT-116 cells monolayer grown in a 96-well plate was treated with 10 µM, 25 µM, and 50 µM of miltefosine, PLGA NP, and miltefosine PLGA NP. The plate was incubated at 37°C with 5% CO₂ and 95% humidity for 24 h. For positive control, 0.1% triton X-100 was added to wells and reincubated the plate at 37°C for 45 min. For controls, cells incubated with triton X-100 (0.1%) and with RPMI alone were taken as a positive and negative control, respectively. Finally, to calculate the amount of LDH released, an equivalent volume of cell supernatant including liberated LDH enzyme was combined with LDH kit reagents (Cytotoxicity Detection kit; Roche Diagnostics, Indianapolis, Indiana, USA). The percent cell cytotoxicity was determined by the following formula:

Cytopathogenicity assays

Amoebae-mediated host cell cytotoxicity was determined by performing cytopathogenicity tests [30,34]. In brief, amoebae (5 × 10⁵) were pretreated with 10 µM, 25 µM, and 50 µM of miltefosine, PLGA NP, and miltefosine-PLGA NP at 30°C for 120 min. Next, the culture containing pretreated amoebae was centrifuged at 2500 g for 5 min, and the pellet was resuspended serum-free RPMI (200 µL). Subsequently, the pretreated amoeba was added to each well of 96-well plate (containing established cell monolayer). The plate was maintained at 37°C with 5% CO₂ and 95% humidity for overnight. Finally, amoeba-mediated host cell death was estimated indirectly by calculating the amount of LDH enzyme liberated into cell media by damaged cells, as reported previously [33,35].

Statistical analysis

All statistical analysis was conducted using two-sample T-test, two-tailed distribution. The data are expressed as the mean and standard error of three times replicated studies each performed in duplicates. All of the analyses and visualizations were done with Graph Pad Prism version 8.0.2 (GraphPad Software; CA; USA). The statistical significance level was set at P ≤ 0.05.

Results

Miltefosine PLGA NP were successfully synthesized with optimal characteristics

Miltefosine PLGA NP and blank PLGA NP were successfully synthesized. The mean (±SD) particles size distribution (Z-average), PDI, and surface charge of formed miltefosine PLGA NP were 145.6 ± 1.98 nm with a PDI of 0.14 ± 0.01 and −31.16 ± 0.50 mV, respectively. For the blank PLGA NP, the mean (±SD) particles size distribution (Z-average), PDI, and surface charge were 148.7 ± 0.91, 0.13 ± 0.01, −29.20 ± 0.86, respectively. Miltefosine PLGA NP and blank PLGA NP showed a mean (±SD) particles size distribution less than 200 nm with a low PDI. To favor a higher cellular uptake (clathrin- and caveolae-mediated pathways), NP with a diameter below 200 nm can penetrate the cells more efficiently compared with larger NP. The low polydispersity values showed that the formed NP were not aggregated and can be efficiently resuspended in a water medium following their lyophilization. The Z-potential values of PLGA NP and miltefosine PLGA NP are almost the same demonstrating that the drug is encapsulated inside the NP and not absorbed on the surface.

The morphology, surface structure, and topography of miltefosine PLGA NP and PLGA NP were illustrated in [Figure 1]. The SEM micrographs of miltefosine PLGA NP [Figure 1A] and B] and PLGA NP [[Figure 1C] and D] showed quite spherical, smooth-shaped particles. The SEM images align well with the reported particles size distribution obtained through the DLS measurements.

Figure 1: SEM images of miltefosine PLGA NP (A, B) and PLGA NP (C, D) synthesized by double emulsion method. SEM micrographs of miltefosine PLGA NP and PLGA NP showed quite spherical, smooth-shaped particles with a mean particles size distribution around 145 nm and 141 nm, respectively.

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Miltefosine was entrapped into PLGA NP with high efficiency but with retarded release

Miltefosine was successfully entrapped in the synthesized PLGA NP, and its EE% (mean ± SD) was 90.61 ± 2.96%. The release of miltefosine from these PLGA NP revealed a biphasic release pattern. A burst release of miltefosine, up to a mean of 18.82%, was observed in the first 2 h followed by a sustained and limited release during the 24 h of the experiment where only 22.6% of the miltefosine was released from the PLGA NP [Figure 2].

Figure 2: Cumulative in vitro release profile of miltefosine PLGA NP at 37°C for up to 24 h. Miltefosine showed a burst release from PLGA NP in the first 2 h, followed by a sustained and limited release for the rest of the 24 h of the experiment up to 22.6%.

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Miltefosine and miltefosine-loaded nanocarrier exhibited potent amoebicidal effects

Results from amoebicidal assays revealed that both miltefosine and miltefosine PLGA NP at 25 µM and 50 µM showed a significant amoebicidal activity against A. castellanii (using Student’s t-test, two-tailed distribution, P < 0.05) [[Figure 3]A and B]. Miltefosine alone at 25 µM eliminated 83%, while at 50 µM abolished 93% of amoebae viability [[Figure 3]A]. The blank PLGA NP did not show amoebicidal effects against A. castellanii. Also, miltefosine PLGA NP at 25 µM and 50 µM killed 36% and 56% of amoeba upon overnight incubation [Figure 3A]. Overall, the drug alone showed a potent amoebicidal effect; however, the released amount of miltefosine from PLGA NP was not significant enough to show a potent antiamoebic activity [Figure 3]A and B. On the other hand, the minimum inhibitory concentration 50% (MIC₅₀) of miltefosine, miltefosine PLGA NP, and PLGA NP was 37.23 µM, 55.26 µM, and 147.2 µM, respectively.

Figure 3: Miltefosine (Mt) and Mt-loaded PLGA NP exhibited an important amoebicidal activity against A. castellanii. Briefly, amoebae (5 × 10⁵) were incubated with 10 µM, 25 µM, and 50 µM of Mt, PLGA NP, and Mt PLGA NP for 120 min at 30°C. For negative control, A. castellanii were incubated in RPMI alone, whereas for positive control, amoebae were incubated with 0.25% SDS. (A) represents amoebicidal effects against A. castellanii; (B) represents the illustrative antiamoebic effects of Mt, PLGA NP, and Mt PLGA NP formulations against amoeba. The data are expressed as the means ± standard errors from several independent experiments performed in duplicate where * represents P ≤ 0.05.

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Miltefosine and miltefosine-loaded nanocarrier significantly inhibited amoebae-mediated host cell death

Miltefosine, PLGA NP, and miltefosine PLGA NP were tested for their cytopathogenicity against human cell lines. The pretreatment of A. castellanii with 25 µM of miltefosine reduced 45% of amoebae-mediated host cell death. At 50 µM, this activity was enhanced and blocked 55% of amoebae (P < 0.05) [Figure 4]. Miltefosine after encapsulation into PLGA NP showed a similar pattern of activity as showed in amoebicidal assays. Miltefosine PLGA NP at 25 µM and 50 µM repressed 26% and 46% of amoebae-mediated host cell death [Figure 4].

Figure 4: Miltefosine (Mt) and Mt-loaded PLGA NP significantly blocked amoebae-mediated host cell death. Briefly, half a million of amoebae were incubated with 10 µM, 25 µM, and 50 µM of Mt, PLGA, and Mt-PLGA for 2 h at 30°C. The pretreated amoebae were subjected to confluent monolayer of human cells. The data are expressed as the means ± standard errors from several independent experiments performed in duplicate where * represents when P ≤ 0.05.

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Miltefosine-loaded nanocarrier and nanocarrier alone showed limited cytotoxicity against human colon epithelial cells

LDH assays were performed to assess the cytotoxic effects of miltefosine, PLGA NP, and miltefosine PLGA NP against human epithelial cell lines. Cytotoxicity results showed that all the PLGA NP as well as the miltefosine PLGA NP presented minimal cell cytotoxic effects against HCT-116 cells [Figure 5], whereas the miltefosine drug alone showed moderate to potent cytotoxicity against human colon epithelial cells. Miltefosine 10 µM, 25 µM, and 50 µM presented 37%, 71%, and 88% of cytotoxic activity against human colon epithelial cells [Figure 5].

Figure 5: Miltefosine (Mt) and Mt-loaded PLGA NP revealed moderate to potent cytotoxicity against human colon cancer cell lines. HCT-116 cells were grown in 96-well plate up to 80%–90% confluency and the cells’ monolayer was treated with Mt, PLGA NP, and Mt PLGA NP at 37°C in 95% humidity and 5% CO₂ for 24 h. For negative control, cells were grown in RPMI alone, whereas cells treated with 0.1% triton X-100 was taken as a positive control. The data are presented as mean ± standard error of three-times independent experiments performed in duplicates. Data were analyzed using Graph Pad Prism software (8.0.2).

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Discussion

Pathogenic free-living amoebae may lead to the development of infections of the CNS and cornea of the eye leading to disabling diseases GAE and AK. GAE is a rare condition but shows a high mortality rate and is typically misdiagnosed [12, 36, 37]. In GAE, A. castellanii infects the CNS and peripheral nervous system in immune-compromised individuals [38]. Typical symptoms of GAE are headache, behavioral abnormalities, seizures, visual disturbances, and focal neurological deficit that develops over months to years [8,39]. However, AK is a painful, debilitating, and potentially blinding condition with optic neuritis, intense pain, inflammation, etc. Also, because of the neutrophil activation, disruption and ulceration of the epithelial tissues have been observed [2, 5, 40]. In this study, for the first time, we report the use of miltefosine and miltefosine PLGA NP as potential therapeutic agents that could be used in the treatment of these diseases.

The usual therapeutic interventions for amoeba infections include the use of hexamidine and propamidine (aromatic diamidines), neomycin (aminoglycosides), ketoconazole and fluconazole (imidazoles), chlorhexidine, and polyhexamethylene biguanide (biguanide) [3,41]. The anticancer drug miltefosine has also been used to treat Acanthamoeba infections [42,43]. Keeping this in mind, in the present study, we aimed to reveal the anti-Acanthamoebic properties of miltefosine and miltefosine-loaded nanocarrier. Previous studies revealed that drugs upon encapsulation into different nanomaterials significantly increased the antimicrobial properties of the drugs [30, 31, 34, 44, 45]. Lipid nanocarriers increased the antitumor effects of miltefosine upon encapsulation [46]. Miltefosine in combination with polyhexamethylene biguanide exhibited a significant antiamoebic activity against A. castellanii [47]. The drug alone as well as drug-loaded PLGA NP was tested against A. castellanii in amoebicidal, cytotoxicity, and cytopathogenicity assays. The drug alone exhibited significant amoebicidal effects both at 25 µM and 50 µM dosage. Also, the drug-loaded PLGA NP showed a significant amoebicidal activity, but the miltefosine PLGA NP had little amoebicidal effects when compared with the drug alone, which can be mainly due to the slower and limited amount of drug being released within the initial 24 h that was equivalent to only 23% of the free drug used.

In this study, miltefosine alone showed a potent amoebicidal activity against A. castellanii, but after encapsulation into PLGA nanocarrier, the activity was reduced because of the significant reduction in the released drug when compared with the free drug. PLGA as a copolymer exists in various copolymer ratios and molecular weights that can significantly alter the drug release profile [48]. Although PLGA drug nanoformulation offers a better cellular uptake and sustains the drug release compared with the free drug, its effect can be limited if insufficient effective drug concentrations were released within the same time frame of applying the free drug. Various other polymeric carriers need to be further investigated to achieve higher drug load and release to improve miltefosine amoebicidal activity.

Additionally, the drug alone and drug-loaded PLGA NP significantly inhibited amoebae to damage human cells upon pretreatment. Chlorhexidine conjugated with gold NPs considerably blocked amoebae-mediated host cell cytotoxicity [49]. Similarly, nystatin and amphotericin B conjugated with silver NPs successfully inhibited amoebae-mediated host cell damage [50]. Miltefosine presented potent cytotoxicity at the highest concentration against human colon cancer cell lines. This is due to the fact that miltefosine has been extensively used as an anticancer drug [46,51]. Interestingly, by treating HCT-116 cells with miltefosine-loaded PLGA NP, the cytotoxic effects have been drastically decreased showing limited cytotoxicity.

Overall, our findings revealed that the drug miltefosine and miltefosine PLGA NP showed important antiamoebic activity against A. castellanii. The miltefosine PLGA NP showed limited cytotoxicity toward human colon cancer cell lines. Miltefosine is an effective drug for treating Acanthamoeba infections, although it can cause cytotoxicity when used at higher doses. As a result, controlled drug release may reduce undesired side effects. Further optimization for miltefosine release from the nanocarrier using various PLGA copolymer’s rations is still required. Additionally, nanomaterials preparations may be employed in aerosol drug delivery, potentially improving the treatment efficacy and dose management of such and other antiamoebic drugs for treating Acanthamoeba infections.

Acknowledgements

The authors acknowledge the American University of Sharjah and the University of Sharjah for their support.

Authors’ contributions

RS and NAK conceived the study amid discussion with JSM and MRQ. NA and RC conducted all investigations and data analysis under the supervision of NAK, RS, JSM, and MRQ. NA and RC wrote the first draft. RS, NAK, JSM, and MRQ finalized the article. All authors read and approved the final article.

Ethical statement

Not applicable.

Financial support and sponsorship

Not applicable.

Conflicts of interest

There are no conflicts of interest.

Data availability statement

Not applicable.

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