Comparative efficacy of Knema retusa extract delivery via PEG-b-PCL, niosome, and their combination against Acanthamoeba triangularis genotype T4: characterization, inhibition, anti-adhesion, and cytotoxic activity

Plant material and extraction

The identity of Knema retusa was confirmed using a method reported by Chuprom et al. (2023). Briefly, heartwood from mature trees in Manong and Kuala Kangsar, Perak, Malaysia, was collected. The wood was air-dried for two weeks, finely ground, and soaked in chloroform (1:5, w/v) at room temperature (RT) for three days. The extract was then filtered, concentrated under reduced pressure at 40 °C, air-dried, and prepared as a 100 mg/mL stock solution in dimethyl sulfoxide (DMSO), which was stored at −20 °C until use.

Synthesis and characterization of PEG-b-PCL

The synthesis of PEG-b-PCL required ring-opening polymerization (Eawsakul et al., 2017). Initially, the moistened MeO–PEG–OH (2 g, 0.4 mmol) was dehydrated by applying heat at 65 °C and followed by vacuum. The ring-opening polymerization of ɛ-caprolactone with a molecular weight of 5 kDa was conducted in a reaction flask to synthesize PCL segment. Then, under an argon atmosphere, anhydrous toluene was introduced to the MeO-PEG-OH mixture. Finally, Sn(Oct)₂ catalyst was added. The entire procedure was conducted below 140 °C. Upon completion of the reaction, the mixture was cooled to RT. The PEG-b-PCL was refined by dissolving it in acetone and allowing it to precipitate in cold diethyl ether. After centrifugation, the PEG-b-PCL block copolymer was separated and lyophilized using a freeze-drying process. This work used PEG-b-PCL with Mn = 9.8 kDa, which was analyzed by nuclear magnetic resonance (¹H-NMR). The ¹H-NMR spectra (Bruker, MA, USA) were recorded on a Bruker AVANCE NEO 500 MHz spectrometer by dissolving 600 µL CDCl₃.

Preparation of KRe-loaded nanoparticles

KRe (drug) was incorporated three different types of nanoparticles (NPs): PEG-b-PCL block copolymer, niosome, and a mixture of PEG-b-PCL and niosome NPs. Following prior studies, (1) drug loading into the PEG-b-PCL block copolymer was achieved through solvent evaporation (Nasongkla et al., 2021). During the preparation process, the initial KRe-to-PEG-b-PCL ratio was 1:9, and the manufacture of KRe-loaded niosomes was identical followed the same procedure as that the PEG-b-PCL block copolymer. (2) For drug loaded in the niosome, the ratio of span 60 to cholesterol was 12.9:7.7 (Miatmoko et al., 2021). Then, 10 mg of KRe was added as an anti-Acanthamoeba model to the solution. (3) The mixture of drug-loaded PEG-b-PCL block copolymers and niosome was fabricated by solvent evaporation. The proportions of the components were as follows: PEG-b-PCL, span 60, cholesterol, and drug were prepared by dissolving in chloroform in the ratio of 90:12.9:7.7:10. The chloroform in these samples was evaporated using a magnetic stirrer overnight after being added dropwise to 10 mL of water using a probe sonicator (Model CV.18; Sonics, Newtown, CT, USA) and sonicated for two minutes at an amplitude of 80%. For the subsequent experiments, samples were refrigerated at 4 °C.

Particle size and charge

After KRe was loaded into three distinct nanoparticle (NP) types, the particle size and surface charges were analyzed using the dynamic light scattering (DLS) and a Malvern Zeta Sizer (Malvern Instruments, Worcestershire, UK) (Eawsakul et al., 2017; Nasongkla et al., 2021; Ongtanasup et al., 2024). Samples were prepared by first performing a 20-fold dilution in water, followed by three successive measurements per sample to reduce the measurement error. The zeta potential was calculated utilizing the Smoluchowski approximation. DLS measurements were conducted over 20 separate 10s runs, with a 10s equilibration interval between each run. Each sample was examined at 25 °C, and a laser wavelength of 633 nm, with a scattering angle of 173°, a medium viscosity of 0.8872 cP, and an index of refraction of 1.33.

Determination of yield, encapsulation efficiency, and drug loading content

NP yield, encapsulation efficiency (EE), and drug-loaded content (DLC) were measured (Eawsakul et al., 2017; Nasongkla et al., 2021). First, NP aggregation was eliminated by centrifugation at 5,000 rpm for 15 min at 4 °C. Next, the free KRe in NPs was identified using a centrifugal filter with a 50 kDa molecular cut-off. To obtain the powder, the solution from the top (KRe-loaded NP) and bottom (free KRe) centrifugal filters were freeze-dried. To verify the encapsulated drug, the powder was weighed, the yield was determined (Eq. (1)), and it was dissolved in a 20:80 combination of ethanol (EtOH) and phosphate buffered saline (PBS). The KRe concentration in the extracted solution was determined, and then the KRe loading content and EE were calculated (Eqs. (2) and (3)) as follows: (1)${\displaystyle \text{\%}Yield=\frac{Weightofnanoparticle}{Theoreticaltotalamoundofnanoparticle}\ast 100}$ (2)${\displaystyle \text{\%}DLC=\frac{AmountofencapuslatedKReinnanoparticle}{Weightofnanoparticle}\ast 100}$ (3)${\displaystyle \text{\%}EE=\frac{AmountofencapsulatedKReinnanoparticle}{InitialamoundofKRe}\ast 100}$

Acanthamoeba culture and anti-Acanthamoeba activity

Based on the biosafety guidelines for scientific research at Walailak University, Thailand (Ref. no. WU-IBC-66-020), all experiments were carried out accordingly. Acanthamoeba triangularis WU19001, a strain from the recreational reservoir at Walailak University, Nakhon Si Thammarat, Thailand (Mitsuwan et al., 2020), and A. castellanii ATCC30010, A. castellanii ATCC50739, and A. polyphaga ATCC30461, were used in this study. Trophozoites of the Acanthamoeba strains were grown in Peptone-Yeast-Glucose (PYG) medium (20 g proteose peptone, 2 g yeast extract, 0.98 g MgSO₄ ⋅ 7H₂O, 0.35 g Na₂HPO₄ ⋅ 7H₂O, 0.34 g KH₂PO₄, 0.02 g (NH4)₂Fe(SO₄)₂ ⋅ 6H₂O, 18 g glucose and 1,000 mL distilled water (DW)] and incubated at RT for 3 days. The mature cyst forms of Acanthamoeba species was cultured in Neff’s medium (containing g/L; 7.45 g KCl, 2.44 g (2-amino-2-methyl-1,3-propanediol), 0.09 g NaHCO₃, 1.97 g MgSO₄.7H₂O, 0.06 g CaCl₂.2H₂O, and DW) at RT for at least 7 days.

To determine the effect of different types of NPs containing KRe (KRe-loaded PEG-b-PCL, niosome and PEG-b-PCL plus niosome) on Acanthamoeba viability, the determination of the minimal inhibitory concentration (MIC) was evaluated using a trypan blue exclusion assay (Mitsuwan et al., 2020; Mitsuwan et al., 2021). Briefly, 100 µL of the KRe NPs serially diluted in two-folded to give a final concentration range (1–0.0625 mg/mL) was added to a 96-well plate. Then, 100 µL of trophozoites (2 × 10⁵ cells/mL) was seeded in each well. Further, 1% DMSO and CHX (0.001–0.128 mg/mL) were used as negative and positive controls. The plates were incubated at RT for 24 h. Then, the MIC values were evaluated. The MIC value was obtained from the enumeration of trophozoites and cysts viability using the trypan blue exclusion method. Viability was investigated under the inverted microscope (Nikon ECLIPSE TE2000-S; Nikon, Tokyo, Japan) based on the principle that the dye can cross the membrane of dead cells with a blue color but not the intact membrane of viable cells with a colorless appearance. For counting, 50 µL of trophozoites and cysts containing the diluted KRe-loaded PEG-b-PCL, niosome, and PEG-b-PCL plus niosome suspension were placed on a new well plate, and 50 µL of 0.2% trypan blue dye was then added. It was incubated at RT for 5 min. The cell suspensions mixed with trypan blue dye (10 µL) were placed on a hemocytometer (Boeco, Hamburg, Germany). Cell viability was then enumerated under inverted microscopy. MIC is defined as the lowest concentration of the tested NPs that inhibits greater than 90% of Acanthamoeba viability. The relative percentage of trophozoites and cysts viability was defined as Eq.(4). (4)${\displaystyle \text{Relative}Acanthamoeba\text{Inhibition}=\left[\frac{\left(Acanthamoeba\text{viability tested by KRe}-\text{Loaded nanoparticles}\right)}{Acanthamoeba\text{viability tested by control}}\right]\ast 100}$

To assess Acanthamoeba’s ability to endure harsh conditions and treatments, and to determine whether it temporarily halts growth, A. triangularis trophozoites (2 × 10⁵ cells/mL) were incubated in NP solutions with 1/2 MIC (0.125 mg/mL) and 1 MIC (0.25 mg/mL). The viability of Acanthamoeba was evaluated at 24, 48, and 72 h using trypan blue staining, as previously described (Mitsuwan et al., 2020).