A synergistic nanoformulation of propolis and chlorhexidine against Acanthamoeba: encapsulation efficiency, release kinetics, and safety evaluation

Reagents and propolis samples for nanoparticle preparation

CS powder was obtained from BFCLAB (Bangalore Fine Chemicals, Bangalore, India) in Ramohali, Bangalore, and absolute ethanol was sourced from Galaxy Chemicals in Pune, Maharashtra, India. Propolis produced by three stingless bee strains: Tetrigona apicalis, Geniotrigona thoracica, and Heterotrigona itama, was collected from a local Bangreen farm in Mueang, in Nakhon Si Thammarat, Thailand (Location: 8°26′38.2″N, 99°54′7.7″E) in February 2023. During the collection period, the weather conditions were characterized by an average temperature of 27 °C and a relative humidity of 92%. Tripolyphosphate (TPP) and Dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Vero cells (ECACC 84113001, RRID: CVCL 0059, Salisbury, UK) were provided by Associate Professor Dr. Chuchard Punsawad, School of Medicine, Walailak University. Dulbecco’s Modified Eagle’s medium was sourced from Merck KGaA, Darmstadt, Germany, and 10% fetal bovine serum, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 2,5-diphenyl tetrazolium bromide) & Ethylenediaminetetraacetic acid (EDTA) was obtained from Sigma Aldrich, St. Louis, USA.

Acanthamoeba cultivation

All cultures were performed following biosafety guidelines and were approved by the Committee of the Biosafety Guidelines for Scientific Research of Walailak University, Nakhon Si Thammarat, Thailand (Ref. No. WU-IBC-66-020). Acanthamoeba triangularis WU19001 (AT), a strain from the recreational reservoir at Walailak University, Nakhon Si Thammarat, Thailand, and A. castellanii ATCC50739 (AC5), kindly provided by Asst. Prof. Dr. Rachasak Bonhook, as well as A. castellanii ATCC30010 (AC3) and A. polyphaga ATCC30461 (AP), purchased from American Type Culture Collection (ATCC), were used in this study. The trophozoites of the Acanthamoeba strain were grown in PYG medium (20 g proteose peptone, two 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) for three days at room temperature (37 °C). The cyst form was cultivated 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 distilled water DW) at 37 °C for at least seven days.

Preparation of ethanolic propolis extracts from stingless bee species

Propolis, or bee glue, derived from three stingless bee strains: T. apicalis, G. thoracica, and H. itama (Figs. 1A–1C), was collected from a Nakhon Si Thammarat, Thailand farm. All samples of propolis were macerated using a mortar and pestle. Then, 50 g of powdered propolis was soaked in 200 mL of 95% ethanol (EtOH) at 1:4 g/v for 2 weeks. The propolis extraction was stirred with a spatula twice a week. The solid residues were discarded using two layers of sterile gauze, and the supernatants were then filtered using Whatman filter paper no. 1 (Whatman, Buckinghamshire, United Kingdom). The ethanolic propolis extracts (EPE) were evaporated under vacuum with a rotary evaporator at 40 °C until dry to extract crude compounds. The dried extracts were preserved at 4 °C and resuspended in 100% DMSO at a concentration of 100 mg/mL before use.

Synthesis of chitosan nanoparticles via ionic gelation with sodium tripolyphosphate

CS-NPs were synthesized using the bottom-up method by dissolving chitosan (two mg/mL) in 1% acetic acid with a magnetic stirrer, followed by filtration with a pore size of 0.45 µm, also known as the ionic crosslinking method. A sodium tripolyphosphate (TPP) solution (0.75 mg/mL) prepared in deionized water was added dropwise with vigorous stirring (1,000 rpm, 15 min) at a rate of one mL/min. A spontaneous formation of the nanoparticles occurred due to the electrostatic attraction between the positive charge on the chitosan’s primary unprotonated amino groups and the negative charge on TPP. The suspension was stirred further for another 30–60 min in order for the suspension to be stable. The purified powder was further characterized (Garg et al., 2019).

Encapsulation of molecular weight-variant propolis and CHX into CS-NPs

The synthesized CS-NPs were loaded with propolis for use in drug delivery applications. Three variations of the propolis drug were tested, differing in molecular weight as follows: Propolis 1 (15.11 g), Propolis 2 (18.73 g), and Propolis 3 (14.48 g). For encapsulation, the propolis extract was dispersed in ethanol (100 mg/mL in 95% ethanol) and added to CS-NPs (dispersed in two mg/mL in deionized water), which were dispersed in water. After mixing both solutions, the mixture was kept in a shaker for 2 h. This solution underwent centrifugation at 800 rpm for 30 mins at 4 °C, and the obtained pellet was further analyzed. Different combinations of drugs used for the encapsulation process are listed in Table 1.

In the formulation containing CHX, a 0.128 mg/mL CHX solution (prepared in deionized water) was added dropwise to the CS-NPs suspension during mixing. It was followed by 2 h of incubation at 150 rpm to ensure uniform loading (Ferreira et al., 2019). In addition to the propolis drug, CHX was used in a few combinations to analyze the binding efficiency. EE was calculated as per Eq. (1) in the manuscript, with quantification via UV-Vis spectrophotometry (Kinglab Model KLUV-2100, India) at 260 nm post-centrifugation (10,000× g, 15 min, 4 °C). The amount of EPE and CHX in CS-NPs was determined indirectly by quantifying the free (unencapsulated) drug in the supernatant after centrifugation, using UV-vis spectrophotometry. Figure 2 presents a schematic diagram of the chitosan–propolis drug encapsulation. (1)${\displaystyle \text{Encapsulation Efficiency}=\frac{\text{Amount of Drug Encapsulated in Nanoparticle}}{\text{Initial Amount of Drug}}\ast 100.}$

Transmission electron microscopy analysis of chitosan nanoparticles

After encapsulation, the chitosan nanoparticles’ internal morphology and structural characteristics were examined using transmission electron microscopy (TEM) (FEI –TECNAI G2-20 TWIN model). TEM provided high-resolution images to assess particle size, shape, and uniformity of the nanoparticle formulations.

pH-responsive drug release kinetics of propolis-loaded chitosan nanoparticles

The characterization of the sustained-release behavior of nanoparticles was conducted under conditions that mimicked human physiology over a specified duration, as the actual drug release kinetics were studied under those conditions. The pH was varied during the study to assess the material’s efficacy. Propolis was incorporated into the CS-NPs at a similar concentration to study the release kinetics by varying the pH from acidic to neutral (pH 3, 5, and 7). Briefly, the nanoparticles with propolis dispersed in phosphate buffer solution (PBS) at pH values of 3, 5, and 7 were used to investigate the drug release kinetics. The NPs were directly dispersed in 50 mL PBS (pH 3, 5, or 7) at 37 °C with gentle stirring at 100 rpm. At each predetermined time interval (every 30 min), a small aliquot (one mL) was withdrawn from the nanoparticle dispersion in the release medium (PBS at pH 3, 5, or 7). The aliquot was then centrifuged at 10,000 × g for 10 min at 4 °C to sediment the nanoparticles, allowing for the measurement of the released drug in the clear supernatant using a Nanodrop (Thermo Scientific Nanodrop 2000 Spectrophotometer; Thermo Fisher Scientific, Waltham, MA, USA) at 260 nm. The separated nanoparticles (pellet from the centrifuged aliquot) were not returned to the release medium. Instead, an equal volume (1 mL) of fresh PBS was immediately added back to the main dispersion vessel to maintain the total volume and sink conditions, preventing drug saturation and ensuring accurate cumulative release profiling. The release kinetics were calculated using the Peppas model for analysis, and the mathematical formula was applied (Saravanabhavan et al., 2019).

Minimal inhibitory concentration determination

The propolis (11 samples; see Table 1) was dissolved in 0.1% acetic acid at a concentration of 100 mg/mL. The minimal inhibitory concentration (MIC) assay was performed as described previously by Chimplee et al. (2024). Briefly, 50 µL of the EPE samples (10% drug-loaded CS-NPs at 100 mg/mL) were serially diluted two-fold to achieve final concentrations (% drug-loaded CS-NPs) ranging from 0.04% to 5%. Then, 50 µL of 2  × 10⁵ cells/mL of trophozoites and cysts were inoculated into each well of a 96-well plate. CHX (0.001–0.128 mg/mL) and acetic acid (0.05%) served as positive and negative controls, respectively. After 24 h of incubation at room temperature, the viability of Acanthamoeba was calculated using Eq. (2). The minimal inhibitory trophozoite concentration (MITC) and minimal inhibitory cystic concentration (MICC) were defined as the lowest concentrations that inhibited > 90% of the viable growth of trophozoites and cysts, respectively. (2)${\displaystyle \text{Viability}\left(\text{\%}\right)=\frac{\text{Survival of Treated Cells}}{\text{Survival of Negative Control}}\mathrm{\ast }100.}$

Minimal parasiticidal concentration determination

The minimal parasiticidal concentration (MPC) was determined as previously described (Chimplee et al., 2024). The concentrations at MITC, MICC, and above were further assessed using parasiticidal activity for 72 h incubation. Viability was counted and calculated using Eq. (2). MPC was the lowest concentration that inhibited > 99.99% of viable growth after 72 h (Chimplee et al., 2024).

Cytotoxicity assay—minimal cytotoxicity concentration

The minimal cytotoxicity concentration (MCC) was performed as previously described (Chimplee et al., 2022; Sama-Ae et al., 2023; Chimplee et al., 2024). The cytotoxicity of propolis and drug combination in CS-NPs was assessed using Vero cells. The cell line was cultured in Dulbecco’s Modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, incubated at 37 °C in an atmosphere containing 5% CO₂.

Once the cells reached 90% confluence, they were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) and incubated again for 5 min. Vero cells (1. 5 × 10⁴ cells/mL) were seeded in 96-well plates and incubated for 24 h before propolis-drug-loaded CS-NPs (0.039–5.00%) treatment. After 24 h, the cytotoxicity was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -2,5-diphenyl tetrazolium bromide) assay. In brief, 100 mL of MTT reagent (0.5 mg/mL) was added to each well, and the cultures were incubated for an additional hour. The MTT reagent was removed and replaced with 100% DMSO to ensure that solubilization was complete. Absorbance at 570 and 650 nm (reference wavelengths) was measured on a microplate reader. The survival percentage was calculated using the following equation: (3)${\displaystyle \text{Survival}\left(\text{\%}\right)=\frac{\mathrm{ABt}570\mathrm{nm}-\mathrm{ABt}650\mathrm{nm}}{\mathrm{ABu}570\mathrm{nm}-\mathrm{ABu}650\mathrm{nm}}\mathrm{\ast }100.}$ ABt and ABu denote the absorbance of treated and untreated cells (0.1% acetic acid), respectively. MCC was the lowest concentration, inhibiting less than 20% cell viability.

Scanning electron microscopy analysis of morphological changes in Acanthamoeba polyphaga cysts after treatment

To observe the morphological changes in treated Acanthamoeba using SEM, the cyst form of A. polyphaga was treated with single EPE or a combination of EPE and CHX at 1.25% and 5%, as previously described (Chimplee et al., 2022; Chimplee et al., 2024). After incubation, cells were collected by centrifugation at 1,520 ×g for 5 min and resuspended in phosphate buffer saline (PBS; Oxoid Holdings, Hampshire, UK). CHX (0.128 mg/mL) and 0.05% acetic acid were used as positive and negative controls, respectively. Samples were fixed with 2.5% glutaraldehyde overnight, dehydrated with a series of graded alcohols (20%, 40%, 60%, 80%, 90%, and 100% ethanol), mounted on aluminum stubs, and allowed to dry using a critical-point dryer. Samples were then coated with gold particles, and the morphology of A. polyphaga cysts after the treatments was subsequently examined under SEM (SEM-Zeiss, Munich, Germany).

Statistical analysis

All the studies were carried out in triplicate, and the data were interpreted using OriginPro Software Version 8 (OriginLab Corporation, Northhampton, MA, USA). Two-tailed Student Independent t-tests were conducted, and the data were presented as mean ± standard deviation, with p < 0.05 considered statistically significant, wherever applicable.

Transmission electron microscopy analysis of chitosan nanoparticles encapsulating EPE

TEM was employed to examine the size, shape, and surface morphology of chitosan nanoparticles (CS-NPs) encapsulating ethanolic propolis extracts (EPE). As shown in (Figs. 3A–3K), the TEM micrographs revealed that the nanoparticles were predominantly of irregular shapes, with a tendency to aggregate. This aggregation may be attributed to electrostatic interactions and hydrogen bonding between chitosan and bioactive compounds in the EPE. Despite the heterogeneity in shape, the nanoparticles remained within the nanoscale range, typically around 20–30 nm in diameter. This nanoscale dimension is critical for enhancing drug solubility, bioavailability, and cellular uptake. The degree of dispersion and clustering varied across the samples, likely reflecting the influence of different EPE-drug combinations on nanoparticle formation. The apparent contrast and defined boundaries observed in the images confirm the successful encapsulation of EPE into CS-NPs. These findings support the potential of chitosan-based nanocarriers for delivering propolis-derived compounds for therapeutic applications.

Encapsulation efficiency of EPE-CHX formulations in chitosan nanoparticles

The encapsulation efficiencies (EEs) of various EPE and CHX formulations with CS-NPs, as mentioned in Table 1, were found to differ under the test conditions. C11 exhibited better drug entrapment than other formulations tested, with a maximum EE of 92.06% (Fig. 4). In contrast, C8 demonstrated the lowest efficiency at 81.38%, suggesting lower drug retention in the NPs. All formulations achieved EEs over 85%, with consistently high values for C1 (90.28%), C2 (88.50%), C3 (89.72%), C5 (89.73%), C6 (89.39%), and C7 (88.54%). The efficiencies of C4 (84.03%), C9 (85%), and C10 (82.98%) were lower than those of the other combinations, which may be related to differences in drug-polymer interactions.

Drug loading capacity of EPE-CHX formulations in chitosan nanoparticles

The drug loading capacity (DLC) of various EPE and CHX formulations with CS-NPs, as mentioned in Table 1, differed under the test conditions. The minimum loading capacity was observed in the C8 combination (54%), while the highest value was obtained in the C11 (61.3%). Only a slight difference was observed between C1 (60%) and C11 values. The remaining combinations fall within the same range from 55% to 59%. C2 (59%), C3 (59.6%), C4 (56%), C5 (59.6%), C6 (59.3%), C7 (59%), C9 (56.6%), C10 (55%). This difference is observed because drug loading capacity is directly related to encapsulation efficiency and follows a similar pattern.

Triphasic drug release kinetics of propolis—chlorhexidine formulations in chitosan nanoparticles

The drug-release kinetics of the chitosan-propolis-chlorhexidine exhibited a linear fitting curve profile with three phases: an early lag phase (0–20% release), followed by a sharp intermediate release phase (20–80% release), and then a plateau phase (80–100% release) (Fig. 5). In controlled drug delivery systems, the release typically follows a multi-phase pattern, which includes an initial burst release, a sustained release phase, and a terminal plateau. This pattern ensures a rapid onset of action followed by prolonged therapeutic levels. The combinations of propolis and CHX (C1-C11) showed distinct drug release profiles over 25 h. Their increased release over time indicates cumulative drug release. For example, combinations like C1 and C2 exhibit a faster release rate, suggesting a stronger initial burst and possibly quicker action. In contrast, combinations from C9 to C11 demonstrate more controlled and sustained release kinetics.

Influence of pH on the release behavior of chitosan-based propolis–CHX nanoparticles

Our investigations also revealed that the release kinetics were significantly influenced by the pH of the surrounding medium (Fig. 6). Drug activity or release was higher in highly acidic conditions (pH 3) but dropped noticeably as the environment became more neutral, as observed at pH 5 and 7. Additionally, propolis, a resin-like compound rich in flavonoids and phenolics, behaved differently depending on pH. These compounds remained more stable and dissolved better in mildly acidic conditions, while they degraded in alkaline environments (Gokce et al., 2014). Chitosan’s swelling and propolis’s chemical stability favor acidic conditions, making this formulation ideal for targeting inflamed or infected tissues, or the stomach, where pH tends to be lower.

MIC and MPC profiles of EPE and CHX nanoparticle formulations against trophozoite and cyst stages of Acanthamoeba

The single EPE-loaded-CS-NPs (C5-C7) exhibited more potent cyst inhibition than trophozoites (Table 2). The T. apicalis CS-NP (C5) demonstrated the most potent activity against A. castellanii ATCC50739 trophozoite inhibition at MITC 5%. In contrast, G. thoracica (C6) and H. itama (C7) loaded-CS-NPs exerted the highest inhibitory effect against A. polyphaga ATCC30461 cyst at MICC 5%. The combination of their EPEs (C1): T. apicalis, G. thoracica, and H. itama, loaded CS-NPs could inhibit various Acanthamoeba species, both trophozoites and cysts. The EPE combination exhibited anti-Acanthamoeba activity against trophozoites of A. castellanii ATCC50739 and A. triangularis at MITC 5%, and against cysts of A. castellanii ATCC30010 and A. polyphaga at MICC 5%. The cyst viability of A. castellanii ATCC50739 was inhibited by greater than 90% when combining T. apicalis and G. thoracica extracts-loaded CS-NPs (C2) at MICC 5%. The combination of EPEs with CHX showed greater activity against cysts than trophozoites. T. apicalis, G. thoracica, and H. itama with CHX-loaded CS-NPs (C8) displayed significant activity against the cysts of A. castellanii ATCC30010 and A. polyphaga at MICC of 5%. Cysts of A. castellanii ATCC30010 and A. triangularis were inhibited by the combination of T. apicalis, H. itama, and CHX (C10, MICC = 5%). Additionally, A. castellanii ATCC30010 cysts were inhibited by the combination of T. apicalis, G. thoracica, and CHX (C9, MICC = 5%). The combination of G. thoracica, H. itama, and CHX (C11) exhibited the best activity against cysts of A. polyphaga at the lowest MICC of 1.25%. All treatments showed amoebicidal activity against both forms at MPC values greater than 5% (Table 2).

SEM analysis of structural damage in Acanthamoeba polyphaga cysts following treatment with propolis-CS nanoparticles and chlorhexidine

EPEs and their CS-NP combinations appeared to have a greater inhibitory effect against A. polyphaga cysts. SEM was utilized in this study to determine how the combination of CS-NPs affected cyst structure (Fig. 7). SEM images displayed the typical morphological characteristics of A. polyphaga cyst, such as polygonal shape, smooth surface, and thin ridges on the wrinkled surface observed in untreated cysts (Fig. 7A) and the negative control (Fig. 7B). Cysts treated with CHX exhibited a rough surface with thick, wrinkled ridges and shrank into a smaller shape, indicating damaged cell characteristics (Fig. 7C). Cysts treated with the combination of EPEs and CHX (Figs. 7D–7F) showed more morphological changes than those treated with a single propolis treatment (Figs. 7G–7L) when compared to the negative control (Fig. 7B). In this combination (MICC, 5%), cysts treated with G. thoracica and H. itama-loaded CS-NPs (C4; P2+P3) shrank and developed thick, wrinkled ridges on their surface (Fig. 7G), which resembled the morphology of cysts treated with single EPE treatments (C5-C7; P1, P2, P3, and Figs. 7D–7F). Cysts became round, lacking the ridge layer on the cell surface, and exhibited small perforations in the walls when treated with the combination of three EPEs (MICC 5% of C1; P1+P2+P3 and Fig. 7H). The combination of all three EPEs with CHX demonstrated more substantial potential for damaging the cyst cell walls, reducing them to small, irregular shapes with perforations and shrinkage (MICC 5% of C8; P1+P2+P3+CHX and Fig. 7I). The combinations of two propolis treatments and a drug at MICC 5% (C10; P1+P3+CHX) caused cysts to exhibit morphological changes characterized by irregular shapes without wrinkled ridges on the surface (Fig. 7K). It was noted that cyst morphology at MICC 5% (C9; P1+P2+CHX) and MICC 1.25% (C11; P2+P3+CHX) shows distinct shrinking cells with thick wrinkled ridges of surface (Figs. 7J and 7L). However, the latter displayed smaller sizes, larger pores, and numerous blunt ends on the cell surface, indicating promising activity with the lowest MICC when their combinations were used.

Cytotoxicity assessment of propolis-chlorhexidine CS-NP formulations on Vero cells

Among all tested formulations, the C11 (P2+P3+CHX) exhibited the lowest cytotoxicity against the Vero cell line, maintaining >80% cell viability at an MCC of 2.5% drug-loaded CS-NP (Table 2). In contrast, C9-C10 (P1+P2/P1+P3+CHX), C1 (P1+P2+P3), C7-C8 (P3 and P1+P2+P3+CHX), C6 (P2), and C2 (P1+P2), C5 (P1) displayed increased cytotoxicity with MCC values as low as 0.313%, 0.156%, 0.078%, and 0.039%, respectively (Table 2).