Evaluation of the Antimicrobial and Embryotoxic Effects of Gold Nanoparticles Synthesized Chemically with 4‑(4’-Chlorophenyl)-2-imino-1,3-thiazino [2,3‑b] Benzimidazole (FDM29) and via Green Synthesis Using Baissea gracillima (liana)

Abstract

The remarkable rise in antibiotic resistance seen in recent years has significantly reduced the effectiveness of most medications. This study aimed at evaluating the antibacterial and toxicological effects of gold nanoparticles (AuNPs), capped with a synthetic heterocyclic compound, 4-(4’-chlorophenyl)-2-imino-1,3-thiazino [2,3-b] benzimidazole (FDM29), and green-synthesized using a leaf extract from Baissea gracillima (B.gracillima-liana) plant. The synthesized AuNPs were characterized by ultraviolet–visible spectroscopy, dynamic light scattering, Fourier transform infrared, transmission electron microscopy, energy-dispersive and diffraction X-ray spectroscopy, supported by ^1H and ^13C nuclear magnetic resonance analysis of the FDM29 ligand. The antibacterial susceptibility of the AuNPs was investigated against Gram-negative (Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) and Gram-positive bacteria (Bacillus subtilis, Mycobacterium smegmatis, and Staphylococcus aureus) using the broth microdilution method. The zebrafish embryo development toxicity test (ZFET) was employed to evaluate the toxicity of the synthesized AuNPs. Characterization indicated that AuNPs were successfully synthesized. The AuNPs were stable and spherical, with sizes ranging from 13 to 84 nm, which fall within the nanometer scale. In addition, the AuNPs effectively inhibited the growth of most bacterial strains, excluding P. mirabilis and M. smegmatis. The findings suggest that capping AuNPs enhances their antibacterial efficacy and reduces their toxicity compared to chemically synthesized uncapped AuNPs. The exposure of zebrafish embryos to the synthesized AuNPs revealed that the pristine AuNPs exhibited more significant toxicity at all concentrations compared to AuNPs-FDM29 and AuNPs-B. gracillima-liana. At 24 h post-fertilization, most eggs had coagulated, and by 48 hpf, the majority embryos showed absence of heartbeat and developmental retardation, thus being considered toxic. The mortality rate followed the order pristine AuNPs > AuNP-FDM29 > AuNP-B.gracillima-liana, at 81.6, 61.9, and 14.76%, respectively, at 0.25 mg/mL. These results indicate that biological functionalization of AuNPs significantly improves their biocompatibility and antibacterial properties. Notably, the green synthesis using B. gracillima-liana leaf extract yielded AuNPs with the lowest toxicity and substantial antibacterial efficacy, outperforming chemically synthesized counterparts. This study presents a novel, plant-based green synthesis approach for generating biocompatible AuNPs with dual antibacterial and low-toxicity profiles, offering a sustainable platform for developing next-generation antimicrobial agents with potential applications in nanobiotechnology, biomedicine, and pharmaceutical sciences.

1. Introduction

The astounding efficacy of antibiotics in killing or inhibiting the growth of bacterial pathogens has saved the lives of millions of people who otherwise would have fallen victim to bacterial diseases. However, the remarkable rise in antibiotic resistance in recent years has significantly reduced the effectiveness of most medications. This is due to the overuse and misuse of antibiotics and poor infection prevention and control. As a result, the rate of disease transmission and the severity of affected patients’ conditions have increased the likelihood of mortality. Gram-positive and harmful bacteria like Staphylococcus aureus and Gram-negative Enterobacteriaceae are among those that have the highest levels of antibiotic resistance. This implies that new infection control methods must have minimal impact on the host and helpful bacteria.

Nanostructured materials hold great promise as antibacterial agents. Metal-based nanoparticles are increasingly used due to their bioactivities. The unique properties of nanomaterials have sparked interest in therapeutic applications. Gold nanoparticles (AuNPs) have recently been widely investigated for diagnostics and novel drug delivery methods. However, there are limitations to the physicochemical synthesis of AuNPs, such as toxic chemicals, duration, stability, hazardous product creation, and large aggregated products. In addition, their potential toxicity risks both human and environmental health. AuNPs can cause oxidative injury, disruption of cellular homeostasis, and deoxyribonucleic acid (DNA) damage. These challenges necessitated the development of an ecologically friendly, energy-efficient, and green manufacturing process based on biological systems (plants and plant-derived products).

Environmental and green synthesis, unlike chemical methods, avoids the use of toxic chemicals. However, it is worth noting that the starting precursor, hydrogen tetrachloroauric acid (HAuCl₄·3H₂O), is typically derived from conventional gold mining processes, which carry environmental burdens such as high carbon emissions and chemical waste. Therefore, while downstream synthesis adopts greener practices, the upstream sourcing of gold salts remains a key consideration in evaluating the overall sustainability of AuNPs production.

In recent years, increasing attention has been given to biologically active compounds from medicinal plants, many of which have been used for centuries by indigenous populations to treat various human ailments. These plant-derived products often contain secondary metabolites with inherent antibacterial properties, making them valuable candidates for nanoparticle synthesis. An essential component of nanotechnology is the dependable, nontoxic, and environmentally friendly chemistry procedure used to create AuNPs. Bacteria, fungi, yeast, and plant extracts are among the vast, diverse, and richest resources that could synthesize metallic nanoparticles (MNPs) as reducing and stabilizing agents, providing advantages over traditional approaches. In the fight against bacteria, heterocyclic compounds have become a potent class of organic secondary metabolites. These compounds’ antibacterial activity has been found to be several orders of magnitude less potent than that of traditional antibiotics. This is due to their poor chemical stability, solubility, and permeability. A possible strategy to address these issues is using biological gold nanoparticles (AuNPs) as a delivery system for heterocyclic compounds. Aside from that, AuNPs’ unique chemical and physical characteristics, such as their size, shape, and enhanced surface area, have therapeutic potential in antibacterial applications.

Pristine AuNPs will be coupled with the synthetic heterocyclic compound 4-(4’-chlorophenyl)-2-imino-1,3-thiazino [2,3-b] benzimidazole (FDM29) and green-manufactured using Baissea gracillina-liana (B. gracillima-liana) plant extract to improve their antibacterial capabilities and reduce their toxicity. The toxicity of the engineered AuNPs will subsequently be evaluated using a zebrafish model to see if they can inhibit the growth of Gram-negative and Gram-positive bacteria.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals and reagents, unless otherwise indicated, were obtained from suppliers and used without further purification. All the chemicals used in the study were purchased from Sigma-Aldrich (USA), namely hydrogen tetrachloroauric acid, trisodium citrate, ethanol, acetic acid, and tween 80.

2.2. Preparation of 4-(4’-Chlorophenyl)-2-imino-1,3-thiazino [2,3-b] Benzimidazole (FDM29)

The preparation of the synthetic heterocyclic compound followed a procedure adapted from Rodriguez, with slight modifications. In a three-necked flask equipped with a magnetic stirrer bar, 2-mercaptobenzimidazole (1.860 mmol), 4-chlorophenylpropynenitrile (1.860 mmol), potassium carbonate (0.372 mmol), and 6 mL of n-butanol were added. The reaction mixture was heated under reflux for 1 h and monitored using thin-layer chromatography (TLC) with a solvent system of ethyl acetate: hexane (1:1). Upon completion of the reaction, the mixture was allowed to cool, and the solvent was evaporated. The resulting solid was recrystallized in n-butanol, yielding 4-(4’-chlorophenyl)-2-imino-1,3-thiazino [2,3-b] benzimidazole (Figure S1) with a mass of 0.43 g. The yield was 63%, corresponding to 1.40 mmol of the compound. The ¹H and ¹³C NMR spectra of the compound are included in Figures S2–S5.

2.3. Collection and Preparation of Plant Extracts

The plant leaves of Baissea gracillima (B.gracillima liana) were obtained from a registered indigenous plant nursery within the Walter Sisulu National Botanical Gardens in Roodepoort, Gauteng, South Africa. The selection of the plant was conducted randomly due to the absence of existing literature regarding its potential for manufacturing gold nanoparticles and because of its cost-effectiveness. Fresh and healthy leaves were collected and thoroughly washed using tap water, followed by deionized water, to eliminate dirt and undesirable visible particles. Subsequently, the leaves were cut into smaller pieces and left to dry at ambient temperature. After drying, the leaves were crushed into a fine powder using a pestle and mortar. The resulting powder was weighed and set aside for later use in creating a leaf extract.

The leaves were subjected to aqueous extraction using a method based on the protocol described by Elia, with some adjustments. To obtain the aqueous extract, a total of 20 g of completely dried leaf powder was added to 250 mL of deionized water and subjected to boiling on a hot plate for 2 h while maintaining a stirring rate of 400 rpm. The liquid obtained was subsequently isolated from the solid components through the process of filtration using Whatman No. 1 filter paper. The resulting filtrate was then filtered through a 0.45 μm PVDF syringe filter (Labfriend, SA) and stored for future use.

2.4. Synthesis of Gold Nanoparticles (AuNPs)

2.4.1. Gold Salt Stock Solution Preparation

To prepare a 10 mM stock solution of the gold (Au) salt, the entire amount (1.0 g) of hydrogen tetrachloroauric acid (HAuCl₄·3H₂O) obtained from Sigma-Aldrich (USA), was completely dissolved in 250 mL of deionized water. The water used for this purpose was obtained from a Mili-Q EQ 7000 Ultrapure water purification system, with a conductivity of 0.055 μS/cm and a temperature of 25 °C, sourced from Merck (SA). The resulting stock solution was then stored in a brown bottle. The required concentration of 1.0 mM was generated by diluting 25 mL of the stock solution to a final volume of 250 mL. Then, the reducing agent, trisodium citrate (Na₃C₆H₅O₇.2H₂O), was prepared by dissolving 0.5 g in 50 mL of distilled water. Since sodium citrate has a limited shelf life, it was freshly prepared prior to each synthesis. The trisodium citrate dihydrate used in this experiment was of 99% purity and was acquired from Sigma-Aldrich (USA).

2.4.2. Synthesis of Pristine AuNPs using the Turkevich Citrate Reduction Method

The production of AuNPs was performed using the approach originally proposed by Turkevich, with few modifications. A 20 mL aliquot of a 1.0 mM stock solution of HAuCl₄·3H₂O was added to a 50 mL Erlenmeyer flask. The mixture underwent agitation by means of a magnetic stirrer (MS200, China (Mainland)) set to a rotating speed of 300 rpm (rpm) placed on a hot plate. The boiling solution was supplemented with 2 mL of 1% Na₃C₆H₅O₇.2H₂O. This addition served the purpose of reducing the Au to form AuNPs. The successful synthesis of AuNPs was indicated by the observed transition in the solution’s color, shifting from yellowish to deep red. After a period of 10 min, the solution was subsequently removed from the heat source and allowed to cool until it reached the ambient temperature. The AuNPs were freeze-dried using Testar, LyoAlfa dryer (Labotec, SA) and stored in a Schott bottle with a blue GL45 cap obtained from Sigma-Aldrich (USA) for future use.

2.4.3. Green Synthesis of AuNPs using Baissea Gracillima (B. Gracillima liana) Leaf Extract

The process of synthesizing AuNPs by green synthesis was achieved by following the method by Lee with minor modifications. A prepared 40 mL aqueous solution of a leaf extract from the B. gracillima liana plant was added into a conical flask. Subsequently, 4 mL of a 1 mM HAuCl4·3H2O solution was added drop by drop at room temperature while maintaining static conditions. After a duration of 10 min, the solution underwent a color change, transitioning from its original state to a pinkish-reddish hue , which serves as an indication of the successful formation of gold nanoparticles (AuNPs). The resulting solution was left for a duration of 60 min to ensure the successful formation of AuNPs. Then, the mixture was subjected to centrifugation at a speed of 5000 rpm for 15 min, to separate nanoparticles of different shapes and sizes. Following centrifugation, the AuNPs were subjected to three rounds of washing with deionized water to eliminate residual biomolecules and contaminants. The AuNPs were subjected to freeze-drying and kept in a screw-cap bottle for further analysis, as previously described.

2.5. Analytical Characterization Techniques

2.5.1. Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectroscopy was employed to elucidate the chemical structure of the synthesized ligand, 4-(4’-chlorophenyl)-2-imino-1,3-thiazino­[2,3-b] benzimidazole (FDM29). Both proton (^1H) and carbon (^13C) NMR spectra were recorded using a Bruker Avance III 500 MHz spectrometer (Bruker Corporation, USA) at 298 K. For sample preparation, approximately 10 mg of FDM29 was dissolved in 0.6 mL of deuterated dimethyl sulfoxide (DMSO). The solution was transferred to a 5 mm NMR tube (Wilmad-LabGlass, USA) for analysis. The ^1H NMR spectra were acquired using a standard one-pulse sequence with a spectral width of 12 kHz, an acquisition time of 2.7 s, and a relaxation delay of 1 s. A total of 64 scans were collected to ensure an adequate signal-to-noise ratio. Chemical shifts (δ) were reported in parts per million (ppm) relative to the residual solvent peak of DMSO at δ_H 2.50 ppm. The ^13C NMR spectrum was obtained using a proton-decoupled pulse sequence with a spectral width of 30 kHz, an acquisition time of 1 s, and a relaxation delay of 2 s, accumulating 1024 scans. Chemical shifts were referenced to the DMSO solvent peak at δ_C 39.52 ppm. NMR data were processed and analyzed using Bruker TopSpin software (version 3.6.2). Peak assignments were made based on chemical shift correlations and comparison with literature values.

2.5.2. Ultraviolet–Visible Spectroscopy

The technique of absorbance spectroscopy is commonly employed for the assessment of the optical characteristics of solutions. The purpose of this study was to verify the existence of the generated nanoparticles by examining their optical characteristics within specific absorbance ranges. The preparation of the sample involved the dilution of a small volume of aqueous nanoparticles (100 μL) with deionized water to completely fill a quartz cuvette. The sample was analyzed within a wavelength range of 200 to 800 nm utilizing a Shimadzu (UV-2450, Japan) spectrometer. Distilled water was utilized as a blank throughout the analysis process.

2.5.3. Dynamic Light Scattering (DLS)

The hydrodynamic size and polydispersity index (PdI) of the nanoparticles were determined using the Malvern Zetasizer Nano-ZS90 device, manufactured by ATA Scientific Instruments, located in Taren Point, Australia. All measurements were analyzed at a temperature of 25 °C. A volume of 100 μL of nanoparticles was diluted with 900 μL of deionized water and thereafter placed into a disposable cuvette (DTS0012) and a disposable folded capillary cell (DTS1070) for the purpose of measuring the hydrodynamic size and zeta potential (ZP), respectively. The PdI was subsequently determined for each hydrodynamic size measurement, which offered explicit details regarding the average dispersity of the nanoparticles that were produced.

2.5.4. X-ray Diffraction (XRD) Analysis

X-ray diffraction (XRD) analysis is a highly adaptable and nondestructive analytical technique employed to investigate various structural characteristics in crystalline powdered samples. To conduct an analysis of the produced AuNPs, the samples were prepared by undergoing a process of fine grinding and homogenization using a mortar and pestle. Following that, the crystallographic arrangement of the prepared samples was confirmed through XRD SmartLab SE instrument, which was equipped with a HyPix-400 detector (manufactured by PANalytical Empyrean, United States) and operated with Cu Kα radiation (with a wavelength of 1.542 Å). The examination was conducted within the angular range of 10° to 90°, utilizing a scanning increment of 5° per minute. The X-ray diffraction measurements were performed using a voltage of 40 kilovolts and a current of 30 mA. The analytical process employed for examining the samples adheres to the principles of Bragg’s Law. Then determination of the crystallinity of the samples was accomplished through a comparison of the X-ray diffraction pattern or diffractogram generated from each sample.

2.5.5. Energy-Dispersive X-ray Spectroscopy (EDS)

The energy-dispersive X-ray spectra of the nanoparticle (NP) samples were acquired utilizing a field emission scanning electron microscope (SEM) (TESCAN, UK) equipped with energy-dispersive X-ray spectroscopy (EDS). This technique is employed to acquire the elemental and chemical composition of nanoparticles. Concisely, the samples were equally positioned onto a carbon tape mounted to a sample holder made of SEM-grade aluminum. The samples underwent carbon coating using a tabletop scanning electron microscope (SEM) sputter carbon cord coater (Quorum Q300T ES, UK) for 45 min. The coated samples were subsequently subjected to analysis at a voltage of 20 kV using the micro X-ray analyzer (EDS Oxford equipment, X-Max, UK) equipped with Aztec software (Oxford), which was coupled to an SEM TESCAN system.

2.5.6. Fourier-Transformed Infrared Spectroscopy (FTIR)

The functional groups of the produced AuNPs were determined by FTIR spectroscopy (PerkinElmer (Pty) Ltd., South Africa). The mid-infrared (MIR) spectra were acquired within the range of 500–4000 cm^–1, employing a resolution of 4 cm^–1 through the utilization of the Bruker Tensor 27 IR instrument. The process was carried out by subjecting the synthesized AuNPs to a coating process using potassium bromide (KBr) at a specific ratio of 1:1000 (AuNPs to KBr) to produce a tablet suitable for analytical purposes. Prior to the analysis of the sample, the machine underwent disinfection using isopropanol, and the background correction was performed using distilled water in conjunction with the ATR unit. The tablet samples were thereafter positioned directly onto the KBr cell and subsequently subjected to measurement. The spectroscopic software OPUS, developed by Bruker Optik GmbH in Ettlingen, Germany, was utilized to preview the live spectrum and collect spectra.

2.5.7. Transmission Electron Microscopy (TEM)

The transmission electron microscopy (TEM) micrographs were acquired with the JEM-2100 TEM instrument manufactured by Jeol in Japan, operating at 200 kilovolts (kV). The samples were produced by dissolving the freeze-dried pristine, FDM29, and B.G AuNPs in deionized water (dH₂O). Each solution of nanoparticles was sonicated and carefully deposited onto a copper grid, which was then positioned on a thin layer of amorphous carbon. Then, the grids with the nanoparticles were left to dry overnight in a closed container at room temperature. TEM micrographs that convey information about the size, shape, and distribution of the nanoparticles were generated. The measurement of the core diameter of the nanoparticles was conducted by analyzing the TEM micrographs with the assistance of the ImageJ software.

2.6. Antibacterial Susceptibility Testing

The antibacterial activity of pristine AuNPs, FDM29-AuNPs, B. gracillima (B.G-liana)-AuNPs, FDM29, and B. gracillima (liana) plant extract was evaluated using the broth microdilution technique. The bacterial strains that were subjected to testing included Proteus mirabilis (P. mirabilis), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), Bacillus subtillis (B. subtillis), Staphylococcus aureus (S. aureas), and Mycobacterium smegmatis (M. smegmatis), which were obtained from Davies Diagnostics, SA. The minimum inhibitory concentrations (MIC) of the produced AuNPs, heterocyclic compound, and plant extract were determined following the methodology described by Fonkui, with minor modifications. The experiment was carried out in accordance with aseptic procedure guidelines within a laminar flow hood (Amadwala Laboratory Systems, SA).

2.6.1. Preparation of a McFarland Standard

A sterile loop was employed to transfer a single colony of bacteria from a Petri plate (Mueller-Hinton Agar) into a 15 mL centrifuge tube containing 5 mL of Mueller–Hinton broth. The tube was then incubated at a temperature of 37 °C for a duration of 30 min. Subsequently, a volume of 100 μL of the bacterial suspension was transferred from the broth cultures into each well of a 96-well microtiter plate. The absorbance at 600 OD was measured to assess the conformity of the suspension to the 0.5 McFarland standard, which is defined by an absorbance value ranging from 0.08 to 0.12 nm.

2.6.2. Resazurin Microtiter Assay

The compounds under investigation were prepared by dissolving them in dimethyl sulfoxide (DMSO) to obtain stock quantities of 1 mg/mL. The experiment utilized 96-well plates, with each well containing 100 μL of Mueller–Hinton broth (Sigma-Aldrich, USA) at the desired concentrations (1, 0.5, 0.25, 0.125, 0.0625, and 0.031 g/mL). Subsequently, an additional 100 μL of the test compound was introduced into wells B to G, resulting in a total volume of 200 μL. To establish the method validity, dH₂O was employed as a negative control in wells A and H. Subsequently, the solutions were then diluted serially, starting from well B and progressing to well G. After performing the serial dilution, a volume of 100 μL was left in each plate. Subsequently, an additional 100 μL of the desired bacteria was added to the appropriate wells (B to G), excluding the negative control wells (A and H). The plates were then incubated overnight at a temperature of 37 °C. 50 μL of resazurin dye (Sigma-Aldrich, USA) was added thoroughly after being left overnight, and the subsequent color shift was observed to acquire the results. Following that, the minimum inhibitory concentration (MIC) for each test compound was determined by assessing the alterations in color observed in the 96-well plates.

2.7. Toxicity Testing using the Zebrafish Embryo Development Test (ZFET)

The OECD Testing Guidelines (TG) 236 were followed for maintenance, breeding, and exposure conditions.

2.7.1. Fish Husbandry and Breeding

Adult zebrafish (Danio rerio) were obtained from a local supplier and maintained in fish tanks that had a volume of 40 L water. The water was prepared by diluting reconstituted water with deionized water to produce a level of hardness that is equivalent to 100–300 mg/L of calcium carbonate. Glass heaters (ViaAqua, Camarillo, China) were used to maintain the temperature at 25 ± 0.5 °C. The light–dark cycle for the zebrafish was maintained at 8–16 h of day-to-night ratio. The high protein food known as TetraMin flakes from Germany was given to the fish three times a day at the following times: 8:00 in the morning, 12:00 at lunchtime, and 15:00 in the afternoon. To guarantee favorable circumstances for the habitat, the parameters of the water, particularly the temperature, were monitored daily. To facilitate reproduction, 5 L tanks were used and maintained at 26 ± 0.5 °C. To prevent the eggs from being eaten by adult fish, the spawn traps were covered with inert wire mesh. In addition, as a spawning stimulus, plastic artificial plants composed of synthetic materials were inserted into the mesh. When the zebrafish were ready for mating, as indicated by the presence of heavily pregnant females, mature male and female zebrafish were put into the breeding tanks at a ratio of 1:2 (female to male) and left in the tanks overnight. 30 min after the beginning of the light, the spawn traps were removed, and the eggs were fertilized. After being cleaned with deionized water, the zebrafish embryos were counted using an inverted light microscope manufactured by Olympus in Hamburg, Germany. The fertilized embryos were separated from the unfertilized embryos, and the fertilized eggs were then used for toxicity analysis.

2.7.2. Zebrafish Embryo Acute Toxicity Test (ZFET)

The 96-h zebrafish embryo acute toxicity test was carried out in accordance with the OECD testing guideline (TG) 236. To prepare testing samples, a quantity of 0.03 g of each sample, namely pristine AuNPs, FDM29-AuNPs, and B. gracillima (liana)-AuNPs, was dispersed in 120 mL of deionized water. The deionized water used for this process was obtained from the Mili-Q EQ 7000 Ultrapure Water Purification System , with a conductivity of 0.055 micro siemens and a temperature of 25 °C, provided by Merck (SA). The resulting solutions were then stored in volumetric flasks and kept for 24 h before use. To determine the lethal concentration (LC₅₀), the testing samples were tested at 0.25, 0.125, 0.063, 0.031, and 0.016 g/mL. To validate the experiment, a negative control was employed using reconstituted water, while a positive control was utilized using 3,4-dichloroaniline (Sigma-Aldrich, USA). Exactly 2 mL of each test sample solution was deposited in a 24-well microtiter plate and pre-exposed in an incubator at 26 °C for 24 h. Following that, embryos were added to each well (20 embryos per concentration/5 embryos per well). All experiments were conducted in triplicate. The plates were incubated at 26 °C and observed for the primary and secondary apical observations every 24 h until 96 h post fertilization (hpf). The apical observations, recorded at approximately 40× magnification, are indicators of lethality, which include embryo coagulation, lack of somite development, lack of detachment of the tail-bud from the yolk sac, and a lack of heartbeat. All observations were carried out using an inverted light microscope with an Olympus C5040 AUD camera (Wirsam Scientific, SA). At the end of the exposure time, the primary end point of the test, such as mortality, hatch rate, and malformations, were assessed based on the outcome in any of the four apical observations collected, and the LC₅₀, defined as the concentration that kills 50% of the embryos during the observation period, was determined.

3. Results and Discussion

3.1. Nuclear Magnetic Resonance (NMR) Spectrometry

The ^1H NMR spectrum of FDM29 (500 MHz, DMSO) exhibits a singlet at δ 9.84 ppm, attributed to the imino (−NH) proton. This chemical shift is consistent with the deshielded nature of imino protons in benzimidazole derivatives, as observed in related compounds. The aromatic region displays multiplets between δ 8.76 and 7.62 ppm, corresponding to the protons of the benzimidazole and chlorophenyl moieties, which agree with patterns reported for similar structures. The ^13C NMR spectrum (126 MHz, DMSO) reveals a signal at δ 154.43 ppm, assigned to the CN carbon of the imino group, comparable to values reported for analogous compounds. Carbons within the benzimidazole and thiazino rings resonate between δ 145.15 and 116.74 ppm, while the chlorophenyl carbons appear in the range of δ 135.69–123.27 ppm, aligning with chemical shifts observed in related structures. The combination of ^1H and ^13C NMR data substantiates the proposed structure of FDM29. The chemical shifts and coupling patterns observed are consistent with the presence of the benzimidazole core, the thiazino ring, and the para-substituted chlorophenyl group. These findings confirm the successful synthesis of 4-(4’-chlorophenyl)-2-imino-1,3-thiazino­[2,3-b] benzimidazole. For comprehensive spectral data, including expanded ^1H and ^13C NMR spectra, refer to Figures S2-S5.

3.2. UV–Visible Spectrophotometry Analysis

Figure presents UV–vis spectra confirming the synthesis of AuNPs by the various methods employed. Peaks were observed at 527, 531, and 539 nm for pristine AuNPs, FDM29-capped AuNPs, and B. gracillima (liana)-capped AuNPs, respectively. According to Abalaka, previous research indicates that AuNPs have a wavelength absorption range of 500 to 600 nm. Furthermore, Renitta additionally reported comparable findings, noting that the absorbance peaks of AuNPs produced by environmentally friendly techniques were detected at wavelengths of 530 and 550 nm, respectively. This aligns with the results of the present study, supporting the production of AuNPs.

1.

UV–visible (UV–vis) absorption spectra of pristine gold nanoparticles (AuNPs) (black curve), heterocyclic compound FDM29-capped AuNPs (red curve), and Baissea gracillima (liana)-capped AuNPs (blue curve). Data represent mean ± SD from three technical replicates (n = 3).

In the study, AuNPs were capped, and they demonstrated a right shift. The right shift observed on the peaks of the curves provides a hint that the capped AuNPs are slightly bigger than the pristine AuNPs. The capped AuNPs shifted from 527 to 531 nm. According to the study by Carrillo-Cazares et al., the formation of the SPR band and the right shift between the curves may be due to the size during the production of AuNPs and can be explained by the fact that surface functionalization of nanoparticles generally results in increased hydrodynamic size.

When comparing the peak characteristics of the pure AuNPs, FDM29-AuNPs, and B. gracillima-AuNPs, it was noted that the functionalized FDM29-AuNPs and B. gracillima-AuNPs exhibited a broader surface plasmon resonance (SPR) peak compared to the pristine AuNPs. Significantly, the B.gracillima-AuNPs displayed the widest peak, indicating a rise in wavelength. A study conducted by Islam demonstrated similar findings, showing that the use of green-produced AuNPs led to a red shift from 536 to 538 nm as the content of the plant extract. The prominent bands found in the ultraviolet spectra are a clear indication of the surface plasmon resonance (SPR) characteristics of the gold nanoparticles (AuNPs). The magnitude of the SPR peak is generally proportional to the extent of the transformation of gold ions into AuNPs. The results align with prior research, indicating that smaller AuNPs often display a shorter absorption wavelength, while larger AuNPs exhibit a longer wavelength.

3.3. Dynamic Light Scattering Analysis

Table demonstrates the results of the size analysis, providing information on the hydrodynamic average particle size, polydispersity index (PDI), and zeta potential of pristine AuNPs, FDM29-AuNPs, and B. gracillima (liana)-AuNPs analyzed using DLS. Based on the results, pristine AuNPs have a smaller average diameter of 14.3 ± 0.4 nm and a satisfactory PDI of 0.185, as depicted, while the average particle size of FDM29-capped AuNPs and B. gracillima (liana)-capped AuNPs was found to be 20.8 ± 1.2 nm with a PDI of 0.396, and 182 ± 4.5 nm with a PDI of 0.658, respectively. According to the study by Murdock et al., the observed increase in size may be due to the dispersion of nanoparticles in a solution, which leads to their agglomeration and contributes to the larger average size of nanoparticles. Another factor contributing to the somewhat larger average particle sizes is the presence of the hydration layer and polymeric stabilizer. The study by Farkas and Kramar also suggests that the increase in diameter could have arisen due to particle aggregation upon dispersion in plant extracts, or alternatively, it may have been caused by the gradual accumulation of gold nanoparticles over a prolonged duration.

1. Hydrodynamic Size, Polydispersity (PDI), and Zeta Potential of Pristine Gold Nanoparticles (AuNPs), Heterocyclic Compound (FDM29)-Capped AuNPs, and Baissea gracillima (liana)-Capped AuNPs.

Sample	Hydrodynamic size	Polydispersity (PDI)	Zeta potential
Pristine AuNPs	14.3 ± 0.4 nm	0.185	–31.1 mV
FDM29- AuNPs	20.8 ± 1.2 nm	0. 396	–39.7 mV
B. gracillima (liana)- AuNPs	182 ± 4.5 nm	0. 396	–34.4 mV

The PDI quantifies the dispersion of nanoparticles within the range of 0.0–0.5. According to Rao, a sample is considered monodispersed when its PDI value is below 0.5; any number higher than this shows polydispersity. The acquired results indicate monodispersity, as evidenced by the PDI values lower than 0.5 (Table ), which are subsequently observed in TEM micrographs.

From the results, it was observed that the zeta potential for pristine AuNPs was −31.1 mV, FDM29-AuNPs was −39.7 mV, and B. gracillima (liana)-capped AuNPs was −34.4 mV, indicating stability at room temperature. According to the study by Muneer et al., nanoparticles with zeta potential more positive than +30 mV or more negative than −30 mV are normally considered stable. This characteristic suggests that FDM29 and B. gracillima (liana) plant extract are capable of being capping agents and reducing agents in producing their relatively stable nanoparticles as they resulted in stable AuNPs (Table ). These results can further be supported by comparable results from the study conducted by Lunardi, which demonstrated a correlation between AuNPs produced using the chemical method and green synthesis. The chemically synthesized AuNPs exhibited a reduced diameter of 18.33 nm, a PDI value of 0.60, and a zeta potential of −0.6 mV, while the green-synthesized AuNPs exhibited a bigger size of 433 nm, a PDI of 0.23, and a zeta potential of −13.3 mV.

3.4. X-ray Diffraction (XRD) Analysis

Figure demonstrates the XRD patterns for the synthesized pristine AuNPs, FDM29-AuNPs, and B. gracillima (liana)-AuNPs. The results show numerous Bragg reflection peaks obtained at 38°, 44°, 64°, and 77°, which are indexed by planes (111), (200), (220), and (311) for all AuNP samples analyzed, based on the face-centered cubic (fcc) orientation of Au. Due to the capping of AuNPs with plant phytochemicals, the strong X-ray diffraction peaks of 38° in the crystalline phase can correspond to the (111) reflection of the metallic gold with fcc structure. Moreover, comparatively, the obtained peak lists are identical to the reported data of the standard gold metal by the Joint Committee on Powder Diffraction Standards (JCPDS) file no. 01-089-3697. The unassigned peak appearing on pristine (black) and FDM29-capped AuNPs (blue) is due to the crystallization of sodium chloride , which originates from trisodium citrate, used as a reducing and capping agent during synthesis.

2.

X-ray diffraction (XRD) patterns of pristine gold nanoparticles (AuNPs) (black), heterocyclic compound FDM29-capped AuNPs (red), and Baissea gracillima (liana)-capped AuNPs (blue). Patterns shown are representative of three independent measurements per sample (n = 3).

Furthermore, the unassigned peaks appearing on the B. gracillima (liana)-capped AuNPs are due to the crystallization of bioorganic phases on the surface of the nanoparticles. The average size of AuNPs was calculated using the Scherrer formula. The (111) plane of the synthesized AuNPs is observed more intensely than other planes. Therefore, the (111) plane of the AuNPs is used to calculate the average particle size of the Scherrer equation. The average particle sizes for pristine AuNPs, FDM29-capped AuNPs, and B. gracillima (liana)-capped AuNPs were 19, 35, and 44 nm, respectively. The particle size obtained through XRD analysis is further corroborated by the particle size determined from the TEM analysis.

3.5. Energy-Dispersive X-ray Spectroscopy (EDS)

Figure displays the EDS spectra of pristine AuNPs, FDM29-capped AuNPs, and B. gracillima (liana)-capped AuNPs. Figure A displays the EDS spectra that provides information about the elemental composition of AuNPs. The spectra confirm the successful synthesis of Au nanoparticles, as evidenced by the presence of distinct optical absorption peaks at energy levels of 2.2 and 9.7 keV. These peaks correspond to the presence of Au nano crystallites. This is corroborated by the findings obtained by Elbagory. Additional absorption peaks for carbon (C), oxygen (O), sodium (Na), and chlorine (Cl) elements were also observed. Sodium citrate, a reducing agent utilized in synthesis, may be the source of sodium. Chlorine comes from the chloroauric acid that was employed as precure salt, and carbon could have originated from the carbon coating applied to eliminate electrical charges that quickly develop in a nonconducting material under a high-energy electron beam. In the case of AuNPs, oxygen could have come from the deionized water used as a solvent.

3.

Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) spectra of (A) pristine gold nanoparticles (AuNPs), (B) heterocyclic compound FDM29-capped AuNPs, and (C) Baissea gracillim a (liana)-capped AuNPs.

In Figure B, it was also possible to identify adsorption peaks for sodium (Na), carbon (C), oxygen (O), and chlorine (Cl). Since sodium citrate was utilized as a reducing agent, it is possible that sodium itself originated from it. Deionized water and the FDM29 structure are sources of oxygen. Chlorine comes from the FDM29 employed as a capping agent, and the carbon comes from the carbon coating and the FDM29 heterocyclic compound. The elemental profile for B. gracillima (liana)-capped AuNPs is presented in the EDS spectra (Figure C). Furthermore, the effective production of Au nanoparticles is confirmed by the appearance of significant optical adsorption peaks at 2.2 and 9.7 keV, which correlate to the nano crystallization of Au. In addition, adsorption peaks corresponding to sodium (Na), carbon (C), oxygen (O), potassium (K), and chlorine (Cl) elements were also observed. The element chlorine could originate from chloroauric acid, a precursor salt. The sodium, carbon, oxygen, and potassium could originate from the B. gracillima (liana) plant extract used as a reducing and capping agent during the synthesis. Throughout the scanning range of binding energies, no impurities were identified, as all elements were accounted for. This indicates that the synthesized AuNPs are pure.

Overall, B. gracillima (liana) had the lowest Au elemental composition compared to pristine AuNPs and FDM29-capped AuNPs. These results align with the study by Geethalakshmi and Sarada, which demonstrated high Au elemental composition in chemically synthesized AuNPs compared to green-synthesized AuNPs.

3.6. Fourier-Transformed Infrared Spectroscopy (FTIR)

The FTIR spectra of pristine AuNPs, FDM29-capped AuNPs, and B. gracillima­(liana)-capped AuNPs are presented in Figure . The uncapped pristine AuNPs (Figure A) showed peaks at 3431, 2928, 2361, 2347, 1714, and 1384 cm^–1. The broad peak observed at 3431 cm-1 corresponded to stretching vibrations of the O–H bond, which could originate from deionized water used as a solvent during synthesis, while the peak at 2928 cm^–1 is related to C–H stretching. The peak at 1714 cm^–1 corresponds to CO stretching vibrations, and the peak at 1595 cm^–1 represents CC aromatic stretching. According to the study by Rotimi et al., the peaks at 1385 cm^–1 and 1244 cm^–1 are associated with the Au and C–O bonds, confirming the ability of Au salt to reduce nanoparticles. From this spectrum, it can be observed that FDM29-capped AuNPs (Figure B) have a broad peak at 3435 cm^–1, indicating the −OH bond stretching of carboxyl groups, N–H stretching of secondary amides, and a C–H group stretching at 2925 cm^–1. These results are comparable with the study by Elia et al., where the presence of phytochemicals is accounted for by the NH. The FDM29-capped AuNPs spectrum showed a sharper peak at 2853 cm^–1 compared to pristine AuNPs.

4.

Fourier Transform Infrared (FTIR) spectrum of (A) pristine gold nanoparticles (AuNPs) (black), (B) heterocyclic compound FDM29-capped AuNPs (red), and (C) B aissea gracillima (liana)-capped AuNPs (blue). Spectra shown are representative of three independent measurements per sample (n = 3).

These FTIR observations are consistent with the structural features identified by NMR, which confirmed the presence of the imino and aromatic protons and carbons associated with the benzimidazole and thiazino ring systems in FDM29. These functional groups are likely involved in the surface capping and stabilization of nanoparticles. Therefore, it is possible that the functional groups present in FDM29 were capped onto the AuNPs. Other peaks in the same spectrum2853, 1735, and 1385 cm^–1,represent C–H, CO, and C–O, respectively. Based on previous literature, functional groups C–O, CO, and NH from amino acids and proteins have a strong affinity to bind metals to produce highly stable nanoparticles. The C–H stretch and CO stretch are also observed in both FDM29-capped AuNPs and uncapped AuNPs. Despite the resemblance between the two, some absorption peaks shifted in their positions. These shifted positions are assumed to be associated with the functional groups around AuNPs formed, indicating the participation of responsible functional groups in capping the synthesized nanoparticles. The FTIR spectra were also performed on B. gracillima (liana)-capped AuNPs (Figure C). From this spectrum, it is observed that B. gracillima (liana) AuNPs have a broad peak at 3415 cm^–1 attributed to NH and −OH stretching, which is similar peak observed for pristine AuNPs alone at 3431 cm^–1 attributed to OH stretching. Another similarity was found at 2853 cm^–1, and 2926 cm^–1 attributed to C–H group stretching vibrations. This confirms the capping of AuNPs. Moreover, a characteristic peak representing the CO group can be observed at 1719 cm^–1, whereas the peak at 1051 cm^–1 corresponds to C–O. The IR band also indicated different B. gracillima (liana) extract phytochemicals. Comparative findings are also observed in the study by Yallappa et al., where more peaks are observed in the functionalized and green-synthesized AuNPs due to the increase in reducing and capping agents present in the compounds.

3.7. Transmission Electron Microscopy (TEM)

The TEM micrographs and size distribution for the pristine AuNPs, and those capped with FDM29 and B. gracillima (liana) are presented in Figure . The pristine AuNPs exhibited a monodisperse distribution and possess a nearly spherical form (Figure A,B). The analysis of the distribution demonstrated that the manufactured AuNPs exhibit sizes ranging from 8 to 18 nm, averaging 13 nm. This finding supports the hydrodynamic size of the AuNPs obtained by DLS, as depicted in Table , which closely aligns with the TEM measurement of 14.3 ± 0.4 nm. The observed commonalities can be attributed to the fact that these AuNPs were not capped with any distinctive functional groups on their surface.

5.

Micrographs of (A) pristine gold nanoparticles (AuNPs), (C) heterocyclic FDM29-capped AuNPs, and (E) Baissea gracillima (liana)-capped AuNPs, along with their corresponding size distributions (B, D, and F), obtained using Transmission Electron Microscopy (TEM).

The micrograph for the FDM29-capped AuNPs (Figure B) indicates that the nanoparticles possess a spherical morphology and are prone to agglomeration. Furthermore, this is supported by the particle size distribution ranging from 16 to 26 nm, with most of the nanoparticles measuring 20 nm. This finding confirms the hydrodynamic size of the FDM29-capped AuNPs, as determined by DLS and shown in Table . According to the study by Anaraki, the observed minute variation could potentially be attributed to the functionalization and subsequent agglomeration of nanoparticles.

Figure E,F depicts the TEM images of the B. gracillima­(liana)-capped AuNPs, revealing clear signs of agglomeration and their characteristic spherical shape. The size distribution ranged from 60 to 100 nm, with an average size of 84 nm. However, the hydrodynamic size, as measured by DLS, was significantly larger at 182 ± 4.5 nm (Table ), which is a common observation attributed to the presence of the capping agent and nanoparticle agglomeration, both of which are accounted for in DLS measurements. Studies, such as those by Liu, have shown that DLS typically yields larger size measurements due to the inclusion of the capping layer and the liquid-state conditions during synthesis. The B. gracillima (liana)-capped AuNPs exhibit a nonuniform distribution, with variations in size indicating polydispersity, as some particles are larger while others are smaller. This diversity is consistent with the DLS analysis, which reflects the nonuniformity of the nanoparticle sizes. Additionally, the micrographs show partial agglomeration, which is typical for metallic nanoparticles like AuNPs. This phenomenon can be attributed to their elevated surface energy and weak intermolecular forces that facilitate particle attachment. Regardless of the agglomeration, the B. gracillima-capped AuNPs retain their nanometer-scale dimensions and predominantly spherical morphology, supporting their potential efficacy in various biological applications.

3.8. Antibacterial Susceptibility Test

Table presents the minimum inhibitory concentration (MIC) values along with their standard deviations for FDM29, B. gracillima (liana), pristine AuNPs, FDM29-capped AuNPs, and B. gracillima-capped AuNPs against three Gram-negative (K.pneumoniae, P. mirabilis, and E. coli) and three Gram-positive bacterial strains (B. subtillis, S.aureas, M. smegmatis). The findings reveal that among the six bacterial strains tested, P. mirabilis and M. smegmatis demonstrated resistance to all the tested agents. This resistance can be ascribed to a confluence of intrinsic traits and adaptive mechanisms present in these bacterial strains. P. mirabilis is well-known for forming biofilms and developing resistance to various drugs, possibly through mechanisms such as efflux pumps, which actively expel antimicrobial agents. In contrast, M. smegmatis being a mycobacterium characterized by its tough cell wall and inherent resistance features, has the capacity to utilize many mechanisms, including efflux pumps, biofilm formation, and genetic alterations, as means to withstand the test samples.

2. Minimum Inhibitory Concentrations (MIC) and Standard Deviations (SD) of Heterocyclic Compound FDM29, Baissea Gracillima (liana) Plant Extract, and Metallic Nanoparticles (Capped and Uncapped), Against Six Bacterial Strains.

Minimum inhibitory concentration (μg/mL)
Test samples	Gram-negative bacterial strains			Gram-positive bacterial strains
	Klebsiella pneumoniae	Proteus mirabilis	Escherichia coli	Bacillus subtilis	Staphylococcus aureus	Mycobacterium smegmatis
FDM29	500 ± 10	-	125 ± 5	15.63 ± 0.5	15.63 ± 0.5	-
B. gracillima (liana) extract	15.63 ± 1	-	250 ± 10	15.63 ± 1	15.63 ± 0.5	-
Pristine AuNPs	15.63 ± 0.5	-	15.63 ± 0.5	250 ± 10	15.63 ± 0.5	-
FDM29-AuNPs	15.63 ± 0.5	-	31.25 ± 1	15.63 ± 1	62.50 ± 2	-
B. gracillima (liana)-AuNPs	15.63 ± 0.5	-	15.63 ± 0.5	15.63 ± 0.5	15.63 ± 0.5	-

The findings further demonstrate that, among the two synthesis modifications tested, the B. gracillima extract demonstrated the strongest antibacterial potency against both Gram-positive and Gram-negative bacteria, with MIC values as low as 15.63 ± 1 μg/mL against K. pneumoniae and B. subtilis. It effectively inhibited Gram-negative strains with MICs ranging from 15.63 ± 0.5 μg/mL to 250 ± 10 μg/mL, while maintaining an MIC of 15.63 μg/mL against Gram-positive strains. This strong efficacy is attributed to bioactive compounds present in B. gracillima such as flavonoids and alkaloids, known for their potent antimicrobial properties. These compounds disrupt bacterial cell membranes and inhibit essential enzymatic activities, ultimately leading to bacterial cell death. In contrast, FDM29 exhibited lower efficacy against Gram-negative strains, with MICs ranging from 125 ± 5 μg/mL against E. coli to 500 ± 10 μg/mL against K. pneumoniae, while it showed comparable potency against Gram-positive strains at an MIC of 15.63 ± 0.5 μg/mL for both B. subtilis and S. aureus.

Among the synthesized AuNPs, those conjugated with B. gracillima extract were the most effective, achieving the lowest MIC of 15.63 ± 0.5 μg/mL against four bacterial strains. The pristine AuNPs exhibited MIC values ranging from 15.63 ± 0.5 μg/mL against K. pneumoniae to 250 ± 10 μg/mL against B. subtilis. Furthermore, FDM29-conjugated AuNPs consistently inhibited bacterial growth across all tested concentrations (15.63 ± 0.5 to 62.50 ± 2 μg/mL), indicating that the use of a capping agent enhances the antibacterial properties of AuNPs. This finding aligns with previous studies, which have demonstrated that the functionalization of AuNPs with plant extracts or bioactive compounds enhances their antimicrobial efficacy by increasing the interaction with bacterial membranes and improving cellular uptake. , The antibacterial mechanism of gold nanoparticles is believed to involve multiple pathways, such as physical disruption of cell membranes, production of reactive oxygen species (ROS), interference with intracellular enzyme function via interaction with thiol groups, and modulation of gene expression. It has been reported that plant extract-capped AuNPs exhibit greater stability and reduce bacterial resistance by disrupting cell walls, inhibiting biofilm formation, and promoting oxidative stress in bacterial cells. Similar research has highlighted that AuNPs conjugation with organic compounds enhances the nanoparticle’s biocompatibility and makes them effective against both Gram-positive and Gram-negative bacteria. ,

3.9. Zebrafish Embryo Acute Toxicity Test (ZFET)

Figure presents visual representations from the acute toxicity test on zebrafish embryos, assessing the effects of pristine AuNPs, FDM29-capped AuNPs, and B. gracillima (liana)-capped AuNPs. The images depict various apical end points, such as coagulation, malformations, and developmental delays observed at a concentration of 0.063 mg/mL. These observations were recorded at critical time points24, 48, 72, and 96 h postfertilization (hpf). At the 24 hpf, most fertilized eggs exhibited coagulation and malformations across all nanoparticle concentrations. A significant increase in yolk-sac edema was noted in each AuNP sample, which preceded a notable rise in mortality at the higher exposure dose of 0.25 g/mL. Additionally, an increase in developmental abnormalities was observed, particularly affecting the snout, heart, and pectoral fin, as illustrated in Figure . By 48 hpf, a substantial number of viable embryos displayed deformities and lacked a heartbeat, categorizing them as nonviable. The positive control group consistently showed a mortality rate of 100% in all assessments. These findings align with those of Patibandla, who noted similar toxicity effects, and Stine, who also reported comparable outcomes concerning nanoparticle-induced toxicity on embryonic development.

6.

Apical observation of zebrafish embryo development acute toxicity test at 0.063 mg/mL concentration. Images were captured at approximately 40× total magnification.

Following a 96-h exposure period, the results indicated significant toxicity associated with pristine AuNPs, as depicted in Figure A. A notable increase in mortality was observed, particularly at high concentrations, with the highest mortality rate recorded at 0.25 mg/mL, reaching 81.6%. The mortality rates for concentrations of 0.063 mg/mL, 0.125 mg/mL, and 0.25 mg/mL were 61.1%, 47.63%, and 81.6%, respectively. Additionally, fish exposed to AuNP concentrations exceeding 0.063 mg/mL exhibited decreased swimming speed and twitching behavior. The minimum observable impact occurred at a concentration of 0.016 mg/mL, while the subsequent concentration of 0.031 mg/mL resulted in mortality rates of 10.02% and 19.08%, respectively. The lethal concentration 50 (LC50) for pristine AuNPs was determined to be 17.207 mg/mL. Comparative findings are reported in the study by Mikhailova, suggesting that the elevated toxicity is attributed to the chemical production process involving reducing agents, including sodium citrate.

7.

Mortality percentage in the zebrafish embryo toxicity test after 96 h exposure to (A) pristine gold nanoparticles (AuNPs), (B) heterocyclic compound FDM29-capped AuNPs, and (C) Baissea gracillima (liana)-capped AuNPs. Data represent Mean ± SD from triplicate experiments (n = 3), with 20 embryos per treatment group.

In the case of FDM29-capped AuNPs (Figure B), the highest mortality rate was 61.9% at a concentration of 0.25 mg/mL, with developmental delays and behavioral changes similar to those observed in embryos exposed to pristine AuNPs. The mortality rates at lower concentrations (0.063 and 0.125 mg/mL) were 15.05 and 26.4%, respectively. No apical observations were recorded at doses of 0.016 and 0.031 mg/mL. The minimum observed effect was identified at 0.063 mg/mL, resulting in a mortality rate of 15.05%. The LC50 for FDM29-capped AuNPs was calculated to be 0.2 mg/mL (±1.23). The presence of the FDM29 capping agent appeared to reduce the toxicity compared to pristine AuNPs, though embryos were still adversely affected at higher concentrations.

By contrast, B. gracillima (liana)-capped AuNPs (Figure C) exhibited the lowest mortality rates, with a maximum percentage of 14.76% at the highest concentration of 0.25 mg/mL. Toxicity levels remained minimal across all tested concentrations, with no noticeable effects observed at levels below 0.25 mg/mL. Additionally, mortality rates decreased by 2.56% at concentrations of 0.063 and 0.125 mg/mL compared to the negative control group, which had a mortality rate of 5%, suggesting a potential reduction in toxicity. However, it was not possible to calculate the LC50 for B. gracillima (liana)-capped AuNPs due to the lack of significant mortality even at the highest concentration tested. The findings align with the study by Machado, which indicates that the toxicity levels were generally minimal across the various concentrations tested using green synthesis.

To contextualize the concentrations used in this study (up to 0.25 mg/mL), we compared them with recent studies assessing the toxicity of other nanomaterials in zebrafish embryos. Silver nanoparticles, for instance, have induced developmental abnormalities such as axial defects and pericardial edema at concentrations as low as 0.1 mg/L. Similarly, zinc oxide nanoparticles synthesized via green methods caused increased mortality at concentrations above 10 mg/L, while selenium nanoparticles and titanium dioxide-cerium oxide nanocomposites exhibited toxicity at concentrations ranging from 5 to 20 mg/L. , These findings suggest that the exposure doses used in the study fall within the range commonly employed in nanotoxicity assessments and reflect comparable or lower levels of observed embryotoxicity.

4. Conclusions

In this study, AuNPs were successfully synthesized and subsequently coated using both FDM29 compound as well as B. gracillima (liana) leaf extract. These capping agents played a critical role in preventing aggregation, leading to nanoparticles with enhanced colloidal stability. The synthesized AuNPs were spherical, negatively charged, and within the nanometer range, with B. gracillima-capped AuNPs exhibiting the largest size, followed by FDM29-capped and pristine AuNPs. The nanoparticles indicated significant antibacterial activity against Klebsiella pneumonia, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus, while Proteus mirabilis and Mycobacterium smegmatis demonstrated resistance to all produced AuNPs. Notably, both FDM29- and B. gracillima-capped AuNPs display enhanced antibacterial efficacy compared to their respective uncapped forms, indicating a synergistic interaction between the AuNP core and the bioactive capping agents. This suggests that AuNPs can potentially enhance the effectiveness of FDM29 and B. gracillima (liana). Despite the efficacy of AuNPs capped with FDM29, B. gracillima-capped AuNPs exhibited the highest antibacterial performance. The commercially available standard AuNPs were not used as controls, as the study aimed on evaluating the biological efficacy of eco-friendly, plant-mediated AuNPs. However, including standardized nanoparticles in future studies could allow for broader comparative analysis. The toxicity evaluation using the zebrafish embryo model showed that pristine AuNPs were the most toxic, followed by FDM29-capped AuNPs, then B. gracillima-capped AuNPs demonstrating the lowest toxicity profile. Overall, these results confirm that functionalization of AuNPs with biological molecules can significantly enhance their antibacterial efficacy while mitigating their toxicity. The antibacterial activity of AuNPs was enhanced, while their toxicity was reduced upon capping. Although this study emphasized antibacterial activity, the potential antifungal properties of the green-synthesized AuNPs should not be overlooked, particularly given the broad spectrum of phytochemicals and known ability of metal nanoparticles to disrupt fungal cell integrity.

4.1. Ethics Approval

The study involving animals received ethical approval from the Faculty Ethics Committee at the University of Johannesburg under reference number 2023-07-12/Selala_Seopela, confirming compliance with the university’s ethical standards for animal research.

All data supporting the findings of this study are available within the manuscript and the Supporting Information files. Additional details are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03475.

• Chemical structure of FDM29 (Figure S1); Spectroscopic data of FDM29; ¹H NMR spectra and expanded view (Figures S2–S3); ¹³C NMR spectra and expanded view (Figures S4–S5); TEM micrographs of AuNPs, FDM29-AuNPs, and B. gracillima-AuNPs (Figure S6) (PDF)

This research was supported by the National Nanoscience Postgraduate Teaching and Training Platform (NNPTTP) of the Department of Science and Technology (DST), South Africa. The authors gratefully acknowledge this funding, which enabled the successful execution of the project.

The authors declare no competing financial interest.