Microwave-Assisted Biosynthesis of Silver Nanoparticles Using Chlorella sp. for Antibacterial and Cytotoxicity Effects of Breast Cancer Cell Line

TL;DR

This paper shows that using microwave-assisted methods with Chlorella algae can quickly make silver nanoparticles that are effective against bacteria and breast cancer cells.

Contribution

The novel contribution is the microwave-assisted biosynthesis method using Chlorella sp. for producing AgNPs with enhanced antibacterial and anticancer properties.

Findings

Microwave-assisted synthesis reduces reaction time to under 7 minutes and produces smaller, more uniform AgNPs.

AgNPs made with hexane extracts show strong antibacterial activity with minimum inhibitory concentrations as low as 0.31 µg/mL.

AgNPs exhibit concentration-dependent cytotoxicity in breast cancer cell lines, with MDA-MB-231 cells being more sensitive.

Abstract

Microwave-assisted biosynthesis using marine Chlorella sp. extracts provides a green and efficient route for the production of silver nanoparticles (AgNPs). Compared with the conventional method (24 h), microwave-assisted synthesis reduces the reaction time to less than 7 min while producing smaller and more uniformly distributed nanoparticles. AgNPs were synthesized using extracts obtained with different solvents and directly compared with those produced via the conventional method to substantiate the efficiency of the microwave-assisted approach. UV–visible spectroscopy confirmed rapid nanoparticle formation, exhibiting surface plasmon resonance peaks in the range of 405 to 427 nm. TEM analysis revealed predominantly spherical AgNPs with particle sizes of approximately 10 to 20 nm. The XRD and FTIR analyses confirmed their crystalline structure and stabilization by algal-derived functional groups. The biological activities of the AgNPs were dependent on the extraction solvent. AgNPs synthesized using hexane extracts exhibited pronounced antibacterial activity, achieving minimum inhibitory concentrations as low as 0.31 µg/mL. In addition, the AgNP induced concentration-dependent cytotoxic effects in human breast cancer cell lines. IC50 values, determined via dose–response analysis, ranged from 0.18 to 0.67 μg/mL in MDA-MB-231 cells and 1.70 to 8.42 μg/mL in MCF-7 cells. These results indicate a potent cytotoxic profile, with MDA-MB-231 cells exhibiting significantly higher sensitivity to the microwave-assisted formulations. Collectively, these findings highlight microwave-assisted algal-mediated biosynthesis as a sustainable and effective platform for generating bioactive AgNPs with promising antibacterial and anticancer potential.

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1. Introduction

Nanotechnology has enabled the controlled fabrication of metal nanoparticles with tunable physicochemical properties for biomedical applications [1,2,3,4]. Among the various bioresources, Chlorella sp. extracts have attracted growing interest for green synthesis due to their abundant bioreductive metabolites, including proteins and polysaccharides, and are well-recognized for their nutritional and functional properties, reflecting their rich bioactive profile and broad health-promoting potential [5]. However, conventional extraction methods often struggle to fully recover these intracellular components due to the robust nature of the microalgal cell wall. To address this, microwave-assisted processing provides an efficient alternative, promoting rapid volumetric heating and enhanced recovery of intracellular bioactives for nanoparticle synthesis [6]. This study advances the field by systematically evaluating how microwave-assisted extraction optimizes the liberation of Chlorella-derived stabilizing agents, resulting in silver nanoparticles (AgNPs) with superior uniformity and biological potency. These AgNPs exhibit potent antimicrobial activity primarily through membrane disruption, intracellular damage, and interference with essential biomolecules [2,7,8]. Their toxicity toward mammalian cells depends on particle size, surface chemistry, concentration, and exposure time, and previous studies have reported relatively low cytotoxicity at lower concentration ranges under controlled conditions [9,10,11]. In addition to their antimicrobial effects, AgNPs have demonstrated promising anticancer activity through mechanisms such as reactive oxygen species (ROS) overproduction, mitochondrial dysfunction, and apoptosis induction [12]. These effects have been widely explored using human breast cancer cell lines such as human triple-negative breast cancer (MDA-MB-231) and Michigan Cancer Foundation-7 (MCF-7), a human breast adenocarcinoma cell line, which serve as well-established in vitro models for evaluating nanoparticle-mediated cytotoxicity and elucidating cell death pathways [13,14].

In recent years, microwave-assisted synthesis has emerged as an efficient technique for nanoparticle production, enabling rapid fabrication with improved control over size and morphology [15]. Compared to conventional heating methods, microwave irradiation is reported to provide rapid and volumetric heating, which can shorten reaction time and influence particle formation [4]. This approach has been associated with reduced aggregation and improved particle dispersion in several studies [16]. AgNPs can be synthesized through various techniques, such as chemical reduction, photochemical reactions, thermal decomposition, radiation, electrochemical methods, microwave-assisted synthesis, and biological approaches [2]. AgNPs can be produced through multiple methods, including chemical, photochemical, thermal, electrochemical, and biological approaches [17]. This green synthesis approach not only reduces the reliance on hazardous chemicals but also enhances the biocompatibility of AgNPs, making them more suitable for biomedical applications.

AgNPs are extensively applied as nanocarriers for bioactive compound delivery, as well as in diagnostic and therapeutic biomedical applications. Their broad-spectrum antimicrobial, antiviral, and anticancer properties further underscore their potential for therapeutic use [18]. Among the various synthesis methods, biosynthesized AgNPs exhibit particularly strong antibacterial activity due to the presence of diverse bioactive compounds on their surface, which enhance their stability and biological interactions [5]. Algal extracts have been widely reported to act as effective reducing agents for metal ions, facilitating the formation of metallic nanoparticles through biologically mediated reduction processes [19]. In addition, microalgae function as efficient biofactories by actively absorbing and detoxifying metal ions, thereby promoting nanoparticle nucleation and subsequent crystallite growth [20]. Due to their rapid growth, ease of cultivation and manipulation, and high biomass productivity relative to higher plants, algae are widely recognized as efficient biological platforms for the environmentally friendly biofabrication of metallic and metal oxide nanoparticles [21]. Marine microalgae, particularly Chlorella, Isochrysis, and Chaetoceros, possess diverse biochemical profiles rich in metabolites associated with antioxidant defense and antibacterial activity, which contribute to their effectiveness as biological mediators in nanoparticle synthesis [22]. Correspondingly, algae-derived nanoparticles have demonstrated notable antimicrobial efficacy, highlighting their potential for environmentally sustainable and biocompatible nanomaterial production [23,24,25]. Building on these advantages, a wide range of microalgal species have been explored for the eco-friendly biosynthesis of silver nanoparticles, with Chlorella spp. receiving particular attention.

Chlorella spp. are widely recognized and consumed worldwide as functional food supplements due to their rich content of bioactive and nutritional components, including polyphenols, proteins, vitamins, minerals, dietary fibers, and chlorophyll [20,26]. These phytochemicals confer diverse bioactivities such as antioxidant, anticancer, anti-inflammatory, anti-hypertensive, and anti-diabetic effects [18]. Chlorella species are found in both freshwater and marine environments; however, the bioactive potential of marine strains remains comparatively underexplored. In addition, Chlorella spp. are routinely cultivated for aquaculture and human consumption, reflecting their scalability and established safety profile. Marine strains, in particular, remain relatively under investigated in microwave-assisted AgNP biosynthesis, and their distinct metabolite composition may influence nanoparticle formation and biological activity. This study aims to biofabricate AgNPs through a green biosynthetic approach using marine Chlorella sp. extracts prepared with ethanol, hexane, and acetone, employing both conventional and microwave-assisted synthesis methods. The physicochemical properties of the biosynthesized AgNPs were systematically characterized to evaluate their structural features, stability, and overall physicochemical behavior. The antibacterial efficacy of the AgNPs was evaluated against common pathogenic bacteria, including E. coli, P. aeruginosa, S. aureus, and B. subtilis. In addition, the cytotoxicity of AgNPs was assessed in the MDA-MB-231 and MCF-7 breast cancer cell lines, commonly used to evaluate nanoparticle toxicity, to provide insight into their safety profile and biomedical applicability.

2. Materials and Methods

2.1. Materials

Silver nitrate (AgNO_3_, 99.9%, analytical grade) was purchased from POCH (Gliwice, Poland). Sodium hydroxide (NaOH) was purchased from KemAus (Cherrybrook, NSW, Australia). Ethanol (99%) was obtained from Q Chemical (Bangkok, Thailand). Hexane (99%) and acetone (99%) were purchased from Macron Fine Chemicals (Center Valley, PA, USA). Dimethyl sulfoxide (DMSO) was purchased from RCI Labscan (Bangkok, Thailand). The marine microalga Chlorella sp. BIMS-PP0081 was obtained from the Institute of Marine Science, Burapha University (Chonburi, Thailand). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin were purchased from Gibco Invitrogen (Grand Island, NY, USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was purchased from TCI (Tokyo, Japan).

2.2. Preparation of Algal Aqueous Extracts

Chlorella sp. was cultured in Guillard’s F/2 medium [27] at ambient temperature under continuous white fluorescent illumination with an 18:6 h light–dark photoperiod. After 10 days, the cells were harvested by centrifugation using a Z513K centrifuge (Hermle Labortechnik GmbH, Wehingen, Germany) at 4200× g at 4 °C for 15 min. The harvested cell pellets were subsequently dried at 45 °C for 5 h and mechanically pulverized using a mortar and pestle. Five grams of the microalgal powder was mixed with 50 mL of different solvents: 95% (v/v) ethanol, 99% hexane, and 99% acetone and incubated at 25 ± 5 °C for 24 h. Ethanol, acetone, and hexane were selected to represent a polarity gradient. This allowed for the isolation of distinct phytochemical fractions—ranging from polar phenolic compounds and proteins to non-polar lipids—each playing a specific role in the reduction kinetics and the capping-mediated stabilization of the AgNPs. The extracts were then filtered through Whatman No. 1 filter paper and stored at 4 °C in the dark until further use.

2.3. Biosynthesis of Silver Nanoparticles via Algal Extracts

The biosynthesis of AgNPs using Chlorella sp. extracts was carried out using two different approaches: with and without microwave irradiation. For the conventional (non-microwave) method, 10 mL of algal extract was mixed with 90 mL of AgNO_3_ solution at concentrations of either 1 mM or 10 mM, hereafter referred to as 1 or 10, respectively. The reaction mixtures were incubated in the dark at 25 ± 5 °C for 24 h. For the microwave-assisted synthesis, the same reaction mixtures were subjected to microwave irradiation at a power of 800 W for five cycles, each consisting of 30 s of irradiation followed by a 50-s cooling interval. The microwave parameters were adapted and modified from the method reported by Torabfam and Yüce [3] and further adjusted according to the reaction volume and material characteristics used in this study. A control experiment was performed by mixing a AgNO_3_ solution (1 mM or 10 mM) with 0.1 M NaOH at a ratio of 1:4 (v/v, AgNO_3_:NaOH) under continuous magnetic stirring. This mixture was heated to 75 °C for 1 h, allowed to cool to room temperature, and subsequently stirred for 24 h to ensure complete reaction. A visual color change from pale yellow to dark brown in the solution confirmed the formation of AgNPs. After the synthesis and complete reduction of the silver particles, the AgNPs were collected by centrifugation at 4200× g for 30 min. After centrifugation, the supernatant was carefully discarded, and the nanoparticle pellets were washed twice with distilled water to remove residual silver ions and unreacted precursors before drying at 45 °C for 1 h. The designation and sample codes of all synthesized formulations are provided in Table 1.

2.4. Characterization of AgNPs

Twelve AgNP formulations were systematically characterized using physicochemical techniques.

2.4.1. UV–Vis Spectroscopy Analysis

The reduction of Ag^+^ ions was confirmed using a UV–Vis spectrophotometer (UV-1601; Shimadzu, Kyoto, Japan) by monitoring the Surface Plasmon Resonance (SPR) characteristics of the synthesized AgNPs. The absorption spectra were recorded over a wavelength range of 300–700 nm upon completion of the reaction. The transition of the reaction mixture from pale yellow to dark brown served as a visual indicator of nanoparticle formation. While time-resolved spectral measurements were not conducted, the endpoint SPR peaks provided confirmation of the characteristic metallic nature and stability of the resulting AgNPs.

2.4.2. Scanning Electron Microscopy (SEM) Analysis

The morphology of the AgNPs was analyzed by SEM, and their elemental composition was determined by EDX using a Quattro-S ESEM microscope (Thermo Fisher Scientific, Hillsboro, OR, USA). For morphological examination, field emission SEM was performed at 15 kV, while EDX analysis for elemental composition was operated at 20 kV. For SEM images, the samples were sputter-coated with about 15 nm of Au using a Polaron coater system to improve surface conductivity and minimize charging effects. The presence of Au peaks in the EDX spectra was attributed to the sputter coating and did not interfere with the identification of Ag as the primary elemental component.

2.4.3. Transmission Electron Microscopy (TEM) Analysis

The size and morphology of the synthesized AgNPs were further examined by TEM using an HT7700 microscope (Hitachi High-Tech, Tokyo, Japan). A drop of the AgNP suspension was placed onto a 200-mesh copper grid coated with a lacy carbon film and air-dried at room temperature before TEM observation. Particle size distribution was determined using ImageJ software (version 1.54; National Institutes of Health, Bethesda, MD, USA) by measuring at least 100 randomly selected nanoparticles from three independent TEM micrographs.

2.4.4. X-Ray Diffraction (XRD) Analysis

The crystalline structure of the synthesized AgNPs was examined by X-ray diffraction (XRD) using a D8-Discover diffractometer (Bruker, Karlsruhe, Germany). The instrument employed Cu Kα radiation in a θ-2θ configuration and was operated at 40 kV and 30 mA. Diffraction patterns were recorded over a range of 20° to 80°.

2.4.5. Fourier Transform Infrared (FTIR) Analysis

The AgNPs obtained after reduction were subjected to FTIR spectroscopy using an INVENIO-S spectrometer (Bruker, Mannheim, Germany) to identify the chemical bonds and functional groups associated with the AgNPs. FTIR measurement was performed using the potassium bromide (KBr) pellet method. The samples were finely ground and thoroughly mixed with dry KBr powder at an approximate ratio of 1:100 (w/w). The mixture was then compressed into a thin, transparent pellet using a hydraulic press. The pellet was analyzed in transmission mode, and the infrared absorption spectrum was recorded in the range of 400–4000 cm^−1^ with a resolution of 4 cm^−1^.

2.5. Antibacterial Activity

2.5.1. Preparation of Bacterial Inoculum

Four test pathogens, S. aureus TISTR 746, B. subtilis TISTR 1248, E. coli TISTR 074, and P. aeruginosa TISTR 2370, obtained from the Thailand Institute of Scientific and Technological Research (TISTR) Bangkok, Thailand culture collection, were cultivated on Tryptic Soy Agar (TSA) at 37 °C for 18 h [28]. All bacterial strains were cultured for 18 h before testing. For the direct suspension method, bacterial colonies were selected and suspended in sterile 0.85% (w/v) NaCl. The bacterial inoculum was adjusted to an optical density corresponding to approximately of 1.5 × 10^8^ CFU/mL (equivalent to a 0.5 McFarland standard) by measuring the absorbance at 600 nm spectrophotometer (OD_600_ = 1.0) using a spectrophotometer.

2.5.2. Well Diffusion Assay

The agar well diffusion method was used to evaluate the antibacterial activity of AgNPs synthesized from algal extracts. Bacterial cultures were evenly inoculated onto TSA plates using sterile cotton swabs. Redispersed AgNPs powder was prepared in 1% (v/v) DMSO at a concentration of 10 mg/mL and dispensed into the wells. AgNPs synthesized from AgNO_3_ in the absence of algal extracts were used as the comparative control. Streptomycin (2 mg/mL) was included as a positive control, while 1% (v/v) DMSO served as a negative control. After the plates were incubated at 37 °C for 20 h, the diameters of the inhibition zones were measured and expressed in millimeters.

2.5.3. Microdilution Assay

The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of all twelve synthesized AgNP formulations were evaluated against four bacterial strains using a broth microdilution method. Briefly, sterile 96-well microplates were prepared by adding 100 µL of growth medium, 50 µL of bacterial suspension (final concentration at 1.5 × 10^6^ CFU/mL), and 50 µL of AgNP solutions at serially diluted concentrations ranging from 5000 to 0.31 µg/mL (5000, 2500, 1250, 625, 312.5, 156.25, 78.12, 39.06, 19.53, 9.76, 4.88, 2.44, 1.22, 0.61, and 0.31 µg/mL). AgNPs synthesized without algal extracts served as the comparative control. The negative control comprised TSB supplemented with 1% (v/v) DMSO and bacterial inoculum, while streptomycin (2 mg/mL) in TSB containing bacterial inoculum was used as the positive control. The plates were incubated for 24 h at 37 °C. For the MBC determination, 100 µL was taken from each well after 24 h and cultured on agar medium for another 24 h at 37 °C. The MBC was defined as the lowest concentration of AgNPs that resulted in no observable bacterial growth on the TSA plates, indicating a 99.9% reduction in the initial bacterial population.

2.6. In Vitro Cytotoxicity

2.6.1. Cell Culture

For cell culture, the human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured and maintained in high glucose medium DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% of penicillin/streptomycin. The cell cultures were incubated in a CO_2_ incubator at 37 °C with 90% humidity and 5% CO_2_.

2.6.2. Cytotoxicity Assay

To determine cell cytotoxicity, all twelve synthesized AgNP formulations were evaluated using an MTT assay. MDA-MB-231 and MCF-7 cells were seeded into 96-well plates at a density of 5 × 10^3^ cells/well and incubated for 24 h to allow for cell attachment. After incubation, the cells were treated with redispersed AgNP powder (stock prepared in 1% (v/v) DMSO and diluted in DMEM). Prior to treatment, the suspension was vortexed for 1 min to ensure a homogeneous dispersion. The cells were then exposed to final concentrations of 100, 10, 1, and 0.1 µg/mL and incubated for an additional 24 h. Cell viability was subsequently evaluated using the MTT assay. Following the treatment period, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated at 37 °C for 4 h to allow for the formation of formazan crystals. The culture medium was then carefully removed, and the resulting formazan precipitates were solubilized in 100 µL of DMSO. Absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to the untreated control, which was defined as 100% viability, based on the reduction in optical density following AgNP exposure.

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2.7. Statistical Analysis

Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons within each experimental group. Data are presented as the mean ± standard deviation (SD) from at least three independent replicates. Statistical analysis was conducted using SPSS version 22.0; p < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Characterization of AgNP Formation

3.1.1. Visual Observation and UV–Visible Spectroscopy

Biological extracts inherently contain biodegradable and biocompatible constituents—such as proteins, peptides, polysaccharides, and pigments—that act as dual functional agents for the reduction of Ag+ ions and the stabilization (capping) of the resulting AgNPs [24].

In this study, the synthesis of AgNPs was investigated using Chlorella algal extracts prepared in three different solvents (ethanol, hexane, and acetone) mixed with silver nitrate solutions (1 mM and 10 mM) at a 1:9 (v/v, extract:AgNO_3_) ratio. Pure silver nitrate solutions at corresponding concentrations served as negative controls. The reduction of silver ions was visually confirmed by a distinct color change in the reaction mixture from pale yellow to dark brown. The brown coloration of the colloidal solution intensified over a 24-h reaction period, indicating the progressive formation of AgNPs and the excitation of surface plasmon resonance (SPR) vibrations characteristic of silver nanoparticles. UV–Vis spectral analysis confirmed the formation of AgNPs, revealing characteristic SPR absorption bands in the range of 405–427 nm for all samples (Figure 1). The position and intensity of the SPR peaks varied depending on the extraction solvent and synthesis method. As shown in Figure 1a, AgNPs synthesized using ethanolic extracts exhibited a consistent absorption peak centered at ~410 nm. No pronounced peak shifts were observed between the conventional and microwave-assisted methods, which may suggest a relatively uniform nanoparticle formation under these conditions. It should be noted that complementary analyses, such as DLS or zeta potential measurements, were not performed to further confirm colloidal size distribution or stability.

In contrast, AgNPs produced from hexane extracts (Figure 1b) displayed a red shift, with peaks ranging from 421 to 427 nm. This shift toward longer wavelengths suggests the formation of larger particles or increased aggregation. This finding aligns with Das et al. [29], who reported that broader peaks and red shifts in SPR bands are indicative of larger particle sizes or anisotropic growth. However, AgNPs from acetone extracts showed peaks between 408 and 423 nm. Crucially, microwave assisted synthesis resulted in sharper and more intense peaks compared to conventional methods. This enhanced peak sharpness indicates improved monodispersity (size uniformity) and crystallinity. As noted by Sajini and Joseph [30], microwave irradiation often provides better control over particle nucleation and growth compared to conventional heating, leading to superior dispersity. Regarding precursor concentration, samples prepared with 10 mM AgNO_3_ consistently exhibited higher absorbance intensities than those prepared with 1 mM AgNO_3_, indicating a higher yield of nanoparticle production. Overall, the UV–Vis data suggest that while Chlorella extracts are effective reducing agents across all solvents, the solvent type influences particle size, and microwave assistance specifically enhances the uniformity of AgNPs derived from acetone extracts.

3.1.2. SEM Analysis

The morphology of the biosynthesized AgNPs was examined using SEM. The SEM images revealed well-defined and predominantly spherical nanoparticles, with some exhibiting slightly rhomboidal features. The particle size ranged from approximately 10 to 20 nm (Figure 2). The relatively well-defined morphology may be associated with biomolecules present in the algal extract that functioned as reducing and capping agents during nanoparticle formation. As shown in the SEM images (Figure 2a,c), AgNPs synthesized without microwave irradiation exhibited more apparent agglomerated structures with relatively rough and compact surface features. This observation may indicate that particle nucleation and growth occurred more gradually under conventional heating conditions, allowing extended particle–particle interactions that contributed to cluster-like aggregation. It should be noted that SEM analysis was conducted on dried samples, and partial aggregation may also arise during sample preparation. In contrast, AgNPs synthesized under microwave irradiation (Figure 2b,d) displayed a finer and more homogeneous morphology, characterized by a dense but fine-grained texture and a reduced extent of agglomeration compared with the non-microwave counterparts. This behavior can be attributed to the unique characteristics of microwave irradiation, which provides rapid and volumetric heating, leading to burst nucleation within a short time frame. Such conditions favor the formation of smaller particles and limit prolonged growth and aggregation into larger clusters. The SEM observations further support that microwave-assisted synthesis improves structural uniformity and reduces particle agglomeration relative to conventional heating. Nevertheless, some degree of aggregation was still observed, which is commonly associated with AgNPs stabilized by organic biomolecules from algal extracts acting as capping agents, as well as interparticle interactions occurring during sample drying.

The morphological analysis was further supported by EDX, which confirmed the presence of silver (Ag) as the primary element, along with oxygen (O) and carbon (C), indicating organic residues from the biological extract. Minor signals corresponding to chloride (Cl) and sodium (Na) were also detected, likely originating from the algal extract or residual salts involved in the biosynthetic process. The detection of C and O suggests the presence of phytochemical-containing materials on the nanoparticle surface. EDX spectra further supported the successful formation of AgNPs, with a distinct peak around 3 keV that corresponded to the typical signal of metallic silver [31].

In addition to silver, peaks of carbon and oxygen were detected, likely originating from Chlorella metabolites such as proteins, polysaccharides, and phenolic compounds that remained associated with the nanoparticle surface. These biomolecules not only facilitate the reduction of silver ions but may also act as natural capping agents, thereby enhancing particle stability and biocompatibility [32]. The influence of microwave irradiation was evident, as rapid heating and shorter reaction times promoted faster reduction and more homogeneous nucleation, resulting in a relatively uniform size distribution compared with conventional methods. This observation is consistent with previous reports showing that microwave-assisted synthesis favors the formation of smaller and more stable AgNPs when biological extracts are used as reducing agents [33]. Collectively, the SEM and EDX analyses support the role of Chlorella sp. extract functioning as both a reducing and stabilizing agent, in agreement with earlier studies on algal-mediated nanoparticle synthesis [34,35]. Consequently, the antimicrobial performance of the AgNPs can be attributed to the combined effects of synthesis conditions, particle morphology, and surface-associated biomolecules, which collectively regulate silver ion release and interactions with microbial cell walls [36].

3.1.3. TEM Analysis

TEM micrographs revealed that the AgNPs synthesized using algal extracts were predominantly spherical in shape, although some quasi-spherical and rhomboidal shapes were also observed (Figure 3). The spherical morphology of AgNPs provides a larger specific surface area than nanorods or nanowires, thereby enabling more effective antimicrobial inhibition through enhanced interaction, penetration, and reaction with bacterial cell walls [37]. The particle sizes were polydisperse, with a measured range of approximately 3 to 70 nm and an average diameter of 11 nm, as determined using the ImageJ software. The broad distribution suggests that while most particles were in the lower nanometer range 10–20 nm, a proportion of larger aggregates was also present. At lower AgNO_3_ concentrations (1 mM), fewer nanoparticles were formed with a greater tendency toward aggregation. Increasing the concentration to 10 mM, particularly under microwave irradiation, appeared to enhance nucleation, resulting in higher particle density and more defined spherical morphologies. Compared with the conventional method, microwave-assisted synthesis produced smaller particles with improved dispersion. However, additional physicochemical analyses would be required to fully evaluate colloidal stability.

Previous studies have reported that AgNPs synthesized using different Chlorella species typically exhibit spherical morphologies with particle sizes ranging from several tens to a few hundred nanometers, depending on the algal strain and synthesis conditions. For instance, AgNPs derived from Chlorella ellipsoidea were reported to have spherical crystalline structures with relatively larger sizes (220.8 ± 31.3 nm) [38], while smaller spherical particles were obtained from Chlorella minutissima (73.13 nm) [39] and Chlorella pyrenoidosa (25–30 nm) [40]. In the present study, the AgNPs exhibited a comparatively smaller size range of approximately 10–20 nm with predominantly spherical morphology. This reduction in particle size may be associated with the application of microwave irradiation, which provides rapid and uniform heating, thereby facilitating faster nucleation and limiting excessive particle growth.

Importantly, nanoparticles below 20 nm are known to possess enhanced antibacterial activity due to their high surface-to-volume ratio and stronger interactions with microbial membranes. Therefore, the size range achieved in this study is particularly advantageous for potential biomedical applications [41]. The morphological diversity and size observed in TEM was characteristic of biosynthesized AgNPs, where biomolecules and synthesis parameters affect nucleation and growth [42,43]. The rapid and homogeneous heating provided by microwave energy likely enhanced the reduction kinetics of Ag^+^ ions, leading to faster nucleation and limiting particle overgrowth. This is consistent with earlier reports that demonstrated that microwave-mediated synthesis often produces nanoparticles with reduced size and improved dispersity compared to traditional approaches [33].

3.1.4. XRD Analysis

XRD analysis was carried out to determine the crystalline nature of the AgNPs synthesized from Chlorella ethanolic, hexane, and acetone extracts (Figure 4). The diffractograms revealed distinct diffraction peaks located at 2θ values corresponding to the (111), (200), (220), and (311) planes of face-centered cubic (fcc) silver. These reflections are consistent with standard diffraction data for metallic Ag, confirming that the biosynthesized nanoparticles possess a crystalline structure [44]. In addition to the major Ag reflections, several minor peaks were observed in some samples. These weak signals can be attributed to residual organic components or crystalline phases of secondary metabolites originating from the algal extracts, which may remain adsorbed on the nanoparticle surface and act as natural capping or stabilizing agents. Similar observations have been reported in previous studies employing biological extracts for nanoparticle synthesis [45].

When comparing conventional synthesis with the microwave-assisted method (M-labeled samples), differences in the appearance of the diffraction patterns were observed. In general, the microwave-assisted samples exhibited better-defined Ag diffraction features, particularly for the dominant (111) reflection, compared with their non-microwave counterparts. This observation can be associated with the rapid and homogeneous heating generated under microwave irradiation, which promotes fast nucleation and more uniform crystal formation within a short reaction time [33]. It should be noted that diffraction intensity is reported in arbitrary units (a.u.) and can be influenced by factors such as sample loading, packing density, and measurement conditions. Therefore, direct quantitative comparisons of peak intensity or peak width between different patterns should be interpreted with caution. Nevertheless, the observed variations among synthesis methods and extract types suggest that both the heating mode and the phytochemical composition of the algal extracts influence AgNP formation, surface capping, and the resulting crystalline features. Overall, the XRD results confirm that Chlorella extracts act effectively as both reducing and stabilizing agents in the biosynthesis of crystalline AgNPs, while microwave-assisted synthesis contributes to more clearly defined diffraction characteristics under rapid and uniform heating conditions [46,47].

3.1.5. FTIR Analysis

FTIR spectroscopy of the biosynthesized AgNPs revealed several characteristic peaks corresponding to functional groups of algal biomolecules responsible for reduction and stabilization (Figure 5). In the untreated extracts (Figure 5d), strong and broad absorption bands were detected at 3419–3467 cm^−1^, attributed to the -OH stretching of alcohols/phenols and -NH stretching of amines.

After AgNP formation, these bands decreased in intensity, suggesting the participation of hydroxyl and amine groups in Ag^+^ reduction. The peaks around 2913–2925 cm^−1^ and 2846–2854 cm^−1^ corresponded to C-H stretching of alkanes, while bands near 1630–1655 cm^−1^ were assigned to amide I (C=O stretching of proteins) and C=C of aromatic rings. These features indicate the involvement of proteins and polyphenols in stabilizing the nanoparticles [36,37]. Additional signals at 1380–1460 cm^−1^ and 1040–1116 cm^−1^ can be linked to C-N and C-O vibrations, confirming the contribution of amines and polysaccharides as capping agents. Importantly, microwave-assisted synthesis of AgNPs resulted in slight shifts in the hydroxyl, amide, and polysaccharide absorption bands compared with nanoparticles prepared via conventional heating, indicating alterations in functional group interactions during the reduction and capping processes. For example, the -OH stretching region appeared narrower and shifted to lower wavenumbers, while amide and carbohydrate peaks exhibited reduced intensity. These spectral modifications suggest that microwave irradiation enhanced the interaction between algal biomolecules and silver ions, leading to more efficient reduction and stronger binding of functional groups onto the nanoparticle surface. This agrees with previous findings that microwave-assisted processes accelerate reaction kinetics, promote uniform nucleation, and strengthen biomolecule–metal interactions [48].

FTIR confirmed that hydroxyl, amine, carbonyl, and polysaccharide groups from Chlorella sp. extracts played dual roles as reducing and capping agents during nanoparticle formation. The observed shifts and intensity changes in the M-labeled samples indicate that microwave irradiation not only reduced particle size, as confirmed by TEM, but also influenced the degree of biomolecule interaction with AgNPs. This effect likely contributes to the improved stability and potential biological activity of microwave-synthesized nanoparticles [48]. The presence of Chlorella biomolecules such as polysaccharides and polyphenols may further account for the nanoscale stabilization observed. These compounds likely acted not only as reducing agents but also as natural capping materials, preventing uncontrolled aggregation and maintaining particle stability [49].

The selection of extraction solvent is a critical factor influencing the phytochemical composition of algal extracts and, consequently, the nucleation and stabilization of silver nanoparticles. In this study, ethanol (polar), acetone (intermediate polarity), and hexane (non-polar) were chosen to selectively extract different classes of metabolites from Chlorella sp. Solvent polarity determines the abundance and activity of biomolecules involved in silver ion reduction and surface capping. Ethanol efficiently extracts polyphenols, proteins, and polysaccharides containing hydroxyl and amine groups that promote rapid nucleation and effective stabilization. In contrast, hexane primarily recovers lipophilic compounds with weaker reducing potential but possible steric stabilization effects, while acetone extracts a broader range of metabolites with moderate reducing capacity. These solvent-dependent differences are reflected in the FTIR band variations, indicating differential participation of biomolecules during nanoparticle formation. The influence of microwave irradiation further enhanced crystallinity and controlled particle growth, complementing the findings from the TEM and FTIR analyses. These results emphasize that microwave-assisted green synthesis offers an efficient strategy to obtain highly crystalline nanoparticles with potential advantages in stability and biological activity.

3.2. Antibacterial Activity of AgNPs

3.2.1. Agar Well Diffusion Assay

The antibacterial performance of biosynthesized AgNPs derived from Chlorella sp. extracts was assessed using the agar well diffusion assay (Figure 6), as well as MIC and MBC determination (Table 2). The agar well diffusion assay was employed to compare the effects of two AgNO_3_ concentrations (1 mM and 10 mM) and two synthesis approaches (conventional and microwave-assisted) on the antibacterial efficacy of AgNPs prepared from extracts obtained using three different solvents. The results indicated that increasing the AgNO_3_ concentration generally enhanced antibacterial activity, while AgNPs synthesized via the conventional method also exhibited comparable inhibitory effects, with no statistically significant differences observed between the two synthesis methods. Among all samples, the AgNPs synthesized using microwave irradiation with the ethanol extract at 10 mM AgNO_3_ (M10 ChloEt) exhibited the highest antibacterial activity, particularly against P. aeruginosa, producing a zone of inhibition of 20.41 ± 1.17 mm. This was followed by inhibitory effects against B. subtilis (16.71 ± 3.30 mm), S. aureus (12.84 ± 1.39 mm), and E. coli (12.56 ± 0.52 mm). Of all the formulations, P. aeruginosa consistently exhibited the greatest susceptibility, as evidenced by significantly larger inhibition zones compared with other bacterial species (p < 0.05), particularly for AgNPs synthesized via microwave-assisted methods. In contrast, E. coli generally displayed the lowest sensitivity, often showing significantly smaller inhibition zones than P. aeruginosa and B. subtilis, suggesting a relatively higher resistance to AgNP-mediated antibacterial activity.

Interestingly, although Gram-negative bacteria were generally more sensitive to AgNPs than Gram-positive strains, B. subtilis exhibited significantly greater susceptibility than E. coli for several formulations (p < 0.05). This observation may be attributed to differences in cell wall structure, surface charge, and interactions between bacterial envelopes and phytochemical residues capping the AgNPs, which can modulate nanoparticle adhesion and membrane disruption [50].

Furthermore, AgNPs synthesized via microwave-assisted methods produced significantly larger inhibition zones than their conventionally synthesized counterparts at equivalent AgNO_3_ concentrations and solvent systems (p < 0.05). This enhanced antibacterial performance is likely associated with the physicochemical characteristics of the microwave-assisted AgNPs, including smaller particle size, higher crystallinity, and improved surface functionalization, as evidenced by the SEM, TEM, and FTIR analyses. These features collectively promote stronger interactions with bacterial membranes, leading to increased antimicrobial efficacy.

3.2.2. MIC and MBC Analysis

The MIC and MBC results further support the agar diffusion findings (Table 2). MIC values of microwave-assisted AgNPs were as low as 0.31 µg/mL against P. aeruginosa and S. aureus, while MBC values were also reduced, in some cases reaching 0.31 to 1.22 µg/mL, particularly for M10 ChloEt and M10 ChloHe. In contrast, conventionally synthesized AgNPs required higher concentrations to achieve similar inhibitory effects. The ethanolic extract-derived AgNPs consistently showed the best antibacterial activity, suggesting that ethanol-extracted metabolites provided stronger reducing and stabilizing agents compared to hexane or acetone. These results align with earlier reports where microwave-assisted synthesis produced smaller and more bioactive nanoparticles than traditional methods [33]. Similar findings were described by Annamalai et al. [51], who observed concentration-dependent antibacterial effects of AgNPs, with higher AgNO_3_ concentrations leading to increased inhibition. The significant inhibition of P. aeruginosa observed in this study is also consistent with Ouardy et al. [52], who reported enhanced sensitivity of Gram-negative bacteria to algal-mediated AgNPs.

The antibacterial activity of AgNPs is influenced by their size, shape, surface charge, coating, and Ag^+^ ion release rate, with smaller nanoparticles (1–100 nm) providing greater interaction due to higher surface area [2]. Positively charged nanoparticle surfaces are known to promote interactions with bacterial cell membranes, facilitating membrane disruption and the generation of ROS, which ultimately lead to cellular damage [53]. Surface functionalization with microalgal derived biomolecules may further enhance bacterial targeting and adhesion. In the present study, an increase in AgNO_3_ concentration to 10 mM enhanced antibacterial activity, producing larger inhibition zones, particularly against Gram-positive bacteria. However, the potential contribution of released Ag^+^ ions cannot be excluded. The quantitative release of Ag^+^ under the bacterial assay conditions was not determined in this study and should be considered when interpreting the antibacterial results.

The antibacterial action of AgNPs is generally attributed to multiple mechanisms, including the release of Ag^+^ ions, ROS generation, membrane destabilization, interference with cellular metabolism, and inhibition of DNA replication [54]. Microwave-assisted synthesis facilitates uniform heating and accelerates nanoparticle formation, which may contribute to improved antibacterial efficacy. This observation is in line with the chemical composition of Chlorella sp. extracts, which contain bioactive compounds such as phenolics, flavonoids, unsaturated fatty acids, carotenoids, and chlorophyll derivatives. These compounds are known to exert antimicrobial activity by disrupting the bacterial cell wall and plasma membrane, leading to loss of integrity and growth inhibition. The inhibitory mechanism is likely to be the result of a synergistic effect of multiple compounds rather than a single constituent. In agreement, Manklinniam et al. [55] reported that ethanolic extracts of Chlorella exhibited stronger antibacterial activity than extracts obtained with highly polar or non-polar solvents, which is consistent with the present findings. The flavonoid yield varies with the plant species employed and is additionally influenced by both the type and quality of the solvent utilized during the extraction process [56,57]. The combined effect of metallic silver and algal biomolecules therefore enhances the antimicrobial efficacy of the biosynthesized nanoparticles.

3.3. Cytotoxicity

The biomedical relevance of algae-derived nanomaterials has been increasingly recognized, as algae provide bioactive secondary metabolites with reported anticancer potential [58]. The cytotoxic effects of biosynthesized AgNPs prepared using different capping strategies and AgNO_3_ concentrations were investigated in MDA-MB-231 cells using a cell viability assay. As shown in Figure 7, all AgNPs formulations exhibited a concentration-dependent reduction in cell viability. At lower concentrations of 0.1 and 1 µg/mL, limited cytotoxic effects were observed, with cell viability generally remaining between 70 and 80%. At these concentrations, no statistically significant differences were detected among most formulations (p > 0.05), indicating acceptable cytocompatibility at low doses. When the concentration was increased to 10 µg/mL, cell viability decreased significantly for all AgNP formulations (p < 0.05). However, the extent of cytotoxicity varied depending on the capping strategy employed. In particular, AgNPs capped with Chlorella extracts consistently retained higher cell viability compared with those synthesized using other capping methods at the same concentration (p < 0.05).

At the highest tested concentration of 100 µg/mL, a marked and statistically significant reduction in cell viability was observed for all AgNP formulations (p < 0.05), with viability values falling below 20% in most cases. This pronounced cytotoxic response suggests a dose-limiting effect at elevated AgNP concentrations and underscores the influence of surface capping on nanoparticle cell interactions. These findings indicate that Chlorella-capped AgNPs exhibit comparatively improved cytocompatibility toward MDA-MB-231 cells over a wide concentration range, particularly at moderate concentrations (≤10 µg/mL). This behavior may be attributed to the presence of bioactive phytochemicals on the nanoparticle surface, which could modulate Ag^+^ ion release and attenuate oxidative stress.

The cytotoxic effects of each sample on the breast cancer cell lines were concentration-dependent and varied among different formulations (Figure 8), as reflected by their half-maximal inhibitory concentration (IC_50_) values. Among all samples, 1ChloAc exhibited the highest IC_50_ value (8.42 µg/mL), indicating the lowest cytotoxic activity, whereas M10 ChloAc showed the lowest IC_50_ value (1.70 µg/mL), suggesting the strongest cytotoxic effect. Formulations prepared using microwave-assisted synthesis, particularly at higher extract concentrations, consistently demonstrated lower IC_50_ values compared with their non-microwave counterparts. The IC_50_ values of the AgNP controls (1–10 mM) were comparable to those of the most active microwave-assisted samples, further supporting the enhanced cytotoxic potential achieved through microwave-assisted biosynthesis.

The cytotoxicity of the biosynthesized AgNPs was also evaluated in MCF-7 breast cancer cells to assess cell line dependent responses. As presented in Figure 9, a similar concentration-dependent decrease in cell viability was observed for all AgNP formulations. However, the MCF-7 cells appeared to be more sensitive to AgNP exposure than the MDA-MB-231 cells. At a concentration of 0.1 µg/mL, treatment with biosynthesized AgNPs resulted in a moderate reduction in cell viability, with values generally ranging from approximately 55 to 65%. These values were significantly lower than those observed in MDA-MB-231 cells at the same concentration (p < 0.05), indicating increased susceptibility of MCF-7 cells to AgNP-induced cytotoxicity. Statistical analysis revealed significant differences among several formulations at this concentration, suggesting that capping strategy influenced cellular responses even at low doses. With increasing concentrations of 1 and 10 µg/mL, cell viability decreased further for all AgNP formulations (p < 0.05), although the magnitude of cytotoxicity varied depending on the capping agent employed. Notably, AgNPs capped with Chlorella extracts consistently maintained higher cell viability compared with AgNPs synthesized using other capping strategies at equivalent concentrations, with statistically significant differences observed in most cases (p < 0.05). This trend is consistent with that observed in MDA-MB-231 cells, indicating a reproducible protective effect associated with Chlorella-derived capping agents.

At the highest concentration tested (100 µg/mL), all AgNP formulations caused a pronounced decrease in cell viability, with values falling below 10% in most cases, confirming a strong dose-dependent cytotoxic response (p < 0.05). The greater sensitivity of MCF-7 cells compared with MDA-MB-231 cells may be related to intrinsic differences in cellular metabolism and membrane composition, which can influence nanoparticle uptake and susceptibility to AgNP-induced oxidative stress. Collectively, these results highlight the importance of both cell type and surface capping in modulating the cytotoxic effects of biosynthesized AgNPs.

The results indicate that AgNPs prepared under most conditions are biocompatible at lower doses, supporting previous reports that green-synthesized nanoparticles often display reduced cytotoxicity compared with chemically produced AgNPs. The consistently high cell viability in most groups (more than 80%) also suggests that phytochemicals from Chlorella extract may help stabilize the nanoparticles and mitigate their toxicity [59]. In this study, the relatively high cell viability observed in several formulations suggests that phytochemicals originating from Chlorella extracts may play a stabilizing role, limiting excessive Ag^+^ release and moderating nanoparticle–cell interactions. On the other hand, the marked toxicity in the M10 ChloEt group highlights how synthesis conditions strongly influence nanoparticle properties. Microwave synthesis with higher precursor concentration is known to generate smaller and more reactive particles, which can penetrate cells more easily and trigger oxidative stress [60].

The cytotoxic responses of MCF-7 to the tested formulations varied markedly, as reflected by their IC_50_ values (Figure 10). Among the algal-derived samples, M10 ChloAc exhibited the lowest IC_50_ value (0.18 µg/mL), indicating the strongest cytotoxic effect, whereas 1ChloEt showed the highest IC_50_ value (0.67 µg/mL), suggesting the weakest activity. In most formulations, microwave-assisted synthesis resulted in lower IC_50_ values compared with the corresponding non-microwave samples. In contrast, the AgNP control at 1 mM did not reach 50% inhibition within the tested concentration range (IC_50_ > 100 µg/mL), while 10 mM AgNPs exhibited substantially lower cytotoxicity compared with the most active algal-derived formulations.

AgNPs demonstrate anticancer activity primarily through ROS-mediated oxidative stress, which disrupts mitochondrial function and intracellular signaling pathways, ultimately leading to biomolecular damage, apoptosis, autophagy, and the suppression of angiogenesis [61]. In breast cancer cell lines such as MDA-MB-231 and MCF-7, tumor progression is highly dependent on angiogenesis-driven nutrient and oxygen supply; therefore, interference with these processes by AgNPs has been associated with reduced tumor growth [34]. The consistently lower IC_50_ values observed in MCF-7 cells relative to MDA-MB-231 cells may be explained by intrinsic differences in breast cancer subtypes. As an estrogen receptor positive cell line, MCF-7 exhibits greater vulnerability to ROS-induced mitochondrial dysfunction, whereas the more aggressive and stress-resistant MDA-MB-231 cells possess enhanced antioxidant defenses and pro-survival signaling pathways, requiring higher AgNP concentrations to achieve comparable cytotoxic effects. Moreover, the substantially higher IC_50_ values observed for uncapped AgNPs indicate reduced cytotoxic potency compared with Chlorella-mediated AgNPs. This attenuation is likely attributable to the absence of phytochemical-derived surface functionalization, resulting in diminished cellular uptake, less efficient Ag^+^ release, and the lack of synergistic interactions that enhance anticancer efficacy.

In the present study, cytotoxicity assessment was limited to breast cancer cell lines, and normal mammalian cells were not included for direct comparison. Therefore, therapeutic selectivity and biosafety toward non-cancerous cells remain to be further investigated. Nevertheless, previous reports have indicated that appropriately stabilized AgNPs may exhibit selective cytotoxicity toward cancer cells compared with normal cells, which has been attributed to differences in metabolic activity, redox balance, and susceptibility to ROS-mediated damage [62]. Cancer cells generally maintain elevated basal oxidative stress levels and are therefore more vulnerable to additional ROS accumulation, whereas normal cells possess more balanced antioxidant defense systems. Future investigations incorporating normal mammalian cell models are necessary to better define the safety profile and therapeutic window of the synthesized nanoparticles.

The biological responses observed in this study may also be influenced by the intrinsic bioactivity of Chlorella sp. extracts. Marine Chlorella extracts have been reported to exhibit concentration-dependent antioxidant and cytotoxic effects [63], suggesting that algal-derived metabolites may contribute to the overall cytotoxic response in addition to serving as stabilizing agents. Thus, the combined effects of nanoparticle physicochemical properties and algal bioactive compounds likely account for the concentration- and synthesis-dependent cytotoxicity observed. Notably, Chlorella-mediated AgNPs demonstrated favorable cytocompatibility at lower concentrations; however, careful optimization of synthesis parameters, particularly precursor concentration under microwave-assisted conditions, is essential to maintain an appropriate balance between anticancer efficacy and cellular safety.

4. Conclusions

The results confirm that extracts derived from Chlorella sp. are capable of facilitating the environmentally friendly fabrication of AgNPs with well-defined crystallinity and stability while simultaneously providing both reduction and surface stabilization during nanoparticle formation. Comprehensive characterization using UV–Vis, SEM/EDX, TEM, FTIR, and XRD confirmed the nanoscale morphology, crystalline nature, surface plasmon resonance, and biomolecule-mediated capping of the particles. Microwave-assisted synthesis yielded smaller and more uniformly distributed AgNPs with enhanced crystallinity, which was reflected in superior antibacterial activity, particularly against P. aeruginosa. Most AgNP formulations maintained high biocompatibility with MDA-MB-231 and MCF-7 cells at lower concentrations, although cytotoxicity was markedly increased when higher silver salt concentrations (10 mM) were employed under microwave conditions. Taken together, these results highlight algae-mediated, microwave-assisted synthesis as a sustainable and efficient strategy for producing bioactive AgNPs while underscoring the importance of optimizing the starting material concentration and synthesis parameters to achieve a balance between antimicrobial performance and cytocompatibility.