Plant-derived gold nanoparticles functionalized with pheophorbide-a for potent photodynamic therapy against A549 lung cancer cells.

Introduction
Cancer is characterized by the uncontrolled proliferation of abnormal cells that can invade nearby tissues and spread to distant parts of the body1. Despite the development of innovative diagnostic and treatment approaches, cancer remains a leading cause of illness and death in both men and women. Lung cancer is an increasingly prevalent global health concern, associated with high morbidity and mortality. According to the World Health Organization, the estimated number of new lung cancer cases is projected to increase from 2.48 million in 2022 to 4.25 million by 20452. Lung cancer is histologically classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)3. Typically, advanced non-small cell lung cancer carries a poor prognosis, necessitating the development of innovative methods to enhance patient survival rates. The aggressive nature and rapid progression of certain tumors pose significant challenges to current treatment approaches4. However, the efficacy of many existing cancer therapies is limited by non-specific targeting and adverse side effects, which constrain the maximum tolerable dose5.
PDT is an emerging treatment modality demonstrating promise in treating various malignancies, including breast, lung, and skin cancers. PDT employs photosensitizers, that trigger the production of reactive oxygen species (ROS) in presence of molecular oxygen upon laser irradiation. Within tumor cells, ROS inflict oxidative stress on various cell organelles, including the nucleus and mitochondria, leading to targeted damage6. PDT provides several benefits compared to traditional treatments, such as precise targeting, reduced systemic toxicity, and protection of nearby healthy tissue. However, its effectiveness can be restricted by challenges like limited light penetration and low oxygen levels within tumors. Despite these drawbacks, ongoing advancements in photosensitizer development and delivery systems are enhancing PDT’s potential, especially for treating localized and surface-level tumors, including those found in the breast, lung, skin, and oesophagus7–9. To overcome these hurdles, different strategies have been explored to improve the targeting efficiency of PDT. One such approach utilizes nanostructured drug carriers that can passively transport photosensitizers to tumor sites by taking advantage of the enhanced permeability and retention effect10,11. Additionally, plant-derived compounds are being actively investigated for their potential as bioactive agents and novel photosensitizers in cancer therapy. Dicoma anomala (D. anomala), an African medicinal plant, has traditionally been employed in treating diverse ailments and is now under scrutiny for its cancer therapeutic potential12.
In this present study, AuNPs were synthesized from Dicoma anomala through an environmentally friendly green synthesis method and subsequently conjugated with the photosensitizer Pheophorbide-a (Pheo-a) using the thin-film hydration technique. Both the nanoparticles and the resulting nanocomplex underwent thorough characterization, and their cytotoxic potential was assessed in vitro against A549 lung cancer cells under 660 nm laser irradiation at a fluence of 10 J/cm².The selection of a 660 nm wavelength is especially ideal for PDT because it lies within the red-light spectrum, enabling deeper penetration into tissues. This property is crucial for effectively reaching tumours embedded deeper within tissues, thereby improving PDT effect. Pheo-a, the photosensitizer employed in this research, exhibits a strong absorption near 660 nm, which facilitates its activation and promotes the production of ROS. These ROS are responsible for triggering cell death in cancerous tissues.
Additionally, red light undergoes less scattering in biological tissues, which improves the accuracy of PDT by concentrating the treatment on tumor cells and reducing damage to surrounding healthy tissue13–15. The focus of many cancer studies has shifted towards combining more than one therapeutic modalities to enhance efficacy while minimizing the side effects16. Despite the clinical promise of PDT, its broader application in lung cancer remains limited by inadequate photosensitizer delivery, insufficient intracellular ROS generation, and concerns regarding off-target toxicity. Although nanoparticle-based platforms have been explored to address these challenges, many reported systems rely on chemically intensive synthesis routes or lack sufficient selectivity and stability for effective clinical translation17. Moreover, the potential of green-synthesized metal nanoparticles as multifunctional PDT enhancers remains underexplored, particularly in lung cancer models.
In this study, we address this gap by integrating eco-friendly, plant-mediated gold nanoparticles with Pheo-a to construct a stable and biocompatible nanoplatform that enhances light-triggered ROS generation. The AuNPs-Pheo-a conjugate demonstrates improved photodynamic efficacy and selectivity under laser irradiation, while maintaining minimal dark toxicity, thereby reducing damage to surrounding healthy tissues. This work establishes a sustainable and optimized nanotechnology-based strategy that advances PDT performance and provides a practical framework for improving treatment outcomes in lung cancer, including the potential for application in deeper-seated tumors.

Methodology

Plant material acquisition, taxonomic identification, and extraction of D. anomala

Dicoma anomala leaves were collected from the eastern highlands of Zambia (13.6445° S, 32.6447° E). The plant material was authenticated by the Zambia Agriculture Research Institute (ZARI), and its identity was confirmed through an official phytosanitary certificate (SR No: 0006064). A voucher specimen (No. 92040/27/10/2020) has been deposited at the Msekera Research Station herbarium in Zambia18. The collection of Dicoma anomala was conducted under official authorization from ZARI, with all required permits and collection licenses obtained in full compliance with national biodiversity and phytosanitary regulations. The formal botanical identification of the plant was performed by a qualified botanist at the Msekera Research Station, ZARI, P.O. Box 510,089, Chipata, Zambia. This study adhered to the International Union for Conservation of Nature (IUCN) Policy Statement on Research Involving Species at Risk of Extinction and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and Dicoma anomala is not listed as a threatened or endangered species.
The dried leaves were powdered and subjected to extraction using a Soxhlet apparatus with methanol, followed by vacuum drying. For experimental procedures, stock solutions were prepared by dissolving approximately 10 mg of the extract in 10 mL of 1X phosphate-buffered saline (PBS), resulting in a final concentration of 1 mg/mL. Subsequent working concentrations were determined using the formula C1V1 = C2V2.

Eco-friendly synthesis of gold nanoparticles
The green synthesis of AuNPs was performed using a methanolic leaf extract of D. anomala under controlled temperature conditions, following a modified bottom-up method as described by Chota et al.12. To prepare the gold precursor solution, 17 µL of 30% (w/w) HAuCl₄·3 H₂O (Gold(III) chloride trihydrate; Sigma-Aldrich, South Africa) was diluted in 50 mL of deionized water to obtain a final concentration of 0.1 g/L, corresponding to an approximate molar concentration of 2.5 × 10⁻⁴ M (0.25 mM).The synthesis of AuNPs involved mixing 10 mL of this gold ion solution with 1 mL of the plant extract, followed by continuous stirring at 80 °C for 60 min using a magnetic stirrer. The solution changed color to a deep purple or red, indicating nanoparticle formation.
The extract, prepared at a concentration of 1 mg/mL in dd.H2O, acted as a reducing and stabilizing agent for the synthesis of AuNPs. A visible colour change indicated the successful formation and completion of the nanoparticle synthesis reaction. To eliminate any excess plant extract, the resulting solution was centrifuged multiple times at 8000 rpm for 30 min using ddH2O. This was followed by additional washes with 100% ethanol (EtOH) to ensure complete removal of residual water. The resulting pellet was resuspended in ddH₂O, and the gold ion concentration in the solution was measured using a UV-Vis spectrophotometer (Genova 7315 Life Science Spectrophotometer, JENWAY, Staffordshire, UK).

Formulation of AuNPs-Pheo-a nanoconjugate
The synthesis of the AuNPs-Pheo-a nanoconjugate was carried out using a modified thin-film hydration technique. First, AuNPs were placed in a round-bottom flask, and a uniform thin film was created through rotary evaporation at 40 °C for 30 min to gently remove the organic solvent. Afterwards, 1 mM stock solution of Pheo-a was slowly added to the AuNP thin film to promote even distribution and avoid particle aggregation. Pheo-a was introduced at a controlled rate of 1 µL per second while the mixture was continuously stirred at 55 °C, allowing for optimal binding and incorporation of Pheo-a into the AuNPs structure. The method employed is indeed thin-film hydration. Lipid/bilayer-forming components were included in the formulation, and the solvent was evaporated to form a thin film, which was subsequently hydrated to produce the vesicles.
This gradual addition and constant stirring produced a concentrated AuNPs-Pheo-a nanocomplex with a uniform composition. The final assembly involved combining AuNPs, and Pheo-a in 1:1 ratio. The nanoconjugate was kept in liquid form and stored at 4 °C to maintain stability. Keeping the nanoconjugate in a liquid state helps preserve both the structural integrity of the AuNPs the functional properties of the Pheo-a.

UV-vis spectroscopy and determination of entrapment efficacy of Pheo-a in AuNPs-Pheo-a nanoconjugate
The absorbance spectra of the green-synthesized AuNPs conjugated with Pheo-a were analysed using a Genova 7315 Life Science Spectrophotometer (JENWAY, Staffordshire, UK) at the Laser Research Centre, University of Johannesburg. Measurements were taken at room temperature across a wavelength range of 300–800 nm.
The concentration of Pheo-a bound to the AuNPs (entrap efficiency) was measured by analysing the absorbance of the AuNPs-Pheo-a nanoconjugate at 680 nm. Centrifugation was employed as a purification step to effectively separate unbound Pheo-a from the nanoconjugate, ensuring that the final preparation contained only the Pheo-a-loaded nanoconjugates. Thereafter, the obtained UV-Vis spectra were plotted using OriginLab Pro 2018 v9.5.1.
A standard calibration curve was generated using absorbance values from Pheo-a solutions with concentrations ranging between 2 and 20 µg/mL at 680 nm. This was used to determine the concentration of Pheo-a, where (y) corresponds to the absorbance of the sample at 680 nm and (x) represents the calculated Pheo-a concentration.
The Entrapment efficiency (EE) was calculated using the formula19:

Fourier transform infrared spectroscopy (FT-IR) for functional group analysis
FT-IR spectroscopy was employed to identify the functional groups present in AuNPs, Pheo-a, and the AuNPs-Pheo-a conjugate. Spectra were recorded using a PerkinElmer Spectrum 2000 FT-IR spectrophotometer (USA) over the range of 500–4000 cm⁻¹ with a resolution of 4 cm⁻¹. For sample preparation, approximately 3 mL of each solution was first frozen at − 80 °C for 24 h and subsequently freeze-dried for 8 h using an Olabo-FD10S Tabletop Freeze Dryer (Axiology Labs Pty Ltd, University of Johannesburg). The resulting powders were mixed with high-purity (99.9%) FT-IR grade KBr, compressed into thin pellets, and evenly coated with the sample. FT-IR spectra were then recorded to characterize the functional groups and confirm the successful conjugation of Pheo-a onto AuNPs.

Dynamic light scattering (DLS), zeta potential analysis scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS)
DLS was employed to determine the particle size distribution of the nanoparticles, while Zeta potential analysis was used to evaluate their surface charge and electrostatic stability. Both measurements were carried out using a Malvern Zetasizer (version 7.03, Serial No. MAL1049730, Malvern Instruments Ltd., UK). Prior to analysis, the nanoparticle suspension was appropriately diluted in dd. H₂O and subjected to sonication for approximately 1 h to reduce aggregation. Sonication was performed under controlled conditions (pulsed mode and low amplitude) to minimize thermal effects. Similar sonication durations have been reported to improve nanoparticle stability and surface functionalization without inducing significant changes in particle size or morphology7,19,20. The sonicated samples were then vortexed thoroughly to ensure uniform dispersion.
For measurement, 1 mL of the prepared sample was transferred into a disposable folded capillary cell cuvette. The readings for both DLS and Zeta potential were recorded at a controlled temperature of 25 °C and a fixed detection angle of 12°. These analyses provided insight into the colloidal stability and size distribution of the synthesized nanoparticles.
The surface morphology and elemental composition of the AuNPs-Pheo-a were examined using SEM and EDS. For sample preparation, the AuNPs-Pheo-a formulation was freeze-dried for approximately 8 h using an Olabo-FD10S Tabletop Freeze Dryer (Axiology Labs Pty Ltd, University of Johannesburg). The dried sample was then mounted onto carbon tape and coated with a thin conductive carbon layer using an automated carbon cord coater (Q150T E, University of Johannesburg) for about one minute. The carbon-coated sample was placed on the SEM stage for imaging and elemental analysis. SEM and EDS were performed under high vacuum at room temperature using a TESCAN VEGA3 SEM (Department of Metallurgy, University of Johannesburg), operated at an accelerating voltage of 20 kV.
TEM was performed to analyse the particle size and morphology of AuNPs-Pheo-a at the nanoscale level. The analysis was conducted using the FEI Tecnai T12 TEM at the Microscopy and Microanalysis Unit (MMU), University of the Witwatersrand, Johannesburg.
For high-resolution imaging, a small volume of freshly prepared AuNPs-Pheo-a suspension was diluted with deionized water to promote dispersion. The diluted sample was sonicated to minimize aggregation and ensure a uniform nanoparticle suspension. A single drop of the sample was carefully placed on a carbon-coated copper mesh grid (Lot #1240,227, SPI Supplies, Protea Laboratory Solutions Pty Ltd, Johannesburg, South Africa) and allowed to air-dry at room temperature. Once completely dried, the grids were loaded into the TEM for image acquisition. The handling of carbon-coated copper grid was carried out according to the manufacturer’s guidelines to preserve sample integrity to achieve optimal imaging results.

Cell culture and treatment
A549 cell line obtained from ATCC were grown in a T175 culture flask using Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Amphotericin B, and 1% Penicillin-streptomycin. The cells were then incubated at 37 °C, 85% humidity, and 5% CO2. Experimental cells were seeded at a density of 3 × 105 in a 3.4 cm2 diameter culture plates. The cells were treated with AuNPs-Pheo-a with various concentrations (i.e., 20, 40, 60, 80, and 100 µg/mL). Photodynamic therapy experiments were performed using 660 nm diode laser at 10 J/cm2 fluency.

Cell morphology
Morphological changes of the cells were evaluated after 24 h treatment with different concentrations of AuNPs-Pheo-a by using an inverted light microscope (Wirsam Olympus CKX 41).

Adenosine triphosphate (ATP) cell viability analysis
Cell proliferation and ATP levels in A549 cells were assessed using the CellTiter-Glo 3D luminescence assay (Promega, G968A). To induce cell lysis, 50 µL of reconstituted ATP reagent was mixed with an equal volume of cell suspension. The mixture was then incubated in dark at room temperature for 10 min. Subsequently, ATP luminescence was measured using the PerkinElmer VICTOR Nivo microwell plate reader.

MTT cell viability assay and selectivity index (SI) evaluation
Cell viability was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. A549 lung cancer cells and WS-1 normal fibroblast cells were seeded in 96-well plates at a density of 3 × 10⁵ cells/mL and allowed to attach for 24 h under standard culture conditions. Following attachment, cells were treated with increasing concentrations of AuNPs-Pheo-a and incubated under dark conditions or subjected to PDT by laser irradiation, as per the experimental design.
After treatment, the culture medium was replaced with fresh medium containing MTT reagent, and the cells were further incubated for 4 h to allow mitochondrial succinate dehydrogenase in viable cells to reduce the tetrazolium salt to insoluble purple formazan crystals. Subsequently, the formed formazan was solubilized, and the absorbance was measured at 590 nm using a Perkin-Elmer VICTOR3 microwell plate reader. Cell viability was expressed as a percentage relative to untreated control cells.
Dose response curves were generated to determine the half-maximal inhibitory concentration (IC₅₀) values for both cancer and normal cell lines. The selectivity index (SI) was calculated using the formula SI = IC₅₀ (normal cells) / IC₅₀ (cancer cells), providing a quantitative measure of the preferential cytotoxicity of AuNPs-Pheo-a mediated PDT toward cancer cells.

Reactive oxygen species (ROS) assay
To evaluate the PDT-induced oxidative stress, intracellular ROS levels were quantified in A549 cells treated with AuNPs-Pheo-a under dark and laser irradiation conditions using the ROS-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, A549 cells were seeded and allowed to attach overnight, followed by treatment with varying concentrations of AuNPs-Pheo-a. After the designated incubation period, cells assigned to the PDT groups were exposed to laser irradiation, while dark-control groups were kept under identical conditions without light exposure.
Following treatment, both control and experimental groups were washed three times with ice-cold PBS and then incubated at 37 °C for 30 min in serum-free medium containing 10 µM DCFH-DA. After incubation, cells were harvested by trypsinization, collected by centrifugation, and resuspended in PBS. The cell suspensions were transferred to a black 96-well plate, and fluorescence intensity was measured using a VICTOR Nivo multimode plate reader at excitation/emission wavelengths of 485/535 nm. The measured fluorescence intensities were normalized to control, and ROS levels were expressed as fold changes relative to untreated control cells.

Live dead assay
The distribution of viable and non-viable A549 cells was qualitatively assessed 24 h post-treatment with the AuNPs-Pheo-a and AuNPs-Pheo-a mediated PDT, following the manufacturer’s instructions in the LIVE/DEAD assay kit (Cat. No. L3224, Life Technologies Corporation). Briefly, the cells (both untreated and treated) were washed three times with ice-cold 1X PBS (1 mL) and resuspended in 1 mL of 1X PBS. They were then stained with calcein (1 µL) and ethidium homodimer-1 (EthD-1) (1 µL) and incubated for 30 min at room temperature. After incubation, the cells were rinsed three times with 1 mL 1X PBS, resuspended in 1 mL 1X PBS, and visualized using Alexa Fluor 488 and EtBr filters on a Carl Zeiss Axio Z1 live imaging microscope.

Flow cytometry assay
Cell death mechanisms were assessed using an Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) kit (BD Pharmingen), which relies on the binding of Annexin V-FITC and PI to nucleic acids and translocated phospholipid phosphatidylserine proteins. After 24 h of treatment, A549 cells were detached from 3.4 cm² culture plates using 300 µL of TrypLE and incubated for 5 min at 37 °C. The detached cells were then suspended in 500 µL of ice-cold 1X PBS and washed three times by centrifugation at 2200 rpm for 4 min at 4 °C. The resulting pellet was resuspended in 500 µL of 1X binding buffer.
Next, 100 µL of the cell suspension was transferred to sterile flow cytometry tubes, and 5 µL each of Annexin V-FITC and PI were added. The tubes were incubated in the dark for approximately 15 min. After incubation, 400 µL of ice-cold 1X binding buffer was added to each tube, and the mixture was incubated for an additional 30 min in the dark. Finally, the mechanisms of cell death were evaluated using a Becton Dickinson (BD) Accuri C6 flow cytometer.

Statistical analysis
A549 lung cancer cells at passages 18–22 were utilized for all experiments; each conducted in triplicate (n = 3). Mean values from experimental groups were compared to those of untreated control cells. Statistical significance between control and treated groups was assessed using one-way ANOVA with a 95% confidence interval. Significance levels were indicated as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). All statistical analyses were carried out using SPSS version 27, and graphical representations were generated with OriginLab Pro 2018 (v9.5.1).

Results and discussion

UV-vis spectrometry and determination of entrapment efficacy EE% of Pheo-a in AuNP-Pheo-a nanoconjugate
To investigate the absorbance spectra of the plant extract, Pheo-a, and the green-synthesized AuNPs from D. anomala, we employed UV-vis emission spectroscopy. The analysis revealed a notable surface plasmon resonance peak for AuNPs at 540 nm, as shown in Fig. 1A, and a pheo-a peaks at 370 nm and 660 nm. In contrast, the plant extract displayed no peak within the PDT therapeutic window. Figure 1B illustrates the different laser parameters used for PDT irradiation.
The entrapment efficiency of Pheo-a within the nanoconjugate system was calculated based on the concentration of the photosensitizer determined from the standard calibration curve as shown in Fig. 1D. A Pheo-a concentration of 19 µg/mL was obtained in a 1 mL sample, corresponding to a total loaded amount of 0.019 mg. To achieve an entrapment efficiency of Pheo-a, the required mass of nanoconjugate was calculated using the formula as mentioned above. Based on this calculation, 0.0475 mg of nanoconjugate was sufficient to encapsulate 0.019 mg of Pheo-a, resulting in an entrapment efficiency of 39.1 ± 2.34%. This high entrapment efficiency suggests successful incorporation of the photosensitizer into the nanoconjugate system and highlights the potential of this delivery platform for effective photodynamic therapy applications.
A standard calibration curve was generated using absorbance values from Pheo-a solutions with concentrations ranging between 2 and 20 µg/mL at 680 nm as shown in Fig. 1C. The linear equation derived from this curve (y = 0.0202x − 0.1513, with an R² of 0.9536) was used to determine the concentration of Pheo-a, where (y) corresponds to the absorbance of the sample at 680 nm and (x) represents the calculated Pheo-a concentration by using the formula as mentioned above in Sect.  2.5.

Scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS) dynamic light scattering (DLS) and zeta potential analysis
Scanning Electron Microscopy (SEM) was utilized to examine the size, shape, and surface morphology of the biogenically synthesized AuNPs-Pheo-a. The SEM analysis demonstrated that the nanoconjugates exhibited a well-defined morphology, with an average particle size of approximately 96 ± 3.2 nm (Fig. 2). Elemental analysis through EDS revealed prominent peaks corresponding to carbon (C), oxygen (O), phosphorus (P), nitrogen (N), and gold (Au), confirming the successful functionalization of gold nanoparticles with Pheophorbide-a.
Further morphological characterization was carried out using Transmission Electron Microscopy (TEM), which showed that although biogenic AuNPs are primarily spherical, additional shapes such as hexagonal and irregular structures were also observed. The HR-TEM images displayed a heterogeneous mixture of particle shapes and an average particle size of 88.73 nm, highlighting the polydispersity and complex morphology commonly associated with biogenically synthesized nanoparticles.
In this study, the nanoconjugates were characterized for their size and surface charge using Dynamic Light Scattering (DLS) and Zeta potential analysis, as presented in Fig. 2. The AuNPs demonstrated a hydrodynamic diameter of 96 ± 3.2 nm. It is well established that DLS measurements often yield larger hydrodynamic sizes compared to TEM, primarily due to the influence of the surrounding solvent and nanoparticle surface interactions. Consequently, the particle sizes observed through TEM were smaller than those measured by DLS.
The synthesized nanoconjugate exhibited a zeta potential of − 29.5 mV, indicating good colloidal stability due to strong surface charge repulsion. Additionally, the polydispersity index (PDI) of 0.219 suggests a moderately uniform particle size distribution.

Fourier transform infrared spectroscopy (FT-IR) for functional group analysis
As illustrated in Fig. 3, FT-IR spectroscopy confirmed the successful formation of AuNPs-Pheo-a by identifying characteristic functional groups associated with both AuNPs and Pheo-a. Free Pheo-a exhibits characteristic bands of the porphyrin framework, including a broad absorption around 3436 cm⁻¹ attributable to O–H/N–H stretching, bands near 2961 cm⁻¹ due to aliphatic C–H stretching, and a prominent band at ~ 1629 cm⁻¹ assigned to C = O and/or C = C vibrations of the conjugated macrocycle. Additional features in the fingerprint region (≈ 1384 and 644 cm⁻¹) are associated with C–N stretching, CH bending, and out-of-plane porphyrin ring deformations. The AuNPs spectrum shows a broad O–H stretching band at ~ 3435 cm⁻¹ arising from surface-adsorbed water or stabilizing agents, C–H stretching bands at ~ 2920 and 2850 cm⁻¹ from surface ligands, and bands in the 1124–1385 cm⁻¹ region corresponding to C–O/C–N vibrations of capping molecules, confirming nanoparticle stabilization. Upon conjugation, the AuNPs–Pheo-a spectrum retains several characteristic Pheo-a bands but with noticeable shifts and intensity changes, notably in the O–H/N–H (~ 3449 cm⁻¹), carbonyl/amide (~ 1745 and ~ 1634 cm⁻¹), and fingerprint regions, indicating interaction of Pheo-a functional groups with the AuNP surface. Overall, the spectral shifts and emergence of new bands in the conjugate confirm successful chemical interaction between Pheo-a and AuNPs rather than simple physical mixing.

Morphological analysis
The morphology of both untreated and treated A549 cells is depicted in Fig. 4A. After 24 h post-treatment, notable morphological changes were observed in the A549 lung cancer cells. Compared to the control group, cells exposed to laser light exhibited no significant morphological changes (Fig. 4B. Similarly, as shown in Fig. 4C-G dark toxicity groups did not show any alterations in cellular morphology. However, cells treated with the 660 nm laser in combination with AuNPs-Pheo-a exhibited dose-dependent morphological changes, including cell shrinkage, increased cell detachment, and irregular cell shapes (Fig. 4H-L.

Adenosine triphosphate (ATP) cell viability analysis
Figure 5 illustrates the ATP luminescence assay results for A549 cells treated with Pheo-a and AuNPs-Pheo-a under dark and PDT conditions, with appropriate control groups included to isolate treatment-specific effects. Untreated cells (Cells only) served as the negative control to establish baseline metabolic activity. The Cells + Laser group was included to evaluate any potential photothermal or photobiological effects of laser irradiation alone, independent of photosensitizers. Treatment with Pheo-a (Dark) and AuNPs-Pheo-a (Dark) assessed the intrinsic cytotoxicity of the free photosensitizer and the nanoconjugate, respectively, in the absence of photoactivation. The Pheo-a + PDT group was used to determine the photodynamic efficacy of the free photosensitizer, while the AuNPs-Pheo-a + PDT group evaluated the combined effect of nanoparticle conjugation and light activation.
A significant, concentration-dependent reduction in ATP levels was observed exclusively in A549 cells treated with AuNPs-Pheo-a + PDT, indicating pronounced cytotoxicity and a marked loss of cell viability. In contrast, Pheo-a + PDT induced a comparatively modest decrease in ATP levels, while all dark control groups including Cells + Laser and AuNPs-Pheo-a (Dark) showed minimal changes in ATP content. Overall, AuNPs-Pheo-a under PDT emerged as the most effective treatment in inducing cancer cell death while maintaining low dark toxicity, highlighting its promising therapeutic potential.

MTT viability assay and selectivity index
The MTT assay results revealed a concentration-dependent reduction in cell viability following treatment with the nanoconjugate. As shown in Fig. 6, A549 cells treated with AuNPs-Pheo-a alone exhibited only a slight decrease in viability under dark conditions. In contrast, AuNPs-Pheo-a–mediated PDT induced a pronounced and statistically significant reduction in cell viability compared with all other treatment groups, demonstrating strong phototoxic effects. This enhanced cytotoxicity confirms that light activation of the AuNPs-Pheo-a conjugate is essential for maximizing its anticancer efficacy against A549 cells.
Importantly, when the same treatment was applied to the normal WS-1 cell line, no significant decrease in cell viability was observed, indicating minimal toxicity toward normal cells. The IC₅₀ values further support this selectivity, with AuNPs-Pheo-a + PDT exhibiting an IC₅₀ of approximately 39.78 µg/mL in A549 cancer cells, compared to 99.43 µg/mL in WS-1 normal cells. The calculated SI is therefore approximately 2.5, highlighting the preferential cytotoxicity of AuNPs-Pheo-a mediated PDT toward cancer cells. Collectively, these findings underscore the strong therapeutic potential of AuNPs-Pheo-a as a selective and effective photodynamic agent for lung cancer treatment.

Reactive oxygen species (ROS) assay
Figure 7 presents the relative intracellular ROS generation in A549 cells under control and experimental conditions, evaluated in dark toxicity and following PDT with laser irradiation. Under dark conditions, treatment with AuNPs-Pheo-a at increasing concentrations resulted in only a marginal change in ROS levels compared with the untreated cell control. This indicates that AuNPs-Pheo-a alone does not induce significant ROS production in the absence of light, confirming minimal dark toxicity. In contrast, upon laser irradiation, a marked and concentration-dependent increase in intracellular ROS levels was observed in cells treated with AuNPs-Pheo-a + PDT. These findings demonstrate that ROS generation is primarily light-activated and underscore the photodynamic specificity of the AuNPs-Pheo-a system, which remains largely inactive in the dark while efficiently producing cytotoxic ROS upon light exposure.

Live dead assay
In live-dead assay, calcein stains live cells, while ethidium bromide stains dead cells. When the cells were treated with AuNPs-Pheo-a, no significant changes in the staining patterns were observed compared to the control. However, treatment with AuNPs-Pheo-a mediated PDT resulted in a notable occurrence of apoptosis (Fig. 8). This suggests that the combination of AuNPs-Pheo-a and PDT induces a specific cellular response, leading to apoptosis, a programmed cell death process. The absence of significant results with AuNPs-Pheo-a alone might indicate its limited efficacy in inducing cell death. The observed apoptosis with AuNPs-Pheo-a mediated PDT highlights its potential for triggering cell death pathways, which could be further explored for therapeutic applications or understanding cellular responses to treatment.

Annexin V/PI Flow cytometry assay
Flow cytometry analysis using Annexin V/PI staining was performed 24 h after PDT treatment (Fig. 9). Minimal changes were observed in A549 cells exposed to laser alone or to non-irradiated nanoparticles, indicating negligible phototoxicity under these conditions. Treatment with AuNPs-Pheo-a without irradiation slightly reduced the live cell population to 83.0%, with 17.0% in early apoptosis. In contrast, AuNPs-Pheo-a-mediated PDT caused a pronounced effect, decreasing live cells to 48.2%, with 35.3% in early apoptosis and 16.0% in late apoptosis/necrosis. The scattergrams in Fig. 9 depict the distribution of cell populations, and Table 1 summarizes the quantitative analysis across varying concentrations. These results demonstrate that PDT in combination with AuNPs-Pheo-a significantly induces apoptotic cell death, while differentiating early apoptosis from late apoptosis/necrosis provides precise mechanistic insights.

Discussion
PDT is a non-invasive and promising approach for cancer treatment, often used alongside chemotherapy and radiotherapy21. Despite its potential, its effectiveness is limited by factors such as low water solubility of photosensitizers, insufficient oxygen levels in tumor tissues, and restricted light penetration depth8,22. To overcome these limitations, nanoparticles have been employed to facilitate more efficient delivery of photosensitizers into cancer cells, thereby enhancing the overall therapeutic effectiveness of PDT8. AuNPs demonstrate distinctive anticancer effects by disrupting cell membranes, triggering ROS that cause oxidative stress and apoptosis, and interfering with protein and DNA functions, all while exhibiting minimal toxicity to healthy cells7,23,24.
Green nanotechnology is an emerging therapeutic approach in cancer research. D. anomala, a commonly used African medicinal plant, has been traditionally employed to treat various medical conditions, including cancer7. In this study, AuNPs were synthesized using methanol leaf extract of D. anomala. We evaluated the anticancer efficacy of the biosynthesized AuNPs both as a standalone treatment and in combination with Pheo-a mediated PDT in vitro. This research further investigated the novel conjugation of green-synthesized AuNPs-Pheo-a, highlighting their potential to enhance PDT for lung cancer treatment. The findings reveal a promising strategy to overcome existing challenges by developing the AuNPs-Pheo-a nanoconjugate. This was accomplished using the thin-film hydration method alongside an active loading technique, effectively addressing the hydrophobicity of Pheo-a13. Our study investigated the effect of gold nanoparticle-based PDT for lung cancer, by introducing a green and eco-friendly synthesis method utilizing methanolic leaf extract of D. anomala.
Compared with algal extracts such as Polysiphonia sp., Spirulina platensis, and Anabaena flos-aquae previously reported for green nanoparticle synthesis25,26, the D. anomala leaf extract used in this study demonstrated superior control over nanoparticle quality and stability. The D. anomala mediated synthesis yielded approximately ~ 18–22 mg Au per g of dry leaf material, producing AuNPs with an average hydrodynamic diameter of 56 ± 2.4 nm and a PDI of 27.5 ± 3.5%, indicating a relatively narrow size distribution. In addition, the synthesized AuNPs exhibited excellent colloidal stability with no visible aggregation or significant spectral changes over 30 days of storage. In contrast, algal extracts reported in the literature predominantly rely on polysaccharides and pigments as reducing and capping agents, which often result in broader particle size distributions and reduced long-term stability25,27. The enhanced performance of D. anomala leaf extract can be attributed to its high polyphenol and flavonoid content, which provides strong reducing capacity and efficient surface passivation, enabling improved nucleation control and more stable gold nanoparticle formation compared with bulky algal polysaccharides.
Various spectral analyses were conducted to confirm the formation of biosynthesized AuNPs. UV-visible spectroscopy showed that the AuNPs exhibited a characteristic peak at 540 nm, while Pheo-a displayed two distinct peaks at 370 nm and 680 nm. These results align with those reported in previous studies7,22,28. In this study, the encapsulation efficiency of Pheo-a within the AuNPs-Pheo-a nanoconjugates was determined to be 39.1 ± 2.34%. High encapsulation and loading efficiencies are crucial parameters in nanoparticle-based drug delivery, as they directly impact therapeutic efficacy, dosage accuracy, and targeted delivery potential29,30. On the other hand, SEM analysis demonstrated that the biogenically synthesized nanoconjugate AuNPs-Pheo-a possessed a relatively uniform morphology with an average particle size of approximately 96 ± 3.2 nm. Elemental composition confirmed by EDS showed distinct signals for carbon (C), oxygen (O), nitrogen (N), phosphorus (P), and gold (Au), indicating successful conjugation of Pheophorbide-a to the nanoparticle surface.
TEM revealed that while most nanoparticles were spherical, a mixture of hexagonal and irregular shapes was also present. The average size was estimated at 88.73 nm, slightly smaller than that observed via SEM, likely due to sample preparation and measurement differences. The AuNPs showed a hydrodynamic diameter of 96 ± 3.2 nm, larger than TEM measurements due to solvation effects inherent to DLS. A zeta potential of − 29.5 mV and a PDI of 0.219 indicate good colloidal stability and a moderately uniform size distribution. This polydispersity is consistent with previous studies on green-synthesized gold nanoparticles, where phytochemicals from plant extracts influence both nucleation and growth processes, resulting in diverse shapes and sizes12,22,31. The observed size difference between SEM (∼96 nm) and TEM (∼88.7 nm) arises from their different imaging principles. TEM measures the metallic core with high resolution, while SEM is sensitive to surface coatings, aggregation, and topographical effects, which can slightly overestimate particle size. Such discrepancies are common and reflect complementary characterization techniques rather than true size variation32,33.
FT-IR analysis, consistent with other studies, revealed all characteristic peaks confirming the presence of AuNPs and Pheo-a within the AuNPs-Pheo-a spectrum7,22,34,35. FT-IR spectroscopy analysis revealed several characteristic absorption bands indicative of functional groups involved in the synthesis and stabilization of the nanoparticles. The prominent peaks at 1634 cm⁻¹ and 1384 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of the carboxylate (COO⁻) groups, suggesting the presence of carboxylic acid derivatives that may have played a role in capping or stabilizing the nanoparticles. A broad absorption band centred around 3449 cm⁻¹ was attributed to O–H bending vibrations, likely originating from hydroxyl groups or adsorbed water molecules in the aqueous nanoparticle suspension, further indicating a hydrophilic surface environment. The band observed at 1092 cm⁻¹ corresponds to C–O–C stretching vibrations, characteristic of ether linkages, which could arise from polyphenolic or polysaccharide components in the plant extract used for green synthesis. Peaks at 2066 cm⁻¹ and 1745 cm⁻¹ were assigned to S = O and C = C stretching vibrations, respectively. The presence of sulfoxide (S = O) groups may indicate sulfur-containing compounds from the extract contributing to nanoparticle stabilization, while the C = C stretching suggests unsaturated organic moieties, possibly flavonoids or terpenoids, participating in the reduction or surface modification of the nanoparticles. Overall, these functional groups support the successful bioconjugation of phytochemicals to the nanoparticle surface, facilitating both reduction of gold ions and stabilization of the synthesized AuNPs. The rich surface chemistry also implies potential for improved cellular interaction and enhanced therapeutic performance in subsequent biomedical applications.
The morphology of A549 cells in the untreated control and laser-only groups remained largely unchanged, indicating that laser exposure by itself does not cause phototoxic effects. In contrast, cells exposed to the nanoconjugate displayed notable morphological alterations, including cell shrinkage, chromatin condensation, nuclear fragmentation, membrane blebbing, and loss of cell-cell adhesion. Similar findings were reported by Lin et al., who demonstrated that curcumin effectively inhibits cancer cell invasion by modulating key signalling pathways involved in metastasis. It is reported that A549 cells exhibited substantial invasion through the EHS-coated filter in the control group (without curcumin), whereas curcumin treatment markedly suppressed this invasive behaviour. The inhibitory effect was dose-dependent, with greater suppression observed at 20 µM compared to 10 µM, resulting in a 95–98% reduction in cell invasion as quantified36.
The MTT assay revealed a dose-dependent decrease in A549 cell viability following AuNPs-Pheo-a mediated PDT, consistent with earlier studies highlighting the enhanced cytotoxicity of nanoparticle-assisted photodynamic therapy. The absence of phototoxicity in the laser-only group further confirms that the observed effects are primarily attributed to the nanoparticle formulation7,12. These findings are consistent with those of Ibrahim et al. who demonstrated that AuNPs of all sizes significantly reduced the viability of A549 cells37. Similarly, a study by Li et al. aligns with our research by developing a novel photosensitizer (PS)-conjugated hybrid nanoparticle. This hybrid nanoparticle, consisting of AuNP as an efficient energy quencher, polysaccharide heparin, and a second-generation PS, pheophorbide a (PhA), showed significant phototoxicity and high intracellular uptake in vitro compared to free PhA. This correlation supports the potential of AuNP-based hybrid systems in enhancing therapeutic efficacy38.
A decline in ATP production is often indicative of mitochondrial dysfunction, which may arise from factors such as genetic mutations, oxidative stress, or nutrient deficiencies. Inadequate ATP levels can significantly disrupt essential cellular processes and, if prolonged, may ultimately lead to cell death39,40. ATP luminescence measurements were used in this study to evaluate energy levels in both untreated and treated A549 cells. Cells treated with AuNPs-Pheo-a-mediated PDT exhibited a dose-dependent decrease in ATP levels, indicating reduced cell proliferation. These findings are aligned with George et al.41. A similar trend was observed in the study by Uprety et al. where ATP levels in WS1 and A549 cells were measured. No significant ATP proliferation was noted in WS1 cells, indicating nontoxicity complexes in normal cells. However, clear toxicity was observed in A549 cells treated with cobalt complexes, demonstrating their effectiveness against cancer cells42.
The Live/Dead assay showed that AuNPs-Pheo-a alone had minimal effect on cell viability, whereas AuNPs-Pheo-a-mediated PDT induced significant apoptosis, indicating its potential to activate programmed cell death pathways. These findings are similar with the results reported with Chota et al.21. They observed a higher number of live cells in the control group and in cells that received laser irradiation without the PS, while more dead cells were present in the treated group. Another similar study by Kang et al. supports the notion that combinatorial therapy offers significantly higher efficacy compared to individual treatments. Their findings indicate that PDT/PTT treatment using PheoA-HA/AuNPs is highly selective and localized43. This aligns with our live/dead assay results, which demonstrate the enhanced effectiveness and targeted action of our treatment approach.
Understanding the complex mechanisms of cell death is crucial for elucidating disease processes and designing effective therapies. Among the various techniques used to assess cell death, Annexin V-FITC/PI staining is a widely employed method that offers valuable insights into both apoptotic and necrotic pathways19. In this study, treatment of A549 cells with 660 nm laser light alone did not result in any significant change in apoptotic or necrotic activity compared to the untreated control group, indicating that the laser itself has no inherent phototoxic effect on A549 cells. However, when A549 cells were treated with the AuNPs-Pheo-a nanoconjugate, a noticeable increase in both apoptosis and necrosis was observed 24 h post-treatment. Moreover, all experimental groups exposed to varying concentrations of the nanoconjugate showed a statistically significant rise in apoptotic and necrotic cell populations, highlighting a dose-dependent cytotoxic effect.
These findings are consistent with the results reported by Chota et al. where 660 nm laser irradiation alone also no significant impact on cell viability in MCF-7 cells had, confirming the safety of this wavelength in the absence of a photosensitizer. Like the current observations in A549 cells, MCF-7 cells treated with the individual IC₅₀ concentrations of therapeutic agents exhibited a marked increase in both apoptotic and necrotic activity after 24 h. Additionally, all experimental groups in the previous study demonstrated significant elevations in cell death, supporting the efficacy of the nanoconjugate-mediated treatment approach across different cancer cell lines19. Overall, the results underscore the non-toxic nature of 660 nm laser light by itself, while reinforcing the potential of AuNPs-Pheo-a as a potent phototoxic agent capable of inducing cell death through both apoptotic and necrotic pathways in a concentration-dependent manner.
The enhanced therapeutic performance of AuNPs-Pheo-a observed in this study is consistent with a growing body of evidence demonstrating that nanoparticle-based platforms can significantly improve the efficacy of photodynamic and combination cancer therapies compared with conventional formulations. Recent comprehensive reviews highlight that nanoparticles engineered to carry or activate photosensitizers achieve higher intracellular accumulation and stronger ROS generation, leading to improved tumor cell killing and reduced systemic toxicity relative to free photosensitizers alone, a trend also seen in metal, polymeric, and hybrid nano systems reported in the literature44. Moreover, nanocarrier strategies that enhance photosensitizer delivery and tumor specificity have been shown to overcome traditional PDT limitations such as poor solubility and low cellular uptake, increasing therapeutic depth and selectivity45,46. These advantages align with the pronounced phototoxic effects and high selectivity index demonstrated by AuNPs–Pheo-a + PDT in A549 cells, suggesting that metal nanoparticle conjugation can effectively augment ROS-mediated cytotoxicity. Importantly, such nanoplatforms are also being explored to reduce off-target effects and enhance combination modalities (e.g., synergistic PDT/PTT or chemo-PDT paradigms), reinforcing the broader therapeutic utility of nanoparticle-mediated approaches in oncology47. Although AuNP-Pheo-a systems have been previously reported by our group7,22,28, this study is the first to employ D. anomala mediated green synthesis and to systematically evaluate photodynamic selectivity, apoptosis pathways, and cancer-normal cell discrimination. These advancements significantly extend the mechanistic understanding and translational potential of AuNP-Pheo-a-based PDT.
In summary, although the reported studies demonstrate notable advancements in nanoparticle synthesis strategies and targeting approaches, the present work distinguishes itself using a green synthesis route, the evaluation in the A549 lung cancer cell model, and the development of an advanced AuNP-based nanocarrier for photosensitizer delivery. The integration of environment friendly synthesis with efficient photodynamic activation resulted in enhanced anticancer efficacy, high selectivity, and minimal dark toxicity. Collectively, this strategy offers a promising and potentially safer photodynamic therapy platform for lung cancer treatment, with clear advantages in biocompatibility and therapeutic precision over conventional formulations.

Limitation of the study
Despite the encouraging in vitro findings, several limitations of the present study should be acknowledged. First, all experiments were conducted using a two-dimensional (2D) cell culture model, which does not fully replicate the complex tumor microenvironment, cellular heterogeneity, or mass transport limitations encountered in vivo. Therefore, future studies should incorporate three-dimensional (3D) tumor spheroid models and in vivo animal studies to more accurately assess therapeutic efficacy, biodistribution, and biocompatibility. In addition, the current work did not include a conventional chemotherapeutic agent (e.g., cisplatin or doxorubicin) as a positive control in cytotoxicity assays. Although the primary focus of this study was to evaluate the light-dependent photodynamic efficacy and selectivity of the AuNPs-Pheo-a nanoconjugate, future investigations will include clinically approved anticancer drugs to enable direct comparison with standard-of-care therapies.

Conclusions and future perspectives
This study demonstrates that green-synthesized AuNPs conjugated with Pheo-a markedly enhance the efficacy of PDT against A549 lung cancer cells. AuNPs produced using D. anomala leaf extract exhibited excellent physicochemical stability, low dark toxicity, and effective cellular interaction, while their conjugation with Pheo-a resulted in significantly amplified light-activated cytotoxicity. Comprehensive in vitro evaluations including morphological analysis, ATP-based metabolic assays, MTT viability measurements, intracellular ROS generation, Live/Dead staining, and Annexin V/PI flow cytometry collectively confirmed a synergistic therapeutic effect, predominantly mediated through ROS-induced apoptosis. Importantly, the nanoconjugate displayed high selectivity toward cancer cells, with minimal toxicity in normal cells and a favorable selectivity index, underscoring its therapeutic safety profile.
Building on these findings, this work establishes a robust foundation for further translational development. Future investigations should focus on elucidating molecular signalling pathways involved in PDT-induced cell death, refining dosage and irradiation parameters, and validating therapeutic performance in 3D tumor spheroids and in vivo models. In addition, incorporating tumor-targeting ligands, conducting long-term toxicity and biodistribution studies, and evaluating large-scale reproducibility will be critical steps toward clinical applicability. Overall, this green nanotechnology-driven PDT platform represents a sustainable, selective, and biocompatible therapeutic strategy with strong potential to advance toward preclinical validation and, ultimately, clinical translation for lung cancer treatment.