A berberine-functionalized green silver nanoplatform for combined chemo-photodynamic therapy against breast cancer cells

Abstract

Breast cancer remains a leading cause of cancer-related mortality among women, while recurrence, multidrug resistance, and systemic toxicity continue to limit therapeutic outcomes. Berberine (BBR), a natural isoquinoline alkaloid with chemo-photodynamic potential, exhibits promising anticancer activity; however, poor solubility, low cellular uptake, and rapid photodegradation restrict its clinical application. This study developed and evaluated berberine-loaded biogenic silver nanoparticles (BBR-AgNPs) as a multifunctional nanoplatform for enhanced breast cancer therapy. BBR-AgNPs were synthesized using Senna didymobotrya leaf extract and characterised using UV-vis, fluorescence spectroscopy, FTIR, DLS, zeta potential analysis, SEM-EDS, and HR-TEM. Photochemical activity was assessed by generating singlet oxygen and reactive oxygen species (ROS) under 405 and 470 nm irradiation, while biological activity was evaluated in MCF-7 and MDA-MB-231 breast cancer cells. The nanoparticles exhibited a negative surface charge (−32.6 mV) and a hydrodynamic diameter of approximately 300 nm, with size heterogeneity evident from DLS analysis. BBR-AgNPs generated significantly higher ROS levels and enhanced photocytotoxicity compared with free BBR, reducing IC₅₀ values below 2.0 µg mL⁻¹ in MCF-7 cells and to approximately 3.0 µg mL⁻¹ in MDA-MB-231 cells under irradiation. Nanoparticle delivery also enhanced intracellular BBR uptake, mitochondrial depolarization, and apoptosis, supporting the potential of BBR-AgNPs as a combined chemo-photodynamic platform for further investigation in breast cancer models.

Green-synthesized BBR-AgNPs were characterized and evaluated for anticancer activity, revealing laser-enhanced ROS generation, mitochondrial membrane potential disruption, and apoptosis in cancer cells. Created in BioRender. Baidoo, I. (2026) https://BioRender.com/6fol5bx.

1. Introduction

Breast cancer remains one of the most prevalent and lethal malignancies among women globally, representing a major challenge to modern oncology.¹ Despite advances in early detection and systemic therapies, recurrence, metastasis, and multidrug resistance continue to limit long-term survival outcomes. These limitations have driven interest in investigating phototherapies and natural phytochemicals as alternatives to or complement of traditional treatment methods. Phytochemicals are structurally diverse bioactive compounds with multitarget anticancer effects and reduced systemic toxicity. Several chemotherapeutic agents, including paclitaxel, vincristine, etoposide, and camptothecin derivatives isolated from medicinal plants, highlight the continued significance of phytochemicals in cancer treatment.² Other phytochemicals, such as curcumin, resveratrol, and BBR, have demonstrated potential anticancer properties and are under investigation.³ Furthermore, phototherapy has emerged as a minimally invasive, spatially selective approach with minimal side effects, which can overcome some of the limitations of conventional therapies. Two key light-based cancer treatment strategies are photodynamic therapy (PDT) and photothermal therapy (PTT), which utilize light to generate cytotoxic reactive oxygen species (ROS) and produce localized heat, respectively, to induce antitumor effects.⁴ However, the drawbacks of many synthetic photoactive agents have prompted growing interest in naturally derived bioactive compounds with inherent phototoxic or ROS-generating properties, together with the potential environmental advantages associated with their biological origin.⁵

There has been growing interest in phytochemicals with intrinsic chemotherapeutic or phototoxic properties, among which BBR, an isoquinoline alkaloid obtained from many plants of the genus Berberis, Hydrastis, Coptis, and Argemone, can be a potential chemo-phototoxic agent.⁶ BBR has demonstrated wide-ranging chemotoxic anticancer activities through mechanisms involving cell cycle arrest, apoptosis induction, and suppression of metastasis-related signaling pathways.⁷ Moreover, BBR's photoactive property allows for light-induced excitation, generating singlet oxygen (¹O₂) and other ROS that amplify its cytotoxic potential.⁸ This dual chemotherapeutic and photodynamic potential renders BBR a promising candidate for combination therapy; however, its poor solubility, low bioavailability, susceptibility to rapid photodegradation, and rapid efflux limit its clinical translation.⁹ Additionally, its excitation in the near-UV/blue region, where light penetration in biological tissues is limited, limits the depth of phototoxic effects and, consequently, can reduce the translational potential of BBR-based phototoxicity.

These challenges have driven increasing interest in nanotechnology-based delivery systems that can improve the physicochemical stability, cellular delivery, and therapeutic efficacy of bioactive molecules such as BBR.^10–12 Sustainable nanotechnology approaches employing environmentally benign synthesis methods have attracted increasing attention for the development of nanomaterials with potentially favorable environmental and physicochemical characteristics. Nanotechnology has transformed cancer therapy by creating multifunctional nanosystems that integrate drug delivery, imaging, and treatment into a single platform.^13,14 Metallic nanoparticles, particularly silver nanoparticles AgNPs, have gained substantial attention for their unique physicochemical properties, large surface area, and tunable optical behavior. AgNPs exhibit strong surface plasmon resonance (SPR) in the visible region, thereby enabling enhanced light–matter interactions and amplifying local electromagnetic fields.¹⁵ These characteristics make AgNPs ideal candidates for loading or conjugating photoactive agents to augment their photodynamic performance. Moreover, AgNPs exhibit intrinsic anticancer, antimicrobial, and ROS-generating properties, further enhancing their therapeutic potential.^16,17 However, traditional chemical synthesis often relies on toxic reagents, raising concerns about biocompatibility. While biogenic synthesis using plant extracts has emerged as a sustainable alternative, it offers additional advantages, including eco-friendly production, reduced cytotoxicity, improved biocompatibility, and rich surface chemistry for efficient drug loading.¹⁸ Despite the significant therapeutic promise of both BBR and biogenic AgNPs, research exploring their combined application, particularly as an integrated chemo-phototoxic nanoplatform, remains limited.

In our previous study, we successfully demonstrated the green synthesis of AgNPs using Senna didymobotrya leaf extract and reported their promising chemo-phototoxic potential when activated at 470 nm. These biogenic AgNPs not only exhibited superior stability and biocompatibility but also showed significantly enhanced phototoxic activity compared to chemically synthesized AgNPs when photoactivated.¹⁹ Building upon these findings, the present study employed biogenic AgNPs as nanocarriers to load and deliver BBR, aiming to enhance its stability, cellular uptake, and therapeutic efficacy through combined chemo-photodynamic effects against breast cancer cells.

2. Materials and methods

2.1. General

All analytical-grade reagents and solvents were used as supplied without additional purification. Cell culture reagents included high-glucose Dulbecco's Modified Eagle's Medium (DMEM; D5796, SIGMA® Life Sciences), fetal bovine serum (FBS; F0679, SIGMA® Life Sciences), Hanks' Balanced Salt Solution (HBSS; 55037C, SIGMA® Life Sciences), 0.25% TrypLE solution (Gibco™, 12563-029, Thermo Fisher Scientific), penicillin–streptomycin (P4333, SIGMA® Life Sciences), and amphotericin B (A2942, SIGMA® Life Sciences). Additional analytical materials, silver nitrate (AgNO₃; ACS reagent, ≥99%, 209139) and the Cell Proliferation Kit I (11465007001, Roche Diagnostics), were procured from Merck. Berberine chloride (10006427) from Cayman Chemical Company. Annexin V-FITC apoptosis detection kits (BMS500FI-100), Singlet Oxygen Sensor Green (SOSG; S36002), and the ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; D399) were obtained from Thermo Fisher Scientific.

2.2. Synthesis of berberine-loaded silver nanoparticles

The biosynthesis of green AgNPs was carried out following our previously reported protocol, whereby 5 mL of Senna didymobotrya aqueous extract of leaves was mixed with 10 mL of 1 mM AgNO₃ solution and stirred at 150 rpm in the dark at ambient temperature for 24 hours until a dark brown colloidal suspension of AgNPs was obtained.¹⁹ Further, for BBR loading, a 0.5 mg mL⁻¹ BBR solution in water was prepared. 4 mL of 10 mg mL⁻¹ AgNPs was mixed with 1 mL of the 0.5 mg mL⁻¹ BBR solution, and the mixture was stirred at 40–45 °C for 24 h. The mixture was then centrifuged at 10 000 rpm for 30 min, washed with ddH₂O, and the resulting BBR-green AgNPs were stored at 4 °C.

2.3. Physicochemical characterization

2.3.1. Ultraviolet-visible (UV-vis) spectroscopy

The absorption spectra of free BBR, green AgNPs, and BBR-AgNPs were assessed using a Jenway 7315 spectrophotometer. Spectral scans were recorded over a wavelength range of 300–600 nm, at a bandwidth of 1 nm.

2.3.2. Fluorescence spectroscopy

The fluorescence properties of free BBR, green AgNPs, and BBR-AgNPs were examined using a fluorescence spectrophotometer (Model RF-6000, S/N: A40245901964SA). Distinct excitation wavelengths were selected to match the absorption maxima of each sample: 420 nm for free BBR, 465 nm for green AgNPs, and 423 nm for BBR-AgNPs. Emission spectra were recorded in the 400–800 nm range with an interval of 1 nm and slit widths of 3 nm for both excitation and emission monochromators. Spectra were baseline-corrected and normalized to eliminate inner-filter effects and solvent background interference.

2.3.3. Fourier transform infrared (FTIR) spectroscopy

Functional groups present in the free BBR and on the surface of the synthesized BBR-AgNPs were identified using an FTIR spectrophotometer (PerkinElmer-Spectrum 100). Before analysis, approximately 5 mL of each sample was frozen at −80 °C for 24 h, then lyophilized using an FD10S tabletop freeze dryer (Olabo, Axiology Labs Pty Ltd, Department of Chemical Sciences, University of Johannesburg) for ∼8 h. The freeze-dried nanoparticle samples were subsequently blended with FTIR-grade potassium bromide (KBr, 99.9%, 3 N) and compressed into transparent pellets using a hydraulic press before analysis. FTIR spectra were then acquired over the 400–4000 cm⁻¹ range to identify the characteristic vibrational bands associated with functional groups involved in BBR-AgNPs synthesis and surface stabilization.

2.3.4. Dynamic light scattering (DLS), polydispersity index (PDI), and zeta potential

The hydrodynamic diameter, PDI, and surface charge (zeta potential) of the synthesized BBR-AgNPs were analyzed using a Zetasizer Nano ZS (v7.03, MAL1049730, Malvern Instruments Ltd, UK). Measurements were conducted to assess colloidal stability across pH ranges (4.5–5.0, 6.5–6.8, and 7.2–7.4) and in a physiological medium. For pH-dependent studies, NP samples were prepared in phosphate buffer adjusted to the desired pH. Each sample was diluted appropriately to minimize multiple scattering effects, sonicated for approximately 1 h to ensure homogeneous dispersion, and vortexed before measurement. To assess changes in nanoparticle size and surface charge in the presence of serum proteins, BBR-AgNPs were dispersed in 10% FBS-supplemented HBSS (pH 7.4) to mimic in vitro culture conditions. After incubation at 37 °C for 1 h, samples were gently vortexed and analyzed under identical measurement settings. Measurements were performed at 25 °C and 12° detection angles in triplicate, with results reported as mean ± SD.

2.3.5. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS)

Surface morphology and elemental composition of the BBR-AgNPs were examined using SEM coupled with EDS. Lyophilized BBR-AgNPs samples were mounted on carbon tape and then coated with a thin carbon layer using a Q150T E carbon cord coater for ∼1 min to enhance conductivity. SEM imaging was conducted under high vacuum at room temperature using a TESCAN VEGA3 (Department of Metallurgy, University of Johannesburg) operating at 20 kV. EDS spectra were recorded to confirm the presence of elemental silver and to detect other elements associated with NP surface stabilization.

2.3.6. High-resolution transmission electron microscopy (HR-TEM)

HR-TEM was performed on a JEOL JEM-2100 operated at 200 kV. Fresh BBR-AgNPs dispersions were sonicated for 30 min, and a drop of each sample was deposited onto carbon-coated copper grids and allowed to air-dry in the dark before imaging. Micrographs were acquired at multiple magnifications to assess particle morphology and size. Particle size measurements were conducted using ImageJ software (NIH, USA) based on analysis of more than 100 NPs per sample.

2.4. Stability assessments under physiological and long-term storage conditions

The colloidal stability of free BBR and BBR-AgNPs was evaluated under both biologically relevant and storage conditions to determine their temporal behavior and stability. For physiological stability studies, both free BBR and BBR-AgNPs were dispersed in four different media: Milli-Q water, phosphate-buffered saline (PBS, pH 7.2–7.4), HBSS, and 10% FBS-supplemented HBSS. Samples were maintained at 37 °C to mimic physiological conditions and monitored at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h. For long-term storage stability, freshly synthesized NPs were stored at 4 °C (pH 7.4) in Milli-Q water and PBS, and their optical stability was examined at extended time points of 0 h, 3 h, 6 h, 12 h, 24 h, day 2, day 3, day 4, and day 20. These intervals were selected to capture both early and prolonged changes in SPR behavior that reflect aggregation, sedimentation, or degradation during long-term storage at lower temperatures. In both studies, the optical properties of samples were recorded using a Victor Nivo® Multimode Plate Reader. Spectral scans were acquired over the 300–600 nm wavelength range with a 1 nm bandwidth. Temporal shifts or broadening in the SPR absorption bands were used to assess stability, dispersion uniformity, and colloidal persistence under each test condition.

2.5. Berberine association with AgNPs and drug loading capacity

The BBR association/encapsulation efficiency (EE%) and loading capacity (LC%) were determined using the method reported by Cambraia et al., with slight modifications.²⁰ For EE% calculation, the BBR-AgNPs were washed and centrifuged at 10 000 rpm for 30 min to separate the nanoparticle-associated fraction from the supernatant. The concentration of unbound BBR in the supernatant was determined using a JENWAY 7315 Spectrophotometer. BBR concentration was quantified using a standard calibration curve generated from the absorbance at 344 nm using serial BBR dilutions of 0.003125, 0.00625, 0.0125, 0.025, 0.05, and 0.1 mg mL⁻¹. The amount of BBR associated with the AgNPs was calculated by subtracting the total unbound BBR recovered in the supernatants from the initial amount added during loading. The resulting EE% represents the proportion of the initially added BBR recovered as nanoparticle-associated material after the specified washing and centrifugation procedure, including BBR adsorbed or otherwise bound to the AgNPs. The present protocol does not quantitatively distinguish weakly adsorbed BBR from more strongly surface-associated BBR; therefore, the EE% should not be interpreted as definitive evidence of physical encapsulation within the AgNPs. The purified BBR-AgNPs pellets were subsequently freeze-dried to obtain their dry weight for loading capacity calculations. In the present study, EE% represents the percentage of the initial BBR dose recovered as nanoparticle-associated BBR after the loading and washing procedure, including BBR that is adsorbed or otherwise bound to the AgNPs. It is calculated relative to the total amount of BBR initially used during formulation, according to the following equation:

1

Loading capacity indicates the amount of BBR associated with the nanoparticles relative to the total recovered nanoparticle mass, and is calculated as the ratio of the associated BBR mass to the total nanoparticle mass, according to the following equation:

2

2.6. Berberine release studies

The release profile of free BBR from BBR-AgNPs was evaluated using a dialysis bag diffusion method, as previously described with modifications.²¹ A pre-activated dialysis membrane (molecular weight cutoff 14 kDa) was soaked in distilled water for 30–60 min before use. 2 mL of either free BBR solution in water (0.5 mg mL⁻¹) or BBR-AgNPs suspension (10 mg mL⁻¹) was introduced into the membrane, and both ends were securely tied to prevent leakage. The dialysis bags were immersed in 100 mL PBS (pH 7.4) maintained at 37 ± 0.5 °C under continuous stirring at 100 rpm to simulate physiological conditions. At 0, 0.5, 1, 2, 4, 8, 12, and 24 h, 1 mL of the release medium was withdrawn and replaced with an equal volume of fresh PBS. The relatively large release-medium-to-sample-volume ratio and periodic replacement of the release medium were used to minimize accumulation of released BBR and maintain sink-like release conditions. The concentration of BBR released into the medium was quantified by measuring absorbance at 344 nm using a JENWAY 7315 Spectrophotometer. Quantification was performed using a standard calibration curve prepared from serial dilutions of BBR in PBS (pH 7.4). Blank PBS samples were used to zero the instrument before each reading. The cumulative percentage of BBR release was calculated based on the amount of BBR detected at each time point relative to the total BBR content using the following formulas:Amount of BBR released (mg) = conc of BBR (mg mL⁻¹) × dissolution bath volume × dilution factor

2.7. Irradiation parameters

Photodynamic irradiation was performed using continuous-wave semiconductor diode lasers at 405 nm and 470 nm. The irradiation conditions, including laser output power, power density, fluence, and exposure time, were selected based on the absorption characteristics of berberine and the BBR-AgNPs and are summarized in Table S1.

2.8. Photothermal response measurement

BBR-AgNPs were freshly prepared at a concentration of 5 mg mL⁻¹ in Milli-Q water. Photothermal stimulation was carried out by irradiating the samples for 5 min at both wavelengths. Thermal imaging was performed in real-time using a FLIR E4 Infrared Camera (FLIR Systems, Wilsonville, OR USA), and temperature measurements were recorded at 0 and 5 minutes intervals. Baseline (pre-irradiation) and post-irradiation temperatures were compared to evaluate the net temperature change.

2.9. Singlet oxygen (¹O₂) and ROS quantification in cell-free system

¹O₂ and total ROS generation were assessed in PBS using SOSG and H₂DCF, respectively.¹⁹ H₂DCF was prepared by hydrolysis reaction using H₂DCFDA according to Cathcart et al.²² Briefly, 0.5 mL of 1 mM H₂DCFDA in ethanol was added to 2.0 mL of 10 mM NaOH, and the mixture was incubated at room temperature for 30 min. The hydrolysate was neutralized with 10 mL of 25 mM sodium phosphate buffer (pH 7.2), kept on ice, and used fresh on the same day. For ROS measurements, either Rose Bengal (RB) (5 µg mL⁻¹, positive control) or 5 µg mL⁻¹ of BBR and BBR-AgNPs was added to 3 mL of the prepared H₂DCF solution in a Petri dish (35 mm diameter). To monitor the generation of 1O2, 5 µM of SOSG was added to 3 mL of sodium phosphate buffer (pH 7.4) in a Petri dish, followed by the addition of the same concentration of either RB or both AgNPs. RB, BBR-AgNPs, and free BBR were irradiated at 405 nm and 470 nm for different times of 0.5, 1, 2, 3, 4, 5, 10, 13, 15, 18, 20, 25, and 30 min. At each time point, 200 µL of the irradiated solution was withdrawn and transferred to a black-walled 96-well plate for fluorescence measurement using a Victor Nivo® Multimode Plate Reader, with excitation/emission of 504/525 nm for SOSG and 488/520 nm for H₂DCF. Data were background-corrected and expressed as the change in fluorescence intensity (F_t/F₀) as a function of irradiation time, where F₀ and F_t represent the fluorescence intensities before and after irradiation, respectively.

2.10. Cell lines and culture conditions

The MCF-7 (ATCC® HTB-22™) and MDA-MB-231 (ATCC® HTB-26™) human breast cancer cell lines were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both the cell lines were maintained in T-75 tissue culture flasks using DMEM supplemented with 10% FBS and 1% of each antibiotic solution. Cells were maintained at 37 °C in a humidified atmosphere supplied with 5% CO₂. Cells were sub-cultured at 70–80% confluency, and experiments were conducted using cells between passages 20 and 40.

2.10.1. Treatment

To evaluate both chemotoxic and phototoxic effects, MCF-7 and MDA-MB-231 cells were seeded in 96-well plates and treated with BBR-AgNPs at concentrations of 1.25, 2.5, 5, 10, and 20 µg mL⁻¹ for 24 h. Both cell lines were treated with free BBR at concentrations of 0.003125, 0.375, 0.75, 1.5, 3, and 6 µg mL⁻¹ for 24 h. The concentration ranges were selected to capture the dose–response behavior of each formulation while accommodating the expected difference in effective concentration between free BBR and the BBR-AgNP formulation. Phototoxicity assessment involved treatment at the same concentrations, followed by irradiation with 405 nm and 470 nm lasers to a total light dose of 5 J cm⁻². Control groups consisted of an untreated control (no NP/BBR and irradiation) and a light control (irradiation without NP/BBR treatment).

2.10.2. MTT cell viability assay

Cell viability following each treatment was assessed using the Cell Proliferation Kit I (MTT). After 24 h of NP treatment and irradiation, the culture medium was aspirated from each well, and a working solution of MTT (10% MTT reagent prepared in serum-free medium) was added. The plates were incubated for 3 h at 37 °C in a humidified incubator with 5% CO₂ to allow mitochondrial reduction of MTT to formazan crystals. Without removing the MTT-containing medium, 100 µL of solubilization buffer was added directly to each well. The plates were incubated overnight at 37 °C to ensure complete dissolution of the formazan crystals. Absorbance was recorded at 540 nm using a VICTOR Nivo™ Multimode Plate Reader (PerkinElmer, USA). Cell viability was calculated as a percentage of untreated controls, which were considered 100% viable.

2.10.3. Cell morphology assessment

Cellular morphology was examined to evaluate the effects of BBR-AgNPs treatment, with and without irradiation, on MCF-7 and MDA-MB-231 cells. Cells were seeded in 35 mm plates and either left untreated or treated with AgNPs at IC₅₀ and IC₈₀ concentrations for 24 h to evaluate cytotoxicity. To assess phototoxicity-induced cellular morphological changes, cells were incubated for an additional 24 h after irradiation at 470 nm, then imaged. After incubation, the culture medium was removed, the cells were gently rinsed with PBS to remove debris and non-adherent cells, and the cells were observed using a Wirsam Olympus CKX41 inverted brightfield microscope equipped with an Olympus SC30 digital camera. Images were captured to monitor treatment-induced alterations relative to untreated control cells.

2.10.4. Annexin V/PI assay

Cells were treated with IC₅₀ concentrations of BBR-AgNPs for 24 h to evaluate chemo-toxic potential. To assess phototoxicity-induced cell death, cells were incubated for an additional 24 h after irradiation. Untreated controls and light controls were included. Following treatment, cells were harvested, washed twice with cold PBS, and resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells per mL. Annexin V-FITC and PI were added according to the manufacturer's instructions, and samples were incubated for 15 min at room temperature in the dark. Stained cells were analyzed using a flow cytometer (Becton Dickinson (BD Accuri™ C6)) with BD C-Sampler analysis software.

2.10.5. Intracellular uptake studies

MCF-7 and MDA-MB-231 cells seeded in 35 mm plates were treated with BBR at 0.375 and 0.75 µg mL⁻¹ and with BBR-AgNPs at 1.25 and 2.5 µg mL⁻¹ for 24 h in the dark. These concentrations were selected based on the lowest cytotoxic doses identified from the MTT assay. Post-treatment, cells were washed twice with PBS, released, centrifuged (5000 rpm, 3 min), and washed again. Pellets were lysed in 0.1 M NaOH + 0.1% SDS for 30 min on ice, centrifuged (10 000 rpm, 10 min, 4 °C), and supernatants were collected. Absorbance spectra (300–700 nm) were recorded, and intracellular concentrations were determined from a standard curve of BBR in SDS-NaOH solvent. Uptake was normalized to cell number for each sample.

2.10.6. Intracellular ROS quantification

MCF-7 cells were treated with free BBR (IC₅₀ 2.9 µg mL⁻¹), green AgNPs (IC₅₀ 3.02 µg mL⁻¹), and BBR-AgNPs (IC₅₀ 1.6 µg mL⁻¹). Similarly, MDA-MB-231 cells were treated with free BBR (IC₅₀ 4.9 µg mL⁻¹), green AgNPs (IC₅₀ 4.88 µg mL⁻¹), and BBR-AgNPs (IC₅₀ 3.2 µg mL⁻¹). After 24 h of treatment, the culture medium was aspirated, and cells were washed once with 100 µL of HBSS to remove residual BBR and nanoparticles. Subsequently, 100 µL of H₂DCF-DA working solution (25 µM in FBS-free DMEM prepared from a DMSO stock) was added to each well, and the plates were incubated for 45 min at 37 °C in the dark to allow intracellular uptake and deacetylation.²³ Following incubation, the cells were washed with 100 µL of phenol red-free HBSS, which was discarded, then replaced with 100 µL of fresh phenol red-free HBSS and subsequently irradiated at 470 nm. Immediately after irradiation, fluorescence intensity was measured using a Victor Nivo® Multimode Plate Reader (PerkinElmer, USA) at Ex/emission wavelengths of 485/535 nm. After recording the ROS fluorescence, HBSS was removed and replaced with 100 µL of Hoechst 33342 solution (5 µL mL⁻¹ in HBSS), and the mixture was incubated for 15 min in the dark. Plates were then re-read at Ex/Em 355/460 nm to normalize H2DCF fluorescence to Hoechst fluorescence for cell number. Data were expressed as relative fluorescence units (RFU) with respect to the untreated control.

2.10.7. Mitochondrial membrane potential (ΔΨ_m) assay

ΔΨ_m was assessed using the cationic dye JC-1, which shifts from green (monomer) to red (aggregate) fluorescence depending on mitochondrial polarization.²⁴ Cells were seeded in 96-well plates and allowed to adhere for 24 h before treatment. The following day, cells were treated with BBR, green AgNPs, and BBR-AgNPs at the concentration mentioned in Section 2.10.6. Two experimental conditions were included: (i) non-irradiated dark controls and (ii) irradiated groups. Irradiated plates were returned to the incubator for 2 h post-irradiation to allow cellular responses to stabilize. Subsequently, the medium was removed, and 50 µL of JC-1 dye working solution was added to each well, followed by incubation for 1 h at 37 °C in the dark as per the manufacturer's instructions. After staining, 50 µL of assay buffer was added to each well, and the plates were gently mixed to ensure uniform distribution. Fluorescence was measured using a Victor Nivo microplate reader at Ex/Em 490/525 nm (green, JC-1 monomers) and Ex/Em 540/590 nm (red, JC-1 aggregates). The red/green fluorescence ratio was calculated as an indicator of ΔΨ_m integrity. Following JC-1 measurements, Hoechst 33342 staining (5 µL mL⁻¹ in HBSS, 15 min) was performed for nuclear fluorescence measurement at Ex/Em 355/460 nm in both irradiated and non-irradiated plates. Changes in ΔΨ_m in each group were normalized with Hoechst fluorescence for cell number.

2.11. Statistical analysis

Each experimental group was tested in triplicate, and the experiments were repeated independently on three separate occasions. Pairwise comparisons between experimental and corresponding control groups were performed using Student's T-test at a 95% confidence level. Data are presented as mean ± standard deviation²⁵ and were analyzed using OriginPro 2018. IC₂₀, IC₅₀, and IC₈₀ values were determined by nonlinear regression analysis in OriginPro 2018. p-Values of <0.05 (*), <0.01 (**), and <0.001 (***) were considered statistically significant.

3. Results and discussion

3.1. Synthesis of BBR-AgNPs

The formation of green AgNPs was visually confirmed by a color transition from pale yellow to dark brown. At the same time, subsequent BBR loading produced a yellowish-brown shift, indicating successful nanoconjugate formation. A schematic illustration of the synthesis process is provided in Scheme 1.

Scheme 1. Schematic representation of the synthesis of green silver nanoparticles (AgNPs) and berberine-loaded AgNPs (BBR-AgNPs) using an aqueous leaf extract of Senna didymobotrya.

3.2. UV-vis spectral analysis

UV-vis absorption spectroscopy is a widely recognized method for confirming the successful synthesis of AgNPs, particularly by observing their characteristic SPR.^26,27 SPR arises from the collective oscillation of conduction electrons in response to incident light and typically occurs between 400 and 500 nm for AgNPs, depending on particle size, morphology, and dielectric environment.^28,29 As shown in Fig. 1A, free BBR exhibited two characteristic absorption maxima at 345 nm and 422 nm, corresponding to π → π* transitions of its isoquinoline alkaloid chromophore and consistent with previously reported BBR spectra in aqueous media.^30–32 Following green synthesis, the AgNPs displayed a well-defined and broad SPR band centered at 466 nm (Fig. 1A), confirming the successful reduction of Ag⁺ to Ag⁰ by phytochemicals present in Senna didymobotrya extract.¹⁹ Earlier investigations by Ider et al.³³ and Saxena et al.³⁴ demonstrated that AgNPs generally exhibit a distinct SPR absorption band within the 380–500 nm region of the visible spectrum, arising from collective electron oscillations at the nanoparticle surface. The presence of a similar SPR band in the current study aligns well with these previous findings.³⁵ It confirms the successful formation of largely spherical AgNPs, a conclusion further supported by TEM analysis. Moreover, the red-shifted and broadened SPR peak observed for the green-synthesized AgNPs is a recognized characteristic of plant-derived nanoparticle formation, in which phytochemical capping typically produces broader absorption bands and shifts the SPR to longer wavelengths.^36–38 Additionally, the BBR-AgNPs displayed a distinct SPR band at 467 nm, accompanied by a weak shoulder peak at 353 nm, indicating effective surface modification and BBR interaction with the NP interface. The emergence of this secondary shoulder also suggests spectral coupling between the intrinsic electronic transitions of BBR and the plasmon resonance of the AgNPs, providing further evidence of successful drug loading. A comparable observation was reported by Bhanumathi et al., who found that biogenic AgNPs synthesized from Syzygium cumini seed extract showed an SPR peak near 428 nm, and subsequent conjugation with berberine introduced additional absorption peaks at 230, 265, 348, and 425 nm.³⁹

Fig. 1. Optical and spectroscopic characterization of free berberine, green AgNPs, and BBR-AgNPs. (A) UV-vis absorption spectra showing characteristic plasmonic and berberine absorption peaks. (B) Fluorescence emission spectra demonstrating fluorescence quenching following BBR conjugation onto AgNPs. (C) FTIR spectra indicating functional group interactions involved in nanoparticle formation and BBR functionalization.

3.3. Fluorescence spectroscopy

Fluorescence analysis further distinguished the optical behavior of free BBR from that of its NP-bound forms (Fig. 1B). Free BBR, excited at 420 nm, displayed a strong emission peak centered at 501 nm, consistent with its known π–π* electronic transitions and characteristic fluorescence profile reported in previous studies.⁴⁰ In contrast, the green AgNPs and BBR-AgNPs did not exhibit measurable emission, and the sharp features observed near 420–470 nm are due to excitation light rather than fluorescence.

Thus, the results showed distinct fluorescence from free BBR, which was completely quenched upon binding to AgNPs. The disappearance of fluorescence in the BBR-AgNPs formulation can be attributed to several well-established quenching mechanisms that occur when fluorophores are positioned in proximity to metallic nanostructures.^41–43 The observed fluorescence quenching may be attributed to an efficient non-radiative energy-dissipation pathway via nanometal surface energy transfer. Upon binding, the excited BBR molecules may undergo non-radiative energy transfer to the AgNPs surface, particularly in NPs larger than 5 nm, thereby quenching fluorescence. The plasmonic fields of AgNPs may further suppress radiative decay by altering the local electromagnetic environment and promoting plasmon-mediated non-radiative relaxation. Additionally, adsorption-driven interactions such as π–π stacking, electrostatic interactions, and charge transfer between BBR's aromatic rings and the metallic surface can also facilitate ground-state complex formation, collectively resulting in the loss of measurable fluorescence in the BBR-AgNPs formulation. These findings suggest that BBR fluorescence is effectively suppressed upon binding to AgNPs, supporting strong surface interactions and further corroborating successful drug loading.

3.4. FTIR analysis

The FTIR spectra provided molecular-level insights into the chemical interactions involved in the conjugation (Fig. 1C). FTIR spectra of BBR-AgNPs confirmed successful conjugation of BBR onto the NP surface. Free BBR exhibited characteristic peaks at 3460, 2058.8, 1632.7, 1508.6, and 1385.5 cm⁻¹, corresponding to O–H/N–H stretching, C C/C C stretching, aromatic C O/C C vibrations, and C–N stretching, respectively, consistent with literature reports.³⁹ In the BBR-AgNPs spectrum, these bands were retained but shifted slightly to 3427.6, 2056, 1630.9, 1506.8, and 1384.2 cm⁻¹, indicating interaction of BBR functional groups with the AgNPs surface. Notably, comparison with our previously reported green AgNPs,¹⁹ revealed corresponding bands at 3441, 1633, and 1385 cm⁻¹, which were attributed to phytochemical functional groups coordinated to the AgNPs surface; the observed shifts upon BBR loading therefore indicate interaction of BBR with these Ag-associated binding sites. The band at 1508 cm⁻¹, characteristic of aromatic C C vibrations of BBR, was absent in the green AgNPs but retained in the BBR-AgNPs with a slight shift to 1506.8 cm⁻¹, confirming successful drug loading and preservation of the BBR structure. The downward shifts in O–H/N–H and aromatic C C vibrations suggest hydrogen bonding, electrostatic interactions, and phytochemical-mediated capping typical of green-synthesized AgNPs systems. These spectral modifications confirm successful loading and stabilization of BBR on the green AgNPs.^39,44,45

3.5. Particle size distribution and zeta potential analysis

Representative zeta potential and DLS profiles of BBR-AgNPs under physiological conditions are shown in Fig. 2A and B, respectively, while the corresponding values are summarized in Table 1. Following BBR loading, the nanoparticles exhibited a hydrodynamic diameter of 300.7 ± 9.0 nm at physiological pH (7.2–7.4), with a PDI of 0.385 ± 0.014 and a strongly negative zeta potential of −32.6 ± 0.7 mV. The negative zeta potential suggests electrostatic repulsion between particles, although the PDI and HR-TEM observations indicate that some degree of particle heterogeneity and aggregation is present. At pH 5.88, the nanoparticles showed a marked reduction in hydrodynamic diameter (208.5 ± 2.6 nm) and PDI (0.288 ± 0.013), whereas at pH 6.54 the values remained close to those observed under physiological conditions (hydrodynamic diameter 298.6 ± 7.8 nm; PDI of 0.366 ± 0.012). The reduction in size and dispersity at pH 5.88 may reflect pH-dependent changes in the surface chemistry and interparticle interactions of the BBR-functionalized nanoparticles; however, the precise mechanism underlying this behavior was not directly investigated.^46,47 This pH-dependent behavior may be relevant to nanoparticle performance in acidic biological environments, although its consequences for cellular uptake were not directly evaluated in the present study.^48,49

Fig. 2. Physicochemical and morphological characterization of BBR-AgNPs showing (A) zeta potential distribution, (B) hydrodynamic particle size distribution measured in phosphate buffer at 25 °C, (C) scanning electron microscopy image, (D) energy dispersive X-ray spectroscopy spectrum, (E) high-resolution transmission electron microscopy image, and (F) high-resolution transmission electron microscopy-derived particle size distribution histogram.

Table 1. Particle size, PDI, and zeta potential of BBR-AgNPs under different pH and serum conditions.

Nanoparticle	pH	Particle size (d, nm)	PDI	Zeta potential (mV)
BBR-AgNPs	5.88	208.5 ± 2.6	0.288 ± 0.013	−13.2 ± 0.3
	6.54	298.6 ± 7.8	0.366 ± 0.012	−32.6 ± 0.9
	7.54	300.7 ± 9.0	0.385 ± 0.014	−32.6 ± 0.7
BBR-AgNPs (10% FBS)	7.4	383.7 ± 5.8	0.631 ± 0.014	−9.57 ± 0.6

Compared with our previously reported green AgNPs, which exhibited a hydrodynamic diameter of 96.9 ± 1.7 nm and a zeta potential of −26.3 ± 1.53 mV under physiological conditions,¹⁹ BBR-AgNPs showed a marked increase in hydrodynamic size following BBR loading. This increase may reflect the formation of an additional surface-associated organic layer as well as increased particle association after BBR functionalisation. The hydrodynamic diameter measured by DLS (300.7 ± 9.0 nm) was substantially larger than the HR-TEM particle diameter (57 ± 10.2 nm). This difference is expected because HR-TEM measures the dry, electron-dense nanoparticle cores, whereas DLS measures the hydrodynamic size of light-scattering entities in dispersion, which can include the hydration layer and surface-associated biomolecules. In addition, the moderate aggregation observed by HR-TEM and the PDI of 0.385 ± 0.014 suggest some degree of particle heterogeneity and association in dispersion.^50–52 Therefore, the DLS value should be interpreted as the apparent hydrodynamic size of the dispersed nanoparticle population rather than the diameter of individual AgNP cores.

To evaluate protein corona formation, BBR-AgNPs were analyzed in 10% FBS-supplemented HBSS. Under serum conditions, the hydrodynamic diameter increased substantially to 383.7 ± 5.8 nm, while the zeta potential decreased to −9.57 ± 0.6 mV, accompanied by a higher PDI value of 0.631 ± 0.014. Compared with the previously reported green AgNPs in 10% FBS (132.6 ± 0.81 nm; −8.48 ± 0.48 mV).¹⁹ The greater increase in hydrodynamic size observed for BBR-AgNPs is consistent with increased association of serum proteins with the nanoparticle surface, although protein adsorption was not directly quantified.^19,53 Although serum exposure reduced the magnitude of the zeta potential, the nanoparticles retained a net negative surface charge. However, the increased hydrodynamic diameter and PDI under serum conditions indicate greater size heterogeneity and particle association in the presence of serum proteins.^54–56

3.6. Surface morphology and elemental composition by SEM-EDS

Scanning electron microscopy-energy-dispersive X-ray Spectroscopy (SEM-EDS) analysis combines high-resolution imaging of NP morphology with elemental composition profiling, providing critical insights into particle size, shape, stability, and surface chemistry relevant to nanomedicine applications.^57–59 The SEM micrographs (Fig. 2C) revealed that BBR-AgNPs exhibited denser, more clustered morphologies, consistent with the presence of an organic coating and enhanced interparticle interactions arising from BBR conjugation. The EDS spectra (Fig. 2D) confirmed the presence of elemental silver (Ag), with a strong Ag peak at approximately 3 keV, characteristic of metallic silver. Additionally, the EDS profile showed Ag (36.47 wt%), accompanied by a higher carbon content (45.61 wt%) and notable oxygen and nitrogen peaks (11.92 wt% and 2.66 wt%, respectively) (Table S2), confirming the presence of organic molecules from BBR on the AgNPs surface.¹⁹ Other elements, such as Cl, F, and Na, were also detected, likely residual components from the synthesis medium.

3.7. Size and morphology evaluation using HR-TEM

The morphology and particle size of BBR-AgNPs were analyzed using High-Resolution Transmission Electron Microscopy (HR-TEM). The representative micrograph (Fig. 2E) and corresponding particle size distribution histogram (Fig. 2F) revealed that BBR-AgNPs displayed large and more heterogeneous particle morphology. The particles appeared mostly spherical with irregular shapes and moderate aggregation, likely due to surface modification upon BBR loading. Green-synthesized NPs often exhibit irregular or polydisperse morphologies, as the heterogeneous phytochemical composition of plant extracts results in uncontrolled reduction kinetics and variable capping interactions during NP growth. This lack of uniformity in nucleation and stabilization commonly results in non-spherical, anisotropic, or aggregated structures in biogenic AgNPs, as widely reported.⁶⁰ The calculated mean particle diameter is 57 ± 10.2 nm. For comparison, our previous study reported that the green AgNPs exhibited a mean particle diameter of 39 ± 8.1 nm,¹⁹ indicating that the observed morphological changes are associated with BBR loading. The observed results are consistent with earlier studies, whereby spherical shapes and size ranges of 15–30 nm and 35–50 nm were reported for biogenic AgNPs and BBR-AgNPs.³⁹

3.8. Stability of AgNPs under long-term storage and physiological conditions

The stability of the BBR and BBR-AgNPs was assessed under long-term storage and physiologically relevant conditions to determine their suitability for biomedical applications. UV-visible spectroscopy was used to monitor spectral changes, as shown in Fig. 3 and 4. During 20 days of storage at 4 °C (pH 7.4), BBR-AgNPs maintained their characteristic SPR band alongside the BBR absorption features, with only minor variations in peak intensity in both PBS (Fig. 3A) and water (Fig. 3B), indicating good colloidal stability. This behavior is consistent with the previously reported stability of green AgNPs synthesized using the same system.¹⁹ In contrast, free BBR (Fig. 3C and D) remained stable in water but exhibited pronounced spectral distortions in PBS by day 20, suggesting reduced chemical stability under buffered conditions.

Fig. 3. UV-visible absorbance spectra of BBR-AgNPs, and free berberine at 4 °C for up to 20 days (A) BBR-AgNPs in PBS; (B) BBR-AgNPs in water; (C) free berberine in PBS; (D) free berberine in water.

Fig. 4. UV-visible absorbance spectra of BBR-AgNPs, and free berberine under physiological conditions at 37 °C for up to 72 hours. BBR-AgNPs in PBS (A), and 10% FBS-supplemented HBSS (B); and free berberine in PBS (C), and 10% FBS-supplemented HBSS (D).

Under physiological incubation at 37 °C for 72 h, BBR-AgNPs retained well-defined absorption features in PBS (Fig. 4A), while slight peak broadening was observed in 10% FBS-HBSS (Fig. 4B), likely due to interactions with serum proteins. Such spectral changes are consistent with protein corona formation and have been widely reported for NPs in biological media. Notably, similar behavior has been observed for green AgNPs under comparable conditions,¹⁹ indicating that BBR functionalisation does not compromise NP stability.

By comparison, free BBR (Fig. 4C and D) showed minimal spectral changes across physiological media over 72 h. Collectively, these findings demonstrate that BBR-AgNPs exhibit good long-term and physiological stability, while association with the AgNPs improves the stability profile of BBR, particularly under storage conditions, without adversely affecting nanoparticle behavior in biological environments.

3.9. Berberine association with AgNPs and loading capacity of BBR-AgNPs

The amount of BBR associated with the AgNPs and the corresponding loading capacity were determined spectrophotometrically using a standard calibration curve of BBR (Fig. S1). The calculated apparent association/encapsulation efficiency (EE%) and loading capacity (LC%) were 90% and 30%, respectively (Table S3), based on the amount of BBR remaining associated with the AgNPs after the loading and washing procedure. The high EE% indicates that a large proportion of the initially added BBR remained associated with the AgNPs after the loading and washing procedure, suggesting substantial interaction between BBR and the nanoparticle surface. The high apparent BBR loading may arise from multiple interactions between BBR and the phytochemical-functionalized AgNP surface, including electrostatic attraction, hydrogen bonding, and π–π interactions. In particular, the cationic nature of berberine and the negative surface potential of the biogenic AgNPs provide a plausible electrostatic driving force for BBR association with the nanoparticle interface.^61,62 The loading capacity of 30% indicates substantial BBR loading relative to the recovered nanoparticle mass and supports the feasibility of AgNPs as a BBR delivery platform. High LC values are generally desirable because they can enable delivery of a greater therapeutic payload with a lower amount of carrier material.⁶³ The relatively high apparent BBR association efficiency observed in the present study may be attributed to the high surface area of the AgNPs and the multiple interactions between BBR and surface-associated phytochemical components. Compared with previously reported BBR-loaded polymeric and liposomal nanocarriers, which have reported EE values in the range of approximately 60–85%,^64,65 the 90% apparent association efficiency obtained here indicates efficient incorporation of BBR into the AgNP-based formulation. The high association may reflect the combined contribution of the AgNP surface area and interactions between BBR and phytochemical-derived surface functional groups. Overall, the EE% and LC% values support the ability of the green-synthesized AgNPs to associate with and carry BBR, supporting further investigation of the formulation as a chemo-photodynamic delivery platform.

3.10. In vitro berberine release studies

The in vitro release profiles of free BBR and BBR-AgNPs at physiological pH (7.4) are presented in Fig. 5. Both formulations exhibited a rapid initial release phase during the first few hours, followed by a slower release pattern that approached near-complete release after 24 h. Approximately 90% cumulative release was achieved within 4 h for both systems, although BBR-AgNPs showed a slightly slower release behavior during the early stages of incubation. This suggests that a portion of the BBR molecules was associated with the nanoparticle surface through intermolecular interactions such as electrostatic attraction, hydrogen bonding, and adsorption onto the phytochemical capping layer surrounding the AgNPs.

Fig. 5. Cumulative release profiles of free BBR and BBR-AgNPs in PBS (pH 7.4, 37 °C) over 24 h. Data are presented as mean ± SD from three independent experiments, with each experimental group tested in triplicate.

The relatively rapid release kinetics indicate that most BBR molecules were weakly associated with surface rather than deeply entrapped within a carrier matrix, resulting in rapid desorption into the surrounding medium. Similar release behavior has been reported for BBR-loaded metallic nanoparticle systems, where drug release is primarily governed by adsorption–desorption equilibrium at the nanoparticle surface.^66,67 Previous studies have also shown that nanoparticle conjugation improves the physicochemical stability, cellular interaction, and intracellular delivery of BBR despite relatively rapid release kinetics.^12,68 Despite the comparable release profiles, nanoparticle conjugation may still improve therapeutic performance by enhancing BBR dispersion, cellular interaction, intracellular uptake, and ROS-mediated photodynamic activity under irradiation conditions. These findings therefore suggest that the enhanced anticancer efficacy of BBR-AgNPs is likely associated more with nanoparticle-mediated delivery and plasmon-assisted photodynamic effects than with sustained drug release alone.

3.11. Photothermal activity

Infrared thermographic analysis showed that BBR-AgNPs can induce minimal photothermal conversion efficiency under visible-light irradiation. Deionized water exhibited a negligible rise in temperature under both irradiation conditions, increasing only from 17.5 °C to 18.4 °C (ΔT ≈ 0.9 °C) at 405 nm (Fig. 6A and B) and from 12.1 °C to 12.8 °C (ΔT ≈ 0.7 °C) at 470 nm (ΔT ≈ +0.7 °C), consistent with its minimal light absorption. BBR-AgNPs showed measurable temperature elevations, rising from 17.6 °C to 19.5 °C (ΔT ≈ 1.9 °C) under 405 nm excitation (Fig. 6C and D) and from 11.2 °C to 14.0 °C (ΔT ≈ 2.8 °C) under 470 nm irradiation (Fig. 6G and H), indicating modest photon absorption and plasmon-mediated light-to-heat conversion. Compared with our previously reported green AgNPs, which exhibited only a minimal temperature increase of approximately 0.8 °C under 470 nm irradiation,¹⁹ BBR-AgNPs demonstrated enhanced photothermal behavior following berberine loading. This improvement may be attributed to increased optical absorption and plasmon–molecular interactions between surface-bound BBR and the AgNPs, which can facilitate more efficient energy dissipation as heat. BBR-AgNPs showed detectable but limited photothermal conversion capability under visible-light excitation. The ΔT values under both 405 nm and 470 nm irradiation indicate measurable light-to-heat conversion; however, the temperature increases (<3 °C) were substantially below the range generally associated with effective photothermal cytotoxicity.⁶⁹ These findings suggest that photothermal heating is unlikely to be a major contributor to the observed photocytotoxicity under the irradiation conditions used.

Fig. 6. Infrared thermographic images illustrating the photothermal behavior of water and BBR-AgNPs under laser irradiation. (A and B) Deionized water before irradiation (0 min) and after 5 min of exposure to 405 nm laser light, respectively. (C and D) BBR-AgNPs at 0 min and after 5 min irradiation with a 405 nm laser, respectively. (E and F) Deionized water before (0 min) and after 5 min irradiation with a 470 nm laser, respectively. (G and H) BBR-AgNPs before irradiation (0 min) and after 5 min of exposure to 470 nm laser light, respectively.

3.12. Singlet oxygen (¹O₂) and ROS generation efficiency

The photochemical analysis revealed clear wavelength-dependent differences in ¹O₂ and ROS generation, and in all assays, BBR-AgNPs consistently outperformed free BBR. The photochemical generation efficiency of free BBR and BBR-AgNPs mediated ¹O₂ and ROS was assessed using two fluorescence probes: SOSG, which is specific for ¹O₂, and H₂DCF, which detects total ROS. Upon reaction with photochemically generated ¹O₂ or ROS, these probes are converted into highly fluorescent species. The change in fluorescence intensity (F_t/F₀) of SOSG and H₂DCF as a function of irradiation time was plotted to compare the ¹O₂ and ROS generation at both 405 nm and 470 nm. In the SOSG assay at 405 nm (Fig. 7A), all samples showed a progressive rise in fluorescence from 0 to 30 min, with Rose Bengal (RB) yielding the highest ¹O₂ production (F_t/F₀ ≈ 5.3 at 30 min). BBR-AgNPs showed a relatively higher fluorescence change (F_t/F₀ ≈ 3.2), representing a 2.1-fold increase over free BBR (F_t/F₀ ≈ 1.5); however, at 470 nm (Fig. 7B), overall ¹O₂ generation of both free BBR and BBR-AgNPs was lower and negligible. In contrast, in our previous study, green AgNPs, with a pronounced absorption band near 470 nm, generated a detectable level of ¹O₂ upon irradiation (F_t/F₀ ≈ 1.69).¹⁹ In the ROS assay, H₂DCF fluorescence was markedly higher in all samples than in the SOSG control. At 405 nm (Fig. 7C), ROS production was substantially higher for BBR-AgNPs (F_t/F₀ ≈ 150), showing roughly a 2.3-fold increase compared with free BBR (F_t/F₀ ≈ 1.6). When irradiated at 470 nm (Fig. 7D), BBR-AgNPs reached an F_t/F₀ value of approximately 270 after 30 min, compared with approximately 160 for free BBR. Green AgNPs exposed to 470 nm light also exhibited ROS production, reaching an F_t/F₀ value of 210.1.¹⁹ Thus, at 470 nm, BBR-AgNPs represented an enhancement in ROS generation compared to green-AgNPs (1.2-fold) and BBR (1.7-fold). The wavelength-dependent photochemical behavior of the BBR, green AgNPs, and BBR-AgNPs reflects their intrinsic absorption properties and NP-BBR interactions. At 405 nm, where BBR exhibits a stronger absorption band, BBR-AgNPs produced relatively higher ¹O₂ signals than free BBR, which could be due to the enhanced intersystem crossing and improved triplet-state population facilitated by AgNPs–plasmon coupling.^70–72 In contrast, both free BBR and BBR-AgNPs generated negligible ¹O₂ at 470 nm, whereby BBR has a lesser absorption coefficient than 405 nm; however, green AgNPs produced measurable ¹O₂ at 470 nm due to their intrinsic plasmon resonance in this region.¹⁹ ROS formation followed a different trend: with 405 nm excitation, BBR-AgNPs showed a 2.3-fold increase in ROS generation compared with free BBR. This enhancement may be related to BBR association with the AgNP surface, which can alter the local photophysical environment and potentially reduce BBR aggregation. Interactions between photoexcited BBR and the plasmonic AgNP surface may also facilitate charge-transfer processes and influence excited-state dynamics, although these processes were not directly measured in the present study.⁷³ The increased H₂DCF response is consistent with a greater contribution from non-singlet-oxygen ROS, potentially involving electron-transfer pathways at the BBR-AgNP interface.^71,72,74 However, direct measurements of interfacial electron transfer were not performed, and this pathway should therefore be regarded as a mechanistic interpretation rather than a definitive assignment.

Fig. 7. Time-dependent generation of singlet oxygen (¹O₂) and reactive oxygen species (ROS) by Rose Bengal, free berberine, and BBR-AgNPs under 405 nm and 470 nm irradiation in a cell-free system. (A) SOSG fluorescence under 405 nm excitation. (B) SOSG fluorescence under 470 nm. (C) H₂DCF fluorescence at 405 nm. (D) H₂DCF fluorescence under 470 nm irradiation. Data are presented as mean ± SD from three independent experiments, with each experimental group tested in triplicate.

Several studies have shown that photo-excited BBR undergoes both type I and type II photochemical pathways, which are strongly influenced by the polarity, oxygen content, and pH of the surrounding medium, as well as by the functional groups involved in its photochemical activity.⁷⁵ Consistent with 405 nm results, the increment in DCF fluorescence was significantly higher than SOSG fluorescence at 470 nm excitation for free BBR, which can be explained by studies indicating that polar environments favor electron-transfer-driven Type I ROS, thereby suppressing ¹O₂ formation.⁷⁶ Under 470 nm excitation, BBR-AgNPs generated the highest ROS signal (F_t/F₀ ≈ 270), surpassing both free BBR (F_t/F₀ ≈ 160) and green AgNPs (F_t/F₀ ≈ 210.10) by 1.7-fold and 1.2-fold, respectively. This enhanced ROS output is consistent with an increased contribution from Type I-like oxidative processes, particularly because the H₂DCF response was substantially greater than the ¹O₂ signal at 470 nm. The underlying contribution of interfacial electron transfer or excited-state interactions between BBR and AgNPs remains to be established experimentally. These findings indicate that BBR-AgNPs produce a predominantly ROS-associated photodynamic response under the tested conditions, with a comparatively greater contribution from H₂DCF-detectable ROS at 470 nm. Further spectroscopic and photophysical studies would be required to directly establish the contribution of interfacial electron transfer and excited-state stabilization to this response.^77,78

3.13. In vitro anticancer activity: chemo and phototoxic effects

Both free BBR and BBR-AgNPs demonstrated concentration-dependent reductions in cell viability in MCF-7 and MDA-MB-231 breast cancer cells under dark and irradiated conditions (Fig. 8 and 9). In all treatment groups, irradiation enhanced cytotoxicity compared with corresponding dark conditions, confirming photoactivation-dependent therapeutic effects. However, BBR-AgNPs consistently produced markedly greater photocytotoxicity than free BBR, particularly under 405 nm irradiation. Although the tested concentration ranges differed between free BBR and BBR-AgNPs, the ranges encompassed the respective IC₅₀ values for the major treatment conditions, allowing comparison of their concentration–response behavior while avoiding excessive toxicity that could obscure dose-dependent effects.

Fig. 8. Cytotoxic and photocytotoxic effects of free berberine in MCF-7 (A and B) and MDA-MB-231 (C and D) cells under dark, 405 nm, and 470 nm irradiation conditions. Bar graphs show concentration-dependent changes in cell viability, while dose–response curves illustrate IC values under different treatment conditions. Data are presented as mean ± SD from three independent experiments, with each experimental group tested in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control group.

Fig. 9. Cytotoxic and photocytotoxic effects of BBR-AgNPs in MCF-7 (A and B) and MDA-MB-231 (C and D) cells under dark, 405 nm, and 470 nm irradiation conditions. Bar graphs show concentration-dependent changes in cell viability, while dose–response curves illustrate IC values under different treatment conditions. Data are presented as mean ± SD from three independent experiments, with each experimental group tested in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control group.

For free BBR (Fig. 8), moderate phototoxic effects were observed in MCF-7 cells. Under 405 nm irradiation, the IC₅₀ decreased from approximately 5.0 ± 1.5 µg mL⁻¹ under dark conditions to 2.9 ± 0.3 µg mL⁻¹, while an IC₈₀ value of 5.9 ± 0.3 µg mL⁻¹ was achieved only following irradiation (Fig. 8B). In contrast, under dark conditions, 80% inhibition was not reached within the tested concentration range, indicating relatively weak standalone cytotoxicity. Under 470 nm irradiation, free BBR also showed enhanced cytotoxicity compared with the dark treatment, although the effect was less pronounced than that observed at 405 nm. In MDA-MB-231 cells (Fig. 8C and D), free BBR displayed substantially weaker activity overall. Under 405 nm irradiation, an IC₅₀ value of approximately 4.9 ± 1.9 µg mL⁻¹ was obtained, whereas only IC₂₀ values of 1.2 ± 0.9 µg mL⁻¹ and 1.9 ± 0.9 µg mL⁻¹ were achieved under 405 nm and 470 nm irradiation, respectively (Fig. 8D), indicating limited efficacy against the more resistant triple-negative MDA-MB-231 phenotype.

In contrast, BBR-AgNPs (Fig. 9) exhibited substantially enhanced chemo-photodynamic activity in both breast cancer cell lines. In MCF-7 cells (Fig. 9A and B), BBR-AgNPs reduced the IC₅₀ from 2.8 ± 0.2 µg mL⁻¹ under dark conditions to 1.6 ± 0.1 µg mL⁻¹ following 405 nm irradiation. Furthermore, the IC₈₀ value decreased from 7.2 ± 0.2 µg mL⁻¹ under dark conditions to 3.0 ± 0.1 µg mL⁻¹ after irradiation, demonstrating strong photoinduced enhancement of cytotoxicity. Under 470 nm irradiation, BBR-AgNPs also displayed marked photocytotoxicity, although the effect was slightly less pronounced than under 405 nm exposure. Similar trends were observed in MDA-MB-231 cells (Fig. 9C and D), where the IC₅₀ decreased from 7.0 ± 0.03 µg mL⁻¹ under dark conditions to 3.2 ± 0.02 µg mL⁻¹ following 405 nm irradiation. Likewise, IC₈₀ values decreased from 10.7 ± 0.04 µg mL⁻¹ to 8.4 ± 0.02 µg mL⁻¹ upon irradiation, confirming enhanced phototoxicity despite the greater resistance of MDA-MB-231 cells.

The enhanced photocytotoxicity observed for BBR-AgNPs compared with free BBR is consistent with the significantly higher ROS generation demonstrated in Fig. 7, particularly under photoirradiation. The greater reduction in IC₅₀ and IC₈₀ values following nanoparticle functionalization suggests that AgNPs conjugation improved both intracellular delivery and photodynamic activation of BBR, thereby amplifying oxidative stress-mediated cell death. Furthermore, compared with our previously reported green AgNPs, which exhibited comparatively moderate photocytotoxic effects under similar irradiation conditions,¹⁹ BBR functionalization substantially enhanced therapeutic efficacy in both breast cancer cell lines. The stronger response observed in MCF-7 cells relative to MDA-MB-231 cells is consistent with the greater ROS generation and mitochondrial depolarization observed in MCF-7 cells, suggesting greater susceptibility to oxidative photodynamic stress under the conditions tested. The lower intracellular BBR levels measured in MDA-MB-231 cells may also contribute to its weaker response, although the present study does not establish the specific molecular basis for these cell-line differences.

3.14. Treatment-induced cellular morphological alteration

A qualitative assessment of cellular morphology following BBR-AgNPs treatment, with and without irradiation, is presented in Fig. 10. Untreated and light control cells for both the cell lines (Fig. 10A, D, G, and J) retained their normal morphology and close intercellular contact, indicating that laser exposure alone did not compromise viability. On the other hand, BBR-AgNPs induced characteristic signs of cytotoxicity, including cell rounding, shrinkage, and detachment at IC₅₀ concentrations (Fig. 10B, E, H, and K), which is more pronounced at higher concentrations (IC₈₀) (Fig. 10C, I, F, and L). The features are consistent with apoptotic or necrotic morphological changes reported in AgNPs-treated breast cancer cells.^79,80 Compared with the unirradiated groups, cells irradiated after BBR-AgNPs treatment showed enhanced cell death at both IC₅₀ and IC₈₀ concentrations, likely due to light-induced ROS generation and oxidative stress.

Fig. 10. Brightfield microscopic images of MCF-7 (A–F) and MDA-MB-231 (G–L) cells following treatment with BBR-AgNPs under dark and irradiated conditions. (A and G) Untreated controls; (B and H) BBR-AgNPs at IC₅₀; (C and I) BBR-AgNPs at IC₈₀; (D and J) light control (470 nm); (E and K) BBR-AgNPs at IC₅₀ + irradiation; (F and L) BBR-AgNPs at IC₈₀ + irradiation. Scale bar = 50 µm, 200× magnification.

3.15. Treatment-induced cell death

The apoptotic response of breast cancer cells following treatment with BBR-AgNPs was evaluated using Annexin V-FITC/PI double staining and analyzed by flow cytometry (Fig. 11). The scatter plots (Fig. 11A–H) and corresponding quantitative histograms (Fig. 11I–J) illustrate the percentages of live, early apoptotic, late apoptotic, and necrotic cells under different treatment conditions. For MCF-7 cells (Fig. 11A–D, and I), the untreated control and light control groups showed minimal apoptosis, with over 90% of cells remaining viable, indicating that light exposure at 470 nm alone did not induce cytotoxicity. Treatment with BBR-AgNPs at the IC₅₀ concentration under dark conditions significantly increased both early (about 12%) and late apoptotic (about 54%) populations compared to the control, while the necrotic fraction remained below 1%. Upon irradiation at the IC₅₀ dose, the proportions of both early apoptotic cells (about 14%) and late apoptotic cells (about 48%) remained high, with a slight decrease in viable cells. Similarly, in MDA-MB-231 cells (Fig. 11E–H, and J), untreated and light-control groups exhibited >90% live cells. BBR-AgNPs at IC₅₀ treatment under dark conditions triggered significant apoptosis, with about 46% late apoptotic and about 12% early apoptotic cells compared to untreated controls. Following irradiation, apoptotic cell populations remained markedly high (about 49% late apoptotic, about 13% early apoptotic), whereas necrotic populations remained <1%.

Fig. 11. Annexin V-FITC/PI analysis in MCF-7 and MDA-MB-231 cells treated with BBR-AgNPs. (A–D) MCF-7 and (E–H) MDA-MB-231 cells: (A and E) untreated control; (B and F) light control (470 nm); (C and G) BBR-AgNPs at IC₅₀ dose; (D and H) BBR-AgNPs at IC₅₀ concentration followed by irradiation. (I and J) Percentage of live, apoptotic (early and late), and necrotic cell populations for all experimental groups in MCF-7 (I) and MDA-MB-231 (J). Data are presented as mean ± SD from three independent experiments, with each experimental group tested in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control group.

The predominance of apoptotic rather than necrotic cell death suggests that BBR-AgNPs primarily activated regulated cell death pathways rather than causing nonspecific membrane disruption. The strong apoptotic response observed in both cell lines closely correlates with the enhanced ROS generation shown in Fig. 7, respectively, supporting ROS-mediated mitochondrial apoptosis as a central mechanism underlying the nanoplatform's anticancer activity. Excessive ROS accumulation can induce mitochondrial dysfunction, cytochrome c release, and activation of downstream caspase signaling pathways, ultimately promoting apoptotic cell death.^81,82 Furthermore, the slightly enhanced apoptosis observed under irradiation conditions supports the role of photodynamically generated ROS in amplifying oxidative stress and apoptotic signaling. Similar apoptosis-dominant responses have been reported for berberine-loaded nanocarriers and plasmonic nanoparticle systems, where enhanced intracellular ROS production triggered mitochondrial-mediated apoptosis in breast cancer cells.⁶⁸ Notably, the substantial apoptotic responses observed in both MCF-7, and MDA-MB-231 cells indicate that BBR-AgNPs retain cytotoxic activity across these molecularly distinct breast cancer models, including the more treatment-resistant triple-negative MDA-MB-231 phenotype.

3.16. Intracellular uptake of BBR and BBR-AgNPs

The intracellular uptake of BBR in MCF-7 and MDA-MB-231 cells following treatment with free BBR and BBR-AgNPs was quantified using spectrophotometric analysis derived from the BBR calibration curve. As shown in Fig. 12A and B, both treatment groups showed a concentration-dependent increase in cellular uptake; however, BBR-AgNPs-treated cells exhibited greater BBR uptake than cells treated with free BBR. In MCF-7 cells (Fig. 12A), uptake of BBR increased from 0.19 ± 0.04 µg per 10⁶ cells to 0.41 ± 0.06 µg per 10⁶ cells at 0.375 µg mL⁻¹ and 0.75 µg mL⁻¹ free BBR treatment, whereas BBR-AgNPs exposure resulted in significantly higher uptake of BBR, 0.64 ± 0.09 µg per 10⁶ cells and 0.86 ± 0.11 µg per 10⁶ cells at 1.25 µg mL⁻¹ and 2.5 µg mL⁻¹ BBR-AgNP treatment, respectively. A comparable pattern was observed in MDA-MB-231 cells (Fig. 12B), whereby BBR uptake increased from 0.13 ± 0.02 µg per 10⁶ cells to 0.22 ± 0.05 µg per 10⁶ cells in the free BBR-treated group, while BBR-AgNPs treatment reached significantly higher levels of intracellular BBR concentration, 0.31 ± 0.04 and 0.44 ± 0.07 µg per 10⁶ cells. Overall uptake in MDA-MB-231 remained lower than in MCF-7, which may reflect differences in cellular phenotype and uptake characteristics between the two cell lines; however, the specific molecular determinants of this difference were not investigated in the present study. Both cell lines showed a 2- to 3-fold increase in intracellular BBR uptake following NP-mediated delivery compared with free BBR.

Fig. 12. Cellular uptake, intracellular ROS generation, and mitochondrial membrane potential (MMP) analysis in breast cancer cells treated with free BBR, green AgNPs, and BBR-AgNPs under dark and irradiated conditions. (A and B) Intracellular berberine uptake in MCF-7 and MDA-MB-231 cells, respectively. (C and D) Relative intracellular ROS levels in MCF-7 and MDA-MB-231 cells, respectively. (E and F) Relative mitochondrial membrane potential (MMP) measured using the JC-1 assay in MCF-7 and MDA-MB-231 cells, respectively. Data are presented as mean ± SD from three independent experiments, with each experimental group tested in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared with corresponding control groups.

3.17. Intracellular ROS generation

Intracellular ROS production was quantified using the DCFH-DA assay following exposure to BBR, green AgNPs, and BBR-AgNPs under both dark and irradiated conditions (Fig. 12C and D). All treatments increased ROS levels in MCF-7, and MDA-MB-231 cells compared with untreated controls. Even without irradiation, each treatment induced a gradual elevation in ROS, with BBR-AgNPs eliciting the strongest response, with increases of approximately 2.6-fold and 2.3-fold in MCF-7 and MDA-MB-231 cells, respectively. Irradiation at 470 nm further amplified ROS generation across all groups. In MCF-7 cells, BBR, green AgNPs, and BBR-AgNPs increased ROS levels by roughly 4.0-, 5.7-, and 7.2-fold, while in MDA-MB-231 cells, the corresponding enhancements were 3.7-, 5.0-, and 6.1-fold relative to the control. These outcomes align with established evidence that NP-mediated oxidative stress disrupts mitochondrial integrity, leading to DNA damage and growth inhibition.^83,84 Consistent with this, El-Hussein and Hamblin reported that photo-activated AgNPs induce apoptosis in lung cancer cells via ROS-driven mitochondrial dysfunction and membrane potential collapse, suggesting a broadly applicable phototoxic mode of action across tumor models.^85,86

3.18. Mitochondrial membrane potential analysis

Mitochondrial membrane potential (ΔΨ_m) disruption was evaluated using the JC-1 assay in MCF-7 (Fig. 12E) and MDA-MB-231 (Fig. 12F) cells under dark and irradiated conditions. As shown in Fig. 12E and F, untreated and light-only controls maintained high red/green fluorescence ratios in both cell lines, confirming that 470 nm irradiation alone did not significantly affect mitochondrial integrity. Free BBR induced moderate but significant reductions in ΔΨ_m in both breast cancer models. In MCF-7 cells, free BBR reduced the relative MMP ratio from 1.00 in the control to 0.78 under dark conditions and further to 0.70 following irradiation, indicating enhanced mitochondrial depolarization after photoactivation. A similar trend was observed in MDA-MB-231 cells, where free BBR reduced ΔΨ_m to 0.90 and 0.86 under dark and irradiated conditions, respectively, although the effect remained less pronounced than in MCF-7 cells.

More substantial mitochondrial depolarization was observed following treatment with green AgNPs and BBR-AgNPs, particularly under irradiation conditions. In MCF-7 cells, green AgNPs reduced the relative MMP ratio to 0.66 under dark conditions and to 0.42 after irradiation, whereas BBR-AgNPs produced the greatest mitochondrial dysfunction, decreasing ΔΨ_m to 0.45 in the dark and to 0.31 following 470 nm irradiation. Similarly, in MDA-MB-231 cells, green AgNPs reduced ΔΨ_m to 0.71 and 0.53 under dark and irradiated conditions, respectively, whereas BBR-AgNPs caused the greatest depolarization, lowering the MMP ratio to 0.59 in the dark and 0.41 following irradiation. The significantly greater mitochondrial membrane collapse observed for BBR-AgNPs under irradiation strongly correlates with the elevated ROS generation shown in Fig. 12C and D, supporting ROS-mediated mitochondrial dysfunction as a major mechanism underlying the enhanced photocytotoxicity of the nanoparticles. Excessive ROS accumulation disrupts mitochondrial membrane integrity, impairs electron transport, and activates apoptotic signaling pathways via cytochrome release and caspase activation.^66,87 Furthermore, the greater ΔΨ_m loss observed in MCF-7 cells compared with MDA-MB-231 cells is consistent with the greater photocytotoxicity and ROS generation in the former cell line, suggesting subtype-dependent differences in susceptibility to oxidative mitochondrial damage.

4. Limitations of the study

The primary aim of this study was to evaluate the anticancer and photodynamic efficacy of BBR-AgNPs in breast cancer models. Consequently, cancer selectivity and cellular targeting were beyond the scope of the present investigation. Future studies will be needed to determine the therapeutic selectivity and safety profile of the nanoplatform. The present work is therefore considered an in vitro proof-of-concept study of BBR-AgNP activity in breast cancer models rather than a definitive assessment of cancer versus normal cell selectivity.

The DLS characterization available for the present study was based on the intensity-weighted size distribution provided in the outsourced analysis report; number- and volume-weighted distributions were not available. The DLS results were therefore interpreted together with the PDI and HR-TEM observations to account for particle heterogeneity and association in dispersion. In addition, the calculated BBR association efficiency and loading capacity represent nanoparticle-associated BBR recovered after centrifugation and washing rather than definitive encapsulation within an internal matrix. The current loading procedure does not distinguish weakly adsorbed BBR from more strongly surface-associated BBR. Nevertheless, the release and spectroscopic data support substantial interaction between BBR and the biogenic AgNP surface.

Although the ROS findings are consistent with a contribution from Type I-like oxidative processes, the present experiments do not definitively distinguish Type I from Type II photochemical pathways. More detailed investigation of radical intermediates, interfacial charge-transfer processes, and excited-state dynamics would be required to establish the dominant photochemical mechanism. Similarly, the irradiation wavelengths used in this study, 405 and 470 nm, have limited tissue penetration compared with longer-wavelength excitation, which may restrict the application of the current approach to superficial or externally accessible tumors. Future studies could therefore investigate deeper-penetrating excitation strategies, including two-photon or upconversion approaches.

Finally, although BBR-AgNPs demonstrated enhanced combined chemo-photodynamic activity, a formal pharmacological synergy analysis, such as Combination Index or isobologram analysis, was not performed. The observed enhancement is therefore not interpreted as formally established pharmacological synergy. In addition, the molecular basis of the differential sensitivity observed between MCF-7, and MDA-MB-231 cells was not directly investigated, although the higher intracellular BBR levels, ROS generation, and mitochondrial depolarization observed in MCF-7 cells are consistent with greater susceptibility to oxidative photodynamic stress under the conditions tested. These limitations define important directions for future investigation while not altering the central finding that BBR association with biogenic AgNPs enhances BBR uptake, ROS-associated photodynamic activity, mitochondrial dysfunction, and apoptosis in the two breast cancer models studied.

5. Conclusion

This study developed and evaluated BBR-AgNPs synthesized using Senna didymobotrya leaf extract as a nanoplatform for combined chemo-photodynamic treatment of breast cancer cells. Physicochemical characterization confirmed nanoparticle formation and BBR association with the AgNP surface, while the stability studies demonstrated persistence of the nanoparticle optical characteristics under the tested storage and physiological conditions. The formulation showed a high apparent BBR association efficiency of 90% and a loading capacity of 30%, supporting substantial association of BBR with the biogenic AgNPs.

BBR-AgNPs demonstrated enhanced cytotoxic and photocytotoxic activity in both MCF-7 and MDA-MB-231 cells, with irradiation producing greater effects than the corresponding dark conditions. The enhanced activity was accompanied by increased intracellular BBR uptake, elevated ROS generation, mitochondrial membrane depolarization, and predominantly apoptotic cell death. The relatively small temperature increases observed during irradiation indicate that photothermal heating was unlikely to be a major contributor to the observed photocytotoxicity under the conditions tested. The comparatively greater response of MCF-7 cells was consistent with higher intracellular BBR accumulation, ROS generation, and mitochondrial depolarization in this model.

These findings demonstrate that association of BBR with biogenic AgNPs enhances BBR uptake and ROS-associated photodynamic activity and promotes mitochondrial dysfunction and apoptosis in the two breast cancer models investigated. The study therefore supports BBR-AgNPs as a promising in vitro chemo-photodynamic nanoplatform for breast cancer research. Further studies using non-malignant cell models, deeper-penetrating excitation strategies, and more detailed photophysical and pharmacological analyses will be required to establish therapeutic selectivity, define the dominant photochemical mechanism, evaluate translational applicability, and determine formal combination effects.

Author contributions

Conceptualization- I. B., and P. S.; writing—original draft preparation, I. B., writing—review and editing, P. S., and B. P. G.; supervision, P. S., and B. P. G.

Conflicts of interest

The authors confirm that the research was carried out without any involvement in commercial or financial relationships that could be perceived as a possible conflict of interest.

Data availability

The datasets supporting the conclusions of this article are available within the article and its supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6na00593d.