Synergistic combinatorial anticancer potential of Tamoxifen with Naringin and Diosmetin in MCF-7 breast cancer cells and their liposomal delivery

Abstract

Breast cancer continues to remain a significant issue due to resistance to endocrine therapies, including tamoxifen. Naringin and Diosmetin (natural flavonoids) have anticancer effects that may synergistically enhance therapeutic efficacy when used with Tamoxifen. However, their inadequate solubility and bioavailability require the formulation of specialised delivery systems. This research aimed to determine the synergistic anticancer potential of dual (Tamoxifen + Naringin, Tamoxifen + Diosmetin) and triple (Tamoxifen + Naringin + Diosmetin) combinations, followed by evaluation of their liposomal co-delivery as a formulation strategy. The MTT test evaluated cytotoxic activities in breast cancer cell lines (MCF-7, T47D). IC₅₀ values were obtained for Tamoxifen (9.38 ± 1.87 µM), Naringin (12.34 ± 1.23 µM), and Diosmetin (22.7 ± 1.76 µM). The combination index study showed a more significant synergism when Tamoxifen was combined with Naringin than with Diosmetin. Protein expression analysis revealed significant downregulation of Bcl-2 (p < 0.01) and Bcl-xL (p < 0.01) and upregulation of cleaved caspase-3 (p < 0.01) in the combination-treated groups. Liposomal formulations were developed and characterized based on the identified synergistic effects of combinations and showing particle sizes ranging from 150 to 210 nm, with a PDI of less than 0.3. Zeta potential readings ranging from − 19 to − 29 mV indicated stability, whereas FE-SEM pictures revealed a spherical shape. In vitro release conformed to the Higuchi kinetics for both dual and triple liposomal delivery systems (r² ≥ 0.98). The findings reveal that dual and triple combinations of Tamoxifen with flavonoids synergistically improve cytotoxicity, decrease anti-apoptotic signaling, and activate caspase-dependent pathways. Therefore, the results indicate the synergistic biological effects of Tamoxifen–flavonoid combinations and suggest that nanocarrier-based co-delivery may serve as a beneficial formulation approach for future therapeutic development.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-37954-5.

Introduction

Breast cancer is one of the most important global health challenges, being the most often diagnosed cancer among women and a primary cause of cancer-related mortality globally^1,2. Despite substantial breakthroughs in early identification and the availability of different treatment options such as chemotherapy, radiation, hormonal therapy, targeted therapy, and immunotherapy, achieving satisfactory long-term outcomes remains challenging^3,4. A key limitation of traditional treatment exists in the establishment of drug resistance, non-selective toxicity, systemic side effects, and inadequate absorption of anticancer drugs^5,6. In this context, the co-delivery of natural bioactive substances and known chemotherapeutic medicines employing nanocarrier systems has emerged as a promising technique for boosting therapeutic responses and limiting side effects^7–9. Tamoxifen is a selective estrogen receptor modulator and has been an integral component in the care of estrogen receptor-positive breast cancer for decades. Tamoxifen treatment is typically coupled with problems such as developing resistance, endometrial hyperplasia, and a higher risk of thromboembolic events^10–12. On the other hand, Naringin and Diosmetin are plant-based flavonoids abundantly found in citrus fruits and have gained increased attention in cancer research due to their versatile pharmacological effects. Naringin has powerful antioxidant, anti-inflammatory, and anticancer properties, and preclinical data support its capacity to affect various signaling pathways involved in cell cycle control, apoptosis, angiogenesis, and metastasis^13–15. Also, Diosmetin has exhibited strong anticancer potential via suppression of cell proliferation, induction of apoptosis, and regulation of important signaling pathways. These pathway are PI3K/Akt and MAPK to make it a suitable option for combination treatment^16,17. These two flavonoids target signaling pathways related to tamoxifen resistance, such as PI3K/Akt, MAPK, NF-κB, and Bcl-2 family proteins that control apoptosis. Naringin induces mitochondrial apoptosis and causes ER⁺ breast cancer cells to be more sensitive to endocrine therapy^15,18,19. Diosmetin decreases proliferation by inhibiting Akt/MAPK and has been connected to cross-talk with ER pathways^20–22. Their multitargeted effects enhance the receptor-selective approach of tamoxifen. It provides a strong molecular framework for determining synergistic potential. The combinatory use of Tamoxifen with Naringin and Diosmetin provides a promising potential due to synergistic interactions between phytochemicals and standard drugs. This idea of combination therapy is validated by the emerging knowledge that cancer is a multifaceted illness driven by complex molecular interactions. Single-agent treatment is generally insufficient to completely remove malignant cells especially owing to adaptive resistance mechanisms. Combination treatment can induce synergistic effects by combining drugs with different modes of action and boost therapeutic indices while delaying the establishment of resistance^23–25. The Chou–Talalay approach and similar models have been frequently utilised to study drug–drug interactions and yield quantitative insights into synergism, additivity, or antagonism^26–28. Within this concept, the co-delivery of Tamoxifen with Naringin and Diosmetin appears as a beneficial treatment approach. It works by combining the receptor-specific action of Tamoxifen with the multitargeted, redox-modulating, and pro-apoptotic potential of Naringin and Diosmetin. A key difficulty remains the successful delivery of these drugs to the tumor microenvironment. Conventional oral or parenteral formulations generally fail to deliver prolonged, specific release. Thus, it reduces the therapeutic advantages of combination regimens^29–32. Nanocarrier-based drug delivery approaches, such as liposomes, provide a potent strategy to this challenge^33,34. Liposomes are biocompatible, biodegradable vesicles designed for encapsulating both hydrophilic and lipophilic drugs, preventing them from degradation, extending systemic circulation, and permitting controlled release^34–37. Their versatility in co-encapsulating different drugs further improves their value in combination treatment^38–40.

This research extensively assessed the biological activity and combination profiles of free Tamoxifen, Naringin, and Diosmetin to find effective and synergistic combinations applicable to breast cancer treatment. Following the results of these combination experiments, a liposomal co-delivery method was studied as a formulation approach to improve drug stability and facilitate controlled release. This systematic approach facilitated the rational selection of potent drug combinations based on biological responses, and was followed by the assessment of a liposomal formulation strategy for combination delivery.

Materials and methods

Chemicals and reagents

The drug Tamoxifen (99.77%) was received as a gift sample from CYNO Pharmaceuticals, Solan, H.P., India. Naringin and Diosmetin were procured from TCI Chemicals, Japan, with a purity above 95%. Egg lecithin (AR, > 95.0%) was obtained from TCI Chemicals, Japan, and cholesterol (AR, 99%) was purchased from Sisco Research Laboratory, Maharashtra, India. Dulbecco’s Modified Eagle Medium (DMEM, high glucose), foetal bovine serum (FBS), antibiotic-antimycotic solution (100×), and Trypsin-EDTA were acquired from Gibco, USA. Phosphate-buffered saline (PBS, pH 7.4) and other cell culture reagents were procured from HiMedia, Mumbai, India. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DCFH-DA (2′,7′-dichlorofluorescin diacetate), and AO-EB were procured from Sigma-Aldrich, USA. All other solvents and compounds used were of analytical grade.

Cell lines and culture conditions

MCF-7 cells were purchased from the National Center for Cell Sciences (NCCS), Pune, India. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high glucose; Gibco, USA) supplied with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotic–antimycotic solution (Gibco, USA). Cultures were incubated at 37 °C in a humidified environment containing 5% CO₂. Subculturing was done using 0.25% Trypsin-EDTA (Gibco, USA), and phosphate-buffered saline (1× PBS; Himedia, Mumbai, India) was used for washing.

T47D cells were purchased from the National Center for Cell Sciences (NCCS), Pune, India. The cells were grown in Roswell Park Memorial Institute medium (RPMI-1640; Gibco, USA) supplied with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotic–antimycotic solution (Gibco, USA). Cells were stored at 37 °C in a humidified incubator with 5% CO₂. Trypsin-EDTA (0.25%, Gibco, USA) was utilised for subculturing, and cell culture grade phosphate-buffered saline (1× PBS; Himedia, Mumbai, India) was used for washing.

Determination of IC₅₀ values of Tamoxifen, Naringin, and Diosmetin

The cytotoxic properties of the compounds Tamoxifen, Naringin, and Diosmetin were assessed in human breast cancer cell lines MCF-7 and T47D using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test, as specified by Choupanan et al. (2019) with minor changes^41–45. This test involves the conversion of MTT to purple formazan by mitochondrial enzymes in living cells.

Cytotoxicity assessment in in MCF-7 and T47D cell lines

MCF-7 and T47D cells were plated at a density of 5 × 10³ cells per well in 96-well culture plates and incubated at 37 °C in a humidified environment containing 5% CO₂ for 24 h to facilitate cell adhesion. Following incubation, the medium was substituted with new media containing successive dilutions of Tamoxifen, Naringin, and Diosmetin at doses between 0.2 and 100 µg/mL. The treated cells were incubated for a further 24 h under identical circumstances.

After treatment, 10 µL of MTT solution (5 mg/mL in PBS, pH 7.2) was introduced to each well and incubated for 4 h. The medium was eliminated, and the formazan crystals were dissolved in 150 µL of DMSO. The maximum concentration of DMSO in all treatment and vehicle control wells were below 0.1% (v/v). Plates were incubated at 37 °C for 20 min, and absorbance was measured at 570 nm using a microplate reader (Fluostar OPTIMA, BMG LABTECH, Germany). The absorbance from blank wells containing only the medium was deducted from all sample observations prior to data processing.

Drug combination studies with Tamoxifen

Drug combination studies were performed to study the possible synergistic effects of Tamoxifen when combined with flavonoids, utilizing Naringin and Diosmetin as co-agents to study the possible synergistic effects of Tamoxifen^27,46,47. Dual combinations (TMX + NRG, TMX + DMT) and triple combinations (TMX + NRG+DMT) were assessed only prior to finding individual IC₅₀ values in both MCF-7 and T47D cell lines. MCF-7 cells were chosen for extensive examination of interactions, mechanisms, and liposomal assessment based on their comparable sensitivity profiles. The combination experiments were developed based on fixed molar ratios calculated from the IC₅₀ values of the different drugs, as assessed by the MTT assay on MCF-7 breast cancer cells. In the beginning, dual combinations of Tamoxifen with Naringin and Tamoxifen with Diosmetin were studied at fixed ratios of 1:1, 1:2, and 1:5. For each fixed ratio, serial dilutions of the combinations were made over a concentration range of 0.2–100 µg/mL, ensuring that both compounds were present in the required ratio at all tested concentrations. Considering preliminary results indicating improved effectiveness at the 1:2 ratio, a ternary (triplicate) combination including Tamoxifen, Naringin, and Diosmetin in a 1:2:2 ratio was also examined under the same dose range to explore possible three-drug synergistic effects. Cell viability following 48-hour treatment was measured using the MTT test, and the percentage of viable cells relative to untreated controls was determined. The resulting dose–response data were analyzed using CompuSyn software (ComboSyn Inc., Paramus, NJ. USA. https://www.combosyn.com), which applies the median-effect concept as established by Chou and Talalay. This software determines whether the interaction between Tamoxifen and the flavonoids is antagonistic (CI > 1), additive (CI = 1), or synergistic (CI < 1) by computing the Combination Index (CI) for each combination data point based on the mass-action law. The CI was determined at several effect levels (fraction affected, Fa, ranging from 0.2 to 0.9) to evaluate whether synergy was constant across the whole response range. In addition to the CI, the Dose Reduction Index (DRI) was also determined for each agent in combination. The DRI indicates the amount to which the dose of each drug can be decreased in combination relative to its single-agent IC₅₀, while still achieving the same therapeutic effect (Fa). All combination studies were performed in triplicate and data were provided as mean ± SD. This systematic approach allowed for rigorous examination of the combinatory potential of Tamoxifen with Naringin, and Diosmetin, both individually and in triple combination type.

Molecular and cellular mechanism analysis

Intracellular reactive oxygen species (ROS) analysis

Intracellular ROS levels were evaluated using the DCFDA technique to analyse oxidative stress generated by Tamoxifen, Naringin, Diosmetin, and their combinations⁴⁸. MCF-7 cells were treated for 24 h, followed by incubation with 10 µM DCFDA for 30 min at 37 °C. The degree of fluorescence was measured (Ex: 485 nm, Em: 535 nm) to assess ROS production (Zeiss Axiovert 25, Germany). Quantitative evaluation of fluorescence intensity from microscopic images was carried out using ImageJ software (https://imagej.net/ij/ NIH, USA) and normalized to the relevant untreated control for each test. Thus, free-compound and combination-treatment datasets have different normalization baselines and are not directly comparable between experiments.

Apoptosis assay

Apoptotic cell death was visualized using AO/EB staining after 24-hour treatment⁴⁹. Cells were washed, stained with acridine orange and ethidium bromide, then studied under a fluorescence microscope for apoptotic characteristics (Zeiss Axiovert 25, Germany). Quantitative evaluation of fluorescence intensity from microscopic images was performed using ImageJ software (https://imagej.net/ij/ NIH, USA) and normalized to the relevant untreated control for each test.

Western blot analysis

Cells underwent two washes with ice-cold PBS and then lysed using RIPA buffer (company) with a protease inhibitor, before western blot analysis⁵⁰. The lysates were vortexed and centrifuged at 10,000 rpm for 10 min. BCA was used to calculate protein concentration with a Protein Assay kit (Thermo Fisher Scientific, Waltham, MA) according to the procedure of manufacturer and applying a BSA standard curve. 30 microgrammes of protein were separated using a pre-cast SDS-PAGE gradient gel (4–12%, Bio-Rad, Hercules, CA) and then transferred to a PVDF membrane (Bio-Rad, Hercules, CA). Membranes were incubated with the primary antibody overnight at 4 °C and with the secondary antibody for 2 h at room temperature. Chemiluminescence was identified via the Thermo Fisher Scientific iBright 1500. The antibodies used from Cell Signalling Technology are Bcl-2 (124) Mouse mAb #15,071, Bcl-xL (54H6) Rabbit mAb #2764, Cleaved Caspase-3 (Asp175) Antibody #9661 and β-Actin Antibody #4967.

Preparation of liposomal formulations

Liposomes were prepared by applying the ethanol injection method, following rotary evaporation and sonication with minimal alterations to previously published protocols⁵¹. Egg lecithin (150 mg) and cholesterol were employed as lipid components. Initially, the lipids were dissolved in 10 mL of heated absolute ethanol (~ 60 °C) in a 50 mL round-bottom Quick-fit flask. To this lipid-ethanol solution, 20 mL of Milli-Q water preheated to 70 °C was added immediately, resulting in the instantaneous creation of a milky dispersion due to liposome production. The ethanol was evaporated under lower pressure using a rotating evaporator at 40 °C. Further kept at 4 °C for additional characterization and application.

Several molar ratios of egg lecithin to cholesterol were examined to optimize the formulation from 6:1, 7:1, 8:1, 9:1, and 10:1. The total lipid concentration of the formulation was kept constant at 7.08 mg/mL. Each formulation was determined for particle size, PDI, and encapsulation efficiency. Additionally, the influence of sonication on particle properties was examined by applying bath sonication for varied durations: 0, 5, 10, and 15 min. Upon determining the optimized lipid composition and processing parameters, the drugs were co-encapsulated into the liposomes at a specified ratio of 1:2 (Tamoxifen: phytoconstituents)⁵².

Characterization of liposomes

Pre-formulation studies

Fourier-transform infrared spectroscopy (FTIR) and powder X-ray diffraction (XRD) investigations were done to examine the chemical compatibility of the selected lipids and active pharmaceutical substances (Tamoxifen, Naringin, and Diosmetin)⁵³.

FTIR analysis FTIR spectra of individual substances (egg lecithin, cholesterol, Tamoxifen, Naringin, and Diosmetin and their physical mixes were acquired using an FTIR spectrometer of 4000–400 cm⁻¹. Characteristic peaks were examined to assess any substantial chemical interaction or shift.

XRD analysis Powder XRD was used to evaluate the crystallinity of pure pharmaceuticals and excipients as well as their physical combinations. XRD patterns were acquired using Cu-Kα radiation over a 2θ range of 5°–80°.

Analytical method development for simultaneous Estimation

Before drug loading, a simultaneous estimation method was developed and validated for Tamoxifen, Naringin, and Diosmetin utilising the UV-Vis spectrophotometric (Spectro Photo meter UV-Vis (LAMDA 35, Perkin Elmer) absorptivity coefficient method⁵⁴. Absorbance values were obtained at their respective λmax values, and the method was verified as per ICH standards for linearity, accuracy, precision, and specificity⁵⁵. This procedure was employed for the quantitative estimation of drug content in all subsequent examinations.

Particle Size, PDI, and zeta potential

Particle size, polydispersity index (PDI), and zeta potential of the liposomal formulations were studied using the Dynamic Light Scattering (Malvern Instrument Pvt Ltd ZEN 3600)⁵⁶. The liposomal formulation was suitably diluted with double-distilled water before analysis in a 1:10 ratio to limit the chance of multiple scattering and to ensure accurate readings. All DLS study were conducted using double-distilled water as the dispersant (ionic strength = 0 mM) at 25 °C. Observations were taken at a backscattering angle of 173° and the viscosity. The refractive index of water was automatically applied by the instrument software. The zeta potential was computed using Smoluchowski approach as suited for aqueous dispersions with low ionic strength. The hydrodynamic diameter (Z-average) and PDI provided insights into the average vesicle size and distribution uniformity, respectively. A lower PDI value showed homogeneity in the vesicle population. Zeta potential measurements were also carried out utilising the same instrument to analyse the surface charge of the liposomes. It acts like an indicator of colloidal stability. A high positive or negative zeta potential value is considered important for sustaining dispersion stability by preventing particle agglomeration through electrostatic repulsion.

Field emission scanning electron microscopy

The morphology of liposomes was analysed with FE-SEM (Zeiss Gemini 300, Carl Zeiss, Germany). A small drop of liposome dispersion was applied to a clean conductive substrate, air-dried, and then mounted to aluminium stubs using carbon tape. Samples were sputter-coated with a thin coating of gold (~ 5 nm) to improve conductivity. Imaging was conducted under high vacuum at an accelerating voltage of 15 kV, with a working distance of 7.6 mm and a magnification range of 30–100,000×.

Encapsulation efficiency (EE%) and drug loading (DL%)

Encapsulation efficiency was assessed by separating free (unencapsulated) medication using ultracentrifugation at 15,000 rpm for 45 min at 4 °C. The supernatant was tested using the validated UV method to assess the amount of free drug⁵⁷.

In-vitro drug release study

The dialysis‑bag diffusion technique examined the in vitro drug‑release profile of the liposomal formulation⁵⁸. The properly specified volume of liposome dispersion, comprising a known amount of each drug, was put into a regenerated‑cellulose dialysis membrane with a molecular‑weight cut‑off of 12–14 kDa pre‑soaked in phosphate‑buffered saline (PBS). The quantity of drug in 1 mL of each liposomal dispersion, determined by the initial formulation concentration, was regarded as its initial mass (M₀) for all release computations. The sealed bag was immersed in 50 mL of PBS (pH 7.4) maintained at 37 °C and stirred at 100 rpm with a magnetic stirrer to produce sink conditions and uniform hydrodynamics. 1 mL samples were removed from the release medium at predefined time points from 0, 1, 2, 4, 6, 8, 10, 12, and 24 h, and the medium was instantly replaced with an equivalent volume of fresh pre‑warmed PBS to maintain consistent volume and concentration gradients. Sink conditions were confirmed by comparing the saturation solubility of each drug to the total drug loaded within the dialysis bag. Since the dissolution medium volume (250 mL PBS, pH 7.4) above the minimum sufficient volume (≥ 3× saturation volume) for Tamoxifen, Naringin, and Diosmetin, the system-maintained sink conditions during the experiment. Drug content in each aliquot was assessed by the previously validated UV‑spectrophotometric absorptivity method, and the cumulative percentage of drug released was determined and plotted against time to develop the release profile over 24 h. The release data were analyzed with the zero-order, first-order, Higuchi, and Korsmeyer–Peppas models, and rate constants (k) were derived from the slope of the linearized plots by conventional regression analysis for the evaluation of drug release kinetics. Each experiment and model fit was conducted in triplicate, and goodness-of-fit (r²) values together with residual plots were utilized to evaluate the accuracy of the kinetic models.

In vitro evaluation of liposomal formulations

Cytotoxicity assessment

MCF-7 breast cancer cell lines were seeded into 96-well plates at an optimum density and incubated overnight to allow cell adhesion. The cells were subsequently treated with individual flavonoids, Tamoxifen, and their combined liposomal formulations for 48 h. After the treatment time, cell viability was evaluated using the MTT test. Absorbance was determined at 570 nm, and results were reported as the percentage of inhibition relative to untreated control cells⁵⁹.

Statistical analysis

Results are presented as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA followed by Tukey’s multiple comparison test for the majority of datasets. The expression of cleaved caspase-3 was evaluated by one-way ANOVA with Dunnett’s post-hoc test for assessing each treatment group against the control. Differences were considered significant at p < 0.05 (*), highly significant at p < 0.01 (**), very highly significant at p < 0.001 (***), and extremely significant at p < 0.0001 (****).

Result and discussion

Tamoxifen, naringin, and diosmetin reduced the viability of MCF-7 and T47D cells, and MCF-7 showing higher sensitivity

The IC₅₀ values of Tamoxifen, Naringin, and Diosmetin were examined in MCF-7 and T47D cells after 48 h of treatment (Fig. 1). Tamoxifen showed the maximum cytotoxicity with IC₅₀ values of 9 µM in MCF-7 and 8 µM in T47D cells. Naringin revealed IC₅₀ values of 12 µM and 66 µM in MCF-7 and T47D, respectively, while Diosmetin displayed comparably lower activity with IC₅₀ values of 22 µM in MCF-7 and 77 µM in T47D cells. The findings suggest that while Tamoxifen remains the most potent. Both Naringin and Diosmetin had substantial cytotoxic effects with increased sensitivity observed in MCF-7 cells. The observations highlight the potential of flavonoids as bioactive substances with fundamental anticancer action.

Fig. 1.

% Cell viability of compounds in breast cancer cell lines.TMX: Tamoxifen; NRG: Naringin; DMT: Diosmetin. (A) MCF-7 cell lines (B) T47D cell lines. Data are expressed as mean ± SD from three independent experiments. Statistical significance compared with the untreated control group: p < 0.001 (***), and p < 0.0001 (****).

The dosage (row factor) and drug type (column factor) significantly influenced cell viability in MCF-7 cells (F₁₀,₆₆ = 734.6, p < 0.0001 for dosage; F₂,₆₆ = 58.02, p < 0.0001 for drug), with a notable dosage × drug interaction (F₂₀,₆₆ = 9.633, p < 0.0001). It suggests that the three compounds variably impacted viability across varying concentration ranges. The analysis of T47D cells revealed statistically significant effects of dosage (F₁₀,₆₆ = 2989, p < 0.0001), drug (F₂,₆₆ = 3600, p < 0.0001), and their interaction (F₂₀,₆₆ = 218.2, p < 0.0001). It indicates unique dose-dependent cytotoxic responses among Tamoxifen, Naringin, and Diosmetin.

The results also suggest that MCF-7 cells exhibited greater sensitivity to therapy than T47D, likely due to variations in estrogen receptor (ER) and progesterone receptor (PR) expression between these two luminal breast cancer subtypes^60,61. The significant effect of Tamoxifen aligns with its recognised function as a selective estrogen receptor modulator (SERM) that triggers cell cycle arrest and death in oestrogen receptor-positive breast cancer cells^62,63. Due to the increased responsiveness of MCF-7 cells to Tamoxifen, Naringin, and Diosmetin, mechanistic studies that included ROS analysis, AO/EB staining, and western blotting were conducted only in this more sensitive cell line to achieve clearer molecular insights.

Naringin and Diosmetin exhibited quantifiable cytotoxicity, but with reduced potency compared to Tamoxifen. Prior research indicates that Naringin induces anticancer effects through the activation of oxidative stress and mitochondrial apoptosis pathways in breast cancer models^14,64, whereas Diosmetin has been demonstrated to suppress proliferation by regulating PI3K/AKT and MAPK signalling pathways^65–67. Somewhat elevated IC₅₀ values in T47D cells indicate potential disparities in cellular uptake, metabolism, or resistance mechanisms between the two cell lines^68,69.

Dual and triple combinations of Tamoxifen with flavonoids enhanced cytotoxic effects compared to monotherapies

To examine the combinatorial effect of Tamoxifen with flavonoids (Naringin and Diosmetin), combination index (CI) values were obtained using the Chou–Talalay method across multiple molar ratios (1:1, 1:2, and 1:5) in a concentration range of 0.2–100 µg/mL (Table s1 & s2). The CI values determined by CompuSyn software offer a quantitative assessment of pharmacological interactions, where CI < 1 suggests synergism, CI = 1 denotes an additive impact, and CI > 1 reflects antagonism⁷⁰. Across all three ratios, significant patterns of interaction were detected. At the 1:1 ratio, the combination exhibited moderate to strong synergism at mid to high fractional effects (Fa ≥ 0.5), notably in the concentration range of 5–50 µg/mL. Particularly, the 1:2 combination provided the lowest CI values across a broader concentration spectrum, indicating a stronger synergistic interaction. This interaction is particularly at Fa values of 0.5–0.9, where CI continuously remained far below 1 (Fig. 2A and B). Whereas, the 1:5 ratio demonstrated varying synergy and a trend toward additivity or moderate antagonism at lower concentrations (≤ 1 µg/mL), indicating a less favourable therapeutic window when the flavonoid was in large excess. The isobolograms created for Fa = 0.5 confirm the synergistic effect of the 1:2 and 1:1 combination, where the experimental data points consistently fell below the line of additivity, while those for 1:5 showed wider dispersion around or above the additive line. This visualization further supported the finding that a balanced or appropriately flavonoid-rich ratio (1:2) produced the most promising synergy.

Fig. 2.

Synergistic interaction analysis in MCF-7 breast cancer cell lines. T: Tamoxifen; N: Naringin; D: Diosmetin. (A) Fa-CI Plot of TMX + NRG combinations (B) Fa-CI Plot of TMX + DMT combinations (C) Fa-DRI Plot of TMX + NRG combinations (D) Fa-DRI Plot of TMX + DMT combinations.

Regarding this, the Fa-DRI plots demonstrated a dose reduction index (DRI) > 1 for both Tamoxifen and the flavonoids, particularly in the 1:2 combination (Fig. 2C and D). This means that in the presence of one drug, the effective dose required for the other was greatly decreased while preserving efficacy, a key aspect in lowering toxicity in combinational cancer therapy. Taken together, the data underline that Tamoxifen + Naringin in a 1:2 ratio displays the most effective synergistic interaction, with improved cytotoxic activity over Tamoxifen alone. Diosmetin likewise demonstrated synergy but was considerably less efficacious at larger ratios. The dose-dependent and ratio-specific synergy was validated by isobologram and DRI studies to reveal the CI values²⁸. It offers a strong justification for adopting the 1:2 ratio as the optimum combination for further in vitro and in vivo assessments.

The results indicate a distinct ratio-dependent synergy between Tamoxifen and flavonoids, with the 1:2 combination (Tamoxifen: Flavonoid) yielding the most potent synergistic interaction across various fractional effects (Fa 0.5–0.9) (Tables s3 & s4). This corresponds with the growing acceptance in cancer research that therapeutic synergy is significantly based on optimised drug ratios, and even little variations could affect outcomes or alter interactions towards antagonism⁷¹. Recent research on combination cancer therapies underlines that balanced drug ratios frequently generate the most powerful antitumor synergy. Whereas skewed ratios may impair efficacy or exacerbate toxicity⁷². The DRI findings show values larger than one to highlight that co-administration significantly improves effectiveness while allowing for lower individual doses. It is a desired attribute in clinical settings that lessens unwanted effects and improves patient tolerance. This dose-sparing effect of combination treatment is increasingly emphasised, since decreasing systemic toxicity is just as critical as preserving therapeutic results²⁸. The observed decreased synergy at the 1:5 ratio (flavonoid in excess) resembles new evidence that overloading one component in treatment combinations might diminish synergistic potential, presumably due to off-target effects or competing inhibition of common pathways⁷³.

Molecular and cellular mechanism analysis

Tamoxifen in combination with Naringin and Diosmetin increased intracellular ROS generation in breast cancer cells

The intracellular ROS levels were evaluated using fluorescence microscopy, and the mean fluorescence intensity was calculated to determine oxidative stress across different treatment groups. The combination treatment of Tamoxifen with flavonoids revealed a considerable change in ROS generation compared to the control and individual therapies (Fig. 3A and B). In the free-compound treatment group, Naringin (7.622 a.u.), Diosmetin (7.226 a.u.), and Tamoxifen (7.289 a.u.) exhibited significantly increased ROS compared to the control (1.52 a.u.), with Naringin yielding the greatest ROS among the individual compounds. In the combination-treatment, fluorescence levels were normalized independently to the appropriate untreated control (set as 1.0 a.u.), and hence cannot be comparisons directly with the free-compound dataset. Within this combination study, the triple treatment (Tamoxifen + Naringin + Diosmetin) generated the highest ROS levels (5.807 a.u.) relative to its own control (1.469 a.u.), showing increased oxidative stress induction compared to Tamoxifen alone (1.336 a.u.), Tamoxifen + Diosmetin (1.885 a.u.), and Tamoxifen + Naringin (2.308 a.u.). These data imply that the co-delivery of Tamoxifen with Diosmetin and Naringin increases ROS-mediated cytotoxicity, potentially contributing to improved apoptosis and cancer cell death.

Fig. 3.

Evaluation of oxidative stress and apoptosis in MCF-7 breast cancer cells. TMX: Tamoxifen; NRG: Naringin; DMT: Diosmetin. (A) DCFDA assay showing intracellular ROS generation in cells treated with TMX, NRG, and DMT; (B) AO/EB dual staining illustrating apoptotic morphology in cells treated with the same compounds; (C) DCFDA assay showing ROS generation in combination treatments; (D) AO/EB dual staining depicting apoptosis in combination-treated cells.

The ROS increases found for Tamoxifen alone and, even higher, for the Tamoxifen + Naringin + Diosmetin combination are physiologically achievable and consistent with recognised functions. Tamoxifen may boost intracellular ROS via mitochondrial and NADPH-oxidase–linked mechanisms, and ROS contributes to its cytotoxic effect in hormone-responsive breast cancer cells^74,75. Flavonoids such as Naringin and Diosmetin can promote oxidative stress and mitochondrial dysfunction, resulting in apoptosis. Recent work in breast cancer models indicates Naringin enhances mitochondrial superoxide, and triggers downstream caspase activation⁷⁶. While Diosmetin has been observed to promote ROS and suppress antioxidant defences, and sensitizes cells to death signals¹⁶. In combination, these drugs likely improve cells through the oxidative threshold that shifts ROS from pro-survival signaling to pro-death functioning, integrating with the redox “double-edged sword” paradigm in cancer⁷⁷. Mechanistically, the enhanced ROS in the triple regimen is consistent with a mitochondria-centric apoptotic cascade (cytochrome-c release and caspase activation), commonly described for natural-product adjuvants in breast cancer^78,79. Together, these findings imply a redox-mediated foundation for the elevated cytotoxicity reported in the combinations, with the triple combination providing the highest oxidative stress compared to monotherapies.

Naringin and Diosmetin combinations with Tamoxifen induced higher apoptotic features compared with single treatments

AO/EB dual staining was conducted to evaluate apoptotic induction and fluorescence intensities were determined for acridine orange (AO), ethidium bromide (EB), and the merged (AO + EB) channels (Fig. 3C and D). The control group revealed low apoptosis with high AO intensity (34.826) and negligible EB staining (1.981) to signify healthy living cells. In Tamoxifen-treated cells, a significant drop in AO fluorescence (14.667) and an increase in EB fluorescence (4.378) were found to indicate early apoptotic processes. Additional effects were observed when combined with Diosmetin or Naringin. Tamoxifen + Diosmetin treated cells showed lower AO intensity (13.349) and increased EB (7.146) to indicate a larger fraction of late apoptotic cells. Tamoxifen + Naringin revealed a more drastic drop in AO signal (4.038) and higher EB intensity (7.496) to confirm greater apoptosis.

The triple combination (Tamoxifen + Diosmetin + Naringin) produced the most significant apoptotic alterations shown by the lowest AO fluorescence (3.05), a high EB signal (7.227), and the lowest total intensity of AO + EB (9.752), indicating extreme late apoptosis. These findings validate that the combination therapy, particularly the triple formulation, markedly increases apoptotic cell death in comparison to Tamoxifen. The addition of Diosmetin considerably boosted EB signal to suggest progression to late apoptosis, while Naringin induced an even stronger apoptosis induction. These data correlate with evidence that flavonoids might increase chemotherapeutic-induced apoptosis via mitochondrial disruption and caspase activation^80,81. The findings underline the increased apoptotic potential of combinatorial approaches and require additional confirmation employing apoptotic protein markers such as cleaved caspase-3 or Bax/Bcl-2 in downstream.

Combination treatments modulated apoptosis-related proteins (Bcl-2, Bcl-xL, cleaved Caspase-3) in breast cancer cells

The expression of apoptosis-related proteins (Bcl-2, Bcl-xL, and Cleaved Caspase-3) in both control and treatment groups was determined by western blot analysis (Fig. 4). β-Actin acted as the housekeeping control to standardise patterns in expression. A significant decrease in Bcl-2 levels was found in the combination-treated groups relative to the control group. All treatment groups exhibited a statistically significant decrease in Bcl-2 expression relative to the control (adjusted p < 0.0001), with the most substantial suppression identified in the triple combination (Tamoxifen + Naringin + Diosmetin). Two-way ANOVA indicated significant effects of treatment (row factor; F(1,20) = 3,302,767, p < 0.0001), protein type (column factor; F(4,20) = 5,764,019, p < 0.0001), and a significant treatment × protein interaction (F(4,20) = 209,447, p < 0.0001). Tukey’s post-hoc test proved that all treatment groups exhibited statistically significant decreases in Bcl-2 expression compared to control (adjusted p < 0.0001), with the triple combination providing the greatest reduction. The reduction of Bcl-2 signifies a suppression of anti-apoptotic signalling, confirming previous research showing that flavonoids such as Naringin enhance the sensitivity of cancer cells to chemotherapy by diminishing Bcl-2 levels^64,82. All treatment groups, including Tamoxifen and Tamoxifen + Diosmetin, exhibited significant downregulation of Bcl-xL compared to the control (adjusted p < 0.0001), with the triple combination yielding the most substantial reduction. Since Bcl-xL is a key regulator of mitochondrial apoptosis resistance, its suppression by the combinational treatments indicates elevated pro-apoptotic potential of the combinations, in line with findings that dual or triple drug–flavonoid combinations may increase therapeutic response by targeting mitochondrial integrity^83,84. The expression of cleaved caspase-3 was significantly increased in the Tamoxifen + Naringin and Tamoxifen + Naringin + Diosmetin groups (adjusted p < 0.0001), with the triple combination producing the most intense activation. Pairwise comparisons indicated that the triple combination demonstrated considerably greater cleaved caspase-3 levels than all other groups, driven by the Tamoxifen + Naringin combination. One-way ANOVA indicated a significant difference across treatment groups (F(4,10) = 855.4, p < 0.0001). Dunnett’s test indicated that cleaved caspase-3 expression was considerably elevated in the Tamoxifen + Naringin and Tamoxifen + Naringin + Diosmetin groups (adjusted p < 0.0001), with the triple combination demonstrating the highest activation. The result aligns with prior research indicating that drug-flavonoid combinations can selectively activate or modulate caspase-dependent pathways based on concentration and interaction profiles^85,86. The combination treatments, mainly Tamoxifen + Naringin and Tamoxifen + Naringin + Diosmetin, effectively inhibited the anti-apoptotic proteins Bcl-2 and Bcl-xL and markedly increased caspase-3 expression, indicating a transition towards apoptosis induction. The findings support the hypothesis that the simultaneous application of Tamoxifen and natural flavonoids can synergistically influence apoptotic signalling pathways, offering a viable approach to surmount chemoresistance in breast cancer.

Fig. 4.

Effect of dual and triple drug combinations on apoptosis-related markers in MCF-7 cells. TMX: Tamoxifen; NRG: Naringin; DMT: Diosmetin. Cells were treated with TMX: NRG (6.25:12.5 µM), TMX: DMT (6.25:12.5 µM), and TMX: NRG: DMT (6.25:12.5:12.5 µM). (A) Western blot analysis of Bcl-2, Bcl-xL, and Cleaved Caspase-3, with β-actin as loading control. (B) Relative protein expression of Bcl-2, Bcl-xL, (C) Relative protein expression of Cleaved Caspase-3. Data represent mean ± SD of three independent experiments. Statistical significance vs. control: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

This study emphasises the regulation of broader apoptotic pathways important to breast cancer through the modulation of Bcl-2 family proteins and caspase signalling, in addition to direct protein-level alterations. The Bcl-2 protein family is crucial in regulating the mitochondrial apoptotic pathway through the modulation of mitochondrial outer membrane permeabilization (MOMP). The overexpression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) is commonly linked to chemoresistance and unfavourable prognosis in breast cancer (Zhao et al., 2021). Their downregulation in the Tamoxifen + Naringin and Tamoxifen + Naringin + Diosmetin groups indicates a compromise in mitochondrial integrity, facilitating cytochrome c release and the subsequent activation of the caspase cascade. This aligns with reports that flavonoids like Naringin and Diosmetin increase mitochondrial dysfunction and facilitate apoptosis when combined with conventional chemotherapeutics^87,88.

The substantial caspase-3 activation seen in the Naringin-containing combinations, notably the triple combination, shows significant involvement of the execution phase of apoptosis. Caspase-3 is the last effector caspase responsible for DNA breakage and apoptotic body formation, and its activation signifies permanent commitment to apoptosis. Naringin has been extensively documented to promote caspase-3 activation via ROS-mediated mitochondrial stress^64,89, which is associated with the high caspase-3 induction seen in the Tamoxifen + Naringin and triple-combination groups. Additionally, the cleaved caspase-3 response in the triple combination (Tamoxifen + Naringin + Diosmetin) may reflect pathway crosstalk, where simultaneous regulation of multiple apoptotic and survival signals could trigger compensatory regulatory feedback⁹⁰.

Mechanistically, the reduction of Bcl-2/Bcl-xL and stimulation of cleaved caspase-3 jointly suggest that the combination formulations are leading cells toward intrinsic death. This is clinically important since intrinsic mitochondrial apoptosis is commonly faulty in Tamoxifen-resistant breast cancer cells^91,92. Flavonoids restore this apoptotic equilibrium by sensitizing cancer cells to pro-apoptotic stimuli, decreasing the threshold for caspase activation. Specifically, Naringin has been shown to influence PI3K/Akt and NF-κB pathways^93,94. Both of which depend on Bcl-2 family members to regulate cell survival and boost the pro-apoptotic action of Tamoxifen. Furthermore, Diosmetin interferes with STAT3 and ERK signaling pathways known to maintain Bcl-2/Bcl-xL expression and accelerate apoptosis induction^95,96. Therefore, the observed modulation of Bcl-2, Bcl-xL, and Caspase-3 suggests the potential of combining Tamoxifen with flavonoids to overcome chemoresistance in breast cancer by targeted modulation of intrinsic apoptotic pathways.

Liposomal formulations of Tamoxifen, Naringin, and Diosmetin were successfully prepared using ethanol injection

Liposomal formulations were effectively developed with the ethanol injection technique, achieved by rotary evaporation and regulated sonication. This approach was selected because of its simplicity, reproducibility, and capacity to generate small, stable vesicles with a restricted size distribution. The application of ethanol as a lipid solvent enabled homogeneous dispersion during rapid injection into the aqueous phase to promote spontaneous liposome formation. The impact of altering the molar ratio of egg lecithin to cholesterol was carefully examined to enhance vesicle stability and drug encapsulation. Within the evaluated ratios, the 9:1 formulation possessed the most advantageous properties, producing liposomes with a Z-average of 156.2 nm and a low polydispersity index (PDI) of 0.214 to signify a moderately narrow size distribution. Enhancing the phospholipid across this ratio produced larger particle sizes and wider PDI values is likely due to enhanced membrane stiffness and decreased vesicle production efficiency. The duration of sonication significantly influenced particle size reduction and the attainment of homogeneity. Bath sonication for 15 min markedly lowered particle size while maintaining vesicle integrity. Thus, a sonication time of 15 min was identified as ideal. The resultant liposomes displayed good physical stability as shown by clean dispersions without aggregation or precipitation upon visual inspection. The results validate that a 9:1 molar ratio of egg lecithin to cholesterol is combined with 15 min of bath sonication was yielded an optimised liposomal formulation appropriate for the co-encapsulation of Tamoxifen and flavonoids (Table s5).

The findings correlate with existing scientific studies comparing hydration vs. microfluidic/ethanolic techniques, which emphasize the convenience and versatility of ethanol injection for nanoliposomes⁹⁷. When cholesterol content increases, bilayer order and elasticity rise, which may reduce membrane permeability. Although beyond an optimum level generally results in larger hydrodynamic diameters and wider dispersion due to packing flaws and lower membrane flexibility limits effective vesiculation⁹⁸. Current biophysical work analyses this process, indicating that cholesterol addition may increase vesicle size until a saturation regime is achieved. The trend observed toward bigger Z-averages and greater PDI with increased cholesterol consequently corresponds with these structure–property relationships⁹⁹. The specified lipid ratio and sonication period are compatible with literature-based predictions for creating stable, nanometer-scale liposomes suited for further drug loading^51,100.

Liposomes exhibited favourable physicochemical characteristics suitable for co-delivery

Preformulation studies

FTIR confirmed drug–excipient compatibility and absence of major chemical interactions

FTIR spectroscopy was used to analyse the potential interactions between Tamoxifen, Naringin, Diosmetin, cholesterol, and phospholipid within their physical combinations. The purpose was to confirm chemical compatibility before formulation.

In the FTIR spectrum of the physical combination including Tamoxifen, Naringin, Cholesterol, and Phospholipid (T + N + C + P), all distinctive peaks belonging to the individual components were conserved. Tamoxifen displayed peaks at 1720 and 704 cm⁻¹, Naringin at 2880, 822, and 703 cm⁻¹, Cholesterol at 1383 and 701 cm⁻¹, and Phospholipid at 1743 and 971 cm⁻¹. The combined spectrum revealed no change or major shifting of these peaks to demonstrate the absence of strong chemical interactions or incompatibilities among the ingredients. Similarly, for the Tamoxifen, Diosmetin, Cholesterol, and Phospholipid (T + D + C + P) mixture, the specific peaks of Tamoxifen (1731, 816 cm⁻¹), Diosmetin (1611, 1502, 814 cm⁻¹), Cholesterol (1666, 1109 cm⁻¹), and Phospholipid (1745, 1461 cm⁻¹) were observed in the combined spectrum without any marked alterations. This further demonstrated the persistence of functional groups and compatibility among these components^101–105. The combination of Tamoxifen, Naringin, Diosmetin, Cholesterol, and Phospholipid (T + N + H + C + P) exhibited a combined FTIR spectrum with different absorption bands for each constituent: Tamoxifen (1731, 977, 768 cm⁻¹), Naringin (1507, 980 cm⁻¹), Diosmetin (1655, 1510, 811 cm⁻¹), Cholesterol (1381, 957, 810 cm⁻¹), and Phospholipid (1744, 971 cm⁻¹). No new peaks were generated, nor were any distinguishing peaks deleted, indicating that the physical combination preserved the chemical integrity of the constituent components. The data collectively suggest that the chosen drugs and excipients exhibit chemical compatibility with no significant chemical interactions occurring in their physical combinations (Figure s1). This establishes a robust foundation for further co-encapsulation in liposomal formulations.

The majority of these standard peaks were present in the FTIR spectra of the liposomal formulations (T + N-LPS, T + D-LPS, and T + N + D-LPS). These peaks were shifted and broadened to indicate interactions between the drug molecules and the lipid components (Figure s2). The decrease in strength or removal of some functional group peaks (particularly O-H and C = O) indicates hydrogen bonding and probable encapsulation of Tamoxifen, Naringin, and Diosmetin inside the phospholipid bilayer. These spectrum modifications suggest that there was no significant chemical incompatibility and that the drugs were physically enclosed inside the liposomal matrix rather than chemically altered.

XRD analysis indicated reduced crystallinity of drugs upon encapsulation

The XRD study was conducted on pure Tamoxifen, Naringin, Dosmetin, cholesterol, phospholipid, and the formulated liposomal preparations (T + N, T + D, and T + N + D) to assess their crystalline or amorphous characteristics (Figure s3 & s4). The diffractogram of Tamoxifen displayed distinct individual peaks at 2θ values of 11.4°, 13.6°, 17.6°, and 18.7°, therefore affirming its crystalline nature. Naringin had strong diffraction peaks at 11.5°, 14.4°, 15.5°, 18.6°, and 26.7°, whilst Diosmetin presented substantial peaks at 12.3°, 13.9°, 14.6°, 25.6°, and 26.8°, both suggesting elevated crystallinity. Cholesterol exhibited crystalline peaks at 12.6°, 15.4°, 16.6°, and 20.7°^106–109.

The XRD patterns of the liposomal formulations (T + N, T + D, and T + N + D) exhibited a significant decrease in peak intensity, characterised by wide diffraction profiles and the lack of the majority of sharp distinctive peaks of the pure drugs. This expansion indicates a shift from a crystalline to an amorphous or molecularly dispersed state following the integration of the drugs into the lipid bilayer. The absence or reduction of drug-specific peaks may be linked to effective encapsulation inside the liposomal matrix, which disturbs the long-range crystalline arrangement^110,111. A few residual peaks identified in the formulations might be owing to the presence of cholesterol in the lipid composition. The results validate that the liposome production technique significantly decreased the crystallinity of the active chemicals, potentially improving their solubility and bioavailability.

A validated analytical method enabled simultaneous quantification of TMX, Naringin, and Diosmetin

A UV-Vis spectrophotometric method utilising the absorptivity coefficient approach was developed and validated for the simultaneous quantification of Tamoxifen, Naringin, and Diosmetin. The individual absorption maxima (λmax) were determined as 242 nm for Tamoxifen, 282 nm for Naringin, and 258 nm for Diosmetin. Calibration curves generated in the concentration range of 2–20 µg/mL for each compound revealed good linearity with correlation values (r²) above 0.998, showing adherence to Beer-Lambert’s law. The absorptivity coefficients (ε) at designated wavelengths were recorded and utilised to calculate the concentrations in mixed solutions. Accuracy assessments at 10 µg/mL support the reliability of the method by demonstrating recoveries of 101.8% for Tamoxifen, 104.1% for Naringin, and 97.5% for Diosmetin. Precision was assessed based on % RSD to confirm an acceptable threshold of < 2% by yielding results of 1.02% (Tamoxifen), 1.04% (Naringin), and 0.97% (Diosmetin). Specificity assessments indicated no interference from solvents or excipients at the targeted wavelengths. The method also displayed good sensitivity, with the limit of detection (LOD) ranging from 0.72 to 0.83 µg/mL and the limit of quantification (LOQ) ranging from 2.20 to 2.52 µg/mL. These numbers demonstrate the capabilities of the method to identify and quantify the substances even at lower concentrations. The entire summary of validation parameters is shown in Table s6. The validated UV-Vis spectrophotometric approach demonstrated linearity, accuracy, precision, specificity, and sensitivity, and was successfully utilised for the quantitative assessment of drug content in formulation development and subsequent analytical evaluations.

Liposomal formulations showed optimal particle size, low PDI, and stable zeta potential

The particle size (Z-average), polydispersity index (PDI), and zeta potential of the produced liposomal formulations were measured using Dynamic Light Scattering (DLS) (Table s7). The blank liposome formulation showed a mean particle size of 128.7 d.nm with a low PDI of 0.116 and a zeta potential of − 16 mV, suggesting a homogeneous and moderately stable colloidal dispersion. The single drug-loaded formulations revealed variation in particle size, with Tamoxifen liposomes having the greatest size of 175.6 d.nm (PDI: 0.233), followed by Diosmetin at 167.2 d.nm (PDI: 0.133), and Naringin at 151.9 d.nm (PDI: 0.142). The comparatively greater PDI of Tamoxifen-loaded liposomes suggests a larger size distribution compared to Naringin and Diosmetin formulations. This could be related to the hydrophobic properties of Tamoxifen, altering vesicle packing and size heterogeneity¹¹². The combination formulations showed a further increase in particle size to indicate the influence of drug co-loading on vesicle shape. The Tamoxifen + Naringin (T + N) combination formulation revealed a size of 172.6 d.nm with a PDI of 0.120 and a zeta potential of − 28 mV. Furthermore, the Tamoxifen + Diosmetin (T + D) formulation had a size of 192.2 d.nm with PDI of 0.184, while the triple combination Tamoxifen + Naringin + Diosmetin (T + N + D) indicated a size of 206.2 d.nm with PDI of 0.134 (Figure s5). The observed increase in particle size with combination formulations is likely due to the trapping of numerous bioactive chemicals as resulted in swelling of the bilayer or changes in lipid packing¹¹³.

Zeta potential values for all drug-loaded and combination formulations were determined as negative, varying between − 19 mV (Tamoxifen) to − 29 mV (T + N + D) (Figure s6). The increase in negative surface charge in the combination formulations may contribute to increased colloidal stability via electrostatic repulsion and thereby lower the possibilities of vesicle aggregation^114,115. Thus, the low PDI values showed that the formulations were monodisperse, and the zeta potential values indicated high stability. The particle size range between 150 and 210 nm is regarded as ideal for passive tumor targeting via the increased permeability and retention effect, making these formulations suitable for future biological investigation.

Field emission scanning electron microscopy imaging revealed uniform spherical morphology with smooth vesicle surfaces

The surface morphology and structural attributes of the formulated liposomes were analysed using FE-SEM, with representative images shown in Fig. 5. All three formulations exhibited distinct, spherical vesicles with smooth surfaces, thus confirming effective liposome production. The particles exhibited uniform distribution with limited aggregation, indicating robust colloidal stability. The spherical form of liposomes observed under FE-SEM has been extensively documented in earlier research, showing that the ethanol injection approach utilised in this study consistently yields vesicular nanostructures^116–118. Such properties are favourable as they promote effective drug encapsulation, prolonged release behavior, and increased contact with biological membranes, which are necessary for the therapeutic efficiency of liposomal delivery systems. It is essential to note that FE-SEM analysis requires the air-drying of liquid liposomal dispersions before imaging. This drying process may induce artifacts or morphological alterations associated with partial dehydration. Notwithstanding this intrinsic limitation, the acquired micrographs provide dependable qualitative insights into the overall particle shape.

Fig. 5.

Scanning electron microscopy (SEM) images of liposomal formulations. (A) Tamoxifen + Naringenin (T + N), (B) Tamoxifen + Diosmetin (T + D), and (C) Tamoxifen + Naringenin + Diosmetin (T + N + D).

Liposomes demonstrated high encapsulation efficiency and acceptable drug-loading capacity

The EE% and DL% of the produced liposomal formulations were assessed using ultracentrifugation to obtain the free (unencapsulated) drug, followed by quantification with a validated UV spectrophotometric technique. The findings exhibited considerable heterogeneity in the encapsulation behaviour of individual drugs and their combinations, based on drug characteristics and formulation complexity (Table s8). Among the dual formulations, T + N displayed a high EE% for Tamoxifen at 82.16% with a DL% of 6.32%. Despite this, Naringin exhibited a significantly lower EE% of 62.88% with DL% of 8.98%, which may be attributed to its greater starting concentration and contact with the lipid bilayer. Furthermore, in the T + D formulation, Tamoxifen encapsulated with an efficiency of 79.73% and DL: 6.13%, but Diosmetin displayed a superior EE% of 76.78% and the highest DL% at 10.97%, showing its great affinity for the lipid matrix. In the triple formulation (T + N + D), Tamoxifen maintained a reasonably high EE% of 78.37% with a DL% of 6.03%. EE% of Naringin showed 58.47% and DL% of 8.35%, whereas Diosmetin kept a good EE% of 74.07% and DL% of 10.58%. The minor decrease in EE% for specific components in the triple formulation might be owing to competitive encapsulation inside the liposomal bilayer by simultaneous drug loading¹¹⁹. However, the total encapsulation efficiencies remained within acceptable ranges for effective distribution, showing that the liposomal system was able to support multi-drug loading without a significant decrease in encapsulation efficiency^120,121. These results suggest the possibility of the liposomal formulation in co-delivery applications notably for combination breast cancer treatment giving a viable technique to boost the bioavailability and therapeutic effectiveness of hydrophobic drugs.

In-vitro release studies showed sustained and controlled release of drugs from liposomal carriers

The in vitro drug release properties of Tamoxifen, Naringin, and Diosmetin from the produced liposomal formulations T + N, T + D, and T + N + D were examined over 24 hours by using dialysis membrane diffusion in phosphate buffer (pH 7.4) (Fig. 6). All release profiles were analysed under maintained sink conditions, providing a consistent concentration gradient during the experiment and ensuring the reliability of the obtained release kinetics. A prolonged and regulated release pattern was seen in all combinations to confirm the efficacy of the liposomal encapsulation in controlling drug release¹²². In the T + N formulation, Tamoxifen demonstrated an initial release of 18.07% at 1 hour, which rapidly expanded to 64.07% at 12 hours and attained a maximum cumulative release of 96.47% at 24 hours. Naringin exhibited a considerably delayed release, with 13.48% at 1 hour and 77.03% at 24 hours. The release kinetics showed that Tamoxifen followed the Higuchi model (r² = 0.9932), indicating diffusion-controlled release. Whereas Naringin exhibited a high correlation with both the first-order (r² = 0.9942) and Higuchi (r² = 0.9946) models, suggesting a combination of concentration-dependent and diffusion mechanisms^123,124. For the T + D formulation, Tamoxifen showed a similar release pattern, with 20.22% released in the first hour and 93.50% by 24 hours. Diosmetin had a substantially slower release, begins at 9.58% (1 hour) and reaching 69.26% at 24 hours. Both Tamoxifen and Diosmetin best fit the Higuchi model (r² = 0.9944 and 0.9905, respectively), further suggesting a diffusion-based mechanism for sustained release from the lipid matrix. In the triple combination (T + N + D), Tamoxifen release was partially lowered compared to dual combinations, with 18.06% at 1 hour and 89.64% at 24 hours. Naringin and Diosmetin followed similar controlled release patterns, releasing 78.52% and 67.26%, respectively, within 24 hours. Tamoxifen, Naringin, and Diosmetin best suited the Higuchi model (r² = 0.9856, 0.9889). Among the three formulations, a combination of three drugs in T + N + D slightly reduced the release of Tamoxifen and Diosmetin, which may be related to enhanced lipid–drug interactions and larger encapsulation loads^125,126. The uniform Higuchi ‘best-fit’ patterns observed across all formulations further support that drug diffusion from the lipid matrix served as the primary mechanism under sustained sink conditions. Korsmeyer-Peppas n exponents (≈ 0.48–0.62) show non-Fickian transport to confirm liposomal carriers utilise diffusion and matrix relaxation mechanisms (Table s9). These results demonstrate that the liposomal approach successfully supplies a regulated and prolonged release of Tamoxifen, Naringin, and Diosmetin, which is beneficial for combination cancer therapy, decreasing the frequency of administration and enhancing bioavailability. The Higuchi model predominated across all formulations, indicating diffusion as the predominant release mechanism from the liposomal matrices^127,128.

Fig. 6.

Cumulative drug release profiles of TMX and flavonoids from liposomal formulations over 24 h. TMX: Tamoxifen; NRG: Naringin; DMT: Diosmetin (A) TMX and NRG from T + N liposomes (B) TMX and DMT from T + D liposomes (C) TMX, NRG, and DMT from T + N + D liposomes. Data represent mean ± SD of three independent experiments.

Liposomal formulations supported enhanced cytotoxic responses in MCF-7 cells while maintaining minimal toxicity toward HEK-293 cells

The viability of the non-cancerous HEK-293 cell line was evaluated after treatment with liposomal formulations under conditions similar to those used in the cancer cell tests. Across the examined concentration range (0.2–50 µM), HEK-293 cells exhibited good viability with all formulations, indicating minimal cytotoxicity to normal cells. Two-way ANOVA demonstrated significant effects of concentration (row factor: F (9,40) = 49.07, p < 0.0001), formulation (column factor: F (3,40) = 72.85, p < 0.0001), and their interaction (F(27,40) = 4.428, p < 0.0001). Post-hoc Tukey comparisons indicate that at elevated concentrations (≥ 6.25 µM), Tamoxifen-liposome resulted in significantly reduced viability compared to the combination liposomes (adjusted p < 0.001 to < 0.0001 at 6.25–50 µM). Although the combination liposomes (T + N, T + D, T + N + D) typically maintained higher viability in HEK-293 cells (Fig. 7A). Significantly, an IC₅₀ value for all liposomal formulations in HEK-293 cells was not attained within the tested dosage range (≤ 50 µM), suggesting that the formulations exhibit much lower toxicity to HEK-293 cells compared to breast cancer cells under the specified circumstances. These results imply adequate cellular tolerance. However, wider biological safety validation involving different normal cell models and functional evaluations would be necessary for a comprehensive analysis.

Fig. 7.

Cell viability (%) of HEK-293 and MCF-7 cells treated with formulations. (A) Cell viability (%) of HEK-293 normal cells and (B) MCF-7 breast cancer cells after treatment with liposomal formulations. TMX-Lipo (Tamoxifen loaded liposome); T + N-Lipo (Tmaxifen+Naringin loaded liposome); T + D-Lipo (Tmaxifen + Diosmetin loaded liposome); T +  N + D-Lipo (Tmaxifen + Naringin + Diosmetin loaded liposome). Data are represented as mean ± SD from three independent experiments. Statistical significance compared with the untreated control: p < 0.001 (***), and p < 0.0001 (****).

The cytotoxic efficacy of the liposomal formulations was assessed in MCF-7 cell lines by comparing their inhibitory effects over a range of concentrations using Tukey’s multiple comparison test (Fig. 7B). T + N + D-liposomes exhibited markedly enhanced cytotoxicity compared to Tamoxifen alone (p < 0.01) at reduced concentrations (0.2–0.4 µM) and in comparison to binary combinations (T + N and T + D). The amplified effect was particularly evident at 0.8–3.12 µM, when T + N + D continuously diminished cell viability more significantly than either single-drug or two-drug liposomes (p < 0.001). The triple-drug liposomal system (T + N + D) exhibited considerably greater potency (p < 0.0001) at elevated concentrations (6.25–50 µM), with mean differences in cell survival surpassing 10–17% relative to other groups. It indicates an enhanced combinatorial effect within liposomal delivery. In the binary formulations, T + N-liposomes exhibited slightly greater inhibitory efficacy than T + D-liposomes at various doses. However, the differences were not statistically significant at most concentrations, except at 25 µM, when T + D maintained a survival advantage (p < 0.01). The findings indicate that Naringin may boost cytotoxicity more significantly when combined with Tamoxifen than with Diosmetin, but both agents have increased efficacy relative to free Tamoxifen alone. Treatment concentrations for liposomal preparations are indicated as Tamoxifen-equivalent concentrations. In dual and triple combination liposomes, Naringin and/or Diosmetin were co-encapsulated at predetermined ratios, with their actual quantities in the formulations determined by their respective encapsulation efficiencies.

Mechanistically, the enhanced cytotoxicity of liposomal formulations may be attributed to several factors. Liposomes boost drug solubility, stability, and cellular absorption, permitting larger intracellular accumulation of Tamoxifen and co-encapsulated phytochemicals^129–131. The T + N + D liposomes displayed the highest efficacy, which might result from the multi-targeted effect of Naringin and Diosmetin. Both are known to produce pro-apoptotic and chemosensitizing effects in cancer cells^16,132. The synergy probably arises from simultaneous modulation (Fig. 8) of estrogen receptor signaling (Tamoxifen)¹³³, oxidative stress and PI3K/Akt inhibition (Naringin)^13,66, and mitochondrial apoptosis pathways (Diosmetin)^16,134, consistent with previous reports highlighting phytochemical-drug synergy in breast cancer therapy¹³⁵. Overall, the results suggest that co-encapsulation of Tamoxifen with phytochemicals in liposomes boosts cytotoxic potential beyond that of free drug or binary systems, with the triple-drug formulation displaying the most noticeable effect. These findings strengthen the rationale for multi-agent liposomal delivery systems as a potential approach for enhancing combination-based anticancer responses and require additional biological validation. Although the liposomal formulations showed better cytotoxic effects under in-vitro conditions, the current biological study of the nanoformulations is limited to MTT-based cell viability assessment. Further research involving additional biological studies and more advanced experimental designs will be essential to completely verify the therapeutic efficacy and mechanistic importance of the liposomal delivery system.

Fig. 8.

Graphical abstract displaying the role of tamoxifen in combination with Naringin and Diosmetin.

Further IC₅₀ values were determined using concentration–response curves to evaluate the cytotoxic efficacy of free drug combinations vs. liposomal preparations. The combinations of Tamoxifen–Naringin (1:2) and Tamoxifen–Diosmetin (1:2) demonstrated reduced IC₅₀ values, indicating enhanced intrinsic cytotoxic efficacy. Liposomal Tamoxifen had a higher IC₅₀, aligning with a regulated drug-release profile. The triple-drug liposomal formulation exhibited a much lower IC₅₀ compared to single- and dual-drug liposomes, indicating enhanced cytotoxic efficacy within the formulation form (Figure s7). Analysis of IC₅₀ values indicates that free Tamoxifen–flavonoid combinations have more potent cytotoxic effects, while liposomal preparations enhance the efficacy of these combinations by increasing stability. Comparable findings have been shown for tamoxifen-based liposomal systems that include natural flavonoids, whereby nanoformulation improves delivery efficacy without exceeding the inherent potency of unencapsulated combinations⁵⁸. The triple-drug liposomal system demonstrated enhanced cytotoxic efficacy relative to single-drug liposomes. It confirms its function as an effective delivery platform rather than a substitute for combination synergy.

Conclusion

The present research shows that the combination of Tamoxifen with the natural flavonoids Naringin and Diosmetin produces a synergistic effect on breast cancer cells by regulating apoptotic signalling pathways. The downregulation of anti-apoptotic markers (Bcl-2, Bcl-xL) and the activation of cleaved caspase-3 indicate the potential of these combinations to overcome the resistance mechanisms often linked to Tamoxifen alone. Using this molecular understanding, liposomal formulations were effectively developed to co-deliver these drugs to exhibit favourable physicochemical properties and prolonged release behaviour. The use of combination therapy with improved nano-formulation offers dual benefits of increasing apoptotic induction and enhancing drug absorption and stability. These findings highlight a promising method that integrates pharmacological synergy with nanocarrier-mediated delivery to enhance anti-breast cancer efficacy. Future in vivo validation and preclinical analysis are necessary for developing this strategy into clinically applicable therapeutic interventions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

P.U: Conceptualization, methodology, visualization, writing—original draft, formal analysis, experimental design, and carrying out experiments & measurements. S.D.P. and S.P.: experimental design, and carrying out experiments & measurements. P.S.: Supervision & Conceptualization. P.R.: Supervision & Conceptualization. D.P. and S.G.: Supervision & Conceptualization. R.R: Conceptualization, Investigation, Supervision, Writing—review & editing. A.G.: Supervision, Writing—review & editing, V.S.L: Writing—review & editing.

Funding

Priyanka Uniyal, Ph.D. Scholar is especially thankful to Research & Development, UPES, Dehradun, for providing funding support as a Junior Research Fellowship (JRF) and yearly contingency.

Data availability

All data analyzed during this study are available from the corresponding author (Anand Gaurav) on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.