Formulation and evaluation of quinoa seed oil loaded folic acid conjugated bovine serum albumin nanoparticles for anti-breast cancer activity

1 Introduction

Cancer is a significant social, public health, and economic challenge in the 21st century, accounting for about one in six fatalities (16.8%) and one in four deaths (22.8%) from noncommunicable diseases (NCDs) globally (Bray et al., 2021). In 2022, female breast cancer was the second most common illness worldwide, with around 2.3 million new cases, or 11.6% of all cancer diagnoses. The disease is the fourth leading cause of cancer-related mortality globally, resulting in 666,000 deaths (6.9% of total cancer fatalities) (Bray et al., 2024). A systematic investigation of the molecular distinctions between cell lines and tumors indicates that breast cell lines, on average, possess a greater number of genetic mutations and a comparable level of epigenetic alterations relative to the cancers from which they originate. The fundamental molecular characteristics of cell lines, however, are consistent with those of tissue malignancies, apart from a few atypical instances where primary markers have diversified into many variants (Dai et al., 2017).

Conventional approaches to cancer treatment include surgical interventions, frequently paired with chemotherapy and radiation as necessary (Satapathy et al., 2023). Despite their effectiveness, both medications present significant adverse effects that limit their application. As a result, these treatments could be more harmful and lead to various unwanted side effects, as well as detrimental effects on healthy cells in the body (Mirhadi et al., 2020). Herbal products may serve as complementary therapies to alleviate adverse effects while also reducing exposure to chemotherapy and radiation (Greenwell and Rahman, 2015). Phytochemicals can be used as chemotherapeutics that are isolated from various plant extracts, as these phytochemicals have shown diverse effects on anti-tumor, anti-inflammation, anti-oxidant, and anti-bacterial activities (Shrihastini et al., 2021).

Chenopodium quinoa Willd. Is a traditional crop indigenous to Bolivia, Peru, and Chile, which has gained popularity because of its salt resistance and high nutritional value (Pulvento and Bazile, 2023). The seeds of this salt-tolerant crop can be utilized to produce puffy cereals or milled into flour for baking purposes. The genus Chenopodium is globally distributed, with over 200 species, with seed coloration ranging from white to gray and black (Park et al., 2017). Quinoa seed oil (QSO) is rich in essential minerals, including polyphenolics (primarily vanillic acid, ferulic acid, and their derivatives) as well as the principal flavonoids quercetin, kaempferol, and their glycosides; vitamins (ascorbic acid, thiamin, riboflavin, and α-tocopherol); trace elements; and macronutrients (protein and carbohydrates) (Shen et al., 2022). QSO has been recognized in several studies as a preventive agent towards multiple ailments, including cancer, allergic reactions, and inflammatory conditions. Furthermore, they may diminish the risk of cardiovascular disorders (Heck et al., 2019). Several additional in vitro studies have demonstrated the anticancer effects of QSO. QSO demonstrates a significant cytotoxic impact on liver cancer cell lines (Mohamed et al., 2019) and human cervical carcinoma cell lines (Paśko et al., 2019). However, the limited solubility of QSO constrains its utilization in the biological domain, especially in cancer therapy. Incorporation of QSO into nanoparticles can augment their therapeutic efficiency against cancer and enhance their antioxidant activity.

Nanotechnology possesses the potential to offer significant opportunities for the advancement of medical innovations across all fields. In recent years, the pharmaceutical industry has focused on enhancing nanotechnology frameworks applicable in numerous medical sectors, notably in drug delivery (Calzoni et al., 2019). In recent decades, polymeric nanoparticles have demonstrated significant potential as therapeutics in biomedical applications, including controlled drug and gene delivery, tissue engineering, and cancer therapy (Nitta and Numata, 2013). An expanding area of research is the application of nanotechnology in conjunction with phytochemicals. Incorporating nanomaterials can enhance the bioavailability, targeting, and regulated release of phytocompounds (Watkins et al., 2015).

Recent breakthroughs in drug delivery system design have emphasized protein-based nanodrug delivery platforms, classified as substances composed of self-assembled protein subunits of a singular protein or a mixture of proteins forming a cohesive structure. Among the extensive array of proteins employed in nanoparticle preparation, bovine serum albumin (BSA) is extensively exploited owing to its bioavailability, non-toxicity, water solubility, and benign degradation compounds (Tarhini et al., 2018). Bovine serum albumin nanoparticles (BSA-NPs) comprise charged amino acids that non-covalently function as transport mechanisms for diverse phenolic acids, including caffeic acid and salicylic acid, thereby promoting their cellular uptake. The surface characteristics of BSA-NPs, including charge, binding sites, and amino acid content, augment their capacity to bind to phenolic acids. Moreover, the electrostatic adhesion of phenolic acids onto the BSA surface is more efficient when the BSA molecules are much smaller, thereby enhancing the regulation of drug dispersion (Ibrahim et al., 2025).

Recently, various investigators have concentrated on folate receptors that specifically target cancerous tumors, which are overexpressed in cancer cell membranes while exhibiting low expression in normal tissues. Folic acid (FA) exhibits several beneficial characteristics that render it suitable for medication delivery: it is biologically compatible, not immunogenic, stable, and nontoxic (Nosrati et al., 2018a). Folic acid (FA) was utilized exclusively as a surface-targeting ligand to enhance folate receptor-mediated internalization of the nanoparticles. The conjugated FA facilitates nanoparticle uptake in folate receptor-overexpressing cells, hence enhancing targeted drug administration without suggesting a direct impact on cancer cell growth.

Recent research has emphasized the therapeutic potential of QSO-based nano formulations for biomedical applications, especially in cancer treatment. Whey protein isolate-gelatin nanoparticles have been shown to augment the water dispersibility and antioxidant efficacy of QSO, hence enhancing its biological application (Lira et al., 2020). Quinoa oil-alginate-loaded nanoemulsions exhibited considerable anticancer activity in chemically generated breast cancer models, indicating the promise of nanoencapsulation methods in enhancing treatment results (El Makawy et al., 2024a). Additional research shown that the nanoencapsulation of quinoa seed oil through oil in water emulsification augmented its antioxidant capacity and inhibitory effects on digestive enzymes, signifying greater bioactivity through nanoscale delivery (Marques et al., 2024). Moreover, quinoa and chia seed oil nanocapsules were observed to promote tumor growth regression by modulating oncogenic markers such as PIK3CA and MYC, exhibiting anti-inflammatory and antiproliferative activities while ensuring hepatorenal safety in breast cancer models (El makawy et al., 2024b). While these findings generally affirm the potential of QSO nano formulations in cancer treatment, tailored delivery methods utilizing folic acid-conjugated bovine serum albumin nanoparticles for breast cancer therapy are still insufficiently investigated.

The present study lies in the development of a folic acid-conjugated bovine serum albumin (FA-BSA) nanoparticle system for the targeted delivery of QSO. To the best of our knowledge, this is the first study to formulate QSO into FA-conjugated BSA nanoparticles and evaluate its targeted anticancer potential against breast cancer cells. While previous reports have described BSA nanoparticles for drug delivery (Ibrahim et al., 2025), and quinoa oil has been studied for its nutritional benefits (Shen et al., 2022), the combination of active targeting with QSO encapsulation in a protein-based nanocarrier represents a novel strategy. The targeted nanoparticles developed herein hold significant potential for selective delivery of hydrophobic bioactive to folate receptor-overexpressing cancer cells, addressing the dual challenges of poor oil solubility and lack of tumor specificity.

2 Methodology

2.1 Materials

We acquired the non-ionic surfactant Polyoxyethylene sorbitan monooleate (Tween 80) from Merck, India (catalog no. 9005-65-6). MTT (catalog no. 298-93-1), DAPI (catalog no. 28718-90-3), propidium iodide (PI) (catalog no. 25535-16-4), RNase (catalog no. 9001-99-4), and rhodamine (catalog no. 81-88-9) were obtained from HiMedia Laboratories, India. The Annexin V-FITC Early Apoptosis Detection Kit (catalog no. E-CK-A211) was procured from eLabScience, India. We acquired the CFSE proliferation kit from ThermoFisher Scientific-Life Science Solutions (catalog no. C34570), India. BSA (catalog no. 9048-46-8) and FA (catalog no. 59-30-3) were procured from Sigma Aldrich Chemicals Private Limited, India. DMSO (catalog no. 67-68-5), N-hydroxysuccinimide ester (NHS) (catalog no. 6066-82-6), N, N'-dicyclohexylcarbodiimide (catalog no. 538-75-0), triethylamine, and chloroform (catalog no. 67-66-3) were acquired from Sisco Research Laboratories Pvt. Ltd. (SRL), India. Double-distilled water was used throughout the experimental procedures (Milli-Q system, Millipore Corporation).

2.2 Isolation of quinoa seed oil (QSO) from quinoa seeds

The Chenopodium quinoa seeds were collected from Kendriya Bhandar, Jamia Hamdard, New Delhi, India, and were further identified by the expert Dr. Fazil Hasan (Assistant Professor, Department of Agriculture, School of Sciences, Noida International University, Greater Noida, U.P., India) with reference number NIU/SOS/AGRI/ID/2026/01. Oil extraction was carried out using the Soxhlet extraction technique (Mufari et al., 2020). It is a continuous extraction of solid and liquid phases. The solid substance designated for extraction is situated in a thimble composed of a material that retains solids while permitting only liquids to permeate, functioning similarly to filter paper. The thimble is thereafter positioned in the extractor. The organic solvent is heated in reflux, causing the produced vapors to boil. As the vapor ascends, it is condensed by the condenser, which subsequently fills the thimble. This procedure is reiterated until the extraction of all components from the solids is complete (Patel et al., 2019).

Briefly, 200 g of quinoa seeds were finely ground using a mechanical grinder, and the resulting powder was sieved through muslin cloth and air-dried to remove residual moisture. The dried seed powder was placed in the Soxhlet extraction chamber; 600 mL of n-hexane was added to a round-bottom flask as the extraction solvent. The Soxhlet apparatus, consisting of the round-bottom flask, extractor, and condenser, was assembled and secured using clamps. Continuous cooling was maintained by circulating water through the condenser. The solvent was heated using an isomantle, allowing n-hexane to evaporate, condense, and percolate through the seed material. Once the solvent level reached the siphon arm, it was automatically returned to the boiling flask, initiating repeated extraction cycles. The extraction process was continued for 6 h. Following completion, the oil extract was collected in the round-bottom flask, and the solvent was removed by evaporation at 60 °C using a rotary evaporator and kept at 4 °C until required.

2.3 GC-MS analysis

For analyses and identification of compounds in QSO, GC-MS (Gas Chromatography-Mass Spectrometry) was performed using a Shimadzu GC-MS-QP 2010 instrument, Mass Hunter\W11N17main.L, established at Jamia Hamdard, New Delhi. An auto injector and mass selective detector were facilitated in the instrument used in this study. The ion source temperature and interface temperature were set at 220 °C and 270 °C, respectively. In addition, the solvent cut time, threshold, and mass range were set to 3.50 min, 1,000 eV, and 40–650 m/z, respectively. A capillary column (Rxi-5 Sil MS) made by the Restek Company in Bellefonte, United States, having specifications of 30 m long, 0.25 m wide, and 0.25 m thick was utilized for the separation of the parts. Parted delivery technique incorporated with split proportion of 10:1, setting thermostat at 260 °C and extracted seed oil injection of 1.0 L, in addition to raising the oven temperature to 300 °C at 10 °C per minute for 16 min, using helium gas as a carrier gas for the investigation. For compound identification, 16.3 and 1.21 mL/min remained for total flow and column flow, respectively. Entries in the National Institute of Standards and Technology (NIST) database and the WILEY8 spectrum library were carried out through a comparison of their compared accumulation time and mass distribution for determination of elements.

2.4 Preparation of blank bovine serum albumin nanoparticles (blank BSA NPs)

Blank BSA-NPs were synthesized using the desolvation technique (Galisteo-González and Molina-Bolívar, 2014). BSA (100 mg) was solubilized in 2 mL of 10 mM NaCl, with the pH adjusted to eight using 0.1 N NaOH. This was succeeded by stirring for 10 min, after which the desolvating agent, 100% ethanol, was added dropwise (1 mL/min) until an opalescent dispersion was formed. Thereafter, the desolvated BSA-NPs were cross-linked with 10 µL of glutaraldehyde at a concentration of 8% (v/v), and the reaction was maintained under continuous moderate magnetic stirring for 24 h at ambient temperature. The resultant colloidal dispersion was subjected to centrifugation at 12,000 rpm for 35 min at 4 °C. The supernatant was subsequently discarded, and the precipitated BSA-NPs were redispersed in 1 mL of PBS (pH 7.4).

2.5 Preparation of FA conjugated BSA NPs (FA-BSA NPs)

Folic acid (FA), one-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and 4-dimethylaminopyridine (DMAP) were introduced into dimethyl sulfoxide (DMSO) under light protection to activate FA by stirring at 50 °C. BSA was ultrasonically dissolved in buffer. The activation liquid of FA was incrementally added and let to react under slow stirring in darkness at ambient temperature. Folate-modified albumin nanoparticles (FA-BSA NPs) were acquired during dialysis against 10 mmol/L NaHCO₃ and ultrapure water (Liu et al., 2022).

2.6 Preparation of QSO encapsulated BSA NPs (QSO-BSA NPs)

A homogeneous BSA solution was prepared using 0.66 g of BSA and 25 mL of water and stirred for 24 h at 25 °C. This solution was added 0.033 g of QSO and stirred for several hours (solution I). Also, Tween 80 (0.75 g) was dissolved in 25 mL PBS at a concentration of 0.1 M and pH of seven and stirred for 30 min at 25 °C (solution II). Then the former solution was added dropwise to the latter solution under stirring. Finally, the BSA nanoparticles containing QSO were obtained. The freeze-dryer was used to gain a solid powder of the formulation (Angourani et al., 2022).

2.7 Determination of QSO encapsulation efficiency and loading capacity

The effectiveness of QSO encapsulation and loading capacity were assessed using a UV-visible spectrophotometer (UV-1800 series, UV Probe 2.62 software, Shimadzu Japan) (Kunjiappan et al., 2018). QSO-BSA NPs (5 mg) were dispersed in phosphate buffer solution (pH 7.4; 50 mL) and centrifuged at 15,000 rpm at 4 °C for 45 min. After centrifugation, the supernatant containing unencapsulated QSO was collected and mixed with ethanol to solubilize the free oil. The concentration of QSO in ethanol was determined using a UV-visible spectrophotometer at 380 nm. The loading capacity (% (W/W) and encapsulation efficiency (EE) were determined using the following formulas:

EE % = Total drug – Free drug Total drug × 100

Drug Loading % = Entrapped drug Total NP weight × 100

2.8 Synthesis of N-hydroxysuccinimide ester of FA (FA-NHS)

The synthesis of FA-NHS was conducted as illustrated by Al-Rahim et al. (2023). All trials were conducted in darkness to prevent the deterioration of FA. FA (1.5 g; 3.40 mmol) was solubilized in a solution comprising DMSO (25 mL), N-hydroxysuccinimide ester (NHS) (0.445 g; 4.08 mmol), N, N-dicyclohexylcarbodiimide (1.05 g; 5.10 mmol), and triethylamine (0.25 mL). The resulting solution was activated in a thermostatic water bath at 40 °C and agitated for 48 h at 80 rpm. The by-product of dicyclohexylurea is eliminated using filtering. The resulting FA-NHS filtrate solution was freeze-dried using a lyophilizer (Martin Christ Germany) for 48 h and thereafter kept in a dark environment.

2.9 Formulation of FA conjugated QSO-BSA NPs (FA-QSO-BSA NPs)

The FA-QSO-BSA nanoparticles were synthesized by conjugating the carboxylic group of folic acid with the amino group of QSO-BSA nanoparticles (Yang et al., 2014). QSO-BSA nanoparticles (5 mg) were dissolved in 3 mL of carbonate/bicarbonate buffer (pH 10.0; 0.1 M). A DMSO solution of FA-NHS (1 mg/198 mL) was incorporated into the aforementioned mixture and agitated for 2–3 h at ambient temperature. DMSO was eliminated by dialysis with water. A Sephadex G-50 column was utilized to isolate unreacted FA-NHS and other by-products from the FA-QSO-BSA nanoparticles. The eluent comprising FA-QSO-BSA nanoparticles were subjected to centrifugation for 30 min at 21,000 rpm and subsequently re-dispersed in 4.0 mL of filtered water.

2.10 In vitro QSO release study

The release of QSO from FA-QSO-BSA NPs was investigated in 0.01 M phosphate buffer solutions (pH 3.2, 5.4, and 7.4) at 37 °C using the dynamic dialysis technique (Kunjiappan et al., 2019). The selection of pH 3.2, 5.4, and 7.4 was intended to simulate different physiological environments relevant to oral and gastrointestinal conditions, tumor-associated acidic microenvironment, and physiological blood pH, respectively (Bachta et al., 2025). FA-QSO-BSA NPs (50 mg) were put in a dialysis bag (3,500 Da molecular weight cut-off) situated in a beaker containing 30 mL of phosphate buffer solution and incubated in a shaker at 37 °C and 150 rpm. The selected medium volume was sufficient to maintain the concentration of released QSO below its saturation level, thereby preserving the concentration gradient required for continuous drug diffusion and avoiding saturation-related effects during release analysis (Gupta et al., 2022). The samples were withdrawn at designated time intervals (30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 till 24 h) and substituted with new medium. The obtained supernatant was diluted with ethanol, and the quantity of QSO released from nanoparticles was measured at 380 nm using UV-visible spectrophotometry. All measurements were performed in triplicate. The cumulative amount of drug release over the time period was plotted. The subsequent formula was employed for computations:Where, A0 = absorbance of the control; A1 = absorbance of the sample.

% Drug release = A 0 − A 1 × 100 / A 0

2.11 Characterization of synthesized FA-QSO-BSA NPs, blank BSA NPs, and FA-BSA NPs

2.11.1 Dynamic light scattering (DLS)

The formulated FA-QSO-BSA NPs, blank BSA NPs, and FA-BSA NPs were analyzed for average size distribution (Z-average), polydispersity index (PDI), and Zeta potential using Zeta Sizer Ver. 7.12 (Malvern instrument) at 25 °C. Prior to the experiment, the samples were diluted with MilliQ water to a concentration of 50% to mitigate impact of several scattering effects. The measurement entailed sending a laser beam with a wavelength of 633 nm through a 2 mL aqueous solution at a detection angle of 90 degrees. The polydispersity index, Z-average, and zeta potential were calculated, and a size distribution by intensity graph was generated. The DLS measurements were conducted in triplicate, and the mean standard deviation was determined.

2.11.2 Transmission electron microscopy (TEM)

The study utilized TEM imaging at the Central Institute Facility, Department of Pharmacy, Jamia Hamdard, New Delhi, India, to analyze the surface morphology of the FA-QSO-BSA NPs, blank BSA NPs, and FA-BSA NPs including size, structure, homogeneity, and roughness. The specimen was placed on a carbon grid and subsequently submerged in 2% phosphotungstic acid (PTA) for 2 min. The grid was desiccated and analyzed by transmission electron microscopy. The image capture utilized a Tecnai G2, 20 S–twin, FEI electron Optics system, equipped with digital photography capabilities and a 35-mm visual apparatus. Results were recorded using a high-resolution digital CCD camera and analyzed using iTEM software.

2.11.3 Fourier-transform infrared spectroscopy (FT-IR)

The QSO, BSA, FA, and FA-QSO-BSA NPs underwent FT-IR examination with Varian 7000 FTIR and Varian 600UMA FT-IR microscopes. The samples were collected on a KBr pellet prior to analysis with the FT-IR spectrophotometer. The materials were evaluated by scanning within the wave number range of 4,000 cm^-1 to 400 cm^-1. The scanning was conducted at a resolution of 2 cm−1, with 16 scans executed for each sample. The data were analyzed with Origin 2021b software.

2.11.4 High-performance liquid chromatography (HPLC)

HPLC analysis was conducted at the Central Instrumentation Facility (CIF), Jamia Hamdard, utilizing a Waters HPLC system with a quaternary pump and managed by Empower™ 2 software (Build 2,154 SPs). Before sample analysis, the HPLC system was purged with methanol to remove residual contaminants and particle matter, thereafter equilibrating with the mobile phase composed of methanol. The samples were prepared by dissolving them in a chloroform: methanol combination (2:1, v/v) and subsequently placed into sampling vials. A volume of 20 µL was utilized for all analyses. The chromatographic run time was established at 15 min for the control QSO, while an extended duration of 30 min was utilized for both FA-QSO-BSA NPs and blank BSA-FA NPs. Chromatographic separation was carried out at a detection wavelength of 310.0 nm with a flow rate of 1.0 mL/min. Chromatograms were documented and analyzed with Empower™ 2 software.

2.12 Cytotoxic activity

2.12.1 Cell culture

The breast cancer cell lines MCF-7 and MDA-MB-231, together with the non-cancerous HEK293 cell lines, were obtained from NCCS, Pune, India. The cells were grown in DMEM medium (HiMedia, Mumbai, India) supplemented with 10% FBS (Fetal Bovine Serum, HiMedia, Mumbai, India) and antibiotics (penicillin and streptomycin) (100X, HiMedia, Mumbai, India) at 37 °C in an environment of 95% air and 5% CO₂.

2.12.2 In vitro cytotoxicity study

The MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) experiment was conducted to evaluate the cytotoxic effectiveness of QSO (QSO was diluted to 5 mg/mL with 10% DMSO in all cell experiments), formulated FA-QSO-BSA NPs, blank FA-BSA NPs (NPs without QSO encapsulation), and the positive control medication doxorubicin on MCF-7 and MDA-MB-231 cell lines compared with untreated (negative control) cell lines. The cytotoxicity of FA-QSO-BSA NPs was assessed against non-cancerous HEK293 cell lines. The cells were inoculated into 96-well culture plates at a density of 1 × 10⁴ cells per well and incubated for 24 h. Subsequently, the cells were subjected to QSO, formulated FA-QSO-BSA NPs, blank FA-BSA NPs, and doxorubicin at various concentrations (20, 40, 60, 80, 100, 150, 200, 250 μg/mL). The cells were subsequently cultured for 24 h. Subsequently, 10 μL of MTT solution (5 mg/mL in PBS) was applied to each well. The plates were then incubated for a further 4 h. The MTT was removed, and the resulting formazan crystals were dissolved in 100 μL of DMSO, followed by gentle agitation. The absorbance was measured at a wavelength of 570 nm using the microplate reader (SpectraMax 340 PC, Molecular Devices, USA) (Ghosh et al., 2025). The investigations were performed thrice, and the results were presented as the percentage of live cells about the medications under evaluation. The IC50 values were calculated by plotting the percentages of cell viability for MCF-7 and MDA-MB-231 cells versus the sample concentrations using GraphPad Prism 9 software.

% Viability = ( Mean Test OD/Mean untreated Control OD ) × 100

2.12.3 Cellular uptake study

To evaluate cellular uptake, two treatment groups were prepared: FA-Rho-B-BSA NPs (Rhodamine-B-loaded FA-BSA NPs) and a Rho-B-QSO physical mixture (Rhodamine-B and QSO at a 1:5 w/w ratio in PBS containing 0.5% DMSO v/v). 1 mg Rhodamine-B was encapsulated in 1 mL FA-BSA NPs through same procedure as described in Section 2.6 and characterize for zeta size, PDI, and zeta potential by DLS (Supplementary Figure S2). MCF-7 and MDA-MB-231 cells (3 × 10^5 cells/well) were seeded in 35 × 60 mm plates on sterile 18 mm round cover slips (Deckglässer) and incubated for 24 h. After incubation, the cells were treated with 17 μL of each formulation, incubated for 3–4 h, and then underwent two to three washes with PBS. The cells were fixed with a 4% aqueous solution of paraformaldehyde, washed with PBS. Image analysis was conducted using ImageJ/Fiji (NIH, UNITED STATES) to evaluate Mean Fluorescence Intensity (MFI). The first raw photographs were subjected to background subtraction using a uniform fixed radius applied consistently across all groups. Images were later transformed into 8-bit format, and a uniform threshold was used across all samples to ensure unbiased segmentation. Whole-cell segmentation was performed using the “Analyze Particles” tool, with size exclusion criteria set to exclude debris while including all intact cells within the field.

The % uptake was calculated as:

Uptake efficiency % = ( MFI FA − Rho − B − BSA NPs ) / ( MFI ​ Rho − B − QSO ) × 100

Fold increase in uptake:

Fold Uptake = MFI FA − Rho − B − BSA NPs / MFI Rho − B − QSO

2.12.4 Nuclear morphology assay

In this investigation, breast cancer cells (3 × 10^5 cells/well) were seeded in 35 × 60 mm plates on sterile 18 mm round cover slips (Deckglässer) and incubated for 24 h. Following incubation, the cells were treated with FA-QSO-BSA NPs and QSO (at the IC₅₀ concentration) and incubated for 24 h. After incubation, both the control cells (untreated) and the cells treated with FA-QSO-BSA NPs and QSO were first fixed with methanol. Subsequently, DAPI (1 μg/mL) was administered to the cells and incubated in the dark at 37 °C for 15 min. The alterations in nuclear morphology were analyzed compared with control cells and determined through confocal microscopy (Leica DMRE) equipped with a confocal head TCS-SPE (Leica, Wetzlar, Germany).

2.12.5 Cell proliferation assay

In this study, cells were seeded at a density of 1 × 10⁶ cells/mL in 25 cm² flasks and incubated for 24 h to facilitate adhesion. Following incubation, the cells were trypsinized and transferred into 15 mL polypropylene tubes, then washed 2–3 times with 1X DPBS. The CFSE dye (5 μM) was added to the solution and incubated at 37 °C for 15 min in a water bath. The substance was subsequently centrifuged at 1,000 rpm for a duration of 5 min. The supernatant was removed, and the pellet was combined with 10 mL of complete media. The mixture was subsequently centrifuged, and the supernatant was discarded. The pellet was meticulously combined and re-suspended in the whole medium. The CFSE-labeled cells were subsequently placed in a six-well plate and incubated for 24 h. After incubation, cells were treated with FA-QSO-BSA NPs and QSO at the IC₅₀ concentration and allowed to incubate for 24 h. Subsequently, the CFSE dye-labeled cells were subjected to cytometric analysis using the BD-LSR II flow cytometer. The CFSE distribution was analyzed utilizing FCS Express Flow Cytometry Software - De Novo Software.

2.12.6 Apoptotic assay

In this investigation, cells were seeded at a density of 1 × 10⁶ cells/mL in 25 cm² flasks and incubated for 24 h to promote adhesion. Following incubation, cells were treated with FA-QSO-BSA NPs and QSO at the IC₅₀ concentration, whereas untreated cells served as controls. Following treatment, both control and treated cells (1 × 10⁶ cells/mL) were harvested and re-suspended in 500 µL of Annexin-V binding buffer. 5 μL of Annexin-V FITC and propidium iodide were administered to each tube. The samples were thereafter incubated in darkness at room temperature for 15–20 min. Apoptosis analysis was performed using a BD LSR II Flow Cytometer, acquiring 25,000 events per sample. The apoptotic distribution was analyzed utilizing FCS Express Flow Cytometry Software - De Novo Software.

2.12.7 Cell cycle arrest analysis

Cell-cycle analysis was performed by evaluating the DNA content in both treated and untreated cells using flow cytometry following propidium iodide (PI) staining. In this experiment, cells were seeded at a density of 1 × 10^6 cells/mL in a 25 cm² flask and incubated for 24 h. After incubation, cells were treated with the IC₅₀ concentration of FA-QSO-BSA NPs and QSO, followed by an additional incubation period of 24 h. The cells were then trypsinized and centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and 4 mL of 70% ethanol was added to the pellet, which was subsequently incubated on ice for 30 min for fixation. Subsequently, the cells were subjected to centrifugation, the supernatant was discarded, and the pellet was reconstituted in 200 μL of PBS. 50μL of RNase was added to the solution and kept on ice for 30 min to enhance RNase activity. Subsequently, 5 μL of propidium iodide was added and incubated in the dark for 5 min. The resulting mixture was subsequently analyzed using the BD-LSR II Flow Cytometer. Each sample documented 25,000 occurrences. Cell cycle arrest was assessed using FCS Express Flow Cytometry Software - De Novo Software.

2.13 Statistical analysis

The study was conducted in three biological and experimental replicates. The GraphPad Prism software (Version 9.0) was utilized to determine statistically significant outcomes (p < 0.05). An unpaired t-test was used to compare two groups, while a one-way ANOVA with a Tukey post hoc test was applied to assess three groups. Data is expressed as the mean ± standard deviation (n = 3).

3 Results

3.1 GC-MS

QSO is a complex combination of notably bioactive constituents, including polyphenols, saponins, betanin, sterols, and unsaturated fatty acids. The predominant fatty acid methyl esters (FAMEs) include linoleic acid (46.67%), palmitic acid (11.87%), oleic acid (10.03%), erucic acid (7.57%), and stearic acid (1.56%). The GC-MS chromatogram had shown 63 active compounds present in the QSO as shown in Table 1. GC-MS reported 9,12-Octadecadienoic acid as the constituent with the highest peak area % of 45.42%, followed by Hexadecanoic acid, methyl ester (11.63%); 13-Octadecenoic acid, methyl ester, (Z) (9.41%); Squalene (6.07%); and 13-Docosenoic acid, methyl ester, (Z) (5.15%), as shown in Figure 1A.

TABLE 1. Compounds identified by GC-MS analysis of Quinoa seed oil.

3.2 Characterization of FA-QSO-BSA NPs

3.2.1 DLS

DLS is a method for ascertaining the particle size distribution (Zeta Size), PDI, and Zeta Potential of FA-QSO-BSA NPs. Figures 1B,C illustrate the mean hydrodynamic diameter of FA-QSO-BSA NPs, approximately 146.4 ± 1.32 nm, a PDI of 0.214 ± 6.1, and a zeta potential of −26.0 ± 5.43 mV. Supplementary Figure S1A demonstrate DLS analysis of QSO-BSA NPs and FA-BSA NPs demonstrating a mean hydrodynamic particle size (Z-average) of 141.1 ± 6.7 and 145.0 ± 3.2, respectively with a PDI of 0.265 ± 1.5 and 0.255 ± 2.3, respectively. Supplementary Figure S1B illustrate zeta potential analysis of QSO-BSA NPs and FA-BSA NPs exhibiting a surface charge of −10.4 ± 4.2 and −10.6 ± 5.9, respectively.

3.2.2 TEM

The TEM image of FA-QSO-BSA NPs illustrates effective nanoparticle synthesis with distinct shapes. The particles generally exhibit a spherical to near-spherical morphology with thick, electron-opaque cores, signifying efficient encapsulation of QSO inside the BSA matrix. The nanoparticles were predominantly discrete; however, some aggregations were noted in specific areas, likely due to sample drying during grid preparation. The FA-conjugated nanoparticles (FA-QSO-BSA NPs) exhibited a smooth and compact surface morphology under TEM, indicating a stable nanoparticle architecture with an average dry core diameter of 50 ± 5.6 nm as observed in Figure 1D. The unconjugated QSO-BSA NPs showed spherical morphology with mean size of 91.8 ± 7.2 nm (Supplementary Figure S1C). The spherical morphology was preserved after conjugation, confirming that the formulation process does not disrupt nanoparticle integrity. Supplementary Figure S1C revealed TEM image of QSO-BSA NPs and FA-BSA NPs revealing predominantly spherical morphology with nanoscale dimensions and relatively uniform particle distribution.

3.2.3 FT-IR

The FTIR spectrum of QSO displayed a significant absorption peak at 1743 cm^-1, indicative of the ester carbonyl (C=O) stretching vibration, with typical aliphatic C–H stretching bands at 2,922 and 2,853 cm^-1. In the spectra of QSO-loaded BSA NPs, prominent Amide I and Amide II bands were seen at 1,650 cm^-1 and 1,540 cm^-1, respectively, affirming the proteinaceous composition of the BSA carrier system. The ester carbonyl peak was seen at 1741 cm^-1, exhibiting a little shift from 1743 cm^-1, which signifies the effective encapsulation of QSO within the nanoparticle matrix without considerable chemical degradation. The FTIR spectrum of FA exhibited distinctive aromatic ring vibrations at 1,604 and 1,483 cm^-1, signifying its structural fingerprint. The spectra of FA-QSO-BSA nanoparticles exhibited the simultaneous existence of these distinctive peaks. A significant widening in the O–H/N–H stretching area (3300–3400 cm^-1) was seen, accompanied by FA fingerprint bands superimposed on the BSA amide backbone, therefore verifying the effective surface conjugation of FA to the QSO-loaded BSA nanoparticles as shown in Figure 1E.

3.2.4 HPLC

The chromatographic profile of the QSO had four prominent peaks at RT 2.053, 2.815, 3.148, and 4.334 min. Conversely, the chromatogram of blank BSA–FA NPs exhibited three distinct peaks with RT of 1.861, 3.109, and 4.278 min, which were ascribed to the constituents of the BSA–FA nanoparticle matrix. The chromatographic analysis of FA-QSO-BSA nanoparticles revealed six peaks. The peaks identified at RT of 1.812, 3.115, and 4.276 min closely align with those seen in blank BSA–FA nanoparticles, indicating the inclusion of the nanoparticle carrier matrix in the formulation. Supplementary peaks identified at retention times of 2.657, 2.833, and 3.343 min exhibited correlation with peaks in free QSO, suggesting the existence of QSO within the nanoparticle formulation as shown in Figure 2A. Minor variations in retention durations may result from interactions between QSO components and the BSA–FA matrix during encapsulation.

3.3 Drug encapsulation efficiency and loading capacity

The encapsulation efficiency (EE) of QSO–BSA NPs was determined using UV–Visible spectrophotometry. The overall QSO-BSA NPs concentration was 1.231 mg, but the quantity of free (unentrapped) QSO quantified in the supernatant was 0.768 mg. The quantity of encapsulated QSO was determined to be 0.463 mg. The entrapment efficiency was calculated to be 37.61% based on these figures. The drug loading percentage was determined by the weight of the encapsulated QSO in relation to the total weight of the nanoparticles. The weight of the entrapped QSO was 0.463 mg, and the overall weight of the nanoparticles was 1.231 mg. Accordingly, the drug loading efficiency was found to be 19.26%. In addition, while optimization of formulation of QSO–BSA NPs significantly improves EE and DL (Supplementary Methods and Results section), the optimized batch was not evaluated in biological assays, and future studies would address this.

3.4 In vitro QSO release study

The QSO was gradually and steadily released from the FA-QSO-BSA NPs, as seen in Figure 2B. QSO was released from NPs in 30 min at 1% (pH 3.2), 2% (pH 5.4), and 4% (pH 7.4). After the first half hour, NPs gradually increased the amount of QSO released, which was approximately 10.5% (pH 3.2), 25% (pH 5.4), and 32% (pH 7.4) after 4 h. The release of QSO from FA-QSO-BSA NPs was significantly impacted by pH variations. After 20 h, there was a noticeable rise in the total amount and rate of QSO released from FA-QSO-BSA NPs (at pH 3.2, 5.4, and 7.4, the release rates were 72.53%, 85.98%, and 94.65%, respectively) (*P < 0.05, ****P < 0.0001). QSO release was more effective at neutral pH 7.4 and at pH 5.4 than pH 3.2, according to the observed experimental results as shown in Figure 2B.

3.5 In vitro cytotoxicity study

The cells were exposed to several concentrations of FA-QSO-BSA NPs, QSO, and blank FA-BSA NPs. It was observed that the FA-QSO-BSA NPs effectively suppressed the proliferation of breast cancer cells at IC50 values of 64.12 μg/mL and 79.14 μg/mL for MCF-7 and MDA-MB-231 (P < 0.05), respectively. However, FA-QSO-BSA NPs did not exhibit any inhibitory effect on non-cancerous HEK293 cells (Figure 3). Considering the cytotoxicity of QSO with respect to IC₅₀ indicates that 150.12 μg/mL and 165.12 μg/mL of QSO were required to eradicate half the population (50%) of MCF-7 and MDA-MB-231 cells (P < 0.05), respectively. Blank FA-BSA NPs did not show any inhibitory effect on MCF-7 and MDA-MB-231.

3.6 Cellular uptake study

A fluorescent lipophilic substance (Rhodamine-B) was added to the prepared FA-BSA NPs (FA-Rho-B-BSA NPs) and QSO (Rho-B-QSO) to ascertain the internalization of FA-Rho-B-BSA NPs and Rho-B-QSO in breast cancer cells. After 4 h of incubation, FA-Rho-B-BSA NPs was effectively absorbed inside the cytoplasm of MCF-7 and MDA-MB-231 cells, as shown in Figure 4. Cells treated with FA-Rho-B-BSA NPs exhibit a greater fold increase in cellular uptake compared to those treated with Rho-B-QSO, with increases of 4.2 in MCF-7 and 3.0 in MDA-MB-231, respectively.

3.7 Nuclear morphology assay

DAPI staining of MCF-7 and MDA-MB-231 cells revealed normal nuclear morphology in control cells, but QSO treated cells resulted in nuclear condensation and structural modifications. Whereas, in FA-QSO-BSA NPs induced more significant chromatin condensation, nuclear fragmentation, signifying increased apoptosis-related nuclear alterations relative to free QSO treatment as shown in Figure 5.

3.8 Cell proliferation assay

The CFSE fluorescent dye was employed to confirm the findings of the MTT assay in the cell proliferation assay. Figure 6 illustrates the proliferation pattern of the FA-QSO-BSA NPs labeled with CFSE in the FA-QSO-BSA NP-treated cells (MCF-7 and MDA-MB-231) compared to the control (untreated) and QSO-treated cells. The cell growth rate was reduced from 99.5% to 73.2% in control and QSO-treated cells, respectively, to 54.8% in FA-QSO-BSA NP-treated MCF-7 cells (*P < 0.001) and from 97.6% to 76.4% in control and QSO-treated cells, respectively, to 53.9% in FA-QSO-BSA NP-treated MDA-MB-231 cells (**P < 0.0001), as observed in Figure 6.

3.9 Apoptotic assay

The FA-QSO-BSA NPs-treated cells exhibited a notable rise in the proportion of PI-Annexin-V FITC + cells (indicating early apoptosis) and a reduction in PI-Annexin-V FITC- cells (indicating viable cells) compared to the control cells. The percentage of cells in the early stages of apoptosis increased from 0.07% to 32.05% and from 0.19% to 27.20%, respectively, in the FA-QSO-BSA NP-treated MCF-7 and MDA-MB-231 cells (P < 0.01) as observed in Figure 7. Whereas, the percentage of cells in the early stages of apoptosis increased from 0.07% to 16.30% and from 0.19% to 6.91%, respectively, in the QSO-treated MCF-7 and MDA-MB-231 cells (P < 0.01).

3.10 Cell cycle arrest analysis

The assay results demonstrated that the percentage of cells in the G0/G1 phase increased from 55.7% in control (Untreated) MCF-7 cells to 82.2% in the FA-QSO-BSA NP-treated MCF-7 cells (P < 0.05). Conversely, the percentage of cells in the G2/M and S phases decreased from 23.5% to 20.8% in control (Untreated) MCF-7 cells to 7.7% and 10.1% in the FA-QSO-BSA NP-treated MCF-7 cells, respectively (P < 0.05). Similarly, the percentage of cells in the G0/G1 phase increased from 55.9% in control (Untreated) MDA-MB-231 cells to 80.7% in the FA-QSO-BSA NP-treated MDA-MB-231 cells (P < 0.05). Conversely, the percentage of cells in the G2/M and S phases decreased from 24.3% to 19.8% in control (Untreated) MDA-MB-231 cells to 8.3% and 11.0% in the FA-QSO-BSA NP-treated MDA-MB-231 cells (P < 0.05), respectively (Figure 8). The findings from the cell cycle study indicate that both cell lines (MCF-7 and MDA MB-231) experienced cell cycle arrest in the G0/G1 phase. In QSO-treated MCF-7 cells, the G0/G1 phase increased to 62.8%, and the S and G2/M phases increased to 20.9% and 16.3%, respectively (P < 0.05). Furthermore, in QSO-treated MDA-MB-231 cells, the G0/G1 phase increased to 66.6%, and the S and G2/M phases increased to 21.1% and 12.3%, respectively (P < 0.05).

4 Discussion

A chemical characterization of the principal lipid components, specifically triacylglycerols, fatty acids, and the unsaponifiable fraction, in a sample of QSO is detected by GC-MS. In this study, GC-MS analysis of QSO identified 63 active compounds. QSO contained a prominent amount of 9,12-Octadecadienoic acid (45.42%); Hexadecanoic acid, methyl ester (11.63%); 13-Octadecenoic acid, methyl ester, (Z) (9.41%); Squalene (6.07%); and 13-Docosenoic acid, methyl ester, (Z) (5.15%). The chemical 9,12-octadecadienoic acid, methyl ester (Z, Z), has significant anticancer efficacy against the A549 human lung cancer cell line. 9,12-octadecadienoic acid, methyl ester (Z, Z), is a bifunctionally unsaturated fatty acid, typically present in plant glycosides. It is an essential fatty acid in the mammalian diet and has a role in the synthesis of prostaglandins and cellular membranes (Ukwubile et al., 2019). Hexadecanoic acid, methyl ester, possesses antioxidant, anti-inflammatory, antihyperlipidemic, and antibacterial properties (Yu et al., 2005). Similar findings have been made regarding the anticancer activity of 13-dodecanoic acid methyl ester E and 13-octadecenoic acid methyl ester (Z, Z) against a prostate cancer cell line with an IC50 value of 32.11 ± 0.41 (Yu et al., 2005), with antioxidant and anticancer properties. 13-Octadecenoic acid, methyl ester (E), has been isolated initially from the leaves of Physalis angulata (Solanaceae) and other plant families. It exhibits anticancer and antioxidant properties (Mizushina et al., 1996). Squalene has demonstrated preventive effects against several carcinogens. A protective effect was discovered when squalene was administered before and/or during carcinogen exposure. Experimental investigations have demonstrated that squalene can successfully impede chemically induced skin carcinogenesis in animals (Smith, 2000). Ganesan and Manivel (2018) evaluated the anticancer potential of squalene isolated from the leaves of Canthium coromandelicum against liver cancer cells (HepG2). Results demonstrated a concentration-dependent reduction in cancer cell viability, with an IC50 value of 100 μg/mL and a marked decrease in cell survival to 32.8%. The findings suggest that squalene exhibits significant anticancer activity.

In our investigation, QSO was encapsulated in BSA-NPs and conjugated with FA to improve solubility and anti-breast cancer efficacy. The incorporation of target-specific ligands into nanoparticle systems enables the precise identification and binding to receptors on cancer cells, hence enhancing the targeting efficiency of the carrier and the therapeutic index of the conjugated medication. Albumin’s surface, comprising carboxyl and amine groups, facilitates covalent interactions with desired ligands, enabling diverse surface modifications, enrichments, and functionalization of albumin nanoparticles (Ibrahim et al., 2025). Folate receptors are markedly overexpressed in several human cancer cells, such as those in breast cancer, while exhibiting modest expression in healthy organs (Siwowska et al., 2017). Folic acid is non-immunogenic, stable throughout a wide spectrum of pH levels and temperatures, and possesses a distinctive capacity to bind to folate receptors following conjugation with drug-loaded albumin nanoparticles. Upon binding to the receptors, folate is absorbed into the cell via the endocytic route. A little decrease in pH (to around 5) alters the receptor’s conformation, resulting in the dissociation of folate from the receptor and the subsequent release of the loaded drug into the cell (Alpers, 2016). In our study, the FA-QSO-BSA NPs demonstrated an average particle size of 146.4 ± 1.32 nm. Prior studies have demonstrated that nanoparticles measuring 100–250 nm can efficiently aggregate in malignant tissues owing to the permeability of tumor vasculature. For example, the hydrophobic bexarotene (BEX) was encapsulated onto a BSA surface and then conjugated with folic acid. The resultant nanocomposite had a diameter of roughly 200 nm, signifying a limited size dispersion (Qi et al., 2014). This size closely resembles that of our nanocomposite (FA-QSO-BSA NPs) developed in our research. Given the extensive vascularization linked to breast malignancies, nanoparticles of this dimension are anticipated to effectively traverse the cell membrane through both active targeting via folic acid (FA) receptors and passive targeting mechanisms (Tseng et al., 2008). Additionally, a prior study verified that A549 cells showed strong folic acid binding, improving BEX’s selectivity and effectiveness while encouraging apoptosis and lowering the drug’s toxicity to healthy cells (Srivastava and Prajapati, 2020).

The polydispersity index (PDI) indicates the consistency in the size distribution of nanoparticle populations, ranging from 0.0 to 1, where 0 signifies an ideal uniform distribution of particle sizes and one denotes significant polydispersity with various particle size groups. For a nanoparticle, PDI values of 0.2 and less are predominantly documented in studies (Gaur et al., 2023). The PDI value was 0.214 ± 6.1, indicating a reasonably narrow variation of particle sizes. The statistics demonstrated that the PDI value of our NPs falls within the uniform size range.

Our findings verified that the zeta potential of the FA-QSO-BSA NPs was −26.0 ± 5.43 mV. This number, ranging from +30 to −30 mV, indicates that the nanoparticles are stable. Zeta potential is a critical parameter for the stability of nanoparticles in a colloidal form (Zhang et al., 2008). The zeta potential of blank FA-BSA-NPs was measured at −10 mV. Following loading with QSO, the zeta potential significantly shifted to −26 mV. This increase in negative surface charge is attributed to the ionizable free fatty acids present in QSO. QSO is rich in unsaturated fatty acids, particularly linoleic acid (C18:2, ∼55%) and oleic acid (C18:1, ∼15%), which contain free carboxyl groups (Mohamed Ahmed et al., 2021). At physiological pH (7.4), these carboxyl groups (pKa ∼4.8–5.0) undergo deprotonation, generating additional negative charges at the NPs and water interface. Furthermore, the incorporation of hydrophobic oil may induce minor conformational changes in BSA. Ballicu et al. (2026) demonstrated that BSA undergoes conformational rearrangement upon nanoparticle interaction, exposing buried anionic residues and contributing to the net surface charge. The elevated zeta potential (negative/positive) of the drug-loaded nanoparticles suggested the presence of robust repulsive forces that enhanced particle stability and inhibited nanoparticle aggregation.

The functional features, particularly physical form and size, significantly influence uptake, elimination, residence in circulation, and cellular penetration of the nanoparticles (Sabourian et al., 2020). Nanoparticle shape significantly influences circulation time in the bloodstream, retention inside blood vessels, and uptake by cancer cells (Vagena et al., 2025). A study on the preparation of gold nanostructures of comparable size but varying shapes: nanospheres, nanodisks, nanorods, and nanocages, revealed that nanospheres exhibited the longest circulation time in the bloodstream and the highest altogether uptake in breast cancer in mice (Xu et al., 2023). Similarly, Huang et al. (2012) investigated the size-dependent infiltration of spherical gold nanoparticles in breast cancer cells, tumor spheroids, and murine tumors, demonstrating that smaller gold nanoparticles exhibited superior cellular uptake, deeper tumor penetration, and enhanced accumulation due to improved uptake characteristics, with localization in both the cytoplasm and nucleus; additionally, TEM analysis of spheroid cellular microstructure revealed that surface-adjacent cells remained healthy, resembling monolayer cultures, whereas central cells showed apoptotic or injury-associated features, including marked chromatin compaction, segregation, and nuclear fragmentation. This was a significant benefit of the nanocarrier drug delivery technology. TEM examination verified the effective synthesis of FA-QSO-BSA nanoparticles exhibiting a well-defined spherical to near-spherical shape and consistent nanoscale dimensions. The existence of electron-dense cores signifies effective encapsulation of QSO inside the BSA matrix and indicates that FA conjugation did not undermine nanoparticle integrity, hence affirming structural stability. Minor aggregation found in some places is likely attributable to drying artifacts during grid preparation rather than formulation instability. Our result aligns with the study by Ibrahim et al. (2025).

The FTIR results validate the effective encapsulation of QSO inside the BSA NPs and conjugation of FA. The distinctive ester carbonyl peak of QSO at 1743 cm^-1 and its little shift to 1741 cm^-1 in the NP spectra signify successful entrapment without chemical degradation, implying physical encapsulation rather than structural modification of the QSO. The significant Amide I (1,650 cm^-1) and Amide II (1,540 cm^-1) bands substantiate the proteinaceous structure of BSA and affirm the creation of nanoparticles. The retention of these amide peaks following oil loading indicates the structural integrity of the carrier system. The emergence of FA-specific aromatic ring vibrations at 1,604 and 1,483 cm^-1 in the conjugated nanoparticles, along with the widening in the O–H/N–H stretching region (3300–3400 cm^-1), corroborates effective surface functionalization. The presence of typical peaks for BSA, QSO, and FA indicates successful formulation development, validating encapsulation efficiency and stable FA conjugation while preserving nanoparticle architecture. Our findings are aligning with the earlier findings of Sadeghzadeh et al. (2023).

In the present study, HPLC analysis was primarily performed to characterize and confirm the presence of QSO and to ensure formulation integrity rather than for routine quantification during entrapment and release studies. The chromatographic results demonstrate that QSO was successfully encapsulated in the FA-conjugated BSA nanoparticle system without causing significant chemical changes to its bioactive components. The retention of characteristic QSO peaks in the nanoparticle chromatogram, as well as peaks corresponding to the blank FA-BSA carrier, confirms the coexistence of both QSO and BSA matrix components. Small changes in retention times are typically ascribed to intermolecular interactions between the encapsulated QSO and the protein matrix, which affect elution behavior while preserving structural integrity. These findings are in line with reports showing that chromatographic profiling is a dependable method to confirm encapsulation efficiency and formulation stability. Similar interpretations have been described in studies by Jithan et al. (2011).

The FA-QSO-BSA NPs exhibited an encapsulation efficiency of 37.61% and a drug loading efficiency of 19.26%. The encapsulation efficiency suggests favorable interactions between QSO and the BSA matrix, which may enhance drug entrapment and reduce drug loss during nanoparticle fabrication. Previous studies have reported that the physicochemical properties of BSA nanoparticles, including their multiple binding sites and affinity toward hydrophobic molecules, contribute to effective drug encapsulation and stability (Hemlata et al., 2025). Furthermore, adequate drug loading is important for improving therapeutic efficacy while minimizing carrier burden. Consistent with previous study by Tazhbayev et al. (2020) concluded that the loading efficiency of anticancer drug hydroxyurea was higher for the BSA-derived particles in comparison to the HSA-derived drug carrier.

In our study, the drug release characteristics of FA-QSO-BSA NPs have demonstrated a progressive and prolonged release pattern, illustrate efficient encapsulating of QSO inside the nanoparticle matrix. The pH of the physiological medium is the primary determinant of the active compound’s release from the NPs and necessary for long-term release in in vivo research. Lazer et al. (2022) found the similar results of increased drug release from FA-CS/ALG-PTX NPs (FA-conjugated chitosan-coated alginate nanoparticles) under basic circumstances.

FA-QSO-BSA NPs displayed concentration-dependent cytotoxicity and exhibited enhanced growth inhibition in breast cancer cell lines relative to free QSO and doxorubicin, whereas the blank NPs exhibited no cytotoxic effects, thereby confirming that the observed activity was due to QSO rather than the carrier system. This enhanced activity may be attributed to the combined effects of folic acid-mediated cellular uptake, improved intracellular delivery of QSO through the nanoparticle carrier, and the sustained release characteristics of the formulation. The nanoencapsulation strategy likely increased the uptake of QSO at the cellular level, thereby amplifying its antiproliferative effects. Consistent with previous reports by El Makawy et al. (2024a) and El makawy et al. (2024b), these findings support the development of QSO-based nano formulations as adjuvant therapies for breast cancer, with potential applications in chemoprevention, combination regimens, and toxicity reduction strategies. Significantly, FA-QSO-BSA NPs did not inhibit normal HEK293 cells, demonstrating selective cytotoxicity and possible safety benefits compared to traditional chemotherapeutics, which frequently lack tumor selectivity. Nosrati et al. (2018) documented the enhanced anticancer efficacy of the MCF-7 cell line when administered Chrysin-BSA NP in contrast to chrysin alone. Nosrati et al. (2018) further showed that BSA serves as an exceptional nanocarrier for delivering therapeutic medicines to the target region.

The therapeutic efficacy of drug-loaded nanoparticles is contingent upon their internalization and prolonged retention within cancer cells (Hattori and Maitani, 2004). Although in vitro and in vivo biological processes may differ significantly, an in vitro study may yield preliminary data demonstrating the benefits of nanoparticles (Zheng et al., 2010). FA-QSO-BSA NPs facilitated intracellular transport, as demonstrated by the increased uptake of FA-Rho-B-BSA NPs in the cytoplasm of MCF-7 and MDA-MB-231 cells. Consequently, our findings indicate the improved tumor cell targeting efficacy and cellular uptake of FA-QSO-BSA NPs via FA receptor-mediated endocytosis. Zhao et al. (2010) had reported similar results: the cellular uptake of fluorescein isothiocyanate-labeled PTX-FA-BSANPs (paclitaxel-loaded FA-conjugated bovine serum albumin nanoparticles) was investigated to illustrate the internalization of the nanoparticles into cells and their targeting efficacy. After a 12-h incubation with PTX-FA-BSANPs, the cells exhibited near-saturation of fluorescence. The targeting of folate-decorated nanoparticles is both substantial and successful.

A primary rationale for utilizing a cytotoxic drug is its capacity to cause apoptosis and cell death in cancer cells, as apoptosis occurs without eliciting inflammatory reactions (Satchell et al., 2003). Mechanistically, Annexin-V FITC/PI analysis demonstrated a significant increase in early apoptosis (Annexin V+/PI−) in FA-QSO-BSA NP-treated cells relative to free QSO. Moreover, FA-QSO-BSA NPs elicited substantial G0/G1 phase arrest, inhibiting cell cycle progression and facilitating programmed cell death. Azizi et al. (2017) reported that albumin-coated copper nanoparticles induced apoptosis and G1 phase cell-cycle arrest in breast cancer cells, where albumin primarily served as a non-toxic carrier to enhance nanoparticle delivery. CFSE-based proliferation studies indicated decreased fluorescence intensity, signifying lower cell division subsequent to FA-QSO-BSA NP treatment. DAPI labeling validated apoptotic nuclear condensation and morphological changes in FA-QSO-BSA NP treated cells. The results demonstrated a considerable reduction in cell size and membrane blebbing, all of which suggested DNA condensation as examined under a confocal microscope (Prasannaraj and Venkatachalam, 2017). Although, imaging at different time point of intervals upon treatment with FA-QSO-BSA NPs would provide more comprehensive interpretation and recommend for future investigations. Similar results had been reported by Majeed et al. (2019). These findings suggest that encapsulation of QSO within FA-conjugated BSA nanoparticles enhance cellular uptake and increases anticancer effectiveness by inducing apoptosis and causing cell cycle arrest while preserving selectivity for cancer cells.

5 Conclusion

In this study, QSO was encapsulated within BSA-NPs conjugated with FA, resulting in FA-QSO-BSA NPs with 139.7 nm in size. Our results show that FA-QSO-BSA NPs have anticancer activity against the MCF-7 and MDA-MB-231 cell lines with lower toxicity to normal cells. The incorporation of QSO into folate-conjugated albumin nanoparticles improves its effectiveness and targeting towards breast cancer cells. In conclusion, FA-QSO-BSA NPs may serve as an effective method for the targeted delivery of QSO to tumors. Nonetheless, more studies and comprehensive analysis are necessary to elucidate the anti-cancer mechanism of the FA-QSO-BSA NPs and to validate their safety and efficacy in targeting breast cancer for human use. Moreover, additional research should be undertaken to assess its safety, alongside assuring it does not damage essential organs. In our subsequent research, we aimed to assess the in vivo behavior of FA-QSO-BSA NPs, including the circulation half-life of the nanoparticles, their distribution to the tumor site and other organs, the rate of QSO release in vivo, the stability of the nanoparticles in the bloodstream, their capacity to reach the target site, overall bioavailability, and elimination pathways.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnano.2026.1852250/full#supplementary-material

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Footnotes

Edited by: Salman Ahmad Mustfa, AstraZeneca, United Kingdom

Reviewed by: Noha Nafee, Kuwait University, Kuwait

Pharvendra Kumar, University of Massachusetts Chan Medical School, United States