Cetuximab-conjugated sodium selenite nanoparticles for doxorubicin targeted delivery against MCF-7 breast cancer cells

Abstract

Aim: To develop and characterize doxorubicin-loaded sodium selenite nanoparticles (SSNP-DOX) and their surface attachment with cetuximab (mAb-SSNP-DOX).

Methods: SSNP-DOX was formulated by gelation and then conjugated with cetuximab to form mAb-SSNP-DOX. Characterization included DLS, SEM, TEM, DSC, Raman spectroscopy and XRD. In vitro, the kinetics of doxorubicin release and cytotoxicity in MCF-7 breast cancer cells were investigated.

Results: The zeta potential for SSNP-DOX and mAb-SSNP-DOX was -14.4 ± 10.1 mV and -27.5 ± 7.28 mV, with particle sizes of 181.3 nm and 227.5 nm, respectively. The formulation intensity was 89.7% for SSNP-DOX and 100% for mAb-SSNP-DOX, with PDI values of 0.419 and 0.251, respectively. SEM and TEM showed that mAb-SSNP-DOX was smooth and spherical. The DSC analysis revealed exothermic peaks at 102.44°C for SSNP-DOX and 144.21°C for mAb-SSNP-DOX, along with endothermic peaks at 269.19°C and 241.6°C, respectively. Raman spectroscopy showed a higher intensity for mAb-SSNP-DOX. The XRD study showed different peaks for each formulation. Both followed zero order kinetics for doxorubicin release. Cytotoxicity studies showed significant effects and high apoptosis in MCF-7 cells for both formulations.

Conclusion: The mAb-SSNP-DOX showed promising properties, more effective doxorubicin release and higher cytotoxicity against breast cancer cells compared with SSNP-DOX.

Plain language summary

Article highlights.

Breast cancer

• Breast cancer is a significant cause of morbidity and mortality worldwide and is characterized by high heterogeneity and treatment resistance. MCF-7 cells are a commonly used human breast cancer cell line in research and a model for studying the efficacy of therapeutic interventions.

Nanoparticle drug delivery system

• Drug delivery systems in the form of nanoparticles improve drugs' therapeutic efficacy and stability while minimizing unwanted side effects by enabling precise delivery to cancer cells. Targeted nanoparticle drug delivery systems, such as those incorporating monoclonal antibodies, further improve treatment outcomes by selectively targeting cancer cells and causing less damage to healthy tissue.

Targeted breast cancer treatment

• The study focused on developing injectable nanoparticle formulations, SSNP-DOX and mAb-SSNP-DOX, which effectively target MCF-7 breast cancer cells. Combining cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), with SSNP-DOX improved the specificity and efficacy of the treatment.

Characterization & physicochemical stability

• The nanoparticle formulations were comprehensively characterized using dynamic light scattering (DLS), differential scanning calorimetry (DSC), Raman spectroscopy, x-ray diffraction (XRD) and nuclear magnetic resonance spectroscopy (NMR). These techniques confirmed the formulations' stability and uniformity, ensuring their suitability for targeted drug delivery.

In vitro performance & enhanced efficacy

• The release profiles of doxorubicin from SSNP-DOX and mAb-SSNP-DOX showed sustained drug release over a prolonged period. In vitro, cytotoxicity studies showed that both formulations effectively suppressed the growth of MCF-7 breast cancer cells. Of the two, mAb-SSNP-DOX was the superior formulation, performing better on all parameters tested.

Target specificity & overcoming drug resistance

• mAb-SSNP-DOX showed increased specificity in targeting breast cancer cells due to cetuximab. This combination of nanoparticles with cetuximab represents a promising approach to overcoming drug resistance in the treatment of breast cancer, offering improved efficacy and targeting of breast cancer cells.

Future directions

• Future research should focus on optimizing these formulations, investigating the underlying molecular mechanisms and conducting extensive in vivo studies to validate the therapeutic potential of mAb-SSNP-DOXstance and conducting extensive in vivo studies to validate these findings.

1. Introduction

Breast cancer is the most common cancer in women and the second most diagnosed cancer worldwide. Over the years, a range of therapeutic approaches have been developed to treat breast cancer, including hormonal therapy, chemotherapy, targeted therapy and immunotherapy. The heterogeneity of breast cancer presents a significant challenge for effective treatment. Additionally, the development of drug resistance, as seen in other cancers, remains one of the major obstacles in managing breast cancer [1–3]. Doxorubicin, a member of the anthracycline family, is currently one of the most effective chemotherapeutic agents for breast cancer treatment [4]. Doxorubicin resistance has proven to be a substantial challenge in improving the clinical prognosis of patients with breast cancer. In addition, doxorubicin appears to promote the progression of breast cancer cell movement and invasion through DCAF13 substrate receptors. Notably, the DCAF13 overexpression in human breast cancer is positively related to disease progression. Further studies have shown that DCAF13 promotes epithelial-mesenchymal transition in human breast cancer cells; however, it does not affect cell proliferation, cell cycle progression, or apoptosis [5]. Another study emphasized the importance of fatty acid-binding proteins (FABPs) and lipid transporters within cells. Fatty acids are transported to distinct organelles, such as the endoplasmic reticulum, mitochondria and nucleus. FABPs facilitates the determination of tumor cells' biological characteristics. The present study highlights that doxorubicin resistance during breast cancer is modulated by the activation of the FABP5/PPARγ (Peroxisome proliferator-activated receptor gamma) and CaMKII (Ca2+/calmodulin-dependent protein kinase) signaling pathway [6]. In contrast, drug resistance development is influenced by alterations in intracellular drug transport, abnormalities in the glutathione system, induction of apoptotic gene expression and impairments in the gene repair pathway [2,7,8].

Nanotechnology based therapeutic advances have been explored by the pharmaceutical industry. Accurate and selective delivery of the therapeutic agents to their respective target sites for less side effects and high efficacy is the prime goal of anticancer drug development. This has been achieved through nanoparticles led targeted drug delivery, which has increased therapeutic efficacy and stability, and reduced adverse side effects [2,9]. Sodium selenite, an inorganic form of selenium, is an essential trace element for both humans and animals, where in humans it serves as a cofactor for numerous enzymes, specifically for glutathione peroxidase. This enzyme has antioxidant properties and plays a pivotal role in protecting the body against the detrimental effects of oxidative stress and exhibits potential efficacy in combating cancer [10–12]. Epidermal growth factor receptor (EGFR) expression is markedly increased in breast cancer, especially triple-negative breast cancer. This factor affects the modulation and maintenance of the basic biological properties of breast cancer, such as its ability to proliferate, invade, metastasize and maintain stem cell-like properties. Notably, EGFR is highly prevalent in breast cancer; however, studies on the efficacy of EGFR inhibitors in treating patients with breast cancer is limited. The remarkable discrepancy between these two factors underscores the need to investigate the resistance mechanisms that contribute to the limited efficacy of EGFR inhibitors in breast cancer treatment. Better understanding of these pathways could aid in improving the response of breast cancer cells to these inhibitors [13]. Cetuximab (C225) is a monoclonal antibody that has potential for cancer therapy. This study focused on the effect of C225 on MCF-7 ATCC breast cancer cells using sodium selenite nanoparticles (SSNP) loaded with doxorubicin (SSNP-DOX) and the surface attachment of cetuximab to SSNP-DOX (mAb-SSNP-DOX) [14,15].

2. Materials & methods

2.1. Materials

Sodium selenite (99%), doxorubicin hydrochloride (98.0–102.0%), sodium tripolyphosphate (85%) and cetuximab were purchased from Sigma-Aldrich. Ejadah Medical Supplies Est, Riyadh, Saudi Arabia, supplied all materials used in this study. The human breast cancer cell line MCF-7 was procured from ATCC (VA, USA).

2.2. Formulation of nanoparticles

Sodium selenite nanoparticles (SSNPs) were formulated using an ionic gelation technique with sodium tripolyphosphate as the crosslinking agent. Hydrogen bonds were formed between sodium selenite and sodium tripolyphosphate, which were supported by water molecules due to the water solubility of both compounds. Doxorubicin hydrochloride was incorporated into the SSNP during the formulation process, resulting in a formulation labeled SSNP-DOX. Briefly a 1% w/v solution of sodium selenite gel was prepared and set aside for 1 hour to stabilize. Simultaneously, solutions of doxorubicin hydrochloride (1% w/v) and tripolyphosphate (1% w/v) were prepared. The sodium selenite gel was stirred on a hot plate using a magnetic stirrer at constant speed (2000 rpm) for 90 min. During the mixing process, doxorubicin and tripolyphosphate were added separately at specific intervals. Sonication was also performed at intervals for 2 min at 100% amplification using a CPX ultra sonicator from (Cole Parmer Instruments Co, USA). After mixing process was complete, the resulting nanoparticle mixture was filtered using a Millex-GV syringe filter unit (0.2 μm, PVDF, Merck KGaA, Darmstadt, Germany). Subsequently, the filtrate was stored in a sterile glass tube with a screw cap and refrigerated at 4°C until further use.

2.3. Antibody tagging

In this study, cetuximab was conjugated to the surface of SSNP-DOX at a concentration of 0.5% (v/v). A solution of cetuximab was prepared and stirred on a heated surface using a magnetic stirrer to form the reaction mixture. SSNP-DOX was added dropwise to the reaction mixture at room temperature with continuous stirring at a constant speed of 2000 rpm. This combination was then kept at a moderate temperature of 40°C for 1 h. The resulting conjugate, labeled as mAb-SSNP-DOX, was filtered using a 0.45 μm CHROMAFIL Xtra PVDF Syringe filter. The filtered injectable mAb-SSNP-DOX was transferred to a sterile glass vial, closed tightly with a screw cap and stored at 4°C until further use.

2.4. Measurement of pH

The pH value measures the acidity or alkalinity of a liquid or colloidal sample. The pH of the nanoparticle reaction mixture was measured using the Oakton pH 700, a benchtop meter (Oakton Instruments, IL, USA).

2.5. Lyophilization process

SSNP-DOX and mAb-SSNP-DOX were lyophilized separately using a Millrock BT85 benchtop freeze dryer (Millrock Technology, USA) [16,17]. A 5% w/v mannitol solution was mixed with the SSNP-DOX and mAb-SSNP-DOX separately at a volume ratio of 2:1 in glass flasks. This mixture was then stored in a freezer at -80°C for 1 day. Subsequently, the mixture in the glass flask was transferred to a freezing tube. A vacuum was created with the opening of the control knob. A constant temperature of -84°C was maintained, and the vacuum pressure was set to 3000 Pa. After freeze-drying for 30 h, the resulting lyophilized nanoparticle powder was removed from the flask and stored at 4°C until further use.

2.6. Preparation of samples for analysis

A 1% (w/v) solution of both SSNP-DOX and mAb-SSNP-DOX was prepared in Millipore water. The solutions were stirred on a hot plate using a magnetic stirrer to obtain a uniform colloidal mixture. Thereafter, injectable colloidal solutions of SSNP-DOX and mAb-SSNP-DOX were filtered through a 0.45 μm Chroafil Xtra PVDF syringe filter. After filtration, the samples were subjected to various analysis. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) was used to analyze liquid formulations. The freeze-dried SSNP-DOX and mAb-SSNP-DOX powders were analyzed using differential scanning calorimetry (DSC), Raman spectroscopy, x-ray diffraction (XRD), nuclear magnetic resonance spectroscopy (NMR), in vitro release profiles and cytotoxicity studies.

2.7. Physicochemical characterization of nanoparticles

2.7.1. Dynamic light scattering analysis

The nanoparticles were physically characterized based on their zeta potential (ZP), conductivity (mS/cm), size (d.nm and z.d.nm) and polydispersity index (PDI). Dynamic light scattering (DLS), the nanoscale particle size (NS) and PDI of each injectable colloidal system were estimated through DLS. The ZP, NS and PDI were determined using a Nano-ZS Zetasizer (Malvern Instruments, UK). Each liquid filtrate was placed in a folded capillary cell without air bubbles in the instrument holder. Colloidal liquid-injectable formulations of SSNP-DOX and mAb-SSNP-DOX were characterized using standard procedures in accordance with Malvern Instruments manual guidelines.

2.7.2. Scanning electron microscopy

The morphological properties and particle sizes of SSNP-DOX and mAb-SSNP-DOX were investigated by high-resolution SEM. The samples were analyzed using an Inspect F50 field-emission scanning electron microscope (Zaragoza, Spain). The microscope operated at an accelerating voltage of 30.0 kV. The particle size distribution was analyzed from the scanning electron microscopy (SEM) images using Image J software.

2.7.3. Transmission electron microscopy

Transmission electron microscopy (TEM) enables high-precision visualization of nanoparticles. Liquid SSNP-DOX and mAb-SSNP-DOX were examined under a TEM (JEOL JEM-1011, JEOL, USA). The samples were placed on a grid coated with a carbon layer. The microscope was operated at 200 kV.

2.7.4. Differential scanning calorimetry analysis

The enthalpy changes due to changes in the physical and chemical properties of the powdered samples of SSNP-DOX and mAb-SSNP-DOX were determined using DSC 214 Polyma instrument (NETZSCH, Germany). This study was performed according to previously described methods [2,16]. Each nanoparticle powder sample was placed in an aluminum pan that was not hermetically sealed, and temperature was increased from 30 to 350°C at the rate of 10°C per min, while the atmospheric airflow was maintained at 10 ml min^-1.

2.7.5. Raman spectroscopy analysis

Nanoparticles are analyzed using Raman spectroscopy. The Raman spectra were recorded with a compact SENTERRA II Raman spectrometer, which is equipped with a 532 nm laser excitation source from Bruker. The laser power was carefully adjusted to 2 mW to allow for interaction with the sample. This specific laser calibration was set because it provides sufficient energy for the generation of Raman scattering from the nanoparticles without damaging or altering their structure. Therefore, parameters should be calibrated carefully to obtain accurate and detailed information regarding nanoparticle composition and properties.

2.7.6. X-ray diffraction analysis

The crystalline structures of the SSNP-DOX and mAb-SSNP-DOX lyophilized powder samples were evaluated using x-ray diffraction (XRD) analysis via an Ultima IV x-ray diffractometer (Rigaku, Japan). The XRD diffractograms were obtained at 2θ in the range of 2–50° with a Cu Kα excitation source using a wavelength of 1.54056 Å at a voltage of 45 kV and a current of 0.8 mA. A scanning range of 2θ/θ was selected and scanning speed of 10 min^-1 was employed.

2.7.7. Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy was used to evaluate lyophilized powdered samples of SSNP-DOX and mAb-SSNP-DOX. The samples were prepared in deuterated water. ¹H and ¹³C NMR data were obtained using a Brucker-Plus NMR spectrometer at 400 MHz to obtain ¹H-NMR and at 100 MHz for ¹³C NMR in deuterated chloroform solvent with tetramethylsilane as an internal standard.

2.8. Preparation & validation of standard cure

A novel method was established for doxorubicin standardization as reported earlier [2]. Subsequently, the working standard solutions of concentrations 1000, 500, 250, 125, 62.5, 31.25, 15.6 and 7.8 μg/ml were prepared in Millipore water via serial dilution of the stock standard solution in Millipore water. A calibration curve was generated by measuring the absorbance of the produced standard dilutions at four different wavelengths and comparing them to a transparent blank (405, 450 and 490 nm) using a UV/visible spectrophotometer. The method was validated by determining the linearity at specific wavelengths, which was consistent with Beer-Lambert's law. The standard curve was prepared by plotting the absorbance values at λ_max against doxorubicin concentrations.

2.9. Encapsulation efficacy

Entrapped doxorubicin was extracted from 5 g of each lyophilized formulation of SSNP-DOX and mAb-SSNP-DOX by suspending it in 10 ml of 0.1 N HCl solution in a flask on a hotplate with a magnetic bead for 30 min. The reaction mixture was centrifuged at 2000 rpm and the supernatant was maintained at 2°C. The concentration of doxorubicin was determined by extrapolating from the standard calibration curve of the drug. The encapsulation efficiency (EE) and drug loading (DL) were calculated using the following equations:

$$\begin{array}{c}\mathit{EE}(\%)=\\ \frac{\left(\mathit{Amount}\mathit{of}\mathit{drug}\mathit{incorporated}\right)-\left(\mathit{Amount}\mathit{of}\mathit{free}\mathit{drug}\mathit{after}\mathit{extraction}\right)}{\left(\mathit{Amount}\mathit{of}\mathit{drug}\mathit{incorporated}\right)}\times 100\end{array}$$

$$\begin{array}{c}\mathit{DL}(\%)=\\ \frac{\mathit{Total}\mathit{amount}\mathit{of}\mathit{drug}\mathit{incorporated}\mathit{in}\mathit{nanoparticles}}{\mathit{Total}\mathit{weight}\mathit{of}\mathit{nanoparticles}}\times 100\end{array}$$

2.10. In vitro release profile

The release studies of SSNP-DOX and mAb-SSNP-DOX were conducted in triplicate in a dialysis bag (DB). Approximately, 500 mg of each lyophilized formulation of SSNP-DOX and mAb-SSNP-DOX was placed in a DB individually, and the DB was immersed separately in 50 ml of phosphate buffer saline, pH 7.4 at 37°C with a magnetic bead stirring at 1000 rpm for 7 h. The first sampling was performed at 30 min to assess the burst-release phase. Subsequently, 3 ml of the medium was withdrawn every 1 h and replaced with an equivalent volume of fresh medium. The samples were analyzed by UV/visible spectroscopy at 490 nm, and the release pattern was determined by plotting the OD graph against doxorubicin concentration.

2.11. In vitro cytotoxicity study

This study was performed according to the methods described in a previous study [2,18]. MCF-7 ATCC breast cancer cells were cultured in RPMI-1640 medium supplemented with a sodium bicarbonate buffer (2.0 g/l, pH 7.4). The medium also contained 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a Heraeus CO₂ incubator (Germany) at 37°C, 90% humidity, and 5% CO₂. Cells were treated with 100 μg/ml of different concentrations of SSNP-DOX and mAb-SSNP-DOX (maximum dose = 100 μg/ml) dissolved in DMSO, with keeping 0.05% DMSO as control. They were seeded in 96-well microtiter plates at a density of 1 × 10⁶ cells/ml in both the treated and control groups. Samples were prepared in triplicates (n = 3) and incubated for 48 h. After incubation, 20 μl of MTT (5 mg/ml) was added to each well and incubated in the dark for 4 h before the medium was removed. The formed formazan crystals were dissolved in 100 μl of DMSO, and the absorbance in each well was measured at 490 nm using a Biotek ELISA reader (ELX 800, USA). The percentage cell viability was calculated after accounting for the controls. The experiment, performed in triplicate, allowed for the calculation of the percentage inhibition of cell proliferation using a specific formula.

$$\begin{array}{c}\text{Growth inhibition}(\%)=\\ \frac{(\text{OD control}-\text{OD treatment})}{\text{OD Control}}\times 100\end{array}$$

2.12. Morphological assessment of apoptosis using double staining method

The impact of SSNP-DOX and mAb-SSNP-DOX on MCF-7 cells were investigated using the dual-staining method using acridine orange (AO) and propidium iodide (PI). The cells were observed under a fluorescence phase-contrast microscope (Nikon Eclipse TE 300, Japan) according to established protocols. The cells, seeded at a density of 1 × 10⁶ cells/ml in a 75-ml culture flask, were treated with different concentrations of doxorubicin (up to 5 μg/ml), SSNP-DOX, and mAb-SSNP-DOX for 72 h. After treatment, cells were centrifuged at 300 × g for 10 min. The supernatant was removed, and the cells were washed twice with PBS. Cell pellets were then stained using 1 μl each of AO (10 mg/ml) and PI (10 mg/ml) in a 1:1 ratio. The freshly stained cell suspension was placed between a glass slide and a coverslip. Observations were performed for 30 min until the fluorescence subsided using a fluorescence phase contrast microscope (Nikon Eclipse TE 300, Japan). The criteria for cell identification were as follows: healthy cells exhibiting green nuclei with intact structures, cells in the early stage of apoptosis showing bright green nuclei with chromatin condensation, cells in the advanced stage of apoptosis exhibiting dense orange chromatin condensation areas, and (iv) secondary necrotic cells recognized by their orange but intact nuclei [19].

2.13. Statistical analysis

The statistical analysis was performed using Prism 9 software (GraphPad Instat, USA). A one-way ANOVA followed by Tukey's post hoc test was used for the analyses. Statistical significance was determined at p < 0.05 for all comparisons.

3. Results

Table 1 shows the properties of nanoparticles measured by dynamic light scattering (DLS). The physicochemical characteristics of SSNP-DOX and mAb-SSNP-DOX are shown in Figure 1A & B. The observations exhibited distinct characteristic peaks clearly confirming the different formulations. ZP value for SSNP-DOX and mAb-SSNP-DOX was -14.4 ± 10.1 and -27.5 ± 7.28 mV, respectively. The zeta average size of SSNP-DOX and mAb-SSNP-DOX was 181.3 and 227.5 z. d. nm, respectively. The diameter of SSNP-DOX was measured to be 248.2 ± 110.5 d. nm. The injectable nanoparticle formulations, SSNP-DOX and mAb-SSNP-DOX exhibited different polydispersity index (PDI) values, with SSNP-DOX having a PDI of 0.419 (45.9%) and mAb-SSNP-DOX having a PDI of 0.251 (53.9%) (Figure 1C & D). Figure 1C shows the size distribution of SSNP-DOX with three distinct peaks. This indicates that 4.2% of the particles had a larger size exceeding 4 μm, suggesting particle aggregation. In this study, the conductivity of SSNP-DOX was recorded at 0.243 mS/cm, which is notably lower compared with the conductivity of mAb-SSNP-DOX, measured at 24.3 mS/cm. The Y-intercepts of the SSNP-DOX and mAb-SSNP-DOX were 0.895 and 0.919, respectively. These values indicated that both formulations were excellent (Table 1).

Table 1.

Physical characterization of nanoparticles.

Characteristics	Zeta potential (mV)	Zeta average size (z.d.nm)	Size (d.nm)	% Intensity	Y intercept	pH	PDI	% PDI	% mass (d,nm)	Conductivity (mS/cm)	Crystal trials
SSNP-DOX	-14.4 ± 10.1	181.3	248.2 ± 110.5	89.7	0.895	6.3	0.419	45.9	59.5	0.243	Not suitable
mAb-SSNP-DOX	-27.5 ± 7.28	227.5	275.6 ± 149.3	100	0.919	7.2	0.251	53.9	100	24.3	Not suitable

mAb-SSNP-DOX: Monoclonal antibody surface linked sodium selenite nanoparticle loaded with doxorubicin; SSNP-DOX: Sodium selenite nanoparticle loaded with doxorubicin.

Figure 1.

Dynamic light scattering analysis. (A) Zetapotential analysis of SSNP-DOX (B) Zetapotential analysis of mAb-SSNP-DOX (C) Size distribution analysis of SSNP-DOX (D) Size distribution analysis of mAb-SSNP-DOX.

The morphological characteristics of the lyophilized SSNP-DOX and mAb-SSNP-DOX were analyzed using SEM, as illustrated in Figure 2, panels A–C. These particles have irregular crystalline shapes and typically form clusters. Figure 2 panels D–F show that the morphology of mAb-SSNP-DOX consists of smooth and discrete particles. TEM analysis of the injectable colloidal nanoparticles is presented in Figure 3. Panels A–C depict particles with an irregular spherical shape, while panels D-F reveal particles with a smooth surface and a distinct surrounding zone, which may suggest the attachment of cetuximab to the nanoparticles. The thermal properties of SSNP, SSNP-DOX, and mAb-SSNP-DOX were analyzed and compared, revealing both exothermic and endothermic peaks for each formulation (Figure 4A). Exothermic peaks, occurring at 238.22, 102.44 and 144.21°C for SSNP, SSNP-DOX and mAb-SSNP-DOX, respectively. The endothermic peaks observed at 231.6, 269.19 and 241.6°C for SSNP, SSNP-DOX and mAb-SSNP-DOX, respectively. Figure 4B shows the Raman spectra of SSNP, SSNP-DOX and mAb-SSNP-DOX. The analysis revealed a notable difference in Raman intensity among the samples. Specifically, the mAb-SSNP-DOX showed an increased Raman intensity compared with SSNP and SSNP-DOX. Interestingly, the Raman intensity of SSNP-DOX was observed to be lower than that of SSNP. In the XRD analysis, different nanoparticles were identified based on their specific diffraction peaks at different 2θ angles (Figure 4C). SSNP showed characteristic peaks at 2θ values of 8.33, 11.98, 12.5, 13.98, 14.98, 16.98, 18.88, 32.78 and 40.36. In contrast, SSNP-DOX exhibited unique peaks at 2θ values of 19.64, 20.3, 21.8, 23.66, 26.76, 29.68, 36.98, 41.4 and 55.64. In addition, mAb-SSNP-DOX nanoparticles exhibited peaks at 2θ values of 20.94, 21.32, 26.36, 27.72, 29.18, 37.64, 41.04 and 61.8. Proton NMR analysis (¹H NMR) revealed detailed features in the fingerprint region of SSNP-DOX observed between 0.99 and 1.01 ppm. The most notable proton peaks were found at various locations: 3.32, 3.68, 3.72, 3.74, 3.82, 3.83, 3.86 and 4.92 ppm, as shown in Figure 5A. Furthermore, ¹³C-NMR showed distinct peaks between 47.27 and 71.23 ppm (Figure 5B). The ¹H NMR spectrum of mAb-SSNP-DOX exhibited distinct characteristics in the fingerprint area, specifically between 1 and 1.06 ppm (Figure 5C). ¹³C-NMR spectrum of mAb-SSNP-DOX showed distinct fingerprint region between 47.32 to 71.33 ppm (Figure 5D).

Figure 2.

Scanning electron microscopy study. (A–C) Morphology of lyophilized SSNP-DOX, (D–F) Morphology of lyophilized mAb-SSNP-DOX.

Figure 3.

Transmission electron microscopy study. (A–C) Morphology of injectable colloidal formulation of SSNP-DOX. (D–F) Morphology of injectable colloidal formulation of mAb-SSNP-DOX.

Figure 4.

Nanoparticle structural analysis. (A) Differential Scanning Calorimetry analysis (B) Raman spectra (C) X-ray diffractometer analysis.

Figure 5.

Nuclear magnetic resonance analysis. (A)¹H-NMR spectrum of SSNP-DOX (B)¹³C-NMR spectrum of SSNP-DOX (C)¹H-NMR spectrum of mAb-SSNP-DOX (D)¹³C-NMR spectrum of mAb-SSNP-DOX.

Doxorubicin was effectively encapsulated in nanoparticles, achieving an entrapment efficiency of 94.6 ± 0.81% and a loading capacity of 93.7 ± 2.51%. The results shown in Figure 6 demonstrate the sustained release of doxorubicin from both SSNP-DOX and mAb-SSNP-DOX. In this study, the 2-h release rates were 19.8% for SSNP-DOX and 17.5% for mAb-SSNP-DOX. Doxorubicin and sodium selenite exhibited cytotoxic effects against MCF-7 breast cancer cells, with IC₅₀ values of 3.2 ± 0.29 μg/ml and 1.1 ± 0.2 μg/ml, respectively. In comparison, SSNP-DOX and mAb-SSNP-DOX showed cytotoxicity with IC₅₀ values of 3.4 ± 0.35 μg/ml and 5.2 ± 0.42 μg/ml, respectively, demonstrating significant cytotoxicity against breast cancer cells, particularly for mAb-SSNP-DOX (Figure 7). The morphological assessment of apoptotic alterations in MCF-7 ATCC breast cancer cells is presented in Table 2. Both SSNP-DOX and mAb-SSNP-DOX induced apoptosis. Figure 8 shows morphological changes after treatment with mAb-SSNP-DOX. Delayed apoptosis, chromatin condensation and secondary necrosis upon treatment of the cells with mAb-SSNP-DOX was observed at a concentration of 5.2 μg/ml.

Figure 6.

In vitro release profile of doxorubicin from SSNP-DOX and mAb-SSNP-DOX.

Figure 7.

Dose response curve of nanoparticles.

mAb-SSNP-DOX: Cetuximab surface linked sodium selenite nanoparticles loaded with doxorubicin; SSNP-DOX: Sodium selenite nanoparticle loaded with doxorubicin.

Table 2.

Apoptosis study on nanoparticle treatment.

Group	Time (h)	Viable (%)	Appearance
Control (no treatment)	48	86.33 ± 4.37	Early apoptosis (%)	Late apoptosis (%)	Necrosis (%)
SSNP-DOX	12 h	60.33 ± 4.51	28.67 ± 2.31	6.67 ± 3.06	4.33 ± 0.58
	24 h	20.67 ± 3.51	26.67 ± 2.89	39.33 ± 2.08	13.33
	48 h	7.33 ± 4.04	24 ± 3	59.67 ± 5.51	9 ± 3.46
mAb-SSNP-DOX	12 h	66.33 ± 4.51	25.33 ± 8.5	7 ± 3.46	1.33 ± 0.00
	24 h	18.67 ± 1.53	27 ± 7.21	40 ± 1	14.33 ± 0.58
	48 h	6.33 ± 3.21	27.33 ± 5.51	60 ± 9.17	6.33 ± 2.31

The percentages of viable early and late apoptosis and secondary necrotic cells were increased after MCF-7 cells were treated with samples in a time-dependent manner. Apoptosis increased significantly (*p < 0.05) in a time-dependent manner compared with control with all samples.

Figure 8.

Apoptosis study. Fluorescent photomicrograph of AO/PI double-stained MCF-7 cells under ×40 magnification. The cells were treated with mAb-SSNP-DOX with their IC₅₀ values for 12, 24 and 48 h. (A) Control (untreated) cells showed a normal structure with no remarkable features of apoptosis and necrosis. (B) At 12 h, AO was interconnected with fragmented DNA (bright green) (C) An orange color, representing late apoptosis, was noticed at 24 h. (D) at 48 h, secondary necrotic cells (bright red color) were noticed.

EA: Early apoptosis; LA: Late apoptosis; NE: Necrosis; VI: Viable cell.

4. Discussion

Chemotherapy involves the use of cytotoxic drugs to treat cancers. This approach targets and eliminates cancer cells but also affects non-cancerous cells that are near cancer cells [2]. Chemotherapy resistance is a major obstacle in cancer treatment. Several factors such as the diversity of tumor cells (tumor heterogeneity), genetic variations in patients and other novel mechanisms that remain to be elucidated can cause drug resistance [20]. Consequently, cancer cells acquire resistance through various cellular and molecular processes, leading to inadequate treatment [21]. Biodegradable nanoparticles surface-bound to monoclonal antibodies can be used to achieve targeted drug delivery. This showcases a substantial improvement in the therapeutic effects of cancer treatment [22,23].

The size and ZP of nanoparticles are crucial factors that determine their biological effects. ZP quantifies the charge distribution on nanoparticles' surface, influencing particle stability and interactions with biological membranes. In the present study, the shift of ZP from -14.4 to -27.5 mV indicates the stable formulation of nanoparticles following cetuximab coating. According to a previous study, SSNP produced using tripolyphosphate as a crosslinking agent showed a ZP value of -20.1 mV and an average nanoparticle size of 189 d.nm [9]. The size of the SSNP-DOX increased because of drug loading. The SSNP-DOX formulation exhibited considerable hygroscopicity after lyophilization. Notably, the mAb-SSNP-DOX showed no signs of hygroscopicity, indicating that the protein attachment was successful and compatible with the lyophilization process. A previous study has highlighted the significance of lyophilization and protein storage [24]. Another study reported that the lyophilization of therapeutic proteins can lead to aggregation [25]. Consistent with previous work, the current investigation demonstrated that nanoparticles coated with cetuximab (mAb-SSNP-DOX) exhibited aggregation but remained smooth because the particles did not display hygroscopic features.

The efficacy of nanoparticles targeting cancer cells is determined by factors such as size, shape, charge, hydrophobicity and hydrophilicity. Positively charged nanoparticles have an advantage due to their strong interactions with negatively charged cell surfaces, which improves cellular uptake through active diffusion. On the other hand, negatively charged nanoparticles are advantageous because they can penetrate the cell pores more easily. Nevertheless, the size of these nanoparticles is a decisive factor for the extent of passive diffusion [26,27]. Studies have suggested that active targeting can be used in conjunction with passive targeting based on enhanced permeability and retention to increase the aggregation and retention of nanomedicines in tumors. Furthermore, nanoparticles with sizes of 80–300 nm are beneficial for the internalization of cancer cells via endocytic pathways [28–30]. Previous studies proposed focusing on two crucial elements while determining the size of particles. First, the particles should be sufficiently large to prevent their elimination by the kidney or their penetration into capillaries. Second, they should be sufficiently small to evade phagocytosis and eliminate the reticuloendothelial system [31–33]. Based on the evaluation criteria, the average sizes of SSNP-DOX and mAb-SSNP-DOX were ideal for targeting breast cancer cells. Notably, nanoparticles with a size ranging from 30–200 nm showed improved biodistribution and faster diffusion within the targeted breast cancer area [34]. Previous studies have shown that smaller particles, especially those smaller than 80 nm, can penetrate tumors via passive diffusion. In addition, the high pressure of the interstitial fluid in the tumor can pump these small particles back into the bloodstream [35–37].

The PDI is an important indicator for assessing the uniformity of the size distribution of nanoparticles. A lower PDI reflects more uniform particles, while a higher PDI indicates a broader range of particle sizes. In this study, the SSNP-DOX formulations exhibited a higher PDI, likely due to the hygroscopic nature of the particles, a property that was further enhanced by the lyophilization process. On the other hand, the PDI of was more optimal, as the protein coating effectively reduced the hygroscopic properties of SSNP-DOX, mAb-SSNP-DOX resulting in a more uniform particle size distribution. Interestingly, the conductivity of mAb-SSNP-DOX was significantly higher than that of SSNP-DOX. This enhancement is likely due to the mAb coating on SSNP-DOX, as the lyophilization process did not impact its conductivity. In contrast, lyophilization caused a reduction in conductivity for SSNP-DOX, as the particles became hygroscopic. Additionally, particle mobility within the colloidal system is a key factor influencing conductivity [38]. Previous studies have shown that sorafenib-loaded SSNP have notable mobility, with sizes ranging from 200 to 320 nm [9]. In 2018, Niyaz Ahmad and colleagues reported preparation of a special nanoparticle, PEGylated-doxorubicin-loaded-poly-lactic-co-glycolic acid (PLGA), that had a particle size of 183.10 ± 7.41 nm and a zeta potential of -13.10 ± 1.04 mV. They concluded that these nanoparticles showed promising potential for oral delivery, as they exhibited substantially better in vitro and in vivo activities and higher bioavailability than orally administered doxorubicin [38]. As described in the Malvern Manual, the term “Y-intercept” in DLS is a point where the correlation curve crosses the y-axis of the correlation diagram. This intercept is crucial for evaluating the signal-to-noise ratio in a measured sample and thus serves as an indicator of data quality. Normally, the y-intercept is normalized such that a perfect signal has a value of one. A high-quality system was indicated by intercepts above 0.6, with values above 0.9 reflecting the performance of the best systems [39]. A previous study indicated that an optimal batch of chitosan nanoparticles loaded with cisplatin had a Y-intercept value of 0.920, which was regarded as the best system [40]. The morphological characteristics of lyophilized SSNP-DOX and mAb-SSNP-DOX were discrete in both SEM and TEM analysis. In this study, sodium tripolyphosphate was used as the crosslinking agent, resulting in discrete particles. However, particularly in the case of SSNP-DOX, the particles tended to clump together due to lyophilization. In the meanwhile, the morphology of mAb-SSNP-DOX showed smooth and distinct particles suggesting that the protein coating of SSNP-DOX remained intact after lyophilization. Previous studies have shown that nanoparticles with smooth surfaces and uniform distributions can be synthesized using chemical crosslinking methods. In this study, sodium tripolyphosphate was effectively used as a cross-linking agent in the preparation of SSNP for sorafenib tosylate via solvent evaporation. This approach resulted in nanoparticles with well-defined and distinct crystalline structure [9]. Similar observations were made in a previous study [2]. Another study revealed that TEM images of both unconjugated PLGA nanoparticles (PLGA-NPs) and PLGA-NPs conjugated with antibodies displayed a smooth morphology [41].

DSC analyzes the thermal behavior of nanoparticles by tracking the heat flow in connection with material changes at different temperatures. The nanoparticles were subjected to a specific temperature regime that facilitated observation of their phase changes, melting points and crystallization processes. The exothermic and endothermic peaks are essential for understanding heat-absorbing processes such as melting or glass transitions. These results indicated that SSNP-DOX and mAb-SSNP-DOX maintained their thermal stability regardless of whether they were coated with antibodies, emphasizing their robustness under different conditions. A previous study suggested that the endothermic peak of sodium selenite was observed at 99.33, 130 and 145°C [42]. In contrast to the previous study, an endothermic peak for SSNP was observed at 231.6°C. However, the endo thermic peaks shifted from 231.6 to 269.19°C and further to 241.6°C due to doxorubicin loading and antibody protein coating after doxorubicin loading. Raman spectroscopy is an effective technique for the analysis of nanoparticles as it provides valuable information by detecting and analyzing molecular vibrations with high sensitivity. The observed increase in Raman intensity for mAb-SSNP-DOX compared with SSNP and SSNP-DOX indicates that the modification of the nanoparticles with the antibody has significantly influenced their molecular vibrations. The lower intensity of SSNP-DOX compared with SSNP could be due to the lyophilization process, as SSNP-DOX was hygroscopic after formulation in this study, thereby affecting the Raman signal [43,44].

XRD analysis plays a central role in the characterization of nanoparticles and provides valuable insights into their crystal structure, phase composition and size. By analyzing diffraction patterns, XRD helps to determine the crystallographic structure and orientation of nanoparticles, which are essential for understanding their physical and chemical behavior. In this study, different diffraction peaks at specific 2θ angles were observed for different nanoparticle formulations, providing important information about their unique structural properties. The SSNP nanoparticles showed characteristic peaks at different 2θ values, while the SSNP-DOX and mAb-SSNP-DOX formulations exhibited different diffraction peaks, highlighting the effects of doxorubicin encapsulation and antibody modification on the crystal structure of the nanoparticles. Interestingly, the observed diffraction patterns for these nanoparticles differed from those reported in previous studies on sodium selenite, highlighting the specificity and effectiveness of XRD in discriminating between different nanoparticle compositions [45]. NMR analysis plays a crucial role in nanoparticle research by providing comprehensive insights into the molecular structure, composition and surface properties of nanoparticles. In this study, ¹H NMR analysis provided detailed information on the structural features of SSNP-DOX and revealed specific proton environments within the nanoparticle formulation. In addition, ¹³C NMR analysis effectively mapped the distribution of chemical groups in the nanoparticles, with the spectrum showing distinct peaks. Previous studies showed that the ¹H-COZY NMR spectra of doxorubicin exhibited a significant shift, with the protons being shifted [46]. In this study, both ¹H and ¹³C NMR techniques confirmed the crystalline structure of the nanoparticles.

The release rate of doxorubicin from SSNP-DOX was slightly higher than that from mAb-SSNP-DOX. This result contrasts with a previous study in which 29% of doxorubicin was released from iron oxide nanoparticles within 2 h [47]. Another study showed that cetuximab-coated thermosensitive liposomes released 70% of the encapsulated doxorubicin within 24 h [48]. However, the current study focused on a release period of 7 h, during which 67.56% of the doxorubicin was released from SSNP-DOX and 60.2% from mAb-SSNP-DOX. The release was consistently sustained in both formulations, with r² values above 0.98, indicating high linearity. These results suggest that both coated and uncoated nanoparticle formulations are promising for controlled drug release. An in vitro cytotoxicity investigation showed that SSNP-DOX effectively suppressed the growth of MCF-7 human breast cancer cells. This could be attributed to the synergistic cytotoxic effects of doxorubicin and sodium selenite. However, the quantity of doxorubicin discharged from mAb-SSNP-DOX exhibited a substantially prolonged duration compared with the in vitro cytotoxicity examination conducted on MCF-7 breast cancer cells. Additionally, mAb-SSNP-DOX had a slightly higher IC₅₀ of 5.2 μg/ml compared with SSNP-DOX's IC₅₀ of 3.4 μg/ml, and its delivery mechanism was more efficient, particularly due to its high specificity for EGFR [49]. The observed apoptotic alterations in MCF-7 ATCC breast cancer cells upon treatment with SSNP-DOX and mAb-SSNP-DOX formulations highlight the efficacy of these nanoparticle-based drug delivery systems in inducing cell death. The morphological changes, including delayed apoptosis, chromatin condensation and secondary necrosis, particularly after treatment with mAb-SSNP-DOX, indicate a controlled apoptotic response that could be beneficial for cancer therapy. Previous studies indicated that cisplatin loaded chitosan nanoparticles at a concentration of 3.50 μg/ml resulted in delayed apoptosis, chromatin condensation and secondary necrosis in the cells. However, cisplatin loaded chitosan nanoparticles-coated monoclonal antibodies exhibit little cytotoxicity and fail to induce any cytotoxic effects in MCF-7 cells [2]. This suggests that the doxorubicin-loaded nanoparticles, especially when combined with cetuximab, are more effective in targeting and killing breast cancer cells. This is important because breast cancer cells often exhibit increased EGFR expression. However, EGFR inhibitors alone are not effective for the treatment of breast cancer. mAb-SSNP-DOX is beneficial owing to its targeted approach, which allows it to selectively attack cancer cells. Therefore, mAb-SSNP-DOX is an excellent injectable formulation against breast cancer cells.

5. Conclusion

The current study demonstrated that the injectable nanoparticle formulations of SSNP-DOX and mAb-SSNP-DOX exhibited effective cytotoxicity against MCF-7 breast cancer cells. In particular, the SSNP-DOX formulation coated with cetuximab (mAb-SSNP-DOX) showed a superior inhibitory effect. DLS, DSC, Raman spectroscopy, XRD, NMR spectroscopy, in vitro release profiles and in vitro cytotoxicity studies showed that mAb-SSNP-DOX was a better formulation than SSNP-DOX. The in vitro studies show significant cytotoxic effects and the ability to induce apoptosis in breast cancer cells, highlighting the potential of mAb-SSNP-DOX to overcome resistance mechanisms. Future research should focus on optimizing the nanoparticle formulations to improve their specificity and efficiency further, investigating the detailed molecular mechanisms of drug resistance and conducting extensive in vivo studies to validate these findings.

Funding Statement

The authors gratefully acknowledge the funding of the Deanship of Graduate Studies and Scientific Research, Jazan University, Saudi Arabia, through Project Number: RG24-M014.

Author contributions

SS Moni: conceptualization, experimentation, investigation, validation and drafting the article; S Mohan, Y Riadi, RW Areshyi, HA Sofyani, FA Halawi, MQ Hakami: experimentation, validation; SI Abdelwahab, IA Aljahdali, B Oraibi, AM Farasani, OY Dawod: funding resources; ME Elmobark, HA Alfaifi, AH Alzahrani, AA Jerah: managing resources and supervision.

Financial disclosure

The authors gratefully acknowledge the funding of the Deanship of Graduate Studies and Scientific Research, Jazan University, Saudi Arabia, through Project Number: RG24-M014. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors declare that no conflict of interest is associated with this study, either financially or non-financially.

Data availability statement

Data presented in this study is available from the corresponding author upon reasonable request.