Facile fabrication of TPGS-PCL polymeric nanoparticles for paclitaxel delivery to breast cancer: investigation of antiproliferation and apoptosis induction

Abstract

Breast cancer is one of the most devastating cancers that have no cure. Many therapeutic and diagnostic strategies have been extensively studied in the past decade. Cancer nanotechnology has developed as a promising approach in preclinical examinations, allowing for the early exposure of primary tumors and metastases and the efficient destruction of cancer cells. The current study aims to fabricate and analyze polymeric nanoparticles for breast cancer therapy established on a copolymeric block of α-tocopheryl polyethylene glycol 1000-b-polycaprolactone (TPGS-b-PCL) incorporating paclitaxel (PTX) (termed as T-b-P@PTX). Characterization of the physical, chemical, and structural properties and the in vitro biological activity of TPGS-b-PCL (T-b-P@PTX) obtained by the nanoprecipitation method. The in vitro cytotoxicity of the nanoparticles was explored by MTT assay using MCF-7 breast cancer cells in a time-dependent manner. The IC₅₀ values T-b-P@PTX for MCF-7 cells 543.7 ± 2.58, 130.2 ± 3.54, 66.99 ± 3.43 in 24, 48, 72-h. Biochemical experiments showed that T-b-P@PTX has a strong ability to induce cell death and to be internalized in the MCF-7 cells. Ethidium bromide (EB)-acridine orange (AO) and nuclear DAPI staining methods investigated the morphological changes of the cells. Considering all the advantages, Paclitaxel-loaded T-b-P@PTX polymeric nanoparticle is a promising approach for treating breast cancer.

Keywords:

• Breast cancer
• nanocarrier
• paclitaxel
• polymeric nanoparticles
• tPGS

1. Introduction

Breast cancer is the second-leading cause of cancer-related mortality among women [1]. There is an immediate need for innovative primary and metastatic breast cancer screening methods to improve patient survival rates [2]. Using biomarker target-specific imaging probes for image-based diagnosis and therapy monitoring is a potential approach to increasing the sensitivity and specificity of cancer cell imaging. Positron and single photon emission tomography are now employed for cancer detection using targeted radionuclide probes. While nuclear imaging technologies are very sensitive, they have drawbacks [3–5]. These include a lack of anatomic localization, adequate tumor lesion resolution, and the need for costly and time-consuming radiochemistry [6]. The radiotracer’s half-life also hinders time and dynamic-resolved imaging, and it might not be able to catch the biomarker-targeting drug when it reaches and accumulates in the tumor [7]. Clinical oncology has extensively used magnetic resonance imaging because of the technology’s exceptional spatial resolution and capability to capture anatomical characteristics in three dimensions [8–10]. The American Cancer Society has recommended breast magnetic resonance imaging as a diagnostic tool complementary to mammography for initial breast cancer diagnosis in women at high risk for this illness [11]. Despite breast cancer MRI's excellent understanding in identifying tiny breast tumors, a key difficulty is its limited specificity when utilizing a nontargeted contrast agent like gadolinium chelates, leading to a high false-positive rate and, in some cases, wasteful biopsy and mastectomy [12–14].

Recently, it has been demonstrated that tumors may be targeted by biocompatible and functionalized nanoparticles, emitting radioactive, magnetic, and optical signals to improve the specificity and sensitivity of noninvasive tumor imaging [15–17]. In vivo MRI of tumors has been proven possible using imaging probes in previous research [18]. Nanoparticles with extended blood flow duration, reactive functional groups, and wide surface area for loading numerous or different tumor-targeting ligands are ideally suited for receptor-targeted imaging. But before, we could use nanoparticle imaging probes in medicine [19–21]. These challenges include finding appropriate imaging biomarkers, getting enough of the probe into the body, and creating imaging probes with good enough contrast enhancement and signal amplification to use [22].

Research into polymeric nanoparticles as promising nanocarriers for chemotherapy drugs has been conducted as an alternate to standard chemotherapy with the potential for fewer adverse effects and increased therapeutic efficacy [23–25]. Natural polymers like alginate, gelatin, and chitosan; or synthetic polymers like poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactide) (PLA), and polycaprolactone (PCL) can be used to fabricate polymeric nanoparticles [26]. Biodegradable polymers can be employed, breaking down into harmless byproducts the body can flush out [27]. Among the earliest synthetic polymers to find usage in the medical field was polycaprolactone [28]. European and North American regulatory bodies have sanctioned its usage, and it decomposes in nature and is compatible with living organisms [29]. Its poor crystallinity might be beneficial in therapeutic settings where controlled drug release is needed since it could delay the release of those pharmaceuticals [30, 31].

Polymer nanoparticles with surface modifications may have a longer circulation period [32–35], which may aid in the enhanced permeability and retention (EPR) effect, to describe the unique pathophysiological phenomenon of the solid tumor vasculature [36]. TPGS also lengthens the half-life of anticancer medications in circulation by decreasing their absorption by the mononuclear phagocyte system thanks to PEG interactions improving the drug’s permeability and bioavailability [37–39]. It has also been observed that TPGS can promote medication penetration and accumulation into the tumor microenvironment, limit tumor metastasis, and stimulate apoptotic pathways [40]. Paclitaxel-loaded polymeric nanoparticles with a T-b-P@PTX matrix were fabricated for this study (Figure 1.). Their morphological and physicochemical features described the resulting system, and in vitro activity was evaluated. Ethidium bromide-acridine orange and nuclear DAPI staining methods investigated the morphological changes.

Figure 1. Graphical representation of fabrication of copolymeric block of α-tocopheryl polyethylene glycol 1000-b-polycaprolactone (TPGS-b-PCL) incorporating paclitaxel (termed as T-b-P@PTX).

2. Experimental section

2.1. Materials

Poly-lysine, TPGS, and stannous 2-ethylhexanoate were bought from Sigma-Aldrich (USA). ε-caprolactone and resazurin sodium salt were bought from TCI. Paclitaxel (PTX), DAPI and acridine orange, and ethidium bromide (AO-EB) were bought from Beyotime (Shanghai, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Beyotime (Nantong, China). RPMI-1640 medium, penicillin, streptomycin (10000x), and trypsin were purchased from Yeasen, Shanghai, China.

2.2. Synthesis of PCL-block-TPGS (T-b-P@PTX)

Using stannous octanoate as a catalyst, ε-caprolactone (CL) was ring-opened polymerized by TPGS to fabricate the T-b-P@PTX copolymer. At 140 °C for 2-h in a dry nitrogen environment, 3.0 g of TPGS were dehydrated in a round-bottom (RB) flask. After adding 20.0 g of CL and 0.02 g of stannous octanoate, the mixture was kept at 130 °C in an N2 environment for over a day. The reaction products were brought down to RT, diluted in dichloromethane (DCM), and precipitated in cold ethanol (4 °C). Products were regained by filtering, and solvents were removed using low-temperature vacuum evaporation overnight. The result was a fine, white powder.

2.3. Fabrication of PCL-block-TPGS (T-b-P@PTX) nanoparticles

The nanoprecipitation process was modified to develop the nanoparticles [41–43]. We used a magnetic stirrer to combine acetone (30 mL), paclitaxel (6 mg), and T-b-P (90 mg) to fabricate an organic phase (600 rpm). An aqueous phase comprising 40 mL of PBS and TPGS was rapidly poured into the receiving solution. We removed the solvent by agitating the nanoparticle suspension at a constant temperature (25 °C) for 24-h.

2.4. Characterization of nanoparticles

2.4.1. High-resolution transmission electron microscopy (HRTEM)

High-resolution transmission electron microscopy (HRTEM) system (JEM-2100, JEOL) was utilized to examine the surface morphology of T-b-P@PTX.

2.4.2. Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was detailed on a Rigaku TG8101Da instrument.

2.4.3. Fourier transform infrared (FT-IR)

The nanoparticle system functional groups and chemical bonds were characterized by Fourier transform infrared (FT-IR) spectroscopy (VERTEX 80v).

2.4.4. Hydrodynamic parameters

Malvern Zetasizer Nano ZS90 was utilized to determine dynamic light scattering (DLS) and zeta potential. The nanoparticles’ stability was established by observing the particle size, polydispersive index, and percentage of encapsulation for 1 month in storage at −5 °C.

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

To calculate the encapsulation efficiency, the nanoparticle’s solution (500 µL) was placed in an ultrafiltration and centrifuged for 15 min at 3000 g. Nonencapsulated paclitaxel was dosed according to the formula, yielding a percentage estimate of the encapsulated drug’s content in the previously reported protocol [44]. $$\text{Encapsulation efficiency}=\text{Total PTX}-\text{Non}-\text{encapsulated PTX}/\text{Total DTX}$$

We calculated the loading capacity of the nanocarriers used to encapsulate the drug by solving the following equation in the percentage of the previously reported protocol [45]. $$\text{Loading capacity}=\text{Total PTX}-\text{Non}-\text{encapsulated PTX}/\text{Freeze}-\text{dried formulation mass}$$

2.6. Cell line culture and cytotoxic assay

The MCF-7 breast cancer cell line and HBL100 human normal cell line were purchased from Peking Union Medical College Hospital (Beijing, China). Cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum and penicillin − streptomycin (1%) at 37 °C with 5% CO₂.

An MTT assay measured the viability of cells treated with free PTX, free T-b-P, and T-b-P@PTX. Human breast carcinoma (MCF-7) cells and HBL100 human normal cell line were planted into 96-well plates at a density of 1 × 10⁴ cells per well and incubated in 5% CO₂ at 37 °C. After 24-h, 48-h, and 72-h incubation, the culture medium was replaced with 100 µL of fresh medium containing different concentrations of free PTX, free T-b-P, and T-b-P@PTX. The cells were further incubated for 4-h. MTT solution (0.5 mg/mL, 20 µL) was pipetted into each well and cultured for 3 h. Finally, 100 µL of DMSO was added to each well, and the plate was shaken for 5 min. The absorbance at 490 nm was obtained utilizing a microplate reader. The cell viability was determined according to our previous study [46–49].

2.7. Acridine orange/ethidium bromide (AO/EB) double staining assay

MCF-7 cells were planted in 6-well plates (1 × 10⁵ cells per well) and incubated for 24 and 48-h. Subsequently, the cells were cultured with fresh medium containing free PTX, free T-b-P, and T-b-P@PTX (IC₅₀ concentration). MCF-7 cells incubated with fresh culture medium were served as a control group. After incubation for 8 h, all floating and adherent cells were collected and washed twice with cold PBS. The apoptotic cells were detected by AO (10 mg/ml) and EB (10 mg/ml). After being fixed with 4% paraformaldehyde, the number of migrated cells was counted using a fluorescence microscope [50–52].

2.8. DAPI staining assay

MCF-7 cells were planted in 6-well plates (1 × 10⁵ cells per well) and incubated for 24 and 48-h. Subsequently, the cells were cultured with fresh medium containing free PTX, free T-b-P, and T-b-P@PTX (IC₅₀ concentration). MCF-7 cells incubated with fresh culture medium were served as a control group. After incubation for 8 h, all floating and adherent cells were collected and washed twice with cold PBS. The apoptotic cells were detected by DAPI stain (2 μg/ml). After being fixed with 4% paraformaldehyde, the number of migrated cells was counted using a fluorescence microscope [53–55].

2.9. Statistical analysis

All data were presented as mean ± standard deviation (SD). The experimental results were analyzed by one-way analysis of variance (ANOVA) by using GraphPad Prism software. The p-value of < 0.05, 0.01, and 0.001 was considered a statistically significant difference and marked with *, **, and ***, respectively.

3. Results and discussion

3.1. Characterization of nanoparticles

The data achieved were similar to those obtained by Bernabeu and collaborators [56]. This indicated that the technique adopted for PCL-TPGS copolymer fabrication was successful [57]. The reaction yield was determined by dividing the product produced by the number of reagents used, which was 93.4%. The standard molecular weight (Mw) of the produced copolymers was determined to be 16,320 g/mol using the ratio of the signals integrals acquired in steps 4.1 and 3.6.

3.1.1. Thermal gravimetric analysis

Using TGA, we could ascertain that 392.72 °C, 391.05 °C, and 336.56 °C were the corresponding temperatures at which PCL, TPGS, and PCL-TPGS began to lose mass (Figure 2(a)). The calorimetric curves showed that the synthesized copolymer was more thermally fragile and, as a result, more susceptible to changes in its physical state than regular PCL and TPGS. Since the DSC results are consistent with TGA's, and the peaks show evidence of first-order phase transitions (fusion), we may infer that the synthesis’s product is highly pure (Figure 2(b)).

Figure 2. (a) Thermogravimetric analysis (TGA) of PCL, TPGS, and TPGS-PCL. (b) Differential scanning calorimetry (DSC) plots of PCL, TPGS, and TPGS-PCL.

3.2. Development of T-b-P@PTX nanoparticles

3.2.1. Hydrodynamic parameters

It is crucial to offer their accumulation inside the tumor microenvironment via the EPR effects [58]. Previous works have indicated that the nanoprecipitation process for creating polymeric nanoparticles with low particle size, polydispersity index, and excellent encapsulation efficiency [59]. The obtained formulations had a size distribution function (SDF) of 132 ± 15 nm, a particle size distribution index (PDI) of 0.11 ± 0.03, an encapsulation efficiency of 97.1 ± 1.0%, and a loading capacity of 4.99 ± 0.33%, making them well suited for enhancing nanoparticle accumulation in the tumour microenvironment via the EPR effect. Polymeric nanoparticles with a small particle size (190.5 ± 19.3 nm) and PDI (less than 0.09) were fabricated by Tureli and colleagues using the same fabrication method [60]. Box Behnken Design was used to optimize the concentrations of the polymer, the quantity of organic solvent, and the surfactant, resulting in lower particle size (145.25 ± 19.87 nm) and PDI (0.115) values. This research also stressed the significance of getting small particles for intravenous injection to prevent blockage of blood capillaries. They also demonstrated that paclitaxel, another taxane used to treat solid tumours, could be encapsulated efficiently by their nanosystem (95.34%). In addition to the high loading capacity rates of 4.99 ± 0.33% achieved by Mitta and colleagues [61], our nanoparticles also demonstrated excellent encapsulation efficiency rates of 97.1 ± 1.0%. Previous investigations of systems transporting taxanes with comparable chemical structures and physicochemical features did not find values as high as those obtained here [62]. The zeta potential of the formulated product was −30.3 ± 2.4 mV. Zeta potential is crucial for enhanced nanoparticles stability due to electrostatic repulsions. The PEGs further aid the steric stability of nanosystem from TPGS that coat their surface. As a result, the two mechanisms can work together to stabilize the new system. Particles having negative surface charges are also said to be non-cytotoxic and non-genotoxic. We also measured the nanoparticle’s stability in storage at 4 °C by evaluating the particle size, PDI, and EE% in the time function. There was no change in drug release throughout the month (5.3 ± 0.4% was released). To show that nanoparticles did not aggregate, the size distribution continued unimodal throughout all time intervals studied. There was no discernible variation in average encapsulation throughout the first 24-h. The following 30 days saw a little drug release, but there were no differences between the various times.

3.2.2. Morphological characterization of the nanoparticles

TEM images were collected to learn more about the nanoparticles’ appearance and three-dimensional structure, which will help us better predict how they could act in living organisms. Images not only corroborate that T-b-P@PTX particles are around the same size as measured by NTA and DLS but also show that nanoparticles morphologies are both spherical and regular, suggesting that they may contain pores on their surfaces (Figure 3(a and b)).

Figure 3. Nanoparticles morphological characterization. (a) TEM images of (TPGS-b-PCL@PTX) (termed as T-b-P@PTX). Scale bar 100 nm. (b) Particle size distribution of T-b-P@PTX by dynamic light scattering (DLS). (c) nanoparticle tracking analysis (NTA) analysis of T-b-P@PTX.

3.2.3. Diffuse light scattering (DLS)

The findings from NTA were consistent with those obtained from DLS. Taking the total particle count into account, NTA produces the findings. Equal to the function of the light scattered intensity is the diffuse light scattering (DLS). Using NTA, we were able to consolidate the observed average (171.3 ± 0.9 nm), type (146.6 ± 9.3 nm), and variation of the particle size (45.5 ± 2.6 nm) (Figure 3(c)). Results from the experi­ment showed very tiny populations with somewhat larger than expected dispersion. A low polydispersity value indicated that the mean size distribution stayed below 200 nm. The span index of 0.69 ± 0.04 is lower than the low PDI values found with DLS (Figure 4(a–c)).

Figure 4. The stability of nanoparticles. (a) Particle size distribution. (b) Polydispertive index (PDI). (c) Ratio of drug encapsulation as a function of time.

3.2.4. FT-IR analysis

The FTIR spectra verified properties shown by DSC tests. Effective encapsulation of PTX was demonstrated by the absence of the PTX-specific stretching band at 565 cm⁻¹ (-CH) in T-b-P@PTX (Figure 5(a)). However, in the combination, all components’ signals were picked up with a degree of transmittance directly related to their concentration. To the hydroxyls of paclitaxel, we owe the strong signal at 3305 cm⁻¹.

Figure 5. (a) Fourier-transform infrared spectroscopy (FTIR) spectral analysis of PTX, physical mixture, drug-free nanoparticles, and nanoparticles loaded with PTX (T-b-P@PTX). (b) DSC curves of PTX, TPGS, copolymer (TPGS-PCL), physical mixture, drug-free nanoparticles, and PTX-loaded nanoparticles (T-b-P@PTX).

3.2.5. Thermogravimetric analysis (TGA)

To evaluate the formulation components’ thermal behaviors and drug and carrier interactions, the thermograms of pure PTX, T-b-P, and the T-b-P@PTX physical combination were examined (Figure 5(b)). The endothermic peak for PTX was recorded at 166.75 °C, while a related signal was shown in the mixture at 156.19 °C. The solubilization of paclitaxel in TPGS, a solubility enhancer, may be one interaction in the mixture contributing to the lowered glass transition temperature. Similar calorimetric profiles were observed for nanoparticles with and without the medication. Since paclitaxel and the physical mixture exhibit an endothermic signal, its absence from T-b-P@PTX shows that the drug is present in the nanostructure as an amorphous or solid solution. Beginning at a temperature of 213.20 °C, the endothermic signal can be observed in both nanoparticles; this is due to the phosphate buffer used to isotonic and alter the pH of the formulations.

3.3. Cytotoxicity assays

By measuring the cytotoxicity of free T-b-P alone, free PTX, and T-b-P@PTX. We found that free T-b-P was as biocompatible as T-b-P@PTX at the same nanoparticle concentrations (Figure 6(a)), as evidenced by the great cellular viability of MCF-7 cells. However, cytotoxicity studies using the commercial paclitaxel formulation demonstrated decreased cell proliferation (Figure 6(c)). Besides, cell viability was significantly decreased relative to controls at the earliest time point and lowest concentration. High-level cytotoxicity (more than 50% loss in cell proliferation) was shown at doses of paclitaxel larger than 10 nM, consistent with the results of earlier research [63, 64].

Figure 6. (a) Cell proliferation of free T-b-P nanoparticles was examined with HBL100 human normal cell line by different concentrations and incubation periods. (b) T-b-P@PTX and (c) PTX were examined with MCF-7 breast cancer cells by different concentrations and incubation periods (24, 48, 72-h). GraphPad Prism 8.0 assessed statistical significance: > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± standard deviation (SD).

The results of the T-b-P@PTX cytotoxicity experiments were consistent with those of the commercial product (Figure 6(b)). T-b-P@PTX showed superior in vitro efficacy contrasted to the paclitaxel formulations at PTX doses after 24-h, 48-h, and 72-h incubations, with a statistically significant reduction in cell viability compared to controls. Free T-b-P alone for non-cancerous HBL100 cells shows a proliferation ratio of more than 80%, which reveals that the T-b-P nanoparticles don’t affect the non-cancer cells. The IC₅₀ concentration of free PTX for MCF-7 cells was 1.06 ± 6.14, 0.019 ± 4.87, 0.078 ± 6.47 in 24, 48, and 72-h. The IC₅₀ concentration of T-b-P@PTX for MCF-7 cells was 543.7 ± 2.58, 130.2 ± 3.54, 66.99 ± 3.43 in 24, 48, 72-h. Assessing the cytotoxic of paclitaxel nanoparticles with paclitaxel solution against MCF-7 cell lines utilizing various polymeric blocks. In contrast to the results reported by Sanna and coworkers [65], our TPGS/PCL-based nanoparticle functioned better than the commercial drugs formulations [66, 67]. These results show that TPGS enhances cytotoxicity by inhibiting P-gp synergistically with paclitaxel [60]. These results are constant with earlier studies of docetaxel cytotoxicity, which displayed high cytotoxicity (>50% reduction in cell viability) at concentrations greater than 10 nM [68].

3.4. Cellular morphology analysis by dual staining assay

Apoptotic cells may be distinguished from normal, necrotic, and early and late apoptotic cells using AO/EB fluorescence staining. The AO/EB dual staining technique quantifies and qualitatively analyses apoptosis [69]. In a nutshell, AO is shown by living and nonliving cells and exhibits greenish fluorescence after connecting with DNA. In contrast, nonliving cells take up EB and emit red fluorescence. Accordingly, the nuclei of living cells are a healthy shade of green. In contrast, early apoptotic cells are a brilliant green with fragmented or condensed chromatin, and late apoptotic cells are a dismal shade of orange with similarly distorted chromatin. According to earlier studies [70], free T-b-P and free PTX triggered less apoptosis in MCF-7 cells after 24 and 48-h of treatment. This study used a straightforward approach based on microscopic interpretations of cells stained with AO/EB to determine the free PTX, free T-b-P, and T-b-P@PTX in MCF-7 cells triggered cell death. Free PTX and T-b-P-exposed cells were uniformly green when stained with AO/EB (Figure 7). Treatment of MCF-7 cell lines with IC₅₀ concentration of T-b-P@PTX for 24 and 48-h resulted in the morphological hallmarks of apoptosis (Figure 7). Early apoptotic cells were shown by yellow fluorescence, while late apoptotic cells were primarily found in the T-b-P@PTX-treated MCF-7 cells. Additionally, MCF-7 cell lines treated with free PTX, free T-b-P, and T-b-P@PTX for 24-h showed signs of necrosis.

Figure 7. Dual acridine orange/ethidium bromide (AO/EB) staining shows the induction of apoptosis in the MCF-7 cells after being treated with T-b-P, PTX, and T-b-P@PTX in a time-dependent manner. Scale bar 40 µm. GraphPad Prism 8.0 was used to assess statistical significance: ***p < 0.001. Data are presented as mean ± standard deviation (SD).

3.5. Effect of chromatin condensation

Apoptosis, triggered by cell exposures, is characterized by a cascade of distinctive morphological differences, including chromatin breakdown, DNA fragmentation, nuclear condensation, and abnormalities in the cellular membrane [71]. Using a DAPI staining test, we analyzed the effects of free PTX, free T-b-P, and T-b-P@PTX on the nucleus of MCF-7 cells subjected to apoptosis. After staining, healthy cells with double-stranded DNA show blue nuclei due to DAPI's specific interaction with DNA, cells treated with free PTX and free T-b-P show a smaller number of apoptotic cells on treatment with 24 and 48-h. Chromatin condensation reduction of nuclear construction and the production of apoptotic bodies were detected in the T-b-P@PTX -treated MCF-7 cells. In contrast, the control cells displayed healthy nuclei with reduced condensed chromatin after 24 and 48-h of exposure to T-b-P@PTX, as determined by DAPI staining (Figure 8). These results showed that 24 and 48-h treatment of MCF-7 cells with T-b-P@PTX might cause chromatin and DNA damage.

Figure 8. DAPI staining shows the induction of nuclear damage in the MCF-7 cells after being treated with T-b-P, PTX, and T-b-P@PTX in a time-dependent manner. Scale bar 40 µm. GraphPad Prism 8.0 was used to assess statistical significance: ***p < 0.001. Data are presented as mean ± standard deviation (SD).

4. Conclusion

This paper describes the first successful use of paclitaxel-encapsulated T-b-P@PTX nanoparticles for breast cancer treatment. According to the results of our characterization, paclitaxel was successfully enclosed in a nanosystem suitable for promising drug delivery. Cells widely took up the established nanoparticles, allowing them to release their deadly payload. Testing for cytotoxicity demonstrates that the nanostructured release mechanism may significantly reduce cell viability in MCF-7 cell lines. Ethidium bromide-acridine orange and nuclear DAPI staining methods investigated the morphological changes. As a result, the anticancer effects of the proposed system hint at the possibility of enhancing traditional cytotoxic therapy in humans, and more research is needed to guarantee clinical safety and efficacy.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data are included in the submission/manuscript file.