The MNPs were synthesized using a modified protocol given elsewhere (Chang et al., 2016; Cheng et al., 2017b; Wei et al., 2017). CTAB (1.82 g, 5 mmol) and NH₄F (3 g, 81 mmol) were dissolved in water, and the mixture was vigorously stirred at 80°C. This followed the addition of TEOS (9 ml, 8.41 g) dropwise. For the next 6 h, the mixture was subjected to vigorous stirring at 80°C in an oil bath. MNPs were collected by centrifugation (10, 000 × g, for 12 min) and washed with ethanol/deionized water repeatedly. MNPs were dried overnight with the help of a vacuum. Calcination eliminated the surfactant template (CTAB). The MNPs were gradually heated to 300°C with a rise in temperature by 2°C min⁻¹ and then heated to 600°C (1°C min⁻¹). The products obtained were kept for 6 h at 600°C (Cheng et al., 2017a).

With the help of diffusion, the aspirin drug was filled in the MNPs. About 100 mg of MNPs were suspended in absolute ethanol (10 ml). Afterwards, 100 mg of aspirin present in the mixture was stirred at room temperature, in a dark space for 24 h, in accordance with the authorized protocols (Lodha et al., 2012; Liu et al., 2016). Centrifugation was used to collect products. As a result, MNPs loaded with aspirin were lyophilized and were termed as MNP-Asp.

For the coating of core particles with PD, 100 mg particles were suspended in a dopamine hydrochloride solution (50 ml) with a Tris buffer (pH 8.5, 10 mM) at room temperature for 6 h, accompanied with vigorous stirring. The black particles obtained were subjected to centrifugation and washed with water afterwards. Lyophilization was used to dry the NPs with PD coating.

The Michael addition reaction resulted in the linkage of functional ligands to PD-coated MNP surface. These PD-coated NPs (100 mg) were added to the Tris buffer (40 ml, pH 8.6, and 20 mM) along with 200 mg ligands (F−PG-SH or G-SH, PG, Mw = 2000), and 2 mg of TCEP. After vigorous stirring for 6 h at 25°C, the nanocarriers (MNP-Asp-PD-PG-F and MNP-Asp-PD-PG) were obtained with the help of centrifugation, and distilled water was used to wash them for the removal of any residual reactants. Final products were subjected to lyophilization.

Distilled water was used to dilute the dry powder sample, and it was sonicated before the other measurements were performed. TEM (Tecnai G2 F30; FEI Company, Hillsboro, OR, USA) was used to study the images of the surface and shape morphology of NPs. To prepare TEM samples, a specified quantity of particles was suspended in a specified amount of distilled water and sonicated. Prepared MNPs were observed on a carbon-coated Cu grid. At −196°C, the isotherms of N2 adsorption and desorption were recorded using ASAP 2020 accelerated surface area. For 24 h, the MNP sample was degassed by applying vacuum at 120°C. Surface areas were estimated from the data of adsorption along with the method of Brunauer−Emmett−Teller. The sample was subjected to heating at 800°C with a heating ramp of 10°C min⁻¹. With the help of HPLC (LC 1200; Agilent Technologies, Santa Clara, CA, USA), the loading content of drug was calculated for aspirin-loaded NPs according to previously authorized methods (Chang et al., 2016). The calibration curves obtained through spectroscopy and HPLC data helped estimate the residual amounts of aspirin in solution.

The aspirin release from MNPs was studied using an implied modified method (Zhang and Feng, 2006). The lyophilized sample (5 mg) was dispersed in PBS (1 ml; pH 7.4, 5.6, and 2.0) and dialyzed (cutoff size 3,500 Da) against the sample release medium (10 ml). At 37°C, the tubes were incubated in an orbital shaker water bath (100 rpm). The dialysis buffer was replaced with fresh buffer in equal volumes with predetermined time gaps in the 7-day session. HPLC helped in evaluating the amount of aspirin present in the supernatant.

Dulbecco’s modified Eagle medium (DMEM) was used to culture MCF-7 cells, accompanied with antibiotic penicillin (100 IU ml⁻¹), fetal bovine serum [10% (v/v)], and streptomycin (100 μg ml⁻¹). Cell culture was performed in a humid environment at 37°C with 5% CO₂−95% air. For next 24–48 h, the cells were taken in 6-well plates. Either free aspirin or its conjugated NPs (containing approx. 5 μg ml⁻¹ aspirin) were added to the well plates. The in vitro MTT assay helped in detecting the antitumor activity of NPs toward MCF-7 cells (Chang et al., 2016). 96-well culture plates were used to seed MCF-7 cells containing 8 × 10³ cells per well. Then, the cells were subjected to various amounts of free aspirin or aspirin-conjugated NPs (approx. 0.125–2.5 μg ml⁻¹ aspirin) for the next 24 and 48 h. A stock solution [10 μl of MTT (5 mg ml⁻¹)] was added to each well. It was further incubated for 4 h. The medium was removed afterwards, and the formazan crystals formed inside the living cells were solubilized by means of the DMSO amount of 100 μl per well. It was subjected to 10 min of gentle shaking, and the absorbance value was obtained at 590 nm wavelength on a microplate reader (Bio-Rad Model680, United Kingdom). The cell values without drugs and MTT were assigned as 1 and 0, respectively, to get the normalized absorbance value.

Breast cancer cell lines were grown in 6-well plates after treatment with aspirin or modified NPs for 72 h, and then cells were fixed and stained with crystal violet. To quantify staining, acetic acid (1 ml, 10%) was added to extract dye in each well, and the absorbance value was obtained at 590 nm with a reference wavelength at 750 nm (Wang et al., 2015).

The relative uptake of the MNPs (MNP-Asp-PD-PG and MNP-Asp-PD-PG-F) was also monitored semi-quantitatively while measuring aspirin contents in the MCF-7 cells by using HPLC (LC 1200; Agilent Technologies, Santa Clara, CA, USA). The MCF-7 cells were seeded into 6-well plates as 8 × 10³ cells per well. Then, the cells were subjected to MNP-Asp-PD-PG and MNP-Asp-PD-PG-F (approx. 100 μg ml⁻¹) for the next 24 h at 37°C. The cells were washed with three times with ice-cold PBS and trypsinized for 15 min, and media fractions were centrifuged (14, 000 × g), followed by cell pellet lysis by a lysis buffer containing 50 mM Tris, pH 7.5; 200 mM NaCl; 1 mM EDTA, and 0.1% tween 20. The cell lysate was analyzed by HPLC as described above.

All the experiments were repeated at least three-to-five times. The experimental data were expressed as the mean ± standard deviation. Statistical analysis was performed by two-way ANOVA, followed by the graph pad prism. *p < 0.05 represents statistical significance, and **p < 0.01 represents extreme statistical significance.

Scheme 1 illustrates a four-step preparation of MNP-Asp-PD-PG-F MPs as described in Methods. Next, we characterized MNPs by using TEM (Table 1). The obtained size distribution displayed a dynamic range of fictitious NPs about 100–200 nm in diameter that is in line with the EPR (enhanced permeability and retention) effect theory for more penetration and cellular uptake of NPs into tumors (Win and Feng, 2005; Zhao and Feng, 2014; Kalyane et al., 2019). Thus, the obtained larger size and shape of our synthesized MNPs ensure no extravasation in normal tissues, while there is a maximum retention of administered nano-formulation in tumors with reduced adverse effects. The TEM images of MNP-Asp in Figure 1A show that MNP-Asp nanoparticles were slightly rough and porous, while the obtained average size was about 147.00 ± 6.0 nm.

Our MNPs appear chemically and thermally stable with clear, controllable morphology and porosity. Moreover, to reduce the vascular irritation of the loaded drug and improve its water solubility, tumor targeting, and bioavailability (Zhang et al., 2019; Zha et al., 2020), MNP-Asp was further coated with polydopamine (PDA) to develop MNP-Asp-PDNPs through anti-solvent precipitation and surface modification. We obtained the average size of the MNP-Asp-PD, which is 160.7 ± 8.3 nm, as shown by the TEM image of MNP-Asp-PD in Figure 1B. The coating increased the particle size of MNP-Asp-PD by 13 nm compared to MNP@Asp, suggesting the successful coating of PDA.

Then, to render the NPs with hydrophilic and antifouling properties, we coated the MNP-Asp-PDNPs with PEG. TEM images show the resulting MNP-Asp-PD-PG, NPs with a size of 197.4 ± 6.4 nm in Figure 1C. The PEGylated particles can provide various positive influences such as preventing particle aggregation in water, maximizing the particle circulation in the bloodstream and their relativity (Patsula et al., 2019; Lazaro-Carrillo et al., 2020).

Since cancer cells tend to express several FRs on their surface, the FA-modified nanoparticles can facilitate a rapid internalization of drug molecules inside the cancer cells (Ansari et al., 2020). Therefore, we modified the MNP-Asp-PD-PG by coating F, and the resulting particles were designated as MNP-Asp-PD-PG-F. The MNP-Asp-PD-PG-F particle size was found to be 204.4 ± 6.0 nm, as shown through TEM (Figure 1D). The morphology of all NPs appears as uniform and monodispersed spheres of high porosity through TEM images, as shown in Figures 1A–D. The hexagonal mesopore’s diameter was around 2–3 nm. TEM also shows a rough shell around the NPs of MNP-Asp-PD and MNP-Asp-PD-PG-F, confirming the PD film formation.

Herein, we studied the release of drug from MNP-Asp-PD-PG-F. Drug release with respect to various pH values including 7.4 (Figure 2A), 5.6 (Figure 2B), and 2.0 (Figure 2C) was observed at 37°C. A typical biphasic drug release pattern was observed for all of the NPs with FA showing an initial (24 h) aspirin burst release, which was later sustained up to 190 h (Figure 2).

The MNP-Asp-PD−PEG−FA exhibited a control release rate under all tested pH values. The characteristic release pattern of the drug from FA-derived nanoparticles suggests that the PD coating may block the MNPs’ pores and provide effectually controlled drug release. For MNP-Asp-PD−PG−F, the drug release rate increased with elevated time intervals at acidic or basic pH (Figure 2). The results agree with previous reports that the PDA-coated particles at physiological pH could provide retained drug diffusion and sustained release over a more extended period of time while essentially preserving the drug delivery system’s structure. This prolonged transmission probably results from intermolecular interactions between the active drug and functional decorations of NPs (Lim et al., 2020).

To overcome aspirin’s gastrointestinal mucosal damage (Gao et al., 2019; Tang et al., 2020), MNPs were loaded with aspirin to treat breast cancer and the cytotoxic effects were examined while comparing aspirin and aspirin-loaded MNPs through the MTT assay. We observed that aspirin-loaded MNPs (MNP-Asp-PD-PG-F) show 15–25% stronger cytotoxic effects against cancer cells than the free drug (Figure 3). Moreover, this result confirms that synthesized NPs are biocompatible with no observed toxicity toward normal cells and tissues. Our findings agree with parallel studies (Tran et al., 2019) that MNP-Asp-PD-PG-F NPs hold increased cytotoxic and anticancer effects compared to other drug-loaded NP derivatives in this study.

In addition, we noticed that increasing the incubation time of the MNP-Asp-PD-PG-F derivative lowered cells’ viability to <40%, indicating a time-dependent cytotoxic effect that, in turn, shows the enhanced therapeutic effect through this platform based on aspirin in FA-conjugated polydopamine-modified MNPs on the breast cancer MCF-7 cell line (Figure 4). Aspirin and aspirin-loaded/modified MNPs, when applied to HK cells, showed very low toxicity (Supplementary Figure S1). Altogether, these results suggest that the MNP-Asp-PD-PG-F inhibits in vitro cell growth; hence, it could be an attractive direction to the therapeutic strategy of breast cancer.

We also examined the proliferation of breast cancer cells in the presence of the free drug (aspirin) and all the derivatives (MNP-Asp, MNP-Asp-PD, MNP-Asp-PD-PG, and MNP-Asp-PD-PG-F) with equal concentrations and for the same incubation interval (Figure 5).

The results show that MNP-Asp-PD-PG-F increased the inhibition of cancer cell proliferation (around 20%) compared to the free drug or other derivatives. Our results agree with an earlier report in which MSN-quercetin@FA NPs reduce MDA-MB 231 cell viability by arresting the cell cycle and inducing apoptosis (Sarkar et al., 2016). Aspirin inhibits protein synthesis initiation (Kwon et al., 1997), COX2 activities (Kashfi and Rigas, 2005), and TGF-β1-induced COX-2 expression (Zhao et al., 2018). It has also been reported to activate caspases (Dikshit et al., 2006), the ceramide pathway (Chan et al., 1998), the tumor suppressor gene (Mahdi et al., 2006), and oxidative stress (Dikshit et al., 2006).

In the MNPs’ uptake assay, the relative concentration of aspirin in the cell was monitored in the MCF-7 cells. The aspirin concentration in the cell after 24 h was found to be 17% from the cell lysate when given in the form of MNP-Asp-PD-PG, while it was observed as 53% when MNP-Asp-PD-PG-F MNPs were used (Figure 6). This shows that folate plays an important role in the targeted delivery of the aspirin to the breast cancer cell lines. For future studies, the fluorescence and confocal microscopy studies are recommended to complete the elucidation of MNPs’ entry in the cancer as well as in normal cells.