Maleic anhydride (MA, CAS No. 108-31-6), Melamine (Me, CAS No. 108-78-1), and Methyl Thiazolyl diphenyl Tetrazolium bromide (MTT, CAS No. 298-93-1) were provided from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany. Benzoyl peroxide (BPO, CAS No. 94-36-0), Paclitaxel (PTX, CAS No. 33069-62-4), penicillin-streptomycin (Pen-Strep, CAS No. 3810-74-0), and styrene (ST, CAS No. 100-42-5) were provided from Merck (Merck KGaA, Darmstadt, Germany). Before use, ST was purified by distillation at reduced pressure. Solvents and other chemicals such as tetrahydrofuran (THF, CAS No. 401757), dimethylformamide (DMF, CAS No. 68-12-2), methanol (CAS No. 67-56-1), Dimethyl Sulfoxide (DMSO, CAS No. 67-68-5) were purchased from Sigma Aldrich (St. Louis, MO, USA). RPMI (Gibco, Catalog No. 11875093) and Fetal Bovine Serum (FBS, Catalog No. 35050061) were provided from Gibco, Thermo Fisher Scientific, Waltham, MA, USA.

The surface morphology of the synthesized sample and the drug release characteristics were studied on a scanning electron microscope (FE-SEM, Joint Stock Company, Tescan, Brno, Czech Republic) and ultraviolet-visible spectrophotometer (UV-Vis, Biomate5, Thermo Fisher Scientific Inc., Waltham, MA, USA), respectively. X-ray diffraction (XRD) pattern was recorded using XRD machine (PHILIPS, PW1730, Netherland). To obtain thermogravimetric analysis (TGA) profiles using TGA analyzer system (TGA STA6000, PerkinElmer, Inc., Shelton, CT, USA).

The PSMA sample was prepared according to previously reported literature with some adjustments [22]. In a 100 mL flask with a magnetic stirrer, 2 g of maleic anhydride and 2.32 g of styrene, measured with a pipette, were mixed in 50 mL of THF solvent. The mixture was stirred under nitrogen gas at 0°C–5°C for 20 min. Then, 0.0197 g of azobisisobutyronitrile (AIBN) was added, and the mixture was kept at 80°C for 7 h. After the reaction, the volume of THF was doubled, methanol (30 mL) was added, and the mixture was filtered (30 µm, Smooth Paper SM-180) to obtain a white precipitate of the desired copolymer. The obtained sediment was left to dry completely at 30°C for 24 h [23].

The method of modification of PSMA with Me is as follows: 1 g of PSMA was mixed with 50 mL of distilled water. The mixture was then stirred at 50°C for 15 min to form a homogeneous emulsion. Then, Me solution (1.13 g of Me in 50 mL of distilled water with 5 mL of 0.1 M hydrochloride) was added to the mixture and after 4 h of reflux at 50°C, 2N hydrochloride solution was added to the mixture until the pH reached 4–5 using a pH meter. As a result, a white product with a gelatin-like consistency was obtained. The final product was filtered and dried under a vacuum drying oven at room temperature [23].

To synthesis PSMA/Me/Fe₃O₄ magnetic nanoparticles firstly, Fe₃O₄ MNPs were synthesized. For this purpose, in a 250 mL flask, 100 mL of deionized water 5.84 g (0.0216 moL) of Fe (III) and 2.17 g (0.0108 moL) of Fe (II) chloride were added under nitrogen gas and stirred for 3 h at 40°C. Then 10 mL of 25% ammonia was added at 80°C for 30 min under magnetic stirrer [24]. The precipitate was collected by magnet and washed several times with ionized water and dried to constant weight under vacuum. In the second step, 1 g PSMA/Me was added to a 50 mL H₂O/ethanol (50:50 v %) solvent and stirred to obtain a homogeneous mixture. Then 0.4 g Fe₃O₄ nanoparticles was added and stirred overnight. The precipitate was collected by an external magnet and washed with deionized water. The obtained PSMA/Me/Fe₃O₄ composites were dried under a vacuum to reach a constant weight.

To accurately load the anticancer drug PTX into the PSMA/Me copolymer, a series of crucial steps were meticulously followed. First, 1.2 g of PSMA was precisely added to a solution containing PTX at a concentration of 6000 ppm in 12 mL of solvent. The mixture was stirred for 48 h in a light-protected environment at room temperature, at a speed of 80 rotations per minute, until the drug encapsulation reached equilibrium. Following this, the supernatant of PSMA/Me loaded with PTX (PSMA/Me/PTX) was retained after purification to determine the adsorption capacity of PSMA/Me using UV-Vis spectroscopy. Then, based on the obtained absorption values, the encapsulation efficiency (EE%) and loading capacity (LC%) were calculated using the following equations. Subsequently, the product was washed twice with distilled water to remove the free drug, and the product sediment was used for cell efficiency testing. Encapsulation efficiency (EE%) = [(weight of initial drug added-weight of unreleased drug in the supernatant)/weight of initial drug added] × 100.

Loading efficiency (LE%) = [(weight of initial drug added–weight of “unentrapped drug” free in the supernatant)/weight of PSAM] × 100.

To investigate the release profiles of PTX from the PSMA/Me copolymer, a mixture was prepared in a 50 mL Erlen-Meyer flask containing 25 mL of buffer solution. Two distinct pH levels were utilized: 5, 6.5, and 7.4. The experiment was conducted at room temperature. To determine the percentage of PTX released over time, 2 mL aliquots of the buffer solution were periodically (every 30 min) withdrawn from the flask and returned to the flask after determining the concentration. These aliquots were subsequently replaced with an equal volume of fresh buffer solution to maintain a constant medium volume throughout the experiment. The collected aliquots were subjected to analysis using a UV-Vis spectrophotometer. The absorbance of each sample was measured at λ_max = 235 nm to determine the concentration of PTX present in the released fraction. By monitoring the PTX concentration over time, the release kinetics and profile of the drug from the PSMA/Me copolymer could be assessed.

MCF-7 (NCBICode: C135, Pasteur Institute of Iran, Tehran, Iran) and Skov-3 (NCBICode: C209, Pasteur Institute of Iran) human breast and ovarian cancer cell lines were donated from Tabriz University of Medical Sciences, Tabriz, Iran. They were checked for contaminations regarding mycoplasma contamination and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin. The cells were incubated in a controlled incubator. The incubation condition was 37°C with 95% humidity and 5% CO₂. Upon 80% confluency, cells were subcultured into proper T25 and T75 tissue culture flasks using trypsin-EDTA (Gibco) [25].

Cells were initially seeded in 96-well tissue culture plates at a density of 7 × 10³ per well and incubated for 24 h in RPMI 1640 containing 10% FBS and streptomycin/penicillin 1%. The cytotoxic effects of free drug (PTX) and PSMA/Me/Fe₃O₄/PTX were evaluated by exposing the cells to varying concentrations of PTX or PSMA/Me/Fe₃O₄/PTX (1, 2, 4, 8 μM) for 24 and 48 h in RPMI 1640 containing 10% FBS and streptomycin/penicillin 1%. A control group that did not receive any treatment was included for each set. Following the treatment, MTT solution (5 mg/mL) was added to each well and kept at 37°C for 4 h. Next, the cell medium was removed, and formazan was dissolved in DMSO (Sigma-Aldrich) at room temperature. The absorbance (optical density) was measured at 570 nm using an ELISA reader instrument (BioTek800 TS), and cell viability was calculated relative to the control cells. The IC₅₀ value, representing the concentration of PTX or PSMA/Me/Fe₃O₄/PTX required to inhibit cell growth by 50%, was also determined [26].

Data was reported as Means± SD of three sets of experiments. GraphPad Prism (GraphPad Software, Inc., Boston, MA, USA, Ver. 8) was used to analyze data by One-way ANOVA and Tukey post-hoc test. p < 0.05 was considered statically significant.

The surface morphology of the synthesized (PSMA/Me/Fe₃O₄) magnetic nanoparticles was studied by SEM technique and the obtained images are shown in Fig. 1A. As can be seen, the obtained PSMA/Me/Fe₃O₄ illustrated an uneven surface with spherical shapes contains holes between granular moieties that can be useful in drug encapsulation. Compared to TEM, SEM costs less to procure, takes less time to generate an image, requires less time for specimen preparation, and accepts thicker samples that are much larger. Thermal gravimetric analysis (TGA) was performed to study the polymeric materials’ thermal stability. As can be seen in Fig. 1B, from ambient temperature to approximately 100°C, there is a gradual decrease in weight, indicative of low-level volatilization or moisture loss (surface water). However, the most notable change occurs at 350°C, with a sharp decrease in weight, suggesting a significant transformation or decomposition process of the copolymer network. This abrupt shift likely corresponds to a thermal degradation or decomposition event, leading to a rapid release of volatile components or breakdown of molecular structures. Despite indications in the research suggesting complete degradation of the copolymer network at 400°C, only approximately 30% of it seems to have been lost, likely attributed to Fe₃O₄ nanoparticles. Overall, throughout the analysis, the material undergoes a weight loss of up to 30%, indicating a considerable degree of thermal instability or decomposition within the specified temperature range and weight loss occurs continuously in two steps up to 500°C.

The XRD pattern obtained for the PSMA-Fe₃O₄ composite provides valuable insights into its crystalline structure and phase composition (Fig. 1C). The pattern exhibits distinctive peaks corresponding to the crystalline phases present in the composite such as 2θ = 30, 35, 43, 53, 57, 63 peaks. It seems that the crystalline phase in the composite is influenced by the crystalline nature of magnetite nanoparticles and has face-centered cubic lattice structures. The Fe₃O₄ associated with PSMA are discernible, indicating the presence of both constituents in the material, and these peaks are characterized by their positions, intensities, and widths, reflecting the crystallinity and arrangement of atoms within the composite and well-matched with the reference pattern of Fe₃O₄ [25].

The synthesized samples were studied by FT-IR analysis and the FT-IR spectrum of final PSMA/Me/Fe₃O₄ was depicted in Fig. 2. The interpretation of all FT-IR spectra is as follows:

The FTIR spectrum of PSMA reveals distinct absorption bands indicative of its molecular composition and structural features. Within the spectrum, absorption bands are observed between 1450–1500 cm⁻¹ and 580–760 cm⁻¹, corresponding to the stretching of C=C bonds and the bending of C-H bonds in the aromatic ring of the styrene repeated groups. Additionally, strong absorption bands are evident around 1779 ± 1 cm⁻¹ and 1855 ± 1.2 cm⁻¹, attributed to the carbonyl stretching of the anhydride groups (C=O) in the maleic anhydride (MA) repeated groups. Notably, characteristic absorption bands at 1200–1300 cm⁻¹, associated with cyclic C-O stretching in the MA residue, are also detected in the PSMA sample. This suggests the presence of unreacted anhydride rings following polymerization. The simultaneous presence of aromatic alkene (1494, 1603 cm⁻¹) and anhydride carbonyl (1779, 1855 cm⁻¹) bands in the IR spectrum of PSMA confirms the successful copolymerization between Styrene and MA comonomers. Furthermore, the absence of the C=O anhydride ring (1779, 1855 cm⁻¹) and the broadening of peaks around 1620 ± 1.1 cm⁻¹ indicate the conversion of cyclic anhydride groups to amidic carbonyl groups following PSMA amidification with melamine molecules. According to the PSMA-Fe₃O₄ spectrum, the width of the peaks in the region of 3000 to 3500 cm⁻¹ has broadened due to the O-H group, and the peaks have appeared in the region of 580 cm⁻¹ are related to the Fe-O group bound in Fe₃O₄. It confirms the establishment of Fe₃O₄ nanoparticles on PMSA. The interpretation of PSMA and the PSMA grafted by melamine spectra is by our published article [23].

The presence of Me as a cross-linker in the copolymer structure not only may improve the mechanical property but also enhance the drug encapsulation efficiency (EE% = 83%) because of making porosity in the copolymer network. In addition, the LE for PSMA/Me/Fe₃O₄ was 78% according to the formula. The PSAM/Me/Fe₃O₄ drug release profile was studied in acidic (pH = 5 and 6.5) and neutral (pH = 7.2) media using PTX as a therapeutic drug model. The PTX release from PSAM/Me/Fe₃O₄ is shown in Fig. 3. The PTX release was 0.46, 0.43, and 0.48 ppm at pH 5, 6.5, and 7.2, respectively after 48 h. As a result, the PTX release from this polymer in neutral media was higher than in other media. Of course, there are differences between drug release in an acidic environment and a basic one. The reason for this difference can be due to the protonation of functional groups in the polymeric network and drug structure it weakened the interaction between the polymeric network and drug molecules and the fast release was seen in an acidic medium.

MTT cytotoxicity assays were conducted to validate the anticancer effect of PSMA/Me/Fe₃O₄/PTX on MCF-7 and Skov-3 cells. The data indicated a significant difference between the control group and treatment groups, either PTX or PSMA/Me/Fe₃O₄/PTX treated MCF-7 cells after 24 and 48 h treatment (p < 0.05). For instance, as compared to the control group, PTX decreased cell viability in treatment groups (p < 0.05). In addition, there was a significant difference between 1 µM group and 4 and 8 µM groups (p < 0.05) (Fig. 4A). In MCF-7 cells treated with PSMA/Me/Fe₃O₄/PTX, there was a significant a decrease in cell viability value of all groups compared to control group (p < 0.05). The cell viability of 4 and 8 µM groups was low compared to 1 and 2 µM (p < 0.05). After 48 h treatment, compared to the control group, the cell viability was decreased in all groups treated with PTX and PSMA/Me/Fe₃O₄/PTX (p < 0.05) (Fig. 4A). Moreover, the cell viability of the 8 µM group was low compared to 1, 2, and 4 µM groups (p < 0.05). The same results were obtained in Skov-3 cells (Fig. 4A). For example, PTX reduced cell viability significantly in 2, 4, and 8 µM cells compared to control cells (p < 0.05) after 24 h (Fig. 4A). Furthermore, there was a significant difference between 1 µM group and groups treated with 2 and 4 µM PTX (p < 0.05). The cell viability of the 8 µM group was low when compared to 1, 2, and 4 µM groups (p < 0.05). Meanwhile, PSMA/Me/Fe₃O₄/PTX decreased cell viability in all groups after 24 h (p < 0.05). Similar to MCF-7 cells, the cell viability of 4 and 8 µM groups was low compared to 1 and 2 µM in Skov-3 cells (p < 0.05). There was a significant difference between the 4 µM group and the group treated with 8 µM PSMA/Me/Fe₃O₄/PTX (p < 0.05). As shown in Fig. 4A, both PTX and PSMA/Me/Fe₃O₄/PTX significantly led to a reduction in cell viability of Skov-3 cells after 48 h treatment (p < 0.05). There was a non-significant difference between the control group and the 1 µM group in the PTX treatment panel (p < 0.05). In cells treated with PSMA/Me/Fe₃O₄/PTX, compared to the 1 µM group, there was a significant decrease in cell viability of either the 4 or 8 µM group (p < 0.05). The IC₅₀ values of PSMA/Me/Fe₃O₄/PTX for MCF-7 cells were 0.8 and 0.44 μM for 24 and 48 h, respectively. For Skov-3 cells, the values were 1.5 and 1.06 μM for 24 and 48 h, respectively. Table 1 displays the IC₅₀ values for PTX and PSMA/Me/Fe₃O₄/PTX in cells. The curves of IC₅₀ concentration of PTX and PSMA/Me/Fe₃O₄/PTX for 24 and 48 h were presented in Fig. 4B.