Molecular docking analysesThe primary goal of molecular docking is to comparatively analyze sericin and gluten, proteins rich in negatively charged amino acids, as a nanocarrier. Our research focuses on revealing the active domains and specific amino acids within these proteins interacting with phosphotungstic acid, aiming to discern and compare the nature of these interactions and exploring potential implications for biomedical applications. Specifically, sericin exhibits a noteworthy composition with 14% negative aspartic acid content47. Conversely, gluten is characterized by a more substantial presence of negative amino acids, particularly glutamic acid, which constitutes 30–35% of its overall composition48. This investigation is conducted with a specific focus on assessing the interaction of these proteins, specifically their aspartic and glutamic acid residues, with phosphotungstic, thereby elucidating their respective roles in facilitating the coating layer, as a nanocarrier, and augmenting biocompatibility. Also, we focus on the effect of these interactions on zeta potential. Through 250 runs, we analyzed distinct conformational clusters with an RMSD tolerance of 0.5 Å across three grid boxes. The docking computations consistently displayed remarkable performance, with consistently average RMSD values ≤ 1.045. Notably, poses with the highest affinity exhibited lower RMSD values. The analysis highlighted specific amino acids crucial in the PTA-Sericin interaction, including (Asp 209, 202), (Ser 208, 211, 127, 1141, 561, 562, 768), (Arg 292, 399, 515), (Gln 302, 402), (Thr 521), (Asn 295), signifying their significance in the binding process (Fig. 2a). Understanding the dispersion of binding energy within detected clusters were gleaned from the clustering histogram. Cluster 1 in grid box A (Fig. 3a), displaying the lowest binding energy of -4.57 kcal/mol and involving Ser 208 and Asp 209, demonstrated stability. In grid box B (Fig. 3b), a remarkable stability was evident with the lowest binding energy recorded at − 3.53 kcal/mol. This stability was associated with Arg 292, 399, Gln 302, 402, and Asn 295, emphasizing the robustness within this particular region. Meanwhile, in grid box C (Fig. 3c), displayed the minimal binding energy of − 4.59 kcal/mol, involving Ser 561, 562, 768, Arg 515, Thr 521, portraying stability within this region. The concurrent alignment or overlap of these three grid boxes pertaining to sericin was implemented to enhance the precision and accuracy of the docking process.Figure 2Possible binding sites of sericin and gluten. (A) showcases the docking of sericin with PTA, highlighting possible binding sites indicated by boxes 1, 2, and 3, depicted in yellow, purple, and orange, respectively. (B–D) Similarly, purple represents the binding sites for the three subtypes of gluten.Figure 3Most important amino acids involved in interactions with PTA. The clusters with the highest stability in terms of energy and reproducibility, along with the critical amino acids engaged in interactions, have been illustrated. Sericin interactions (A–C) correspond to sericin gridboxes 1, 2, and 3, respectively. Similarly, gluten interactions (D–F) are linked to gliadin, glutenin high molecular weight, and glutenin low molecular weight, respectively.Regarding the PTA-Gluten interaction (Fig. 2b–d), key amino acids crucial for different subtypes were identified. For the Gliadin subtype (Fig. 3d), the lowest binding energy of − 6.44 kcal/mol was associated with stability involving Glu 134, Ser 19, Tyr 16, Gly 20, Gln 14, Pro 115, Lys 147, Val 117. In the Gluten in high molecular weight subtype (Fig. 3e), the lowest binding energy of − 3.87 kcal/mol displayed stability, implicating Thr 499, Gln 569, Ala 568, Leu 567. Similarly, for the Gluten in low molecular weight subtype (Fig. 3f), the most minimal binding energy of − 3.51 kcal/mol was observed, showing stability involving Gln 146, 188, Ser 142, Trp 73. The histogram’s distribution pattern highlighted the varied energetically favorable conformations, emphasizing the importance of these findings in understanding the complex molecular landscape of the studied system. As observed, the most stable clusters, characterized by energy values of − 4.57 and − 6.44 kcal/mol, were those composed of the amino acids, aspartic acid and glutamic acid. Specifically in cluster with the most minimal binding energy of − 4.57 kcal/mol, oxygen atoms at positions 25 and 27 in sericin form hydrogen bonds with serine 208 and aspartic acid 209 of the receptor, respectively. With distances of 3.00 Å and 3.16 Å and binding energies of − 0.7 kcal/mol and − 1.4 kcal/mol, these interactions indicate favorable molecular recognition and binding affinities, highlighting the potential importance of hydrogen bonding in the sericin-PTA complex. The docking analysis of gluten with phosphotungstic acid reveals multiple interactions highlighting the molecular recognition between the ligand and the receptor. Notably, the oxygen atom (O) at position 28 in gluten forms a hydrogen bond with GLU 134 at a distance of 3.07 Å, resulting in a significant binding energy of − 4.0 kcal/mol. Additionally, interactions involving LYS 147, GLY 20, GLN 14, TYR 16, and SER 19 exhibit binding energies of − 5.5 kcal/mol, − 2.1 kcal/mol, − 1.9 kcal/mol, − 2.6 kcal/mol, and − 0.8 kcal/mol, respectively. These findings underscore the diverse hydrogen bonding interactions between gluten and phosphotungstic acid, suggesting a complex network of molecular associations that contribute to the stability and specificity of the ligand-receptor complex. Notably, while the gliadin clusters associated with gluten exhibited the lowest bond energy (− 6.44 kcal/mol), it is essential to underscore that the number of binding sites for PTA within sericin (# denoting the frequency of observed binding events in dlg file of docking) surpassed those observed for the gluten clusters. Moreover, the grid boxes designated as Sericin number 1 and 2 exhibited a greater number of binding sites, whereas grid box 3 demonstrated heightened stability in terms of energy, indicative of a more stable binding environment. The extensive analysis underscores the efficacy of blind-docking calculations in predicting favorable binding conformations, revealing distinctive conformational clusters and pivotal amino acids crucial for interactions within PTA-Sericin and PTA-Gluten complexes. Lower RMSD values associated with high-affinity poses affirm the computational reliability in capturing near-native conformations, elucidating the molecular dynamics governing these interactions and highlighting essential binding sites and amino acids. Moreover, clustering histograms depict diverse energetically favorable conformations, emphasizing the intricate nature of protein–ligand interactions and advocating for exploring varied binding modes in protein–ligand design. Special consideration was given to the influence of negatively charged amino acids, particularly aspartic acid and glutamic acid, as these interactions are crucial in regulating the zeta potential of the nanocarrier45. Significantly, our findings highlight Asp 209, 202, and Glu 134 as key contributors, underscoring their substantial roles in orchestrating these interactions through the formation of hydrogen bonds. These insights offer a deeper understanding of the molecular mechanisms underlying the binding dynamics between sericin, gluten, and phosphotungstic acid, with implications for nanocarrier properties.Development and characterization of protein-based nanocapsulesThe physicochemical characteristics of Fe3O4, MSN, MSN-PTA, MSN-PTA-SER, and MSN-PTA-GLU were investigated in each section. Figure 4, displays DLS measurements for Fe3O4, MSN, and MSN-PTA, revealing average sizes of 37 nm, 71 nm, and 103 nm, respectively, with a Polydispersity Index (PDI) below 0.3. The MSN magnetic nanoparticles exhibited a size of approximately 71 nm, displaying superparamagnetic behavior. The nanoprecipitation method was employed for synthesizing these nanoparticles. To identify the ideal protein concentration for formulating protein nanocapsules, we experimentally tested various concentrations of sericin and gluten. After conducting these inquiries, we opted for the formulation employing proteins at a concentration of 0.1% (w/v) for our experiments45. It’s important to highlight that the protein concentration within these nanocarriers can impact their size, with elevated protein concentrations resulting in a broader spectrum of nanoparticle sizes49,50. Protein coating typically imparts favorable attributes such as biocompatibility, biodegradability, reduced immunogenicity, and lower cytotoxicity to MNPs. Sericin markedly increases drug solubility by a factor of 8–tenfold, contrasting with the water insolubility of monomeric gluten, particularly gliadin51,52. The coating of sericin and gluten on MSN-PTA resulted in a marginal increase in size distribution, with an average size of 251 nm and PDI 0.3 for MSN-PTA-SER, and an average size of 314 nm and PDI 0.34 for MSN-PTA-GLU. These findings indicate that the coating procedure resulted in a marginally increased particle size for the gluten-based particles when contrasted with the sericin-based particles. Notably, sericin-based nanocarriers exhibited a more uniform and smaller size than their gluten-based counterparts. Sericin has been systematically investigated for diverse pharmaceutical applications, encompassing solubility augmentation, release profile adjustments, formulation stabilization, and its utilization as a carrier for therapeutic agents53. Remarkably, sericin demonstrates notable solubility, especially at a low concentration of 0.1%, making it an exceptionally promising choice for anti-solvent utilization54. This feature corresponds to the intrinsic traits of proteins with high water solubility, which have garnered significant interest for their effectiveness in methodologies based on anti-solvent precipitation55. The increased size of the coating seen in gluten compared to sericin may be attributed to wheat gluten’s restricted solubility in water. This trait is often linked to the significant molecular size and aggregation between molecules of gluten, which arise from strong non-covalent bonds, such as hydrogen bonding and hydrophobic interactions56. However, it is well known that particles with dimensions below a certain threshold can evade the body’s natural clearance mechanisms (RES), resulting in an extended period of circulation for MSN-PTA-SER. Consequently, for optimal cellular uptake and evasion of the RES, the nanocarrier size must fall within this specified range57. Our discovery aligns with a prior investigation in which the smallest concentration of sericin (0.1%) resulted in sericin nanocarriers displaying uniformity in size as measured by DLS, indicating appropriateness45,46.Figure 4DLS analysis of (A) Fe3O4, (B) MSN, (C) MSN-PTA, (D) MSN-PTA-SER, and (E) MSN-PTA-GLU. Zeta potential analysis of (F) Fe3O4, (G) MSN, (H) MSN-PTA, (I) MSN-PTA-SER, (J) MSN-PTA-GLU and (K) Overall comparison of zeta potential of mentioned nanoparticles with zeta of sericin and gluten alone.In addition, the zeta potential of Fe3O4, MSN, MSN-PTA was observed at − 9.97, − 14, and − 18.8 respectively at pH 7.4 (Fig. 4). The isoelectric point (PI) of uncoated MNPs has been documented in the literature to be at pH 6.8, specifically for magnetite58. Therefore, at pH 7.4, as expected, the zeta potential for magnetite − 9.97 was obtained. So Fe3O4 do not benefit from the good stability in aqueous solution. Fe3O4 particles coated with a SiO2 shell exhibit a lower PI of approximately pH 3.959. Consequently, MSN particles possess a higher negative charge compared to bare Fe3O4 nanoparticles, with a measured value of − 14. This lower isoelectric point and increased negative charge contribute to the enhanced stability and dispersibility of MSN magnetic nanoparticles. This suggests that improving these properties for Fe3O4 nanoparticles can be easily achieved by applying a SiO2 coating. Additionally, the zeta potential becomes negative as the pH increases to 7.4 due to the rise in OH− ions in the dissociated solution and subsequent deprotonation60. The Keggin anion tends to agglomerate and create less negative surface charge in comparison to MSN. In solutions with a pH below 8, the inclusion of ethanol or acetone stabilizes the [PW12O40]3− anion, thereby mitigating decomposition61. In our investigation, we examined the zeta potentials of sericin and gluten both independently and as coatings on the magnetic core of nanoparticles. At pH 7.4, sericin alone exhibited a zeta potential of − 15.2 mV, while gluten alone displayed a zeta potential of − 14.3 mV. However, the coating of sericin on the MSN-PTA induced a substantial reduction in zeta potential to − 28.5 mV (MSN-PTA-SER), surpassing the reduction observed for gluten-coated nanoparticles, which reached − 23.3 mV (MSN-PTA-GLU) at pH 7.4. This outcome suggests that the sericin coating imparts a more negative charge to the nanoparticles compared to gluten. Molecular docking results align with this observation, indicating more binding sites for sericin, potentially contributing to the stronger negative zeta potential. Despite the low binding sites in gluten, the most stable cluster in terms of energy was associated with the phosphotungstic interaction with the gliadin subunit of gluten. These findings collectively point towards a greater interaction of sericin with the MSN-PTA, emphasizing the significance of the sericin coating in altering the surface properties and potential applications of the nanoparticles. Particles with zeta potential values around ± 30 mV62, notably observed in MSN-PTA-SER nanoparticles, indicate stability, particularly when compared to gluten-coated nanoparticles. Further exploration of the biological and functional implications of these interactions is warranted for a comprehensive understanding of the observed phenomena.Morphological and structural characteristicsThe examination through SEM demonstrated a uniform dispersion of MSN-PTA-SER and MSN-PTA-GLU, displaying a spherical morphology (Fig. 5). The average diameter observed was consistent with the measurements obtained through DLS for Fe3O4, MSN, MSN-PTA, MSN-PTA-SER, and MSN-PTA-GLU, with values of 20.1 nm, 36.1 nm, 63.4 nm, 177 nm, and 241 nm, respectively. The SEM-determined dry particle size was smaller than that obtained by the DLS, which measures in solution and considers the size increase caused by particle hydration63. SEM analysis performed on three powder samples (Fe3O4, MSN, MSN-PTA) revealed no aggregation in the final syntheses, indicating well-dispersed nanoparticles in the final products. The TEM images provided a detailed visualization of the MSN-PTA-GLU and MSN-PTA-SER morphologies, revealing significant size differences between the samples while both retained a consistent spherical shape. Notably, the MSN-PTA-GLU nanoparticles appeared larger than the MSN-PTA-SER nanoparticles, indicating that the inclusion of gluten influences the overall size more significantly than sericin. The core structures of the magnetic nanoparticles were distinctly visible in the TEM images, highlighting the successful incorporation of the MSN-PTA core within the nanoparticle matrix. This core visibility confirms the magnetic nature of the nanoparticles and provides insight into the core–shell architecture of the synthesized materials. Additionally, the nanoparticles exhibited a predominantly spherical shape across all samples. This spherical morphology is crucial for uniformity in application and enhances the consistency of the nanoparticles’ physical and chemical properties.Figure 5SEM and TEM analyses with size distribution assessment performed using Image J. SEM images include: (A) Fe3O4, (B) MSN, (C) MSN-PTA, (D) MSN-PTA-SER, and (E) MSN-PTA-GLU, with detailed size distribution analysis using Image J. TEM images include: (F) MSN-PTA-SER, and (G) MSN-PTA-GLU.Encapsulation efficiency and drug loading capacitiesThe EE% of MSN-PTA-SER and MSN-PTA-GLU were determined to be 94% and 95%, respectively. The higher EE for gluten can be attributed to its larger protein structure, which comprises three subunits. However, under identical conditions, the DL% for both sericin and gluten was found to be 19%. This suggests that despite the high encapsulation efficiencies, the loading capacities for both biopolymers remain at 19%. This discrepancy highlights the distinct behavior of these biopolymers in terms of MSN-PTA release and loading capacities, emphasizing their potential roles and applications in drug delivery systems.FTIR analysisThe synthesis process of the composite was further explored by examining any alterations in the FTIR spectra of Fe3O4, MSN, MSN-PTA, MSN-PTA-SER, and MSN-PTA-GLU (Fig. 6). In the spectrum of Fe3O4, the prominent peak at 582 cm−1 is ascribed to the stretching vibrations of the Fe–O groups within the Fe3O4 nanoparticles. Additionally, the peak observed at approximately 3437 cm−1 is attributed to both the bending and stretching vibrations of –OH groups, originating from both water molecules and surface –OH groups on the particles. In the case of MSN, the F–O adsorption peak is notably evident at 582 cm−1. The most significant alterations, distinguishing it from bare Fe3O4 nanoparticles, include the emergence of a peak at 1076 cm−1 associated with the asymmetrical vibrations of the Si–O–Si group. This peak confirms the successful surface modification with silica. Additionally, the presence of a peak at 886 cm−1 is indicative of the Si–OH bond. The distinctive FTIR bands associated with the Keggin structure are prominently exhibited in pure PTA, featuring peaks at 1080, 987, 889, and 780 cm−1. The observed peaks are associated with distinct vibrational modes: P–Oa, W = Ot, W–Ob–W, and W–OC–W, respectively. These spectral features represent different oxygen configurations within the Keggin framework, including oxygen in the central tetrahedral PO4 (Oa), terminal oxygen linked exclusively to the tungsten atom (Ot), bridge oxygen situated between two separate W3O13 units (Ob), and bridge oxygen within the same W3O13 cluster (OC)64. The FTIR analysis uncovered distinctive peaks in sericin’s molecular makeup. At 3419 cm−1, the stretching oscillations of O–H bonds suggested the existence of hydroxyl functional groups. A singular absorption related to amide vibrations at 1631 cm−1, identified as the amide I band, verified the existence of proteins, signifying the stretching oscillations of C = O bonds. Additionally, peaks ranging from 1540 to 1520 cm−1 were linked to the amide II band, originating from N–H bending vibrations. The amide III region, spanning from 1240 to 1384 cm−1, highlighted the presence of a random coil structure within sericin, involving C–N stretching and N–H bending. In contrast, gluten, the wheat-derived protein complex, exhibited peaks corresponding to various functional groups. Notably, the stretching oscillations of gluten N–H bonds at 3406 cm−1 and O–H bonds hinted at the protein’s hydroxyl groups. The band at 2954–2958 cm−1 aligns with the stretching vibrations of the CH2– group. Additionally, distinctive peaks corresponding to the amide I and amide II bands were detected at 1635 cm−1 and 1575 cm−1, respectively, with an amide III band ranging from 1200 to 1340 cm−1. These bands are consistent with the characteristic features of gliadins.Figure 6The FT-IR spectra of Fe3O4, MSN, MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU.XRD analysisXRD ascertains the crystalline structure of samples. XRD pattern of pure Fe3O4, MSN, MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU nanoparticles illustrated in Fig. 7. Peaks at 30.5°, 35.5°, 43.2°, 53.5°, 57.62°, and 62.5° (2θ) were assigned to (220), (311), (400), (422), (511) and (440) miller indices of Fe3O4, respectively. The XRD spectrum of Fe3O4 indicates that the nanoparticles exhibit an inverse spinel structure, characteristic of magnetite. This structure features a cubic lattice with space group Fd3m65,66. The corresponding ICDD file number for magnetite is 19–629, which matches the observed Bragg peaks67. The results suggest that the synthesis of MSN nanoparticles occurred effectively, preserving the crystal structure of the Fe3O4 core. Furthermore, the presence of broad peaks centered around 23° in the MSN sample indicates the formation of an amorphous silica shell encasing the nanoparticles’ surface. The XRD pattern shows that there are no other impurity phases present. The XRD pattern of the MNP reveals a diffuse scattering or broad hump around 20° (2θ), as depicted in the Fig. 7. This characteristic feature may be ascribed to the presence of amorphous SiO2 materials. In the case of MSN-PTA, the primary XRD peaks are observed at 2θ values of 10.5, 18.3, 23.7, 26.1, 30.2, 35.6, and 38.8. The observed peaks are consistent with the cubic structure characteristic of Keggin-type heteropolyacids, corroborating findings from previous studies (JCPDS#7521–25)68. XRD analysis of silk sericin indicates a broad diffraction peak appearing at 2θ = 18.86°–20°, attributed to the presence of the amorphous organic phase. Additionally, the highest diffraction peak observed at 2θ = 14.2° suggests a transition from the random coil structure to the β-sheet structure within sericin. This transition is likely facilitated by intermolecular hydrogen bonding between the hydroxyl groups of the amino acids present in sericin69. In contrast, gluten exhibited dual diffraction peaks around 2θ = 10° and 2θ = 20°, signaling its amorphous composition and the presence of α-helix structures. The lower 2θ value peak denotes the typical inter-helix spacing, while the higher 2θ value peak indicates the average distance between helix backbones70.Figure 7X-ray diffraction patterns of Fe3O4, MSN, MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU.VSM analysisThe primary hysteresis curve analysis of Fe3O4 and its derivatives, namely MSN, MSN-PTA, MSN-PTA-SER, and MSN-PTA-GLU, was conducted using a VSM (Fig. 8). The magnetization of MNPs were recorded in an applied magnetic field of − 15,000 ≤ H(Oe) ≤ 15,000 at room temperature. The magnetic properties of MNPs and bare MNPs could be seen by magnetic (Ms) attraction. the magnetization curves revealed that the magnetic nanoparticles were superparamagnetic. The magnetization values obtained were 54 emu/g for uncoated Fe3O4, 41 emu/g for MSN, 28 emu/g for MSN-PTA, 6 emu/g for MSN-PTA-GLU, and 5 emu/g for MSN-PTA-SER, as depicted in Fig. 8. The results demonstrate that the coercivity of SiO2-coated and protein-coated Fe3O4 nanoparticles is notably lower than that of the uncoated counterparts. The magnetic behavior of magnetite nanoparticles is known to be influenced by dipole–dipole interactions, which are strongly dependent on the interparticle distance. The SiO2 layer enveloping the Fe3O4 nanoparticles in this study serves as an insulating layer, hindering electron transfer, increasing the distance between nanoparticles, and preventing their agglomeration. Consequently, MSN nanoparticles exhibit reduced coercivity71.Figure 8The VSM examination of Fe3O4, MSN, MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU.TGA analysisTo determine the weight percentage of Fe3O4 in MSN, MSN-PTA, MSN-PTA-SER, and MSN-PTA-GLU, TGA was conducted in an air environment from room temperature to 900 °C. In the pure Fe3O4 graph, only a minimal weight loss of 0.8% was observed, attributed to the removal of adsorbed physical and chemical water. The residual mass percentages were used to estimate the magnetite content in the samples. The TGA results depicted in the (Fig. 9) indicate an increase in weight loss above 200 °C, attributed to the removal of the SiO2 layer coated on the Fe3O4. The estimated weight loss of the SiO2 coating on the Fe3O4 nanoparticles is approximately 9.4%. In the graph related to MSN, no significant difference was observed with the graph of MSN-PTA, however, a decrease of 2.4% was observed in the temperature range of 250 and above, which is related to the weight loss of PTA. The TGA results reveal distinct thermal behaviors for magnetic nanoparticles coated with gluten and sericin. The gluten-coated nanoparticles show a gradual weight loss from 100 to 43% across the temperature range of 0 to 900 °C, indicating continuous decomposition of the gluten coating; in Fig. 9, for MSN-PTA-GLU, the initial weight loss stage (below 140 °C) is attributed to the evaporation of water molecules, while the subsequent stage (about 200 °C) corresponds to the decomposition of gluten. On the other hand, sericin-coated nanoparticles exhibit a more intense weight loss from 100 to 39%, followed by a constant weight percentage up to 900 °C. This indicates a rapid and substantial change or decomposition in the sericin coating at the initial stage, with the material remaining stable thereafter. The differences in weight loss patterns provide insights into the distinct thermal stabilities and decomposition characteristics of the gluten and sericin coatings on the magnetic nanoparticles.Figure 9The TGA examination of Fe3O4, MSN, MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU.In vitro release profileThe MSN-PTA release test was conducted under laboratory conditions for nanoparticles coated with sericin and gluten over a 50-h period at pH 6 and 7 (Fig. 10). At the 25-h mark, the release percentages for MSN-PTA-GLU were 35% at pH 7 and 54% at pH 6. For MSN-PTA-SER, the release percentages were 32% at pH 7 and 65% at pH 6 after 24 h. By the end of the 50-h period, the release of MSN-PTA-SER and MSN-PTA-GLU reached their maximum levels: 40% and 71% at pH 7 and 6, respectively, for MSN-PTA-SER, and 51% and 61% at pH 7 and 6, respectively, for MSN-PTA-GLU. The graphs indicate that MSN-PTA-SER and MSN-PTA-GLU reach a steady state of approximately 70% and 60%, respectively, after 30 h at pH 6. In contrast, at pH 7, they achieve a steady state of around 40% and 50%, respectively, after 35 h. This difference in release profiles can be attributed to the proteins releasing more quickly and efficiently at the lower pH level. These results indicate that both types of nanoparticles exhibit controlled release capabilities. However, nanoparticles coated with sericin demonstrate a higher release capacity compared to those coated with gluten, attributed to the charge reversal property of sericin at lower pH levels. This means that sericin has more charge reversal properties.Figure 10The in vitro release of MSN-PTA at pH value of 7, 6.In vitro biological evaluationCell viabilityAssessing the capability of MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU (at concentrations of 5, 10, 25, 50, 100, 200 μg/mL) on the viability of MCF-7 cells and ZR-75–1 at pH 7.4, the MTT assay was conducted (Fig. 11). After 24 h for MCF-7 (Fig. 11a), a mild reduction in cell viability was noted in the groups treated with MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU compared to the control group. Nevertheless, within 24-h timeframe, this reduction was notably more pronounced for the ZR-75–1 cell group (Fig. 11c). Despite a gradual decrease, no statistically significant differences in cell viability were observed over a 24-h period for both MCF-7 cells and ZR-75–1 when comparing cells treated with MSN-PTA, MSN-PTA-SER and MSN-PTA-GLU, both within each concentration and across different concentrations. Cells subjected to a 48-h treatment exhibited a more pronounced and dose-dependent inhibition. In a 48-h treatment of MCF-7 cells (Fig. 11b) at a concentration of 5 μg/mL, MSN-PTA showed a significant 68% decrease in cell viability (P-value ≤ 0.05), MSN-PTA-GLU exhibited a 64% decrease (P-value ≤ 0.001), and MSN-PTA-SER demonstrated a significant 54% reduction (P-value ≤ 0.0001) compared to the control group. For MCF-7 (Fig. 11b), the decrease and distinction persist until the concentration of 50 μg/mL, which represents the IC50 of MSN-PTA with a statistically significant difference (P-value ≤ 0.05) compared to sericin and gluten-coated particles. At 10 μg/mL (sericin coated IC50), a significant difference (P-value ≤ 0.01) is observed compared to gluten-coated particles, resulting in a 63% reduction in cell viability. Similarly, at 25 μg/mL (gluten coated IC50), a highly significant difference (P-value ≤ 0.0001) is noted compared to sericin-coated particles, accompanied by a 40% reduction in cell viability. Ultimately, for MCF-7 at a concentration of 200 μg/mL nanoparticles coated with sericin, the resulting 25% cell viability did not significantly differ from that observed with gluten-coated nanoparticles at the same concentration. The viability of the ZR-75–1 cell line treated with MSN-PTA-SER exceeded that of the MCF-7 cell line across all doses over a 48-h period (Fig. 11d). This discrepancy, however, led to a notable difference (P-value ≤ 0.05) in the final concentration of 200 μg/mL between sericin and gluten-coated nanoparticles in the ZR-75–1 cell line. In summary, the treatment with MSN-PTA-SER resulted in reduced cell viability for both ZR-75–1 and MCF-7 cells compared to the treatment with MSN-PTA-GLU. These findings suggest that MSN-PTA-SER is a more effective treatment option than MSN-PTA-GLU, and could potentially lead to better outcomes for patients.Figure 11Viability of MCF-7 cells at 24 h (A) and 48 h (B), and ZR-75–1 cells at 24 h (C) and 48 h (D) following exposure to different concentrations of MSN-PTA, MSN-PTA-SER, and MSN-PTA-GLU. Results represent the mean ± SD from three independent experiments, with statistical significance denoted by *P ≤ 0.05 compared to the respective controls.Intracellular uptake and DAPI stainingThe cytotoxicity of the synthesized MSN-PTA-SER and MSN-PTA-GLU nanoparticles was assessed by determining their IC50 values. Subsequently, intracellular uptake and nuclear assessment were evaluated using fluorescence microscopy (Cytation 5 Cell Imaging Multi-Mode Reader). The morphologies of ZR-75–1 and MCF-7 cells were examined after exposure to FITC-labeled nanocarriers for 4 h under acidic conditions (pH 6) (Fig. 12). In our prior investigation, we conducted experiments involving sericin under varying pH conditions to examine drug release dynamics and evaluate cell viability. Results showed that optimal drug release and cellular uptake were observed at pH 6, albeit with a significant reduction in cell viability45,46. Cancer cells typically possess negatively charged surfaces, attributed to the secretion of lactic acid, a metabolic trait resulting from heightened glycolysis rates72. The experimental findings demonstrate the successful cellular uptake of both MSN-PTA-SER and MSN-PTA-GLU following a 4-h incubation period, resulting in their localization within the cytoplasm. This uptake was evidenced by the accumulation of the fluorescent dye within the cellular compartments, leading to a notably enhanced green fluorescence intensity compared to the untreated control samples. The increased efficiency of cellular uptake observed for MSN-PTA-SER can be attributed to its transition from a negatively charged surface to a positively charged one under lower pH conditions, along with its comparatively lower zeta potential. Conversely, at pH 7.4, the negative surface charge of both MSN-PTA-SER and MSN-PTA-GLU impedes their internalization into the cells due to repulsive electrostatic interactions with negatively charged components such as phospholipid head groups, glycans, and membrane proteins73. Conversely, at a lower pH of approximately 6, aligning with the pI of sericin and gluten, the transformation of surface charge to positive for these nanocarriers enhances its affinity for cell membranes, thereby facilitating the facile internalization of MSN-PTA-SER and MSN-PTA-GLU by MCF-7 and ZR-75–1 cells (charge reversal effect). To assess the cytotoxic effects of MSN-PTA-SER and MSN-PTA-GLU, cell nucleus staining was conducted using DAPI. Sericin and gliadin subtype of gluten, have been shown to elevate apoptotic cells and regulate the expression levels of Bax, Bcl‑2, Cyto‑C, and Caspase‑3 in cancer cells, consistent with observations reported in previous studies across diverse cancer cell types74,75. As represented in Fig. 12, significant nuclear changes were detected in MCF-7 and ZR-75–1 cells, as they emit a bright blue fluorescence. It indicated the DNA fragmentation and apoptosis after treatment with IC50 doses of MSN-PTA-SER and MSN-PTA-GLU. Generally, sericin-based nanocarriers exhibited more robust uptake in both cell lines compared to gluten-based nanocarriers. However, gluten-based nanocarriers demonstrated superior cell uptake specifically in the MCF-7 cell line. This outcome can be ascribed to the more negative zeta potential of sericin nanocarriers, suggesting that the enhanced stability of the nanocarrier led to a further reduction in cell viability, as evidenced by the cell viability test.Figure 12Fluorescence microscopy revealed intracellular uptake of FITC-labeled MSN-PTA-SER and MSN-PTA-GLU at pH 6 in MCF-7 cells, alongside DAPI staining for visualizing cell nuclei, presented with a bar scale of 200 nm.
