Controlling cisplatin release by synergistic action of silver-cisplatin on monodispersed spherical silica for targeted anticancer and antibacterial activities

2.1 Preparation of 1–6 wt% Ag/MSS (Antibacterial Study)

For 1 wt% Ag loading, 0.016 g of silver nitrate was dissolved in 30 mL distilled water for 30 min. Then 1.0 g of predried MSS was added, and the solution mixture was allowed to stir (300 rpm) for 12 h. The mixture was then heated at 120 °C for 6 h. The obtained sample was powdered using mortar and pestle and finally calcined in air at 500 ℃ for 3 h (5 °C/min). Likewise, 2 wt% (0.031 g), 4 wt% (0.063 g) and 6 wt% (0.094 g) of Ag was loaded over MSS (1 g) and then calcined in air at 500 ℃ for 3 h (5 °C/min).

Following a similar procedure, 4 wt% Ag (0.063 g) was loaded per gram of different supports such as SBA-16 (Santa Barbara Amorphous), TiZSM-5 (Zeolite Socony Mobil #5), mesosilicalite, halloysite, and Ag-silicalite, respectively. A detailed list of final samples was presented in Table 1.

2.2 1–6 wt% Ag/MSS or structured Silica/Cpt (Anticancer Study)

In the first step, cisplatin (∼30 mg) was mixed in 10 mL of normal saline solution (10 mL) at 40 °C and then stirred (200 rpm) for 20 min. In the second step, 1-6 wt% Ag/MSS samples (600 mg) with Cpt/MSS ratio of 0.05 was allowed to stir overnight in ice cool environment. The solution was then filtered, washed with 5 mL of normal saline and dried at room temperature using vacuum drying method. The filtrate was then analyzed by UV–visible spectroscopy at 208 nm. Prior to analysis, a calibration curve was established at λmax = 208 nm. The analysis of 4 wt% Ag/MSS/Cpt showed a DEE % of 90 % and DLC % of 4.34 %. The drug Cpt entrapment efficiency (DEE%) and loading capacity (DLC%) were measured as: $$\mathit{DEE}\left(\%\right)=\left[\left(\frac{\mathit{Initial\; amount\; of\; Pt\; -\; Pt\; in\; filtrate}}{\mathit{Initial\; amount\; of\; Pt}}\right)\right]\times 100$$ $$\mathit{DLC}\left(\%\right)=\left[\left(\frac{\mathit{Initial\; amount\; of\; Pt\; -\; Pt\; in\; filtrate}}{\mathit{Pt}+\mathit{MSS}}\right)\right]\times 100$$

2.3 Characterization techniques

The phase of 1-6 wt% Ag/MSS and 4 wt% Ag/structured silica were analyzed using benchtop XRD (Miniflex 600, Rigaku Co., Tokyo, Japan). The surface area, pore volume with pore size distribution modifications before and after Ag addition were measured using the nitrogen adsorption technique (ASAP-2020 plus, Micromeritics Instrument Corp., Norcross, GA, USA). The silver nanoparticle chemical coordination was analyzed using DRS-UV–visible spectroscopy analysis (JASCO Corporation, Hachioji-City, Tokyo, Japan). The chemical compositions and oxidation states of the elements in 1Ag/MSS and 6Ag/MSS were studied using Kratos DLD XPS instrument, where C1s peak at 284.8 eV is used as a reference to calibrate the XPS spectra. The hydrodynamic particle size, polydispersity index, and zeta potential of MSS, 1, 4, and 6Ag/MSS samples were measured by Zetasizer Nano ZS (Malvern Panalytical Ltd, Worcestershire, UK). The sample (5 mg) was mixed with 10 mL of PBS buffer (pH 7.4) and sonicated for 30 min. The sample was then transferred into a disposable measuring cell and analyzed. The measurement was carried out three times at room temperature. SEM was analyzed using JSM-6610LV from JEOL Ltd, Tokyo, Japan. Elemental mapping was obtained by energy dispersive spectroscopy (EDS) using Aztec software (Oxford). The morphology of the sample was analyzed using transmission electron microscopy (JEM2100F from JEOL Ltd, Tokyo, Japan).

2.4 In vitro Cpt release study

The drug release analysis of Ag loaded nanoformulations such as 1-6 wt% Ag/MSS, 4 wt% Ag loaded SBA-16, TiZSM-5, Mesosilicalite, Halloysite and Silicalite was studied by Franz cells (PermeGear, USA) (Balasamy et al., 2019). The system was assembled with tumor acidic solvent reservoir, 8 inline cells, solution driving peristaltic pump and sample collector. The setup was constantly maintained at 37 °C using water heater (Julabo GmbH). Firstly, dialysis membrane (MWCO = 14000, Sigma) was activated by washing in phosphate buffer solution. Any air blockages in the tubings were removed by purging with phosphate buffer solution. One square inch sized membrane was placed inside the Franz cells. The Ag loaded nanoformulation was mixed with acidic PBS solution (30 mg/mL) and then placed in the donor site. Then the solution was pumped at the constant volume flow rate of 10 mL/60 min with volume to time ratio of 0.167. The samples were collected at every hour, analyzed and quantified by Cpt calibration curve using JASCO UV–Vis spectroscopy. The release study using each nanoformulation was studied in triplicate.

2.5 Anticancer studies

2.6 Antibacterial activity

The bacterial strains of Pseudomonas aeruginosa ATCC27853 (gram-negative) and Staphylococcus aureus ATCC25923 (gram-positive) were subjected to antibacterial studies. The bacteria were sub-cultured and maintained on Muller Hinton agar (MHA). The stock solution of nanomaterial (8 mg mL⁻¹) was sonicated for 10 min in Mueller Hinton broth (MHB) to obtain the homogenized solution for antibacterial assay. Inoculum was prepared by adjusting the cell number to 1.5 × 10⁸ CFU mL⁻¹ by using freshly grown strains grown for overnight at 37 ⁰C.

3.1 X-ray diffraction and nitrogen adsorption–desorption isotherm

The monodispersed spherical silica with different weight percentages of Ag nanoparticles loadings were characterized by X-ray diffraction (XRD) and N₂ adsorption–desorption technique (Fig. 1a-i). Fig. 1(a-d) shows the X-ray diffraction pattern of 1-6 wt% Ag/monodisperse spherical silica. 1Ag/MSS showed only the broad diffraction pattern with peak height at 2θ = 22.5° characteristics to that of the (1 0 1) plane of amorphous silica. With 2, 4, and 6 wt% loadings, Ag nanoparticles corresponding to the cubic phase were observed with (1 1 1), (2 0 0) and (2 2 0) planes. The nitrogen adsorption–desorption technique was used to study the textural variation (surface area, pore size, and pore volume) of support MSS and Ag impregnated samples (1, 2, 4 and 6 wt% Ag/MSS) (Fig. 1e-i). MSS sample showed a type IV isotherm with hysteresis loop pattern at higher relative pressure p/p₀ > 0.8. The parent silica showed a uniform pore size distribution with a high surface area of 170 m²/g and pore volume of 0.35 cm³/g. The Ag impregnated samples showed a decrease in the volume of nitrogen adsorption compared to MSS indicating a reduction in the surface area and pore structure. For instance, the loading of 2 wt% Ag attributes to 67 % decreases in surface area of MSS to about 56 m²/g. Similarly, a 20 % reduction in the total pore volume of 0.28 cm³/g was observed compared to MSS. These results suggest that the surface area and pores of MSS are accessible for Ag NPs. Further, increasing the Ag loading to 6 wt% had a significant drop in the surface area to about 90 % (17 m²/g) along with pore volume (0.08 cm³/g). However, the average pore diameter of MSS was about 8.3 nm increases with Ag loading to about 20 nm. Yu et al., (2022) have observed that Ag₂O loading on MCM-41 increases the pore diameter compared to support MCM-41 and is attributed to the stacking of silver oxide NPs at the interior and exterior side of the pore channels. In our case, a similar pore expansion shows the deposition of AgNPs at the external pore walls of MSS and contributes to the increase in the pore size.

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Fig. 1

X-ray diffraction of 1-6 wt% Ag loaded monodispersed spherical silica (a) 1Ag/MSS, (b) 2Ag/MSS, (c) 4Ag/MSS and 6Ag/MSS, N₂-adsorption–desorption isotherm of (e) MSS, (f) 1Ag/MSS, (g) 2Ag/MSS, (h) 4Ag/MSS and (i) 6Ag/MSS.

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3.2 In vitro drug release

The release ability of Cpt with increasing Ag loadings over MSS was carried out in a continuous flow system that imitate in vitro drug release (Fig. 2a and b). The system is more accurate than that of using a dialysis membrane, which is a closed system. In this condition, sink and saturation levels were negated due to an open system (continuous flow of buffer solution). The removal and refilling errors were reduced due to semi-automation. The release profiles were reproduced with triplicate runs. In the case of Cpt release (Fig. 2a), the percentage cumulative Cpt release shows the following pattern 2Ag/MSS > 1Ag/MSS > 4Ag/MSS >6Ag/MSS. The quick release of drugs is used for acute diseases, while slow and sustained release is beneficial for cancer therapeutics (Senapati et al., 2018). Such controlled slow release to cancer cells, reduce the toxic effect on normal cells. Interestingly, the cumulative percentage release pattern as observed in Fig. 2a showed an inverse relation with respect to different weight percentage Ag loading. A quick release of Cpt occurs at lower Ag loadings (1 wt% and 2 wt%) on MSS, whereas the release of Cpt reduced significantly with high Ag loadings (4 wt% and 6 wt%). Therefore, 4 wt%Ag loading was chosen due to sustained release and compared with 4 wt% Ag loaded on different structured materials. The release profile over different structured silica shows that SBA-16 with cubic-shaped pores have high Cpt release (∼15 %) within short duration between 0 and 10 h. TiZSM-5 zeolite with micropores, halloysite with nanotube, and mesosilicate with mesopores showed an intermediate Cpt release (3–5 %). In the case of 4 and 6Ag loading on MSS, Cpt remains intact, which indicates a synergistic interaction between Ag and Cpt complex (Fig. 2b).

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Fig. 2

(a) In vitro Cpt release over 1-6Ag/MSS and (b) 4Ag impregnated on different porous structured materials using automated Franz cell system for 10 h.

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The stability experiments of two samples (1 and 4Ag/MSS) were studied for the period of 72 h (dialysis membrane) at pH 5.6 and 6.6, respectively. In our previous study, structured silica such as SBA-16 functionalized with 3-aminopropyltriethoxysilane and polyacrylic acid showed a high percentage cumulative Cpt release at about 60 % (Alomari et al., 2019). Chitosan-modified spinel ferrite/spherical silica nanocomposites also showed similar release profile ranging between 60 and 70 % (Jermy et al., 2020). In this study, 1Ag/MSS showed a maximum Cpt release reaching about 12.7 % for 72 h, while 4Ag/MSS showed a further slow release of about 8 % for 72 h. A high Cpt release at lower Ag loading, while a slow and sustained Cpt release with high Ag correlates with release trend of Franz cell system (Table 2). Increasing the pH 6.6, the Cpt release over 1Ag/MSS increases to a maximum of 33.24 % for 72 h, while with high 4Ag/MSS slightly improved to 4.26 %%, respectively. The Cpt release profile (Fig. 2) and Table 2 clearly show such interactive and release abilities between Ag and Cpt. This evidence indicates that different amount of Ag interacts differently with Cpt. The higher the Ag species, the lower the Cpt release and vice versa.

3.3 Kinetics of drug release using Korsmeyer-Peppas model

The drug release profiles were examined using the Korsmeyer-Peppas model, expressed using the equation: $$R\%=k{t}^n$$ where R% is the drug percentage release at time (t), k and n are the kinetic rate constant and the release exponent, respectively. The obtained fitting parameters together with their 95 % confidence intervals and correlation coefficient (R²) are shown in Table 3. The drug release profiles were also examined using different models including zero order, first order and Higuchi model, expressed using the equations:

Zero order: $$C_t=C_{\infty }+kt$$ First order: $$\mathit{ln}(C_t/C_{\infty })=kt$$ Higuchi model: $$C_t/C_{\infty }=k{(t)}^{0.5}$$ where C_t is the cumulative amount of drug released at time t, C_∞ is the cumulative amount of drug released at infinite time. The drug release constant (k) and the correlation coefficient for the models are presented in Table S2.

For the different loadings of Ag on MSS support, the rate of drug release as observed from the release constant (k) is higher at low Ag loadings (1 wt% and 2 wt%). Also, the diffusion mechanism at all loadings followed the non-fickian diffusion mechanism (0.45 < n < 0.89) except at 6 wt% loading that is slightly less than 0.45 (n = 0.4473 < 0.45). Similarly, for the different loadings of Ag on different supports (SBA-16, Ti-ZSM-5, Halloysite, Mesosilicalite, MSS, and silicalite), both the release constant (k) and the release exponent (n) showed different values signifying that the rate of drug release and diffusion mechanism (fickian or non-fickian) depends on the nature of the support. All the models have correlation coefficient in the range of 0.75 < R2 < 1, depending on the drug formulations. However, among all the models, Korsmeyer-Peppas model still gave the best correlation coefficient for all the formulations. The regression correlation coefficient (R²) is high for the different loadings and different supports, implying that, the Korsmeyers-Peppas model appropriately describes the drug release kinetics.

3.4 Diffuse reflectance UV–Visible spectroscopy (DR-UV Vis)

DR-UV Vis is used to characterize the oxidation state of transition metals. In the present study, the coordination state of different weight percentages of Ag (1-6 wt% Ag) before and after Cpt functionalization was shown in Fig. 3(a-h). In the case of Ag/MSS nanoformulations, Ag⁺, Ag_n^δ+ (nano-sized clusters), and Ag⁰ species at 220 nm, 300 nm, and 400 nm were observed (Aldossary et al., 2022). After the introduction of Cpt, an increase in the absorption band towards the visible region occurred at 221, 268 nm and 400 nm, indicating the presence of tetrahedral, octahedral and polymeric Pt species (Gharibshahi and Saion, 2012, Jermy et al., 2018). In the case of 2, 4, and 6 wt% Ag loading, the intensity of the Ag⁰ band increases indicating the presence of Ag⁰ nanoparticles. Such bands are primarily attributed due to presence of free electrons giving rise to surface plasmon resonance. Conversely, the cisplatin peak intensity band in Cpt reduced with increasing Ag content exhibiting the presence of an effective cohabitation of Pt in Cpt with dominant Ag nanoparticles. The synergistic presence of Pt of Cpt and Ag nanoparticles indicates a dispersion and cohabitation of Pt and Ag nanoparticles on MSS. Fig. S3(a and b) represents the XPS analysis of elemental, binding energy and chemical state of 1Ag/MSS and 6Ag/MSS. The survey spectrum depicts the elements and atomic ratio of 1Ag/MSS and 6Ag/MSS. The high-resolution spectra were presented the energy scan for all elements. The C1s peak at 285 eV was composed of two peaks at binding energy of 284.8 eV and 286.7 eV which related to C = O as depicted in Fig. S3(a and b) (Avishek Roy et al., 2019). The O spectrum in both compositions showed only one peak O 1 s at 532.6 and 532.4 eV. The O 1 s peak shows a slight shift when increasing the loading of Ag due to Fermi level of oxide (Dalby et al., 2007). The fitted spectra of Si 2p showed deconvoluted of four peaks at 103.0, 103.4, 103.5 and 103.8 eV relevant to oxidized silicon which indicated to Si⁴⁺ (Hellen S. Santos et al., 2022). Furthermore, the peak Si 2p intensity increased when increasing the Ag loading. The Ag 3d peaks at 368.4 and 374.5 eV denoted to Ag 3d5/2 and Ag 3d3/2 respectively as seen in Fig. S3(a and b) which respect to the metallic silver. The Ag 3d3/2 deconvoluted to 374.1 and 374.8 eV (Nienke J. Firet et al., 2019).

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Fig. 3

DR-UV Visible spectra of 1-6 wt% Ag/MSS and 1-6 wt% Ag/Cpt containing monodispersed spherical silica (a) 1 wt% Ag/MSS, (b) 1 wt% Ag/MSS/Cpt (c) 2 wt% Ag/MSS, (d) 2 wt% Ag/MSS/Cpt, (e) 4 wt% Ag/MSS and (f) 4 wt% Ag/MSS/Cpt, (g) 6 wt% Ag/MSS and (h) 6 wt% Ag/MSS/Cpt.

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3.5 SEM-EDS analysis

The Ag and Cpt distributions of 1 wt% Ag/Cpt/MSS and 4 wt% Ag/Cpt/MSS samples were mapped using SEM-EDS analysis (Fig. 4a-i). The elemental mapping pattern shows the well-distributed Ag and Cpt species at 1 wt% loading over MSS (Fig. 4a-c). EDS spectra showed the presence of Si, Ag, and Pt of Cpt (Fig. 4d and e). The densification of both species was revealed as Ag loading increases with 4 wt%. Some agglomerations are observed which can be attributed to the cohabitation of Ag and Cpt species leading to some formation of large grains (Fig. 4f-i). SEM-EDX and zeta potential (Table S1) indicate the agglomeration of Ag-Cpt complex and heterogeneity signaling a unique role played by Ag at high loading over MSS. Li et al., (2005) has stated that in Pt-Ag/SiO₂ nanocomposite, co-existence of linear and bridged Pt species occurs. The presence of Ag on the Pt/SiO₂ catalyst changes the Pt surface structure. The Ag enrichment occurs at the catalyst surface and reduces the Pt concerted activities (ensembles). Further, the oxidation property of Pt is also altered with Ag addition. In our study, there exists a multispecies cohabitation and synergistic action between the content of Ag and Pt of Cpt (Figs. 2 and 4).

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Fig. 4

SEM-EDS analysis of (a) 1Ag/MSS/Cpt, (b and c) elemental mapping of elements Si, Ag, and Cpt, (d and e) EDS spectrum of 1Ag/MSS and 1Ag/MSS/Cpt. SEM-EDS analysis of (f) 4Ag/MSS/Cpt, (g and h) elemental mapping of elements Si, Ag, and Cpt, (i) EDS spectrum of 4Ag/MSS/Cpt.

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3.6 High-resolution transmission electron microscopy (HR-TEM)

HR-TEM was used to investigate the effect of 1 and 6 wt% Ag loadings and Pt of Cpt dispersion on MSS. In the case of 1 wt% Ag/MSS/Cpt sample, the monodispersed spherical silica form is retained with a uniform particle size of about 80 nm without any distinct Ag and Cpt nanoparticles on MSS. (Fig. 5a). However, the morphological analysis of 6 wt%Ag/MSS/Cpt at the microscopic level clearly shows the large Ag particles with an average size of about 8 nm was dispersed on MSS (Fig. S1). Interestingly, Pt nanoparticles of Cpt with a particle size of about 3.3 nm were observed as nanosized aggregation around large Ag particles on MSS (Fig. 5b-d). The variation in the Pt particle distributions with increasing Ag content from 1 to 6 wt% clearly shows the cohabitation effect of Ag on Pt species. Moreover, the lattice fringes of Ag and Pt were processed using HRTEM (Fig. 5d and Fig. S4). The larger Ag nanoparticles were confirmed with the lattice d-spacing of 0.24 nm corresponding to the (1 1 1) plane of face-centered cubic crystal structure (Fig. 5d). The smaller Cpt nanoparticles illustrate the d-spacing of 0.22 nm. In Addition, Ag also displayed an inter-fringe distance of 0.22 nm (Fig.S4). The presence of such narrow inter-fringe distance of Ag indicates an effective alloying formation with adjacent lattice plane of bimetallic nanoparticles (Wang et al., 2011).

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Fig. 5

HR-TEM images of (a) 1 wt%Ag/MSS/Cpt and (b-d) 6 wt% Ag/MSS/Cpt at magnification scale of 50, 10 and 5 nm, respectively.

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3.7 Anticancer activity

MTT assay was used to evaluate the cell viability. The influence of different weight percentage Ag loadings on MSS at constant Cpt functionalization over cancer cells such as HeLa and HCT-116 and normal cells embryonic kidney cells (HEK-293) were studied (Figs. 6 and 7). The normal cells were used to evaluate the cytotoxic effect of nanoformulations at the same time HeLa and HCT-116 were used to evaluate the anticancer effect. The impact of nanoformulations on both colon cancer (HCT-116) and cervical cancer (HeLa) cells was examined. The cell viability assay confirmed a significant decrease in the HCT-116 cell viability after the treatments of nanoformulations, except 6 wt% Ag/MSS, where the decrease was not significant (Fig. 6). The cell viability assay confirmed a significant decrease in the HeLa cell viability after the treatments of nanoformulations except Ag-Silicalite, where the decrease was not significant (Fig. 7).

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Fig. 6

Cell viability by using MTT Assay: It shows the impact of treatment of nanoparticles on HCT-116 cells post 48-h treatment. * p < 0.05; ** p < 0.001.

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Fig. 7

Cell viability by using MTT Assay: It shows the impact of treatment of nanoparticles on HeLa cells post 48 h treatment. * p < 0.05; ** p < 0.001.

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We have also calculated the inhibitory concentration (IC₅₀) of nanoformulations on both HCT-116 and HeLa cells. The IC50 values on HCT-116 cells were MSS (23 µg/mL), 1 wt% Ag/MSS (15 µg/mL), 2 wt% Ag/MSS (21 µg/mL), 4 wt% Ag/MSS (26 µg/mL), 6 wt% Ag/MSS (26 µg/mL), and Cpt (13 µg/mL). Whereas on HeLa cells, IC₅₀ were MSS (24 µg/mL), 1 wt% Ag/MSS (13 µg/mL), 2 wt% Ag/MSS (14 µg/mL), 4 wt% Ag/MSS (14 µg/mL), 6 wt% Ag/MSS (15 µg/mL), and Cpt (13 µg/mL). We have also tested the impact of nanoformulations on non-cancerous cells (HEK-293), the average cell viability was 63 % post-treatments (Fig. S2). The cytotoxic impact of NPs post 48 h of treatment was observed and it was found that the average cell viability for Hela cells was 23.22 %, HCT-116 cells was of 43.48 %, and for normal HEK-293 cells, it was 62.00 %. This is the first study demonstrating the cell viability of synthesized nanoformulations against HCT-116 and HeLa cells. We have previously reported the impact of different molecules (nanomaterials and plant extracts) on colon and breast cancer cells (Rehman et al., 2020a). Metal based drug formulation has shown to bypass the drug resistance mechanism involving drug efflux pumps, enzyme (glutathione S-transferases), protein (multidrug resistance-associated protein 1), dysfunction in programmed cell death (apoptosis), alteration in extracellular matrix mechanics, and over expression of hypoxia related protein (hypoxia-inducible factor-1α) (Alavi et al., 2022). AgNPs has been reported to assist in reducing the cancer tissue spread by inhibiting the endothelial cell-specific mitogen (vascular endothelial growth factor) Delivering topoisomerase inhibitor (camptothecin) along with AgNPs is shown to surpass the drug efflux pumps and facilitate apoptosis pathways (Zhan et al., 2017). However, the targeted cellular interaction determines the effectivity of nanoformulations. Based on these observations, we may suggest that synthesized nanoformulations possess an inhibitory effect on HCT-116 and HeLa cells than HEK-293 cells.

3.7.1

3.7.1 Apoptotic cancer cell death

The treatment impact of nanoparticles on HCT-116 cells was studied by staining with DAPI (fluorescent dye) post 48-h treatment. The control cells did not show any inhibitory action on the colon cancer cells (Fig. 8A). However, the treatment of nanoformulations caused a significant decrease in the number of colon cancer cells, as the number of DAPI stained cells was found to be significantly less in the nanoformulations treated cells as compared to control cells (Fig. 8B-H). The decrease in the cancer cells is due to cell death which is due to programmed cell death or apoptosis. In the case of Cpt, a significant number of cancer cell deaths can be observed due to (25 µg/mL) treatment.

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Fig. 8

A-H: Nuclear morphology of HCT 116 cells after stained with DAPI post 48 h treatment. (A) Control cell, (B) (MSS), (C) (1 wt% Ag/MSS), (D) (2 wt% Ag/MSS), (E) (4 wt% Ag/MSS), (F) (6 wt% Ag/MSS), (G) (Ag-silicalite), and (H) (Arrows show the cell membrane disruption, nuclear condensation and fragmentation.

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The cubic structured SBA-16, TiZSM-5, mesosilicalite, and halloysite showed higher Cpt release than 4 wt% MSS sample. Interestingly, the cell viability study using MTT assay on HCT-116 reflected the drug release pattern, where cell viability decreases in the following pattern 1 wt% Ag/MSS > 2 wt% Ag/MSS > 4 wt% Ag/MSS > 6 wt% Ag/MSS. Ag-silicalite, where Ag is incorporated into the silicalite framework, showed less impact on HCT-116 and HeLa cells. In the case of HeLa cells, 1-6 wt% Ag-loaded MSS nanoformulations were found to be more sensitive with less cell viability percentage and apparent morphological damage which could be owed to cell membrane disruption, nuclear condensation, and fragmentation. Ag nanoparticles tend to inhibit cell viability due to oxidative stress. Ag nanoparticles induced cycle cell arrest and apoptosis to cause cancer cell death in bladder cancer cells (Zhao et al., 2018). Ag nanoparticles conjugated with anti-cancer drug induced apoptosis as confirmed by nuclear condensation, DNA fragmentation, and activation of caspase-3 in A549 cells (Zou et al., 2020). In addition, Ag nanoparticles induced apoptosis in the human hepatocellular carcinoma (HepG2) cell line (Ahmadian et al., 2018). Treatment with normal cells (HEK-293) showed a less cytotoxic effect, reflecting the nontoxic effect of MSS and nanoformulations, irrespective of Ag loadings (Fig. S2). SiO₂ alone shown to exhibit a cytotoxic effect on cancer cells such as hepatocellular carcinoma cells (HCC) and MCF-7 cells. For instance, SiO₂ has been reported to induce apoptosis through p53 and Bax activation mediated by oxidative stress (Niu et al., 2019). The cancer cells with leaky vasculature leads to high silica uptake than normal cells causing subsequent toxic effect to cancer cells (Lin et al., 2006). MSS composed of such SiO₂ has shown to have a similar effect of cancer cells (HCT-116 and HeLa) and less effect on normal cells (HEK-293). Further study with DAPI showed a high cell apoptotic effect with Cpt loading on increased Ag-loaded MSS nanoformulations.

3.8 Antibacterial activity

To demonstrate the antibacterial activity of the nanomaterial, bacterial strains P. aeruginosa and S. aureus were used. The synthesized nanomaterial depicted the appreciable bactericidal activity with both organism except silica with no inhibitory action. However, P. aeruginosa was seen to be slightly more susceptible compared to S. aureus. The minimum concentration of nanomaterial for P. aeruginosa and S. aureus is presented in Fig. 9 (A and B). The treatments of nanomaterial showed dose-dependent inhibitory action on the bacterial cells. It was observed that the mono dispersed spherical silica with different percentages Ag (1, 2, 4, and 6 wt%) exhibited varying impacts on the bacterial cells. Growth of both the bacteria gram positive and gram negative were inhibited with the increasing percentage of Ag in MSS (Fig. 9 A' and B').

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Fig. 9

(A and B). Graph presenting the MIC and MBC values for the Ag/MSS nanomaterials. Fig. 9 (A' and B'). Agar plates showing the MIC and MBC of the synthesized nanomaterial. A': P. aeruginosa, B': S.aureus. i) MSS, ii) AgNO₃, iii) 1 wt% Ag/MSS, iv) 2 wt% Ag/MSS, v) 4 wt% Ag/MSS, vi) 6 wt% Ag/MSS [1: 8 mg/mL, 2: 4 mg/mL, 3: 2 mg/mL, 4: 1 mg/mL, 5: 0.5 mg/mL, 6: 0.25 mg/mL, c: control; For AgNO₃ i.e., (ii): 1: 0.125 mg/mL, 2: 0.0625 mg/mL, 3: 0.031 mg/mL, 4: 0.155 mg/mL, 5: 0.0075 mg/mL, 6: 0.00387 mg/mL, c: control]. P. aeruginosa growth inhibition MIC (mean = 1.917917, SD = 3.005982), MBC (mean = 2.2525, SD = 2.926011). S. aureus growth inhibition MIC (mean = 1.835833, SD = 3.042319), MBC (mean = 2.260833, SD = 2.918425).

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The antibacterial activity of the Ag/MSS nanocomposites with varying Ag loading was evaluated to obtain the mass ratio of Ag NPs. The 6wt%/Ag loading percentage showed the best efficacy with both organisms, which could be due to the dispersion of Ag. The inadequate amount of Ag with only 1 wt%Ag loading showed the least efficacy. The bactericidal action of pure AgNO₃, MSS against P.aeruginosa, and S.aureus. has been shown in Fig. 9. The MIC/ MBC of all the ratios and AgNO₃ showed effectiveness, and the pristine silica showed no antibacterial activity. The bacterial inhibition was regulated by Ag, most significantly, Ag/MSS nanomaterial indicated an increased antibacterial activity and stability which could be attributed to the Ag introduced. Using one-way ANOVA, the variation in the antibacterial, using varying concentration Ag loading were found significant (P < 0.001).

This antibacterial nanocomposite attached to the bacterial cell wall and penetrated into the periplasmic space of bacteria cell, generating reactive oxygen species (ROS) that leads to cellular disorganization. Most studies suggested that nanoparticles significantly attach to the cellular surfaces and produce increased levels of ROS, mainly hydroxyl radicals during the interaction of electrons and water under visible light (Shu et al., 2017). AgNPs are known to possess antimicrobial activity than many antibiotics like vancomycin or gentamicin against pathogens MRSA and P. aeruginosa (Mateo and Jiménez, 2022). A significant action of AgNPs was reported against a number of MDR like E. coli, P. aeruginosa, ampicillin-resistant, and Streptococcus pyogenes (Lara et al., 2010). In the current study, the formulated nanocomposites could have influenced the dispersion and release of Ag and facilitated an efficient contact with the bacterial membrane. The higher dispersion of the nanomaterial could also enhance the surface area, making more ROS production from active sites. It has been shown that Ag/SiO₂ composite can release Ag⁺ ions that interact with bacterial cell structure and damage bacterial membrane (Mosselhy et al., 2017). Ag NPs anchored with monodispersed silica promote the damage caused to the cellular surfaces, which could significantly increase the antibacterial and anticancer activity. The current study is crucial for determining the bioactivity mode of operated Ag-monodispersed silica nanocomposites.

In the case of Pt-Ag/SiO₂ nanocomposite, the co-existence of linear and bridged Pt species occurs. The presence of Ag on Pt/SiO₂ catalyst changes the Pt surface structure. Ag enrichment occurs at the catalyst surface and reduces the Pt concerted activities (ensembles). Further, the oxidation property of Pt is also altered with Ag addition (Li et al., 2005). The presence of Pt or Cu with Ag enhances the dissolution of metal species (Ag or Cu), and facilitates a sufficient ion release with improved antibacterial activity (Meister et al., 2022). In our study, there exists a multispecies cohabitation and synergistic action between the content of Ag and Pt of Cpt (Figs. 2 and 4). The electronic transition between Pt surface sites in Pt/bimetallic system is influenced by the inter-atomic distance, coordination and dispersion state (Stamenkovic et al., 2006). Non-alloy metals such as Pt and Ag interactions exhibit a synergistic effect due to their reduction and activation properties. The presence of oxygen functional moieties in the support has the capability to reduce the Pt and Ag salts and increase the bimetal interactions, while the presence of chlorine ions of Pt also increases the mobility of Ag species and interactions with Pt (Chen et al., 2012, Oh et al., 2002). Therefore, the presence of hydroxyl groups of silica could initiate simultaneous reduction and increase the Pt of Cpt with Ag interactions. Moreover, an increase in the Ag content could further increase the bimetal interaction and subsequently control the Cpt release. The Cpt release profile (Fig. 2) and Table 2 clearly show such interactive and release abilities between Ag and Cpt. This evidence indicates that different amounts of Ag interact differently with Pt of Cpt. The higher the Ag species, the lower the Cpt release vice versa. In the case of Ag/MSS functionalized with anticancer Pt-based drug such as cisplatin, a controlled release can be beneficial for overcoming drug resistance, facilitating apoptosis pathways, and DNA damage to cancer cells. Antibacterial activity of metal-based nanoformulations is primarily attributed to the ability of release abilities of metal ions that interact with bacterial membrane. The AgNPs and nanocomposite particle shape and size determine the antibacterial activity. AgNPs with particle size ranging between 5 and 10 nm has been reported to induce cell membrane disruption and disintegration of bacterial chromosome. In the present study, Ag impregnated on monodispersed spherical silica able to control the Cpt release, exhibits better antibacterial and anticancer activity (Scheme 1).

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Scheme 1

Controlling the cisplatin release by synergistic action of silver and cisplatin for targeted cancer therapy.

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