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Synergistic impact of silver on anticancer potential of doxorubicin-loaded chitosan-coated amino-functionalized silica nanoparticles
*Corresponding author: E-mail address: nbedowr@taibahu.edu.sa (N. Bedowr)
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Received: ,
Accepted: ,
Abstract
This study investigates the synergistic effect of silver on the anticancer activity of doxorubicin (DOX) encapsulated in silver-integrated amino-functionalized silica nanoparticles (AFS-NPs) coated with chitosan (DOX@AFS-NPs-Ag-CS). AFS-NPs were synthesized by a co-condensation approach using tetraethyl orthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES) with cetyltrimethylammonium chloride (CTAC) as a template, followed by silver incorporation through silver nitrate (AgNO3) treatment in dark conditions. Doxorubicin was subsequently loaded and stabilized by chitosan (CS) coating. Morphological analysis by scanning electron microscopy (SEM) confirmed spherical, uniform nanoparticles (∼900nm), while X-ray diffraction (XRD), Fourier transform infrared (FTIR), UV-Visible spectrophotometry, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) verified successful synthesis and drug encapsulation. Thermal stability enabled the physical encapsulation of DOX and conferred suitable thermal stability to the formulation for anticancer studies. The formulation showed a drug loading content (LC) of 17% and an entrapment efficiency (EE) of 84%. Release studies revealed pH-responsive release of DOX, achieving ∼94.2% in 24 h under acidic conditions. Importantly, DOX@AFS-NPs-Ag-CS demonstrated significant anticancer activity (60.18% inhibition) against Hep G2 cancer cell lines. These results highlight the potential of this hybrid nanocarrier for effective and targeted cancer therapy.
Keywords
Cancer nanomedicine
Hybrid nanosystem
Liver cancer (HepG2)
pH-responsive drug release
Targeted chemotherapy
1. Introduction
Cancer is a leading cause of morbidity and mortality worldwide. About 8.8 million deaths are annually reported due to cancer, and this places a heavy burden on healthcare systems [1]. Radiotherapy and chemotherapy remain a mainstay of cancer treatment, but conventional chemotherapeutic agents suffer from poor selectivity, systemic toxicity, and the development of multidrug resistance (MDR), which collectively limit therapeutic outcomes and patient quality of life. Doxorubicin (DOX) is one of the most widely used anthracycline chemotherapeutics; however, its clinical utility is constrained by rapid systemic distribution and clearance, low tumor selectivity, and severe dose-limiting toxicities, most notably cardiotoxicity. Furthermore, active drug efflux mediated by P-glycoprotein (PgP) and other resistance mechanisms in tumor cells often necessitate higher DOX doses, aggravating systemic side effects and reducing long-term efficacy.
Nanotechnology-driven drug delivery systems (DDSs) have the potential to overcome these limitations by improving drug solubility, prolonging circulation time, and enabling targeted, controlled, and stimuli-responsive release at tumor sites. Among nanocarriers, silica-based nanoparticles are especially attractive due to their structural versatility: tunable pore size and volume, high surface area, ease of surface functionalization, and favorable biocompatibility [2].
Mesoporous silica nanoparticles (MSNs) and organically modified silica platforms have demonstrated notable advantages in encapsulating chemotherapeutics, protecting drugs from premature degradation, and providing controlled release profiles appropriate for tumor microenvironments. Recent studies on DOX-loaded silica nanoassemblies provide compelling evidence that silica-based carriers can enhance drug loading, achieve pH-responsive release, and improve in vitro and in vivo antitumor efficacy [3,4].
Surface functionalization is a key strategy to optimize silica carriers for DOX delivery. The incorporation of amino functional groups via silane coupling agents not only ensures stable morphology but also increases affinity for DOX through electrostatic interactions and hydrogen bonding, thereby achieving high drug loading capacity. Critically, protonation of amine groups under acidic conditions typical of tumor interstitium (pH ∼5-6) enables pH-triggered release, which promotes preferential drug liberation at the tumor site while limiting systemic release. To further enhance performance, polymeric coatings like chitosan (CS) offer a secondary protective matrix: CS coatings reduce premature leakage, improve colloidal stability, provide mucoadhesive/biocompatible interfaces, and contribute to pH sensitivity through polymer swelling in acidic media [5,6].
Metallic nanoparticles prepared from transition metals have explicit pivotal biomedical applications due to their extraordinary structural properties [7]. Integration of metallic nanoparticles into silica-polymer hybrids provides an additional avenue to augment therapeutic activity. Silver nanoparticles (AgNPs), in particular, possess intrinsic antimicrobial and anticancer properties, mediated by the generation of reactive oxygen species, disruption of cellular membranes, and induction of apoptosis. They can synergize with conventional chemotherapeutics to potentiate cytotoxic effects and help overcome MDR pathways [8]. Combining AgNPs with amino-functionalized silica and a CS coating, therefore, creates a multifunctional platform that aims to (i) increase DOX loading and retention, (ii) enable dual-stage, pH-responsive release (silica-amine + CS), and (iii) enhance anticancer efficacy via AgNP-mediated synergy.
The primary challenge addressed in this study is how to deliver therapeutically effective concentrations of DOX to tumor cells while minimizing systemic exposure and circumventing resistance mechanisms. Specifically, the work targets three interrelated problems: (1) suboptimal DOX loading and premature release from carrier systems, (2) insufficient tumor-selective/pH-triggered release, and (3) limited therapeutic efficacy due to MDR and lack of synergistic modalities. To solve these problems, we developed a hybrid formulation, DOX@AFS-NPs-Ag-CS, composed of amino-functionalized silica nanoparticles (AFS-NPs) synthesized via co-condensation of TEOS and APTES), in situ silver integration, DOX loading, and CS coating. This design intentionally couples high-affinity drug binding (amine groups) with a CS secondary matrix for controlled diffusion and a silver component to provide complementary anticancer action.
Further, encapsulation of DOX-loaded AFS-NPs into CS can significantly enhance drug delivery efficiency by leveraging the combined benefits of silica nanoparticles, surface amino groups, and CS. CS has a tendency to develop a secondary protective matrix around SiNPs, reducing premature drug release. This formulation exhibits pH-sensitive secondary release from the CS matrix and slows down diffusion, leading to sustained and prolonged DOX release. Moreover, AFS-NPs release DOX faster in acidic conditions (tumor microenvironment, pH ∼5-6) due to protonation of amine groups, which may be slowed down due to swelling of CS in acidic pH, further facilitating DOX diffusion.
The hypothesize is that amino functionalization will increase DOX entrapment through favorable electrostatic/hydrogen bonding interactions, that protonation of amine groups and CS swelling will afford pH-responsive and sustained DOX release in acidic tumor conditions, and that AgNP incorporation will synergize with DOX to enhance cancer cell killing and potentially mitigate MDR. The current study evaluates physicochemical properties (size, morphology, surface chemistry), thermal stability, drug loading and entrapment efficiency, pH-dependent release kinetics, and in vitro anticancer activity (HepG2) to validate these design goals.
2. Materials and Methods
2.1. Materials
Hydrochloric acid (37%), sodium hydroxide, cetyltrimethyl ammonium chloride (CTAC) ≥98.0%, chitosan, acetic acid, phosphate-buffered saline (PBS), 3-aminopropyltriethoxysilane (APTES), ethanol (95%), and doxorubicin hydrochloride were purchased from Sigma Aldrich. All chemicals were of analytical grade and used as received.
2.2. Preparation of doxorobicin-loaded silver-amino functionalized silica nanoparticles coated with CS
2.2.1. Synthesis of AFS-NPs
AFS-NPs were synthesized by a previously reported method by T.M. Suzuki [9]. This involved a co-condensation process between TEOS and APTES under basic media (NaOH) using CTAC as surfactant and structure-directing agent. Measured quantities of TEOS (1.25 g) and APTES (0.08 g) were added to a 50% ethanolic solution containing surfactant. The contents of the reaction were stirred for 8 h at room temperature. Afterwards, the contents were allowed to gel for 24 h. The product was filtered, washed, and heated at 318 K for 72 h. CTAC was removed by heating the white powder in ethanol/HCl solution for 3 h. The template-free product was filtered, washed, and dried at 318 K. Moreover, ammonia solution was added to remove any residual chloride ions. Finally, pure AFS-NPs were collected by preparing a suspension in a methanol solution having 1 mL of ammonia (28%) for 8 h. The pure product was further dried at 423 K for 12 h in a vacuum.
2.2.2. Synthesis of AFS-NPs-Ag by direct reduction approach
Over 96 h, the aqueous solution of AgNO3 (30 ml of 1.0 mM) was brought into equilibrium with 0.3 g of AFS-NPs in darkness at a moderate temperature with agitation. The end product was then filtered and rinsed with some extra water. The resultant product (AFS-NPs-Ag) was vacuum-dried for 24 h [10].
2.2.3. Loading of doxorubicin hydrochloride on AFS-NPs-Ag
For this, 6 mL (1mg/mL) of doxorubicin hydrochloride was added to a 100 mL solution of 5 mg of AFS-NPs in distilled water and stirred for 24 h at room temperature. This mixture was centrifuged at 8000 rpm for 15 min at 25°C. The supernatant was collected in a beaker, and drug-loaded nanoparticles (DOX@AFS-NPs-Ag) were washed with distilled water to remove the unloaded drug. The residue was collected in the same beaker containing unloaded drug to measure the loading content (LC) and encapsulation efficiency (EE) of the drug using a UV-Vis spectrophotometer. The given equations (Eqs. 1-2 ) were used to evaluate these properties [11].
2.2.4. Preparation of CS coated formulation (DOX@AFS-NPs-Ag-CS)
The CS coating on DOX@AFS-NPs-Ag was conducted according to a previously reported method [12]. The drug-loaded DOX@AFS-NPs-Ag were mixed in 50 mL of ethanol, and the pH of the mixture was maintained at 3.5 using acetic acid solution. Then, 1.6 g of APTES was quickly added and stirred at room temperature for 3 h. The 200 mL of 2% CS solution was added dropwise to the above mixture and stirred for 10 h at room temperature. Then this mixture was centrifuged at 1000 rpm for 10 min at 37°C. The supernatant was removed, and the CS coated nanoparticles (DOX@AFS-NPs-Ag-CS) were washed with distilled water three times and centrifuged repeatedly. The washed DOX@AFS-NPs-Ag-CS were oven-dried at 30°C for 60 h, and then finely ground well using a mortar pestle.
2.3. In-Vitro DOX release study
To study the drug release, DOX@AFS-NPs-Ag and DOX@AFS-NPs-Ag-CS were dispersed in PBS buffers of different pH values, which were 7.4 and 5.5, and these mixtures were poured into conical flasks. These DOX-loaded formulations containing conical flasks were fixed in the shaking water bath apparatus and heated at 37°C with stirring. Then, a 5mL sample of the mixtures was taken from the different mixtures after predetermined time intervals, and the same amounts of fresh PBS buffers were poured into them separately according to their pH values. The amounts of samples were used to measure the released concentration of DOX at different time intervals to evaluate the release behavior at different pH values, comparing to the normal physiological conditions (pH=7.4) and cancer cell acidic conditions (pH=5.5). The concentration of released DOX was determined by UV-visible spectrophotometry at a maximum wavelength of 485 [13].
2.4. Anticancer activity: MTT assay
To study the anticancer MTT assay, a seeding density of 2 × 104 HepG2 and HEK-293 cells was cultured in separate wells of a 96-well plate. Freshly seeded cells were further incubated for 24 h at 37°C in CO2 (5%). Afterwards, each sample (3 μL) was added to all wells and allowed to incubate for 48 h. After the completion of incubation, 10 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to wells, and plates were incubated for more 4 h. Moreover, dimethyl sulfoxide (150 μL) was also immersed, and the study was conducted at 30-min intervals. Absorbance measurements at 630 nm were recorded for an enzyme-linked immunosorbent assay (ELISA) microplate reader to ascertain cell death. Statistical calculations were made on MS Excel 2016 and GraphPad Prism, and the inhibition ratio was computed by following (Eq. 3) [14].
Where As = Absorbance of treated, Ac = Absorbance of untreated.
2.5. Instrumentation
Fourier transform infrared (FTIR) analysis was performed on a Shimadzu QP2010 instrument in the scanning range 400-4000 cm-1 at a resolution of 4 cm-1. Siemens D5000 diffractometer with Cu-K alpha radiation (λ = 1.54060Å) was employed for XRD analysis at 2θ at room temperature. A SEM Jeol (JSM 6390LA), was employed to study surface morphology operated at 10 kV. Malvern, Zetasizer Nano ZSP was employed to determine size and zeta potential. The samples were dispersed in ethyl alcohol at 25°C and placed in disposable zeta cells. The measurement point was 2.00 mm, with a count rate was 221.3 kcps. The attenuator was 3, and there were 12 zeta runs. The thermogravimetric analysis/ differential scanning calorimetry (TGA /DSC) was performed on SDT Q-600 in a nitrogen environment at a rate of 20 mL/min and heating rate of 10°C/min in a temperature range from 20°C to 600°C. The drug loading and release studied were performed on a UV-Vis spectrophotometer (Azzota SM1200 UV-VIS spectrometer).
3. Results and Discussion
The schematic presentation for the synthesis of DOX@AFS-NPs-Ag-CS has been illustrated in Scheme 1. AFS-NPs were prepared by the sol-gel approach under basic media using CTAC as a template. Afterwards, silver (Ag) nanoparticles were coordinated with AFS-NPs by reduction of silver nitrate, followed by loading DOX. In continuous, CS coated formulation was developed by the interaction of CS at the free amino groups and the hydroxyl group of DOX.
- Stepwise synthesis of chitosan-coated, amino-functionalized silica nanoparticles decorated with silver and loaded with doxorubicin (DOX@AFS-NPs-Ag-CS).
SEM analysis was performed to examine the surface morphology of AFS-NPs, CS, and DOX@AFS-NPs-Ag-CS, as presented in Figure 1. SEM images revealed that AFS-NPs were spherical, smooth, uniform, and monodispersed with an average particle size of ∼900 nm. The micrograph of CS showed a flaky, fibrous, rough, and porous structure, consistent with its natural polymeric nature. These interconnected pores of CS are favorable for enhanced absorption and interactions with other materials.
- SEM images of (a) AFS-NPs, (b) CS, (c) DOX@AFS-NPs-Ag-CS, and (d) zeta potential comparison of AFS-NPs and DOX@AFS-NPs-Ag-CS, confirming morphology and surface modification.
The SEM analysis of DOX@AFS-NPs-Ag-CS indicated a spherical shape with a slightly rough and aggregated surface, confirming the successful coating of CS and loading of DOX onto the AFS-NPs surface. Some degree of agglomeration was observed, which may be attributed to electrostatic interactions between the drug and the carriers. Importantly, the morphology of AFS-NPs was not significantly altered after DOX loading and CS coating, demonstrating that drug encapsulation was efficient while maintaining nanoparticle integrity.
Zeta potential analysis was carried out to evaluate formulation stability. Zeta potential values, whether positive or negative, are considered good indicators of nanoparticle stability. However, formulations with dual charges are generally less stable due to intraparticle charge interactions, which can hinder permeability in biological systems. In this study, AFS-NPs exhibited a zeta potential of +18 mV, attributed to protonation of surface –NH2 groups. For DOX@AFS-NPs-Ag-CS, the zeta potential decreased to +12 mV. This reduction can be explained by the amphoteric nature of DOX, which carries both positive and negative functional groups, leading to partial neutralization of surface charges. Silver NPs did not significantly affect the potential unless present in excess and deposited on the surface.
Notably, CS, being a cationic polymer, helped to maintain a positive zeta potential. Thus, DOX@AFS-NPs-Ag-CS demonstrated moderate stability, ensuring favorable interactions with negatively charged cancer cell membranes and enhancing cellular uptake, while avoiding the excessive cytotoxicity associated with highly positive zeta potential values. The optimized formulation is therefore suitable for effective and controlled DOX release under both physiological and cancer-specific pH conditions.
XRD analysis of AFS-NPs, drug (DOX), CS, and formulation has been presented in Figure 2. XRD is an expedient tool to investigate the degree of crystallinity of the drug after entrapment in the formulation. The characteristic broad peak at 21.66 o confirmed the amorphous nature of AFS-NPs. Crystalline nature of DOX was confirmed due to sharp peaks in the XRD spectrum at 12.5°, 15.66°, 20.5°, 25.27°, and 27.2o [15]. The amorphous nature of CS was verified from two peaks observed at 9.8° and 20.18° [16]. In the case of the final formulation, the XRD spectrum of DOX@AFS-NPs-Ag-CS exhibited all prominent DOX peaks, albeit with slight shifts in position and reduced intensity. The observed peak shift reflects reduced crystallinity and the presence of interfacial interactions among DOX, AFS-NPs, silver, and CS. This partial loss in crystallinity is advantageous, as it indicates physical entrapment of DOX in the hybrid matrix, which facilitates controlled and sustained drug release. Moreover, such structural changes contribute to better formulation stability while enabling pH-responsive release kinetics, where faster release occurs under acidic tumor-like conditions compared to physiological pH.
- XRD spectra of (a) AFS-NPs, (b) DOX, (c) CS, and (d) DOX@AFS-NPs-Ag-CS showing amorphous silica, crystalline DOX, semi-crystalline CS, and successful hybrid integration.
Figure 3 represents the FTIR spectra of AFS-NPs, DOX, CS, and DOX@AFS-NPs-Ag-CS. The AFS-NPs exhibited a peak at 3323 cm-1 due to the overlapping of OH and –NH2 groups. Two peaks at 2959 and 2898 cm-1 were assigned to the methylene group of the propyl chain. A strong peaks at 1026 cm-1 correspond to Si–O–Si asymmetric stretching vibration [17,18]. DOX exhibited an intense stretching band at 3488 cm-1 due to N-H Stretching, and a broad band at 3286 cm-1 was assigned to O-H stretching. The carbonyl group (C=O) of quinone groups demonstrated a peak at 1735 cm⁻1. Stretching peaks due to the C-O of the ether appeared at 1030 cm⁻1. In the IR spectrum of CS, a multiple stretching frequency appeared at 3338 cm-1 was related to the OH and NH2 groups of CS. The peak at 2856 cm-1 was related to the C-H stretching vibrations of the alkyl groups. A band observed at 1583 cm− 1 was assigned to the amide II peak’s N–H in-plane bending. The peak at 1363 cm-1 was related to C-H bending vibrations of CS. An intense peak at 1042 cm− 1 was due to primary hydroxyl groups’ C–O stretching vibration [19,20]. FTIR spectrum of DOX@AFS-NPs-Ag-CS exhibited all the major peaks of functional groups present in silica nanoparticles, DOX, and CS with slight shifting in intensity and position of stretching and bending frequencies. Asymmetric stretching frequency at 1067 cm-1 due to the Si–O–Si group illustrates that the backbone of the formulation is composed of silica nanoparticles. Moreover, two peaks at 672 and 512 cm-1 ensured the presence of silver (Ag) nanoparticles in the formulation. Presence of peaks related to DOX in the IR spectrum of the formulation is evident of the physical entrapment of the drug in the formulation [21-25]. The observed peak shifts can be attributed to hydrogen bonding and electrostatic interactions between the amino groups of CS, hydroxyl/carbonyl groups of DOX, and silanol/amino groups of AFS-NPs. These interactions confirm successful drug–polymer–nanoparticle integration, which contributes to enhanced stability and controlled release behavior of the formulation.
- FT-IR spectra of AFS-NPs, DOX, CS, and DOX@AFS-NPs-Ag-CS confirming amino-functionalization, DOX loading, and CS coating.
TGA was employed to analyze thermal stability and the amount of DOX encapsulated in the formulation, as demonstrated in Figure 4. TGA curve of AFS exhibited initial weight loss of 4.5% from 25-100°C, related to evaporation of adsorbed solvent molecules. The second weight loss (13%) observed at 175-250°C was attributed to decomposition of amino propyl groups followed by rapid degradation (15.6 wt%) [26]. The thermogram of DOX revealed two stages of weight loss. The initial step with a 30.4% mass loss happened from 200 to 270°C, attributed to the removal of HCl. The second step, with a loss of 9.5% was observed between 272 and 388°C, related to the loss of the initial mass of DOX [27]. Thermal analysis of pure CS exhibited an initial weight loss of 8.95% from 25-130°C due to the removal of moisture. Further, the onset of decomposition of pure CS initiated at 263°C, and it was completely decomposed at about 475°C with a weight loss of about 51.37% [24]. TGA of DOX@AFS-NPs-Ag-CS presented four weight loss stages. The first stage of weight loss (12%) exhibited from 25-120°C was linked to evaporation of water and residual solvents. The second weight loss of 14% from 225-340°C was linked to thermal decomposition of the backbone of CS. The third weight loss of 18% from 350-465°C was due to thermal decomposition of DOX. The last weight loss of 15% from 470 to 540 was attributed to the degradation of amino-functionalized groups.
- (a) TGA and (b) DSC curves of AFS-NPs, DOX, CS, and DOX@AFS-NPs-Ag-CS demonstrating thermal stability and composition of the hybrid system.
The DSC curve of AFS-NPs exhibited an endothermic Peak (∼100-150°C) corresponding to the removal of solvent. A broad endothermic transition (∼200-300°C) indicated the degradation of aminopropyl (-NH₂) groups. An exothermic peak (∼350–450°C) represented the thermal degradation of aminopropyl groups. The DSC profile confirmed successful development of AFS-NPs is suitable for drug delivery applications. The DSC curve of pure CS exhibited two broad endothermic peaks at 92°C and 212°C, possibly due to evaporation and molecular degradation of the CS backbone. From the DSC of DOX, it was established that the decomposition temperature matches the DOX melting peak at 220°C, as observed by TGA. DSC thermogram of DOX@AFS-NPs-Ag-CS displayed characteristic endothermic peaks at 85°C linked to the evaporation of residual solvents. A broad peak at less than the melting point of DOX was observed, which evidences the presence of DOX in the formulation with reduced crystallinity. A broad peak around 285°C corresponds to the thermal degradation of formulation [28].
3.1. In Vitro drug release
The loading efficiency of DOX was measured through a UV-visible spectrophotometer using 485 nm as the λmax value of the DOX. The LC% was 17% and EE% was 84% for the CS coated formulation (DOX@AFS-NPs-Ag-CS).
Drug release profile of DOX was investigated at 5.5 and pH 7.4 from DOX@AFS-NPs-Ag and DOX@AFS-NPs-Ag-CS, as presented in Figure 5. At pH 7.4, DOX exhibited a faster release from DOX@AFS-NPs-Ag as compared to DOX@AFS-NPs-Ag-CS. DOX@AFS-NPs-Ag exhibited 9.4% of DOX was released after 1 h, reaching 30.9% at 6 h, and achieved 60.8% cumulative release within 24 h. DOX@AFS-NPs-Ag-CS demonstrated 6.2%, 27.4% and 50.4%. This higher drug release from nanoformulation as compared to CS coated formulation is attributed to reduced solubility of CS at neutral pH. CS established strong interactions with DOX in a neutral environment, possibly due to intercalation of cationic amino groups (-NH₂) of CS with the negatively charged DOX functional groups. Moreover, CS exhibited less swelling and slow diffusion and leading to sustained drug release. On the contrary, DOX@AFS-NPs-Ag lacks a polymer barrier and hence paves a way for faster diffusion, leading to a faster release of DOX.
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In vitro release profiles of DOX@AFS-NPs-Ag and DOX@AFS-NPs-Ag-CS at pH 7.4 and 5.4, showing pH-responsive and sustained release with CS coating.
In contrast, at pH 5.5, the release profile was significantly increased due to the protonation of amino groups in AFS-NPs and functional groups of CS. DOX@AFSi-NPs-CS demonstrated faster release, reaching 94.2% in 24 h, compared to 85.5% exhibited by DOX@AFSi-NPs. At pH 5.5, protonation of amino (-NH2) groups weakens the drug-nanoparticle interactions, leading to a faster release. Further, in the final formulation (DOX@AFSi-NPs-CS), CS, being a pH-responsive biopolymer, got protonated and exhibited enhanced solubility in an acidic environment. CS got swelling, and the diffusion of DOX from the formulation became faster. Moreover, repulsive forces came into operation between CS and DOX, enhancing drug diffusion out of the formulation, resulting in more release.
The outcomes established the potential of DOX@AFSi-NPs-Ag-CS as an effective pH-sensitive drug carrier for tumor-targeted delivery. Under an acidic environment (pH=5.5), the developed formulations presented an efficient drug unloading in tumor environments, making the most promising formulation for sustained drug delivery suitable for application in the tumor microenvironment, while sustained release at pH 7.4 minimizes premature drug leakage.
These findings are consistent with recent reports emphasizing the importance of pH-responsive polymeric coatings in achieving site-specific drug delivery. For instance, a recent study demonstrated that silica-based nanocarriers functionalized with pH-responsive polymers showed sustained release at neutral pH and accelerated release under acidic conditions due to protonation effects [29]. Similarly, a Nature Communications report highlighted that protonation-driven repulsive forces in polymer-drug systems significantly enhance therapeutic release efficiency in acidic tumor microenvironments [30]. Moreover, recent cancer nanomedicine research underscores that CS-based nanocarriers offer dual benefits of biocompatibility and pH-responsive release, making them particularly effective in targeted cancer therapy [31]. The observed performance of DOX@AFS-NPs-Ag-CS in our study aligns with these findings, reinforcing its potential as a tumor-targeted drug delivery platform.
The data obtained from drug release profiles were evaluated for kinetic modeling, as shown in Table 1. It was established that DOX@AFS-NPs-Ag (pH 5.5) showed the highest Higuchi constant (kh = 22.20) and Korsmeyer–Peppas kP = 24.13, indicating a higher release rate. The release data exhibited higher correlation coefficients (R2) for the Higuchi and Korsmeyer-Peppas models compared to zero-order and first-order models, suggesting that the release mechanism was predominantly diffusion-controlled. Moreover, the Korsmeyer-Peppas values (∼0.5) indicated a non-Fickian diffusion mechanism, further confirming the enhanced DOX release at pH 5.5.
| Formulation | pH | Zero-order k0 | R2 (Zero) | First-order K1 | R2 (First) | Higuchi kh | R2 (Higuchi) | Korsmeyer-Peppas kp | Korsmeyer-Peppas (n) | R2 (KP) |
|---|---|---|---|---|---|---|---|---|---|---|
| DOX@AFS-NPs-Ag | 7.4 | 3.645 | 0.956 | 0.0532 | 0.982 | 18.51 | 0.993 | 16.51 | 0.547 | 0.991 |
| DOX@AFS-NPs-Ag-CS | 7.4 | 2.146 | 0.943 | 0.0509 | 0.979 | 11.30 | 0.987 | 10.82 | 0.517 | 0.989 |
| DOX@AFS-NPs-Ag | 5.5 | 3.920 | 0.961 | 0.0471 | 0.985 | 22.20 | 0.996 | 24.13 | 0.466 | 0.994 |
| DOX@AFS-NPs-Ag-CS | 5.5 | 2.474 | 0.948 | 0.0530 | 0.981 | 12.86 | 0.991 | 11.97 | 0.529 | 0.992 |
3.2. Cytotoxicity studies
To analyze the synergic effect of silver on cytotoxicity of DOX, MTT assay of AFS-NPs, DOX@AFS-NPs, DOX@AFS-NPs-Ag, DOX@AFS-NPs-Ag-CS, and unloaded AFS -Ag and DOX@AFS-CS was performed, as shown in Figure 6. In this research, cells of the Hep G2 cell lines were treated with diverse concentrations (25, 50, and 100 ug/L) of all samples. The outcomes of the MTT assay demonstrated that AFS-NPs exhibited cytotoxic effects of 35.16%, 29.93%, and 20.44% at 100, 50, and 25 μg/mL, respectively. The DOX@AFS-NPs-Ag presented a synergic effect of Ag on the cell death of nanoformulation 44.32%, 33.45%, and 23.47% at previously described concentrations. This increase can be explained by the fact that AgNPs are known to induce reactive oxygen species generation, mitochondrial damage, and DNA fragmentation, thereby sensitizing cells to chemotherapeutic agents. Notably, the final formulation (DOX@AFS-NPs-Ag-CS) presented enhanced activity of 60.18%, 54.88%, and 43.45%. However, the formulation with DOX loading AFS-NPs-Ag-CS presented the lowest activities of 29.53%, 21.88%, and 15.72%. The remarkable cytotoxic performance of the developed formulation can be linked to constituents of the formulation. AFS-NPs increase cellular uptake, allowing for more efficient delivery of the DOX. Coating with CS polymers served as a protective environment for the nanoparticles, enhancing the stability and bioavailability of DOX. Moreover, the pH-responsive property of the formulation due to protonation of amino-functionalized silica and CS leads to degradation of the formulation and hence ensures improved and sustained release, leading to enhanced therapeutic efficacy of this novel formulation.
- MTT assay results of AFS-NPs, DOX@AFS-NPs, DOX@AFS-NPs-Ag, DOX@AFS-NPs-Ag-CS, and AFS-NPs-Ag-CS against HepG2 cells, highlighting synergistic cytotoxicity from silver and CS.
In particular, the superior efficacy of DOX@AFS-NPs-Ag-CS can be attributed to three synergistic factors: (i) amino-functionalized silica promoted high DOX loading and facilitated electrostatic interactions with cancer cells, (ii) CS coating improved stability and provided pH-responsive release in acidic tumor environments, and (iii) AgNPs offered intrinsic anticancer potential and further amplified DOX cytotoxicity, leading to a dual-action therapeutic effect. In contrast, the comparatively weak activity of DOX@AFS-CS underscores the critical contribution of silver integration to the overall therapeutic efficacy.
The novel formulation (DOX@AFS-NPs-Ag-CS) proved as a potent anticancer agent, showing significantly better outcomes compared to other tested formulations.
This formulation depicted the potential of combining silica nanoparticles with AgNPs and further coating with CS for improved cancer therapy. These outcomes are consistent with recent literature. In this study, the cytotoxicity of the developed formulation was specifically demonstrated in HepG2 cells. Comparable outcomes have also been reported in other cancer cell models: for example, DOX combined with AgNPs has shown enhanced activity in MCF-7 breast cancer cells and A549 lung cancer cells [32,33]. Likewise, polymer-coated hybrid nanocarriers have exhibited improved drug stability and pH-responsive release in HeLa cervical cancer cells and HT-29 colon carcinoma cells [34,35]. Overall, the cytotoxicity results validate the promising role of DOX@AFS-NPs-Ag-CS as a tumor-targeted delivery system, integrating the benefits of silica, silver, and CS for enhanced therapeutic efficacy.
3.3. Study limitations and future perspectives
While the present study demonstrates the promising potential of DOX@AFS-NPs-Ag-CS as a multifunctional anticancer nanoplatform, certain limitations should be acknowledged. First, the cytotoxicity evaluation was restricted to in vitro HepG2 cell models, which cannot fully replicate the complexity of tumor microenvironments in vivo. Second, although the formulation showed sustained and pH-responsive release profiles, detailed pharmacokinetic and biodistribution studies are needed to confirm its stability, circulation time, and tumor-targeting ability in biological systems. Additionally, the long-term safety of AgNPs and their possible accumulation in healthy tissues requires further investigation to ensure clinical applicability. A brief discussion of potential silver-related risks is also warranted. AgNPs, while offering strong antimicrobial and anticancer benefits, may release ions that cause oxidative stress, DNA damage, and disruption of normal cellular functions under prolonged exposure. To address these concerns, strategies such as dose optimization, surface functionalization, and encapsulation within biocompatible carriers have been proposed to minimize toxicity while preserving efficacy.
Future studies should therefore focus on comprehensive in vivo assessments, including toxicity, therapeutic efficacy across different tumor models, and potential immunological responses. Moreover, expanding this strategy to incorporate active targeting ligands or dual-drug loading could further enhance tumor selectivity and broaden therapeutic applications.
4. Conclusions
In this study, AFS-NPs were successfully synthesized using TEOS and APTES via the sol-gel method, exhibiting monodispersed particles of approximately 900 nm, as confirmed by SEM. Structural characterization through XRD demonstrated that the amorphous nature of AFS-NPs was altered after drug loading and CS coating, while FTIR confirmed the successful synthesis, DOX loading, and CS encapsulation. Zeta potential measurements revealed a positive surface charge, favoring enhanced cellular uptake. The optimized formulation, DOX@AFS-NPs-Ag-CS, achieved a high entrapment efficiency of 84% and demonstrated pH-responsive sustained drug release, with 94.2% cumulative release of DOX at pH 5.5 after 24 h. Cytotoxicity evaluation using MTT assay on HepG2 cells showed that the inclusion of AgNPs exerted a synergistic effect, while the CS coating contributed to stability, protection, and controlled release. Collectively, the final formulation exhibited superior anticancer activity compared to all other tested variants, confirming the benefits of this combinatorial design. Overall, the findings establish DOX@AFS-NPs-Ag-CS as a promising multifunctional nanocarrier capable of addressing critical challenges in cancer therapy, including non-specific targeting, burst release, and limited cellular uptake. This work highlights the translational potential of integrating silica nanoparticles, AgNPs, and CS into a single platform for effective and sustained anticancer drug delivery. Future studies may focus on in vivo validation, pharmacokinetic evaluation, and optimization for clinical applications.
Acknowledgment
The author extends her appreciation to The Department of Chemistry, College of Science, Taibah University, Yanbu 30799, Saudi Arabia.
Credit authorship contribution statement
Noha Said Bedowr: Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Visualization, Writing – Original Draft, Writing – Review & Editing, Resources, Supervision, Project Administration, and Funding Acquisition.
Declaration of competing interest
The authors declares no competing interests.
Declaration of generative AI and AI-assisted technologies in the writing process
The author confirms that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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