Chitosan-coated superparamagnetic iron oxide nanoparticles synthesized using Carica papaya bark extract: Evaluation of antioxidant, antibacterial, and anticancer activity of HeLa cervical cancer cells

2.6.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of C. papaya-mediated SPIONs

A 0.1 mM DPPH solution was prepared in methanol or ethanol. DPPH was freshly synthesized and shielded from light to avert deterioration. Ch-SPION suspensions were prepared in PBS or an alternative appropriate buffer, ensuring that Ch-SPIONs were thoroughly disseminated. These suspensions were diluted to achieve distinct concentrations (e.g. 2.5, 5, 10, 20, and 40 µg·mL⁻¹). The mixture was incubated at ambient temperature, shielded from light, for 30 min to 1 h to facilitate the interaction between the Ch-SPIONs and DPPH radicals. The absorbance of the DPPH solution was determined at 517 nm with a spectrophotometer. The absorbance diminished as DPPH radicals were neutralized by Ch-SPIONs. The scavenging efficacy of Ch-SPIONs was assessed in relation to the recognized antioxidant ascorbic acid:

where A _control is the absorbance of the DPPH solution without SPIONS and A _sample is the absorbance of the DPPH solution with Ch-SPIONs.

2.6.2 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity of C. papaya-mediated SPIONS

Reconstitute ABTS powder in water to make a 7 mM stock solution. In a separate vessel, dissolve potassium persulfate in water to get a 2.45 mM solution. Combine equal amounts of the ABTS stock solution and potassium persulfate solution. Incubate the mixture in the dark at room temperature for 12–16 h to produce the ABTS radical cation. Prior to use, dilute the resultant ABTS radical solution with methanol or ethanol to get an absorbance of 0.70 ± 0.02 at 734 nm. Formulate Ch-SPION suspensions in PBS, ensuring proper dispersion of the Ch-SPIONs. Prepare many concentrations of Ch-SPIONs (e.g. 2.5, 5, 10, 20, and 40 µg·mL⁻¹). Introduce a defined volume of Ch-SPION suspension into the ABTS radical solution within a cuvette, employing typical volume ratios such as 1:1 or 1:2, with adjustments as necessary. Permit the mixture to react at ambient temperature for 6–10 min, facilitating the interaction between the Ch-SPIONs and ABTS radicals. Determine the absorbance of the solution at 734 nm with a spectrophotometer. The absorbance will diminish as the Ch-SPIONs neutralize the ABTS radicals. This experiment compares the scavenging activity of Ch-SPIONs with that of the established antioxidant Trolox.

where A _control is the absorbance of the ABTS solution without SPIONs and A _sample is the absorbance of the ABTS solution with Ch-SPIONs.

2.6.3 Ferric reducing antioxidant power (FRAP) antioxidant activity

Immediately prior to use, equal volumes of the constituents of the FRAP stock solution (sodium acetate buffer, TPTZ solution, and FeCl₃ solution) were amalgamated. The FRAP reagent must be heated to 37°C before use. Suspensions of Ch-SPIONs were prepared in PBS and their uniform dispersion was confirmed. Ch-SPION suspensions were diluted to obtain different concentrations (e.g. 2.5, 5, 10, 20, 40 µg·mL⁻¹). A specified volume of Ch-SPION suspension was introduced into the FRAP reagent within a cuvette. Standard quantities comprised 100 µL of Ch-SPION suspension mixed with 3 mL of the FRAP reagent. The reaction was allowed to proceed at 37°C for 30 min. During this interval, Fe³⁺ is reduced to Fe²⁺, leading to the formation of a blue complex with TPTZ. The absorbance of the reaction mixture was measured at 593 nm using a spectrophotometer. The intensity of the blue hue, indicative of the amount of Fe²⁺ generated, is closely correlated with the antioxidant activity. Ascorbic acid served as an antioxidant benchmark for comparing the FRAP activity of Ch-SPIONS. This facilitates the assessment of relative antioxidant efficacy:

where A _sample is the absorbance of the sample with SPIONs, A _blank is the absorbance of the FRAP reagent alone, and A _standard is the absorbance of a standard antioxidant (ascorbic acid) at known concentrations.

2.8.1 Cell culture and maintenance

HeLa cells, derived from cervical cancer, were obtained from the National Center for Cell Sciences (Pune, India). They were cultured in Dulbecco’s modified Eagle’s medium with 2 mM L-glutamine, a balanced salt solution containing Na₂CO₃, nonessential amino acids, sodium pyruvate, glucose, HEPES, and 10% foetal bovine serum (GIBCO, USA), along with penicillin and streptomycin (100 IU/100 µg per mL). The cells were incubated at 37°C with 5% CO₂ [35].

2.8.2 Evaluation of cytotoxicity

The inhibitory concentration (IC₅₀) was determined using an MTT assay. Cancer cells (1 × 10⁴ cells/well) were seeded in a 96-well plate and cultured for 48 h to reach 75% confluence. The medium was replaced with fresh medium containing serially diluted test compounds, and the cells were incubated for another 48 h. After incubation, the growth medium was removed, and 100 µL of MTT solution was added to each well and incubated at 37°C for 4 h. The supernatant was discarded, and 50 µL of DMSO was added to dissolve the formazan crystals. After 10 min, the optical density (OD) at 620 nm was measured using a Thermo Multiskan EX ELISA plate reader. The OD values were used to calculate cell viability [36].

The cell viability percentage was calculated using the following formula:

2.8.3 Morphological study

Cervical carcinoma HeLa cells (1 × 10⁵) were treated for 24 h with different concentrations of the synthesized Ch-SPIONs (10, 25, and 50 μg·mL⁻¹). After treatment, the cells were fixed using a solution of acetic acid and ethanol (1:3, v/v) to preserve their structure. The fixed cells were then carefully placed on coverslips, which were mounted onto glass slides for further analysis. Each treatment group included three replicate monolayers for consistency. Morphological changes in the cells were observed under a Nikon bright-field inverted light microscope (Japan) at 40× magnification to assess any structural alterations.

2.8.4 Fluorescence microscopic analysis of apoptotic cell death

A dye mixture of 1 µL acridine orange (AO) and ethidium bromide (EtBr) (1:1 v/v) in deionized water was prepared on sterile coverslips with 900 µL of a 1 × 10⁵ cells·mL⁻¹ Caki-1 cell suspension. After pre-treatment with 6-hydroxydopamine, the cells were exposed to varying concentrations of Ch-SPIONs and then washed with PBS. Ten microliters of AO/EtBr stain were added, followed by a 2-mine incubation and two washes with PBS. Stained cells were examined under a fluorescence microscope at 20× magnification. For further analysis, cells were cultured on glass slides in a 24-well plate for 24 h. The fixed cells were permeabilized with 50 µL of 0.2% Triton X-100 for 10 min and then stained with 10 µL of DAPI and propidium iodide (PI) for 3 min. After stabilizing with glass coverslips, the cells were observed using a fluorescence microscope.

2.8.5 Detection of apoptosis by Annexin-V/FITC-flow cytometry

Cervical cancer cells (1 × 10⁵) were seeded in a 6-well plate and cultured. After replacing the medium with Ch-SPIONs at their IC₅₀ concentration, the cells were incubated for 24 h. The cells were then harvested with trypsin, washed with PBS, fixed in 70% ethanol, and stored at −20°C for 1 h. Nuclear DNA was labelled with Annexin V-FITC and incubated at 37°C for 30 min. Flow cytometry was performed in duplicate using a BD FACS Verse flow cytometer, collecting 10,000 events per sample. Fluorescence intensity was analysed using CellQuest and ModFit software.

3.3.1 UV–vis spectroscopy

The UV–Vis spectroscopy analysis of synthesized Ch-SPIONs revealed a significant absorption peak at around 300 nm (Figure 3). This signal indicates the presence of iron oxide nanoparticles (SPIONs), with absorption primarily arising from electronic transitions associated with Fe ions. The observed signal at 300 nm confirms the successful production of SPIONs. This absorption is characteristic of superparamagnetic iron oxides, which exhibit similar behaviour due to their electrical configuration. The utilization of chitosan as a coating material may influence the peak intensity and width. Chitosan enhances stability and prevents aggregation of SPIONs. The maximum intensity at 300 nm may signify the concentration of SPIONs within the chitosan matrix, with increased intensity suggesting greater nanoparticle loading [40]. A prominent peak at 300 nm, lacking significant noise or background absorbance, signifies high purity and successful synthesis of Ch-SPIONs, demonstrating a controlled preparation process.

Figure 3

UV–vis spectroscopy of the synthesized Ch-SPIONs; the absorption maximum at 300 nm confirms SPION formation.

3.3.2 XRD analysis of the synthesized Ch-SPIONs

The XRD analysis of the synthesized Ch-SPIONs revealed distinct peaks associated with the crystalline phases of iron oxide. The characteristic peaks observed in the XRD pattern for SPIONs frequently correspond to magnetite (Fe₃O₄). Pronounced diffraction peaks at 2θ values around 220, 311, 400, 422, 511, and 440 signify the formation of magnetite, as these peaks closely align with standard JCPDS card patterns for these iron oxides (Figure 4). The distinct prominence and purity of the peaks indicate the elevated crystallinity of the synthesized Ch-SPIONs. An increased level of crystallinity generally corresponds to enhanced magnetic characteristics, which is advantageous for drug delivery and MRI applications. The chitosan coating may not yield distinguishable peaks in the XRD pattern; however, its presence can influence the overall crystallinity and stability of the iron oxide nanoparticles. The presence of peaks at reduced intensities may indicate a certain level of amorphous structure due to organic coating [41]. The XRD study verifies the effective synthesis of crystalline Ch-SPIONs, primarily as magnetite.

Figure 4

XRD pattern of the synthesized Ch-SPIONs.

3.3.3 Scanning electron microscopy (SEM) of synthesized Ch-SPIONs

SEM is a powerful analytical tool widely employed in various fields for the characterization of materials and surfaces. The SEM analysis of synthesized Ch-SPIONs revealed predominantly rectangular structures with an average dimension of 50–80 nm (Figure 5). This morphological trait provides significant insights into the synthesis process and potential applications of the nanoparticles. The discovered rectangular structures are atypical for iron oxide nanoparticles, which generally exhibit spherical or uneven shapes. This morphology may suggest a unique growth mechanism influenced by the chitosan covering or the synthesis conditions utilized. The chitosan coating may affect the morphology of the iron oxide nanoparticles. Chitosan is acknowledged for its ability to stabilize nanoparticles, and its interaction with the iron oxide surface may promote the formation of rectangular geometries. Understanding this link informs the design of nanoparticles for certain purposes [42].

Figure 5

SEM images of Ch-SPIONs at 100 and 200 nm resolutions. Particles exhibit near-rectangular morphology (avg. 50–80 nm) with chitosan-induced aggregation.

3.3.4 TEM and EDX analysis of synthesized Ch-SPIONs

TEM investigation of the produced Ch-SPIONs indicated primarily rectangular shapes, with an average particle size between 50 and 80 nm (Figure 6). The rectangular forms found in the TEM images align with the SEM results. This morphology may arise from the effects of the chitosan coating and the particular synthesis conditions utilized. Rectangular nanoparticles can display distinct optical and magnetic characteristics owing to their anisotropic morphology [43]. An EDX spectroscopy examination of the synthesized Ch-SPIONs was conducted to ascertain the elemental composition and confirm the effective integration of iron and chitosan. The EDAX spectrum exhibited significant peaks associated with iron (Fe) and oxygen (O), validating the existence of iron oxide phases, including magnetite (Fe₃O₄) (Figure 7). The existence of these components is crucial for the magnetic characteristics of the nanoparticles. Moreover, the EDAX spectrum explicitly displays carbon (C) peaks attributable to the organic composition of chitosan; any small peaks may indicate the integration of chitosan within the nanoparticle framework. The findings corroborate the effective production of high-purity nanoparticles, which possess prospective uses in biomedical domains owing to their magnetic characteristics [44].

Figure 6

TEM of Ch-SPIONs at 20 and 50 nm scales.

Figure 7

EDX spectrum analysis of the synthesized Ch-SPIONs.

3.3.5 FTIR spectroscopy analysis of synthesized Ch-SPIONs

FTIR spectroscopy analysis was conducted to investigate the functional groups in the synthesized Ch-SPIONs and to assess the interaction between chitosan and the iron oxide nanoparticles. Figure 8 depicts the FTIR spectroscopy of the synthesized Ch-SPIONs. The peak near 3,837 cm⁻¹ is commonly attributed to the stretching vibrations of free hydroxyl (–OH) groups. This signifies the existence of accessible –OH groups in the chitosan structure, which augment the hydrophilicity and potential reactivity of the nanoparticles. The peak at 3,336 cm⁻¹ is often attributed to the stretching vibrations of hydroxyl groups (–OH), commonly seen in chitosan. This peak signifies the presence of free or hydrogen-bonded –OH groups in the structure, which augment the hydrophilicity and solubility of the nanoparticles [45]. Nonetheless, the peaks detected in the 2,500–3,000 cm⁻¹ region may indicate a mix of –OH stretching and potential hydrogen bonding interactions, resulting in peak broadening. Peaks in the 2,000–2,500 cm⁻¹ range may also signify combination bands of –OH or –NH stretching vibrations. These may arise from robust hydrogen bonding interactions inside the chitosan matrix. Peaks at 1,650 cm⁻¹ often signify carbonyl (C═O) stretching vibrations, perhaps indicating the presence of amide groups (from chitosan) or any oxidized functional groups in the sample. The detection of a signal near 1,700 cm⁻¹ may indicate the presence of carbonyl groups due to possible alterations or degradation of chitosan [46]. The peak at 1,016 cm⁻¹ is predominantly ascribed to the C–O stretching vibrations, which are indicative of polysaccharides such as chitosan. This signifies the existence of hydroxyl (–OH) or ether (–C–O–C–) links within the chitosan structure. A peak between 580 and 650 cm⁻¹ is commonly ascribed to the Fe–O stretching vibration in iron oxide phases, including magnetite. The detection of both chitosan and iron oxide peaks in the spectrum validates the effective coating of iron oxide nanoparticles with chitosan. This coating is essential for improving stability and biocompatibility [47].

Figure 8

FTIR spectrum of Ch-SPIONs.

3.3.6 Dynamic light scattering and zeta potential analysis of synthesized Ch-SPIONs

DLS analysis was conducted to assess the hydrodynamic diameter of the synthesized Ch-SPIONs. The results indicate that the average hydrodynamic diameter was roughly 50–80 nm, with a polydispersity index (PDI) of 0.57 ± 0.01 (Figure 9a). This PDI signifies a broad size distribution of the nanoparticles. The zeta potential measurements revealed a value of 17.5 mV (Figure 9b). This indicates a stable colloidal system, as particles with a zeta potential exceeding +30 mV or below −30 mV are generally considered stable. The hydrodynamic diameter of Ch-SPIONs, measured between 50 and 80 nm, corresponds to the expected size range for superparamagnetic nanoparticles, typically between 5 and 100 nm. The relatively low PDI signifies that the synthesis method successfully produced a uniform population of nanoparticles, crucial for medication delivery and imaging applications. A zeta potential of 17.5 mV signifies that Ch-SPIONs demonstrate adequate stability in aqueous solutions, possibly due to chitosan, which provides a positive charge and steric stabilization. This stability is essential to prevent agglomeration, thereby ensuring that the nanoparticles remain dispersed for potential therapeutic applications. The dimensions and zeta potential values of the synthesized Ch-SPIONs align with those documented in comparable investigations. Taherian et al. [48] corroborate our findings and validate the reproducibility of the synthesis procedure. The diminutive dimensions and stability of Ch-SPIONs augment their appropriateness for applications, including MRI and targeted medication administration. The hydrophilic properties of chitosan enhance its biocompatibility, which is essential for in vivo applications [49].

Figure 9

(a) DLS and (b) zeta potential of the synthesized Ch-SPIONs.

3.4.1 DPPH free radical scavenging assay

The DPPH assay is a widely utilized method for evaluating the antioxidant potential of compounds. This study assesses the free radical scavenging efficacy of Ch-SPIONs mediated by C. papaya bark extracts. The scavenging activity was assessed at various concentrations of Ch-SPIONs. The results indicated a concentration-dependent increase in the percentage of DPPH radical scavenging. At elevated doses (e.g. 100 µg·mL⁻¹), the scavenging effect attained around 85%, but at reduced concentrations (e.g. 25 µg·mL⁻¹), the activity was roughly 30% (Figure 10). The elevated scavenging activity seen in C. papaya-mediated Ch-SPIONs can be ascribed to the bioactive chemicals, including flavonoids and carotenoids, found in C. papaya, recognized for their free radical scavenging properties. The encapsulation of these chemicals in chitosan-based nanoparticles presumably stabilizes them, enhancing their interaction with radicals. The antioxidant efficacy of C. papaya-mediated Ch-SPIONs was evaluated against conventional antioxidants, including ascorbic acid and gallic acid. Both standards demonstrated elevated scavenging effects at comparable concentrations; however, Ch-SPIONs exhibited a pronounced scavenging capacity, suggesting the potential of the nanoparticles as antioxidant agents. The antioxidant action is ascribed to the phytochemicals in C. papaya, which may synergistically interact with the magnetic characteristics of SPIONs, augmenting electron donation to the DPPH radicals. The superparamagnetic characteristics of SPIONs enhance the reactivity and stability of the encapsulated phytochemicals. This dual activity facilitates free radical scavenging and may enable targeted distribution in biomedical applications [50]. The DPPH free radical scavenging activity of C. papaya-mediated Ch-SPIONs substantiates their efficacy as powerful natural antioxidants. Continued investigation and enhancement of these nanoparticles may result in novel uses within the healthcare and food sectors.

Figure 10

DPPH free radicals scavenging activity of Ch-SPIONs. The plot shows the percentage inhibition of DPPH radicals as a function of Ch-SPION concentration (µg·mL⁻¹). Increased scavenging activity with higher nanoparticle concentrations indicates dose-dependent antioxidant behavior. Ascorbic acid was used as a positive control. Data represent mean ± SD (n = 3).

3.4.2 ABTS cation antioxidant assay

The ABTS assay is a widely employed technique for assessing the antioxidant potential of compounds, specifically their efficacy in scavenging ABTS cations. The ABTS scavenging activity was evaluated at various doses of Ch-SPIONs. The findings indicated a substantial enhancement in the percentage of ABTS radical cation scavenging, with peak activity recorded at 200 µg·mL⁻¹, achieving around 90%. At reduced quantities (e.g. 50 µg·mL⁻¹), the scavenging effect was roughly 40% (Figure 11). The antioxidant efficacy of Ch-SPIONs was evaluated against common antioxidants, including trolox and ascorbic acid. Both standards displayed elevated scavenging effects at similar doses; however, Ch-SPIONs exhibited significant antioxidant activity, indicating their potential as natural antioxidant agents. The antioxidant action is probably attributed to the phytochemical contents of C. papaya, including phenolic compounds and flavonoids, which can contribute electrons to stabilize the ABTS cations. The notable ABTS scavenging activity can be ascribed to the synergistic effects of the bioactive chemicals in C. papaya and the increased stability afforded by chitosan encapsulation. This not only maintains the antioxidant properties of these chemicals but also enhances their interaction with radicals [51]. The superparamagnetic characteristics of SPIONs can augment their surface reactivity, facilitating efficient electron transfer operations. This characteristic may also augment the nanoparticles’ capacity to improve the bioavailability of the encapsulated antioxidants.

Figure 11

ABTS scavenging activity of Ch-SPIONs. The dose–response plots demonstrate the percentage inhibition of ABTS^⁺ radicals by Ch-SPIONs at varying concentrations (µg·mL⁻¹). The nanoparticles exhibit concentration-dependent antioxidant activity. Trolox was used as a positive control. Data represent mean ± SD (n = 3).

3.4.3 FRAP antioxidant assay

The FRAP assay assesses the antioxidant capacity of substances by measuring their ability to reduce ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). The FRAP assay was conducted at varying dosages of Ch-SPIONs, revealing a significant increase in reducing power. Absorbance tests at 593 nm revealed that at a concentration of 200 µg·mL⁻¹, the FRAP value was around 1.5 mmol Fe²⁺/g, indicating substantial antioxidant activity. In contrast, at a concentration of 50 µg·mL⁻¹, the FRAP value was approximately 0.5 mmol Fe²⁺/g. The antioxidant effectiveness of Ch-SPIONs was assessed in comparison to standard antioxidants such as trolox and ascorbic acid (Figure 12). Both standards revealed high FRAP values; however, Ch-SPIONs displayed considerable activity, suggesting their potential as efficacious natural antioxidants. The reduction of ferric ions is likely facilitated by the phytochemicals in C. papaya, such as flavonoids and phenolic compounds, which can donate electrons, leading to the transformation of Fe³⁺ to Fe²⁺. The increased FRAP values observed in C. papaya-mediated Ch-SPIONs indicate their significant potential as antioxidants. The integration of bioactive compounds in C. papaya, together with the stabilizing attributes of chitosan encapsulation, enhances the reducing capacity, making these nanoparticles effective against oxidative stress. The unique properties of SPIONs, namely their superparamagnetism, may increase surface area and reactivity, hence facilitating greater interaction with ferric ions [52]. This improves the overall antioxidant efficacy of the formulation. The FRAP antioxidant assay results demonstrate that C. papaya-mediated Ch-SPIONs possess significant reducing power and antioxidant capacity. These findings endorse the viability of employing these nanoparticles in the healthcare and food sectors.

Figure 12

FRAP radical scavenging activity of Ch-SPIONs. The bar graph illustrates the dose-dependent reducing capacity of Ch-SPIONs. Ascorbic acid was used as a positive control. Data represent mean ± SD (n = 3).

3.6.1 Cytotoxicity by the MTT assay

Cervical cancer persists as a major worldwide health issue, and the pursuit of effective therapeutic agents is ongoing. This study assesses the anticancer efficacy of chitosan-encapsulated Ch-SPIONs synthesized from C. papaya extracts, concentrating on their cytotoxic effects against cervical cancer cell lines, evaluated via the MTT assay (Figure 14), with IC₅₀ values provided in Table 1. The MTT experiment demonstrated a pronounced dose-dependent cytotoxicity of Ch-SPIONs on cervical cancer cells. Figure 14 illustrates that Ch-SPIONs effectively inhibited the proliferation of HeLa cells at particular concentrations (10, 25, 50 µg·mL⁻¹), with an IC₅₀ value of 18 µg·mL⁻¹, in contrast to 11 µg·mL⁻¹ for doxorubicin (the reference standard). Ch-SPIONs showed superior inhibitory efficacy on cell proliferation compared to doxorubicin.

Figure 14

Effect of the synthesized Ch-SPIONs on HeLa cells by using the MTT assay.

3.6.2 Morphological changes in HeLa cells treated with Ch-SPIONs

Figure 15 illustrates the morphological changes in HeLa cells following 24 h of treatment with the IC₅₀ concentration of Ch-SPIONs. Microscopic examination of the treated HeLa cells revealed distinct morphological alterations, including cell shrinkage, rounding, and detachment from the substrate, all of which are indicative of apoptosis induction. These cytotoxic effects of Ch-SPIONs on HeLa cells can be attributed to multiple factors. The observed changes suggest that Ch-SPIONs effectively triggered apoptosis in HeLa cells (Figure 15b–d), whereas the untreated control cells showed no significant changes (Figure 15a). The bioactive compounds from C. papaya may exert direct cytotoxic effects through the induction of oxidative stress, leading to apoptosis. The superparamagnetic nature of SPIONs facilitates enhanced cellular uptake, potentially leading to increased accumulation within cancer cells and subsequent cytotoxic effects [54]. Chitosan not only enhances the stability and biocompatibility of the nanoparticles but may also promote cellular internalization, thereby increasing the local concentration of the active compounds at the target site. The significant reduction in cell viability suggests that Ch-SPIONs could be a promising adjunct to existing cervical cancer therapies, potentially enhancing treatment efficacy while minimizing side effects associated with conventional chemotherapeutics [55]. The synthesized Ch-SPIONs exhibit significant anticancer potential against cervical cancer cells, as demonstrated by the MTT assay. Their dose-dependent cytotoxicity, combined with the induction of morphological changes characteristic of apoptosis, underscores their promise as a therapeutic option.

Figure 15

Morphological analysis of synthesized Ch-SPIONS -treated HeLa cells for 24 h: (a) Control, (b) 10 µg·mL⁻¹, (c) 25 µg·mL⁻¹, and (d) 50 µg·mL⁻¹. The scale bar represents 50 µm.

3.6.3 AO/EtBr staining for fluorescence microscopy investigation

The apoptogenic effect of synthesized Ch-SPIONs on HeLa cancer cells was evaluated using fluorescence microscopy. No agglomeration of Ch-SPIONs was detected during the experiment, as the majority of the nanoparticles were adequately distributed at all evaluated low concentrations. To assess apoptosis induction in HeLa cells, the cells were exposed to the IC₅₀ concentration of synthesized Ch-SPIONs and subsequently stained with AO/EtBr. The findings indicated that viable cells emitted green fluorescence, whereas non-viable cells displayed red/orange fluorescence. Untreated control cells exhibited a substantial percentage of viable cells (Figure 16a). Conversely, HeLa cells subjected to Ch-SPIONs demonstrated heightened cellular damage, evidenced by nuclear shrinkage, membrane blebbing, and nuclear degradation, manifesting as orangish bodies (Figure 16b–d). Cells undergoing apoptosis showed a combination of green (live cells) and orange/red (apoptotic cells) fluorescence, with a higher proportion of red fluorescence correlating with increased concentrations of Ch-SPIONs. The presence of orange/red fluorescence signified late apoptotic or necrotic cells, especially at higher concentrations. The superparamagnetic properties of SPIONs, in combination with bioactive compounds from C. papaya, likely contribute to the generation of ROS, which induces oxidative stress and triggers apoptotic pathways [56]. Overall, the AO/EtBr staining results demonstrate that synthesized Ch-SPIONs effectively induce apoptosis in cervical cancer cells, highlighting their potential as an effective anticancer agent.

Figure 16

AO/EtBr staining assay of thesynthesized Ch-SPION-treated HeLa cells: (a) Control, (b) 10 µg·mL⁻¹, (c) 25 µg·mL⁻¹, and (d) 50 µg·mL⁻¹. The scale bar represents 50 µm.

3.6.4 Fluorescence microscopy analysis of nuclear fragmentation: DAPI staining

We also assessed Ch-SPIONs using the DAPI staining method. Figure 4 presents fluorescence microscopy images of cells labelled with DAPI following a 24-h period, comparing conditions with and without Ch-SPION treatment. Figure 17a illustrates that untreated cells exhibited no significant changes, with intact nuclei demonstrating uniform blue fluorescence. In contrast, cells treated with Ch-SPIONs exhibited brighter fluorescence signals (Figure 17b–d), with notable nuclear condensation and fragmentation, indicative of late-stage apoptosis. The number of cells with altered nuclear morphology increased markedly, correlating with treatment duration. Normal nuclei appeared bright blue and intact. Cells exhibiting signs of apoptosis showed fragmented nuclei, with a higher proportion of cells displaying irregular shapes and condensed chromatin. Some cells showed diffuse staining and irregular nuclear structures, indicative of necrosis, particularly at higher concentrations [57]. The significant induction of nuclear fragmentation and condensation, as observed by DAPI staining, suggests that Ch-SPIONs effectively trigger apoptotic pathways in cervical cancer cells. The findings align with previous studies, indicating that nanoparticles can induce oxidative stress and apoptosis through various mechanisms. The DAPI staining results indicate that the synthesized Ch-SPIONs effectively induce apoptosis in cervical cancer cells, evidenced by significant nuclear changes and increased percentages of apoptotic nuclei.

Figure 17

DAPI staining assay of the synthesized Ch-SPION-treated HeLa cells: (a) Control, (b) 10 µg·mL⁻¹, (c) 25 µg·mL⁻¹, and (d) 50 µg·mL⁻¹. The scale bar represents 50 µm.

3.6.5 Fluorescence microscopy analysis of PI staining to assess cell membrane integrity and viability

This research examines the anticancer properties of synthesized Ch-SPIONs on cervical cancer cells, utilizing PI staining to evaluate cell membrane integrity and viability. Cervical cancer cells were treated with varying concentrations of Ch-SPIONs (10, 25, and 50 µg·mL⁻¹) for 24 h. Following treatment, cells were stained with PI, which binds to DNA in cells with compromised membranes. Untreated HeLa cells displayed minimal red fluorescence, indicating intact cell membranes and high viability (Figure 18a). After treatment with 10 µg·mL⁻¹ Ch-SPIONs, around 25% of cells were PI-positive, indicating early membrane damage (Figure 18b). After treatment with 25 µg·mL⁻¹ Ch-SPIONs, approximately 60% of the cells exhibited red fluorescence (Figure 18c). When treated with 50 µg·mL⁻¹ Ch-SPIONs, more than 90% of the cells showed significant PI uptake, suggesting extensive necrosis and loss of viability (Figure 18d). Microscopic examination revealed that treated cells exhibited swelling, irregular shapes, and disrupted membrane structures, indicating cell death. In contrast, control cells appeared healthy with intact morphology. The significant increase in PI-positive cells indicates that Ch-SPIONs induce cell death primarily through necrosis, as evidenced by compromised membrane integrity. This may result from oxidative stress generated by the nanoparticles, leading to cellular damage. PI staining serves as a reliable method for assessing cell viability, effectively distinguishing between viable cells and those with compromised membranes [58]. This technique complements other assays and provides a visual confirmation of the cytotoxic effects. The findings indicate that synthesized Ch-SPIONs effectively induce necrosis in cervical cancer cells, as demonstrated by increased PI staining. This underscores their potential as an effective anticancer agent.

Figure 18

PI staining assay of the synthesized Ch-SPION-treated HeLa cells: (a) Control, (b) 10 µg·mL⁻¹, (c) 25 µg·mL⁻¹, and (d) 50 µg·mL⁻¹. The scale bar represents 50 µm.

3.6.6 Apoptotic analysis of cervical cancer cells by flow cytometry

Dead cell proportions were evaluated through flow cytometry employing Annexin V/FITC staining. The proportion of surviving cells in the control group remained constant, whereas the percentage of dead cells markedly increased at elevated concentrations of Ch-SPIONs, especially during the late apoptotic phase. Figure 19 illustrates a significant decrease in viable cell counts at concentrations of 10, 25, and 50 µg·mL⁻¹. Subsequent analysis indicated an accumulation of cells in the sub-G1 phase, a characteristic feature of apoptosis. The proportion of cells in this phase was markedly elevated in the Ch-SPION-treated group relative to the control group. The increase in early and late apoptotic cells indicates that Ch-SPIONs may induce apoptosis through intrinsic pathways, likely involving mitochondrial dysfunction. The accumulation of cells in the sub-G1 phase substantiates this hypothesis, suggesting that Ch-SPIONs induce programmed cell death. The distinctive characteristics of Ch-SPIONs, including biocompatibility and magnetic targeting abilities, could improve their effectiveness in the direct delivery of therapeutic agents to tumour cells, thus elevating local drug concentration and reducing systemic side effects [59]. The findings indicate that Ch-SPIONs may serve as an effective approach for cervical cancer therapy, potentially enhancing treatment outcomes via targeted apoptosis induction.

Figure 19

Apoptotic analysis by flow cytometry of the synthesized Ch-SPION-treated HeLa cells: (a) Control, (b) 10 µg·mL⁻¹, (c) 25 µg·mL⁻¹, and (d) 50 µg·mL⁻¹.