Fe3O4@TiO2 Microspheres: Harnessing O2 Release and ROS Generation for Combination CDT/PDT/PTT/Chemotherapy in Tumours

In the treatment of various cancers, photodynamic therapy (PDT) has been extensively studied as an effective therapeutic modality. As a potential alternative to conventional chemotherapy, PDT has been limited due to the low Reactive Oxygen Species (ROS) yield of photosensitisers. Herein, a nanoplatform containing mesoporous Fe3O4@TiO2 microspheres was developed for near-infrared (NIR)-light-enhanced chemodynamical therapy (CDT) and PDT. Titanium dioxide (TiO2) has been shown to be a very effective PDT agent; however, the hypoxic tumour microenvironment partly affects its in vivo PDT efficacy. A peroxidase-like enzyme, Fe3O4, catalyses the decomposition of H2O2 in the cytoplasm to produce O2, helping overcome tumour hypoxia and increase ROS production in response to PDT. Moreover, Fe2+ in Fe3O4 could catalyse H2O2 decomposition to produce cytotoxic hydroxyl radicals within tumour cells, which would result in tumour CDT. The photonic hyperthermia of Fe3O4@TiO2 could not only directly damage the tumour but also improve the efficiency of CDT from Fe3O4. Cancer-killing effectiveness has been maximised by successfully loading the chemotherapeutic drug DOX, which can be released efficiently using NIR excitation and slight acidification. Moreover, the nanoplatform has high saturation magnetisation (20 emu/g), making it suitable for magnetic targeting. The in vitro results show that the Fe3O4@TiO2/DOX nanoplatforms exhibited good biocompatibility as well as synergetic effects against tumours in combination with CDT/PDT/PTT/chemotherapy.


Introduction
In our modern society, cancer is one of the most serious diseases [1].In cancer disease, ROS play an important role, as light offers distinct advantages, including minimal invasiveness and spatiotemporal resolution [2,3].Through irradiation with lasers, PDT can result in the death of cancer cells by generating cytotoxic ROS from photosensitisers (PS) [4].During PDT, PS drugs generate ROS through a series of photochemical reactions to induce cytotoxicity [5].Traditional organic photosensitisers, such as small-molecule organic PSs like sulphonamide-cyanine dye 7 (Cy7), indocyanine green (ICG), and porphyrins, can be utilised as PSs owing to their remarkable efficacy in generating high levels of ROS.Nonetheless, their potential biomedical applications encounter limitations due to factors such as inadequate photostability and sensitivity constraints influenced by environmental factors [6].In contrast, inorganic materials such as TiO 2 have shown long-term physical and chemical stability [7].TiO 2 has a large band gap energy (3.0 eV for anatase phase and 3.2 eV for rutile phase).PDT relies on the formation of an electron-hole pair upon the absorption of a photon with an energy equal to or higher than the semiconductor's band gap [8].When combined with oxygen or water, these lone electrons form hydroxyl radicals (•OH), hydrogen peroxides (H 2 O 2 ), and super oxides (O 2 − ).The generated ROS can attack virtually all macromolecules, which results in serious damage to cancer cellular components [9].However, the low ROS generation efficacy and the lack of targeting mechanisms remain critical issues in achieving a desirable tumour-suppressing effect and favourable therapeutic outcome [10,11].Moreover, low oxygen levels in hypoxic tumour microenvironments (TMEs) result in low ROS production and might result in poor therapy outcomes for PDT [12][13][14][15][16]. Furthermore, tumour cells are rich in glutathione (GSH), which enables the 1 O 2 produced during PDT to be rapidly reduced, thus diminishing the efficacy of PDT [17][18][19][20][21][22].These two TME-associated factors greatly limit the efficacy of PDT [23].
In order to improve the efficiency of PDT, we successfully prepared a novel composite photocatalytic material (Fe 3 O 4 @TiO 2 /DOX) composed of the typical photosensitiser TiO 2 and enzyme Fe 3 O 4 components (Scheme 1).The Fe 3 O 4 component's catalase-like function can facilitate the breakdown of surplus H 2 O 2 , generating O 2 within the tumour's cytoplasm to improve PDT [12,[31][32][33][34]. Fe 3 O 4 can slowly release Fe 2+ and Fe 3+ , induced by the low-pH environment of tumour cells [35].H 2 O 2 reacts with Fe 2+ to form Fe 3+ and a hydroxyl radical to kill tumour cells.In detail, Fe 3+ doping can transform GSH to glutathione disulphide (GSSH), which decreases the consumption of ROS [23].Remarkably, the heat generated by the photothermal conversion of Fe 3 O 4 @TiO 2 further promotes the efficiency of Fenton-like and photocatalysis reactions.The incorporation of DOX into Fe 3 O 4 @TiO 2 microspheres enhances targeted delivery to cancer cells.Concurrently, these microspheres facilitate the controlled and sustained release of DOX, optimising therapeutic efficacy while mitigating potential side effects.
Schematic illustration of Fe 3 O 4 @TiO 2 /DOX microspheres and the combined CDT/PDT/PTT/Chemotherapy for tumour inhibition.

Preparation of Fe 3 O 4 Microspheres
The synthesis of the Fe-glycerate precursor involved mixing Fe(NO 3 ) 3 •6H 2 O (AR, Macklin., Shanghai, China), glycerol (AR, KERMEL, Tianjin, China), and isopropanol (AR, KERMEL, Tianjin, China) under high-temperature and high-pressure conditions.This process included ultrasonic treatment, dropwise addition into deionised water with vigorous stirring, a 12 h reaction in a stainless-steel high-pressure vessel lined with polytetrafluoroethylene at 190 • C, followed by centrifugation, multiple washes with deionised water and ethanol, and drying for 24 h at 60 • C, while purging air with argon.Subsequently, the Fe-glycerol precursor was heated at a ramp rate of 2 • C min −1 under an argon atmosphere to 500 • C for 3 h, yielding the desired Fe-glycerate substance.These procedures encompassed chemical mixing, high-temperature high-pressure reactions, and subsequent processing steps, ensuring the purity and desired form of the final product.

Preparation of Fe 3 O 4 @TiO 2 Microspheres
In this experimental procedure, 0.3 g of Pluronic F127 (with a molecular weight of 12,600) (AR, Macklin., Shanghai, China) were introduced into 50 mL of water while being stirred continuously for a duration of 200 min.Following this, 60 mg of previously prepared Fe 3 O 4 microspheres were added to the mixture and mechanically stirred for an additional 30 min.Subsequently, 1.5 mL of tetra butyl titanate (AR, Macklin., Shanghai, China) were slowly incorporated into the mixture and stirred for a duration of 1 h.Following the completion of the stirring process, the Fe 3 O 4 @TiO 2 core-shell structures were obtained.To conclude the procedure, the obtained Fe 3 O 4 @TiO 2 core-shell structures were separated via centrifugation and washed multiple times with ethanol to eliminate impurities or residual compounds.

DOX Loading and Release
The encapsulation process of DOX (Doxorubicin) (AR, Macklin., Shanghai, China) into Fe 3 O 4 @TiO 2 involved dissolving 10 mg of DOX in a 10 mL PBS (pH 7.4) solution and suspending 50 mg of Fe 3 O 4 @TiO 2 in this solution.The mixture was stirred in darkness for 24 h, followed by the collection of the supernatant for UV-vis spectrophotometry analysis at 490 nm.The drug loading content within the Fe 3 O 4 @TiO 2 nanoplatform was determined.This encapsulation method assesses the efficiency of DOX binding to the nanoplatform, essential for evaluating its potential in targeted drug delivery and therapeutic applications.DOX loading efficiency was calculated as below: Drug loading efficiency (LE) = (Weight of loaded DOX (mg)/Weight of total DOX (mg)) × 100 Drug loading capacity = (total amount of DOX − Free DOX)/amount of Fe 3 O 4 @TiO 2 /DOX (2) The in vitro investigation focused on the pH responsiveness and drug release characteristics of DOX-loaded formulations in PBS buffers with varying pH levels (pH 5.4 or 7.4).The study involved dispersing 5 mg of DOX-loaded microparticles (Fe 3 O 4 @TiO 2 ) separately into 10 mL volumes of acetate buffers with differing pH values (5.4 or 7.4).The quantification of released DOX in the supernatant was conducted using a UV-visible (UV-vis) spectrophotometer.Following irradiation with a NIR laser for 10 min, the UV-vis absorption spectra of the supernatant were recorded to assess photothermal-triggered drug release.

DOX release(
where M i is the amount of DOX released from Fe 3 O 4 @TiO 2 /DOX at time and M 0 is the total amount of loaded drug in Fe 3 O 4 @TiO 2 .

NIR-Triggered Photothermal Effect of Fe 3 O 4 @TiO 2 Microspheres
To assess the photothermal properties, varying concentrations of Fe 3 O 4 @TiO 2 microspheres were introduced into a cuvette and subjected to irradiation using an 808 nm laser at different power densities.The study also investigated the photothermal effect of Fe 3 O 4 @TiO 2 under distinct laser powers (1, 1.5, and 2 W/cm 2 ).

Intracellular ROS Detection
HeLa (pricella, Wuhan, China) cells were seeded in a 96-well plate at a density of 5 × 10 3 cells/100 µL per well and incubated overnight.Then, the medium was removed and replaced with a medium containing the Fe 3 O 4 @TiO 2 .After treatment, cells were incubated with 10 µM of DCFH-DA at 37 • C for 20 min and then washed twice with PBS.Cells were irradiated for 10 min with UV light of 365 nm.Afterwards, the cell nucleus was stained with DAPI.Finally, the fluorescence images were observed via CLSM (λ ex = 488 nm, λ em = 525 nm).

Cellular Cytotoxicity Assay
The cytotoxicity of the Fe 3 O 4 @TiO 2 and Fe 3 O 4 @TiO 2 /DOX was analysed using the CCK-8 assay.After incubating cells in a culture medium within 96-well plates for 24 h, they were subsequently categorised into distinct groups and exposed to varying samples for treatment.For CCK-8 detection, 10 µL CCK-8 reagent was added to the culture medium 4 h before analysis.

Evaluation of •OH Generation
According to MB degradation under the oxidative conditions, a classic colorimetric method was adopted to detect the production of •OH.Briefly, Fe 3 O 4 @TiO 2 was added into an H 2 O 2 (10 mmol/L) aqueous solution containing MB (15 µg/mL) and GSH (5 mmol/L).The laboratory shaker was employed for incubating the mixture at a speed of 300 rotations per minute (rpm) and a temperature of 37 • C for a duration of 4 h.Subsequently, the determination of the concentration of the MB solution was performed by measuring the absorbance at 644 nm.

Singlet Oxygen Detection
In this experiment, a solution of 1,3-Diphenylisobenzofuran (DPBF) is prepared by dissolving it in dimethylformamide (DMF) with a concentration of 0.6 mg mL −1 .The DPBF solution is then mixed separately with Fe 3 O 4 @TiO 2 , maintaining a Fe 3 O 4 @TiO 2 concentration of 0.5 mg mL −1 .All mixing steps are carried out meticulously under lightfree conditions to prevent interference from external light sources.The UV absorbance of the solution is systematically recorded at 420 nm at various time points over a 40 min duration, utilizing UV-vis spectra analysis for accurate measurement.The experimental design is implemented rigorously to ensure controlled conditions, and the reaction kinetics are monitored accurately to comprehensively investigate the photochemical interactions between DPBF and Fe 3 O 4 @TiO 2 .

Cell Culture and Intracellular Localization Assay
HeLa Cells (5 × 10 3 cells/well) were seeded onto 96-well plate and incubated overnight under 5% CO 2 at 37 • C. The fresh medium containing Fe 3 O 4 @TiO 2 -FITC microspheres and Fe 3 O 4 @TiO 2 -FITC with a magnet (the magnet is positioned beneath the 96-well plate) were added and cultivated for another 6 h.Subsequently, observation was conducted using a fluorescence microscope.
In this experimental protocol, Fe 3 O 4 @TiO 2 -FITC microspheres were synthesised by dissolving 20 mg of Fe 3 O 4 @TiO 2 in 20 mL of PBS and incorporating 1 mg of FITC, followed by overnight stirring in darkness.

Results and Discussions
The procedure for synthesising Fe 3 O 4 @TiO 2 /DOX microspheres is illustrated in Figure 1a.In the primary stage, TiO 2 coatings are applied onto Fe 3 O 4 microspheres using tetrabutyl titanate.Subsequently, the resulting Fe 3 O 4 @TiO 2 microspheres undergo a 24 h stirring process in a DOX solution, facilitating the effective loading of DOX into the Fe 3 O 4 @TiO 2 microspheres.The preparation of Fe 3 O 4 microspheres was carried out in two steps.Hollow spheres containing Fe were first synthesised using the hydrothermal method at 190 • C with Fe(NO 3 ) 3 •6H 2 O as a precursor.Upon annealing at 500 • C, the Fe-containing spheres were completely converted to crystalline Fe 3 O 4 without other phases.These Fe 3 O 4 microspheres are hollow structures assembled via interlaced nanosheets (Figure 1b,c).The diameters of the Fe 3 O 4 hollow spheres were calculated to be 600-1200 nm using Nano Measurer measurements (Figure 1d).The SEM image of Fe 3 O 4 @TiO 2 reveals that the surface of the Fe 3 O 4 is coated, with the nanosheets of Fe 3 O 4 being enveloped (Figure 1e).The SEM and TEM images of Fe 3 O 4 @TiO 2 confirmed the uniform spherical morphology and hollow interior with a diameter of approximately 600-12,000 nm, as shown in Figure 1e-g.These diameters were almost in agreement with those measured from the SEM and TEM images.
Nanomaterials 2024, 14, x FOR PEER REVIEW 6 of 16 steps.Hollow spheres containing Fe were first synthesised using the hydrothermal method at 190 °C with Fe(NO3)3•6H2O as a precursor.Upon annealing at 500 °C, the Fecontaining spheres were completely converted to crystalline Fe3O4 without other phases.These Fe3O4 microspheres are hollow structures assembled via interlaced nanosheets (Figure 1b,c).The diameters of the Fe3O4 hollow spheres were calculated to be 600-1200 nm using Nano Measurer measurements (Figure 1d).The SEM image of Fe3O4@TiO2 reveals that the surface of the Fe3O4 is coated, with the nanosheets of Fe3O4 being enveloped (Figure 1e).The SEM and TEM images of Fe3O4@TiO2 confirmed the uniform spherical morphology and hollow interior with a diameter of approximately 600-12,000 nm, as shown in Figure 1e-g.These diameters were almost in agreement with those measured from the SEM and TEM images.The phase characteristics of the Fe3O4 and Fe3O4@TiO2 architecture were surveyed using XRD (Figure 2a).According to the XRD pa ern of Fe3O4@TiO2, there is a combination of the ( 220), ( 311), ( 400 The phase characteristics of the Fe 3 O 4 and Fe 3 O 4 @TiO 2 architecture were surveyed using XRD (Figure 2a).According to the XRD pattern of Fe 3 O 4 @TiO 2 , there is a combination of the (220), (311), (400), (511), and (440) peaks of Fe 3 O 4 and the (101), (004), and (200) peaks of anatase TiO 2 .Based on the SEM and EDS tests, the elemental compositions of the Fe 3 O 4 microspheres and the Fe 3 O 4 @TiO 2 composite microspheres were determined.The EDS mapping in Figure 2b illustrates the distribution of O, Fe, N, and C within the Fe 3 O 4 microspheres, providing a visual representation of their elemental presence.Moreover, the EDS of the Fe 3 O 4 @TiO 2 composite microspheres (Figure 2c) distinctly reveals the predominance of C, N, Ti, and Fe.The elemental analysis concludes that the Fe 3 O 4 composite microspheres contain O, Fe, N, and C. The presence of C may be attributed to the addition of glycerol and isopropanol during the hydrothermal reaction process, as well as its persistence in the form of C after annealing at 500 • C. The trace amount of nitrogen, N, is a result of the introduction of Fe(NO 3 ) 3 •6H 2 O.The contents of each element are illustrated in Table 1.Additionally, the Fe 3 O 4 @TiO 2 composite microspheres primarily comprise C, N, Ti, and Fe, indicating the chemical composition of the sample.Notably, titanium (Ti) was identified as a significant component within the composite microspheres, suggesting its potential existence as a TiO 2 coating.The zeta potential of Fe 3 O 4 and Fe 3 O 4 @TiO 2 was also tested, and a higher negative zeta potential of Fe 3 O 4 after TiO 2 coating was observed (3.72 mV versus 5.16 mV) (Figure 2d).The change in potential may be due to titanium dioxide coating the surface of ferric oxide.Based on the XPS spectra, the Ti 2p XPS spectrum displays prominent peaks at 464.18 eV (Ti 2p 1/2 ) and 458.48 eV (Ti 2p 3/2 ), indicative of the presence of Ti in the Ti 4+ oxidation state within the Fe 3 O 4 @TiO 2 microspheres (Figure 2i).Furthermore, the analysis of the Fe 3 O 4 @TiO 2 microspheres revealed distinct peaks at 709.57 and 723.20 eV for Fe 2p 3/2 and Fe 2p 1/2 of Fe 2+ , in addition to peaks at 715.4 eV and 728.1 eV corresponding to Fe 2p 3/2 and Fe 2p 1/2 of Fe 3+ (Figure 2h).These results confirm the coexistence of Fe 3 O 4 and TiO 2 within the Fe 3 O 4 @TiO 2 microspheres.The evaluation of ROS generation from Fe 3 O 4 @TiO 2 was performed using various chemical probes, as depicted in Figure 3a.Under UV irradiation, the production of 1 O 2 by Fe 3 O 4 @TiO 2 was assessed using DPBF (1,3-diphenylisobenzofuran).While the free DPBF exhibited no significant alteration in its UV-Vis absorbance spectra, Fe 3 O 4 @TiO 2 displayed a rapid decrease in DPBF absorption upon UV laser exposure, indicating a swift generation of 1 O 2 (as shown in Figure 3b,c).Furthermore, the observed colour changes of the methylene blue (MB) solution from blue to colourless over time suggested the effective generation of •OH by Fe 3 O 4 @TiO 2 (Figure 3d).This phenomenon was attributed to the Fenton-like effect arising from the interaction between released iron ions and H 2 O 2 .The study also uncovered pH-dependent enzymatic activities of Fe 3 O 4 .Under acidic conditions, it exhibited peroxidase activity, leading to the generation of highly toxic  [36][37][38].Moreover, an investigation using dissolved O 2 apparatus demonstrated that Fe 3 O 4 @TiO 2 possessed catalase-like properties at pH 7.4, promoting the decomposition of H 2 O 2 to produce O 2 , as illustrated in Figure 3e.In summary, the comprehensive findings highlight the versatile ROS-generating capabilities of Fe 3 O 4 @TiO 2 , emphasising its potential in the field of tumour therapy.Furthermore, its capacity to generate oxygen by decomposing H 2 O 2 adds significant value to its potential applications in various aspects of tumour treatment.
trum displays prominent peaks at 464.18 eV (Ti 2p1/2) and 458.48 eV (Ti 2p3/2), indicative of the presence of Ti in the Ti 4+ oxidation state within the Fe3O4@TiO2 microspheres (Figure 2i).Furthermore, the analysis of the Fe3O4@TiO2 microspheres revealed distinct peaks at 709.57 and 723.20 eV for Fe 2p3/2 and Fe 2p1/2 of Fe 2+ , in addition to peaks at 715.4 eV and 728.1 eV corresponding to Fe 2p3/2 and Fe 2p1/2 of Fe 3+ (Figure 2h).These results confirm the coexistence of Fe3O4 and TiO2 within the Fe3O4@TiO2 microspheres.4b).In the PTT experiments, it was observed that as the concentration of Fe 3 O 4 @TiO 2 microspheres increased, there was a substantial elevation in the temperature of the solution (Figure 4c).Further, the solution temperature rise was found to increase with the power of the laser beam (Figure 4d).The photothermal stability of Fe 3 O 4 @TiO 2 was further evaluated through several photothermal cycles.The evaluation after five ON/OFF laser cycles indicated the exceptional photothermal stability of Fe 3 O 4 @TiO 2 , showcasing negligible alterations in its performance (Figure 4e).Assessments using infrared thermal imaging displayed temperature increments upon exposure to NIR laser radiation.Figure 4f illustrates the gradual temperature increase in Fe 3 O 4 @TiO 2 (1 mg mL −1 , 1 W cm 2 ) when exposed to 808 laser irradiations.In comparison, Fe 3 O 4 demonstrated a temperature change of 35.3 • C, whereas Fe 3 O 4 @TiO 2 showed a temperature change of 29.2 • C under identical NIR irradiation conditions (Figure 4g).Moreover, the calculated photothermal conversion efficiencies of Fe 3 O 4 and Fe 3 O 4 @TiO 2 were 65.56% and 54.56%, respectively (Figure 4h).
an investigation using dissolved O2 apparatus demonstrated Fe3O4@TiO2 possessed catalase-like properties at pH 7.4, promoting the decomposition of H2O2 to produce O2, as illustrated in Figure 3e.In summary, the comprehensive findings highlight the versatile ROS-generating capabilities of Fe3O4@TiO2, emphasising its potential in the field of tumour therapy.Furthermore, its capacity to generate oxygen by decomposing H2O2 adds significant value to its potential applications in various aspects of tumour treatment.We then explored the photothermal performance (PTT) of Fe3O4@TiO2. Figure 4a depicts the absorption spectra of Fe3O4 and Fe3O4@TiO2 microspheres in the UV-visible range.Both Fe3O4 and Fe3O4@TiO2 microspheres exhibited excellent PTT, as shown in Fig- ure 4b.The TiO2 coating did not obviously weaken the photothermal effect of Fe3O4.The  4c).The reduction in magnetic saturation is attributed to the introduction of non-magnetic TiO 2 during synthesis.Additionally, the homogeneously dispersed Fe 3 O 4 @TiO 2 rapidly adhered to the vial walls, resulting in a clear and transparent solution upon the application of an external magnetic field (MF).Subsequently, upon removal of the magnets, the Fe 3 O 4 @TiO 2 microspheres uniformly dispersed again after gentle shaking, demonstrating their excellent water-dispersive capability.This magnetic responsiveness holds promise for potential applications in magnetically targeted tumour therapy.
N 2 adsorption-desorption isotherm analysis was carried out to measure the specific surface area and pore size distribution to discover the BET-specific surface area and the BJH pore size distribution for the as-synthesised Fe 3 O 4 @TiO 2 (Figure 5a,b).The Fe 3 O 4 @TiO 2 microspheres, with an extensive surface area measuring 84.611 m 2 /g and a pore size distribution within the range from 1 to 2 nm, serve as facilitating factors for promoting and enhancing the loading of drugs.The typical chemotherapeutic drug DOX was selected to evaluate Fe 3 O 4 @TiO 2 as a carrier.Compared to the Fourier infrared absorption spectrum of Fe 3 O 4 @TiO 2 , a red shift was noted in Fe 3 O 4 @TiO 2 /DOX, suggesting the effective incorporation of the chemotherapeutic drug DOX into Fe 3 O 4 @TiO 2 microspheres (Figure 5c).The concentration of DOX in the supernatant was calculated based on the fitting curve shown in Figure 5d.The LC of DOX in Fe 3 O 4 @TiO 2 is 32.2%.The LE of DOX in Fe 3 O 4 @TiO 2 is 85.6%.changes with varying laser power densities for a 1 mg mL −1 Fe3O4@TiO2 solution; (e) photostability test of Fe3O4@TiO2 solution during five cycles under 808 nm; (f) IR thermal photographs of saline and Fe3O4@TiO2 under NIR laser irradiation; (g) photothermal curves of Fe3O4 and Fe3O4@TiO2; (h) photothermal conversion efficiency of Fe3O4 and Fe3O4@TiO2; (i) magnetic hysteresis loops of the Fe3O4 and Fe3O4@TiO2 microspheres (inset of top shows coercive field values (Hc) of samples and inset of the bo om shows their suspensions before and after magnetic separation using an external magnet.
N2 adsorption-desorption isotherm analysis was carried out to measure the specific surface area and pore size distribution to discover the BET-specific surface area and the BJH pore size distribution for the as-synthesised Fe3O4@TiO2 (Figure 5a,b).The Fe3O4@TiO2 microspheres, with an extensive surface area measuring 84.611 m 2 /g and a pore size distribution within the range from 1 to 2 nm, serve as facilitating factors for promoting and enhancing the loading of drugs.The typical chemotherapeutic drug DOX was selected to evaluate Fe3O4@TiO2 as a carrier.Compared to the Fourier infrared absorption spectrum of Fe3O4@TiO2, a red shift was noted in Fe3O4@TiO2/DOX, suggesting the effective incorporation of the chemotherapeutic drug DOX into Fe3O4@TiO2 microspheres (Figure 5c).The concentration of DOX in the supernatant was calculated based on the fi ing curve shown in Figure 5d.The LC of DOX in Fe3O4@TiO2 is 32.2%.The LE of DOX in Fe3O4@TiO2 is 85.6%.Therefore, Fe 3 O 4 @TiO 2 /DOX is shown to be a promising chemo-photothermal nanocarrier due to its capacity for controlled drug release responsive to dual stimuli (pH and NIR).The Fe 3 O 4 @TiO 2 /DOX microspheres demonstrate both pH-responsive and photoresponsive release properties.Lowering the pH from 7.4 to 5.4 notably increases the rate and quantity of DOX release, with a significant difference observed between pH 5.5 and pH 7.4, releasing 42% and 19% of DOX, respectively (Figure 5e).NIR irradiation further accelerates the overall release rate of DOX (Figure 5f).Therefore, Fe 3 O 4 @TiO 2 /DOX is a promising chemo-photothermal nanocarrier due to its capacity for controlled drug release and its response to dual stimuli (pH and NIR).
Figure 6 depicts CLSM images of HeLa cells incubated with Fe 3 O 4 @TiO 2 -FTTC microspheres for 8 h, showcasing distinct blue fluorescence from DAPI staining for cell nuclei and green fluorescence from labelled FITC.The Fe 3 O 4 @TiO 2 was magnetically manipulated by the external magnetic field, showing an obvious green fluorescence signal overlapping with the blue fluorescence signal.The Fe 3 O 4 @TiO 2 group without magnetic field loading exhibited a weak FITC fluorescence signal at 8 h co-incubation.The results presented in Figure 6 demonstrate that the application of a magnetic field enhances the cellular uptake of the Fe 3 O 4 @TiO 2 .
and NIR).The Fe3O4@TiO2/DOX microspheres demonstrate both pH-responsive and photo-responsive release properties.Lowering the pH from 7.4 to 5.4 notably increases the rate and quantity of DOX release, with a significant difference observed between pH 5.5 and pH 7.4, releasing 42% and 19% of DOX, respectively (Figure 5e).NIR irradiation further accelerates the overall release rate of DOX (Figure 5f).Therefore, Fe3O4@TiO2/DOX is a promising chemo-photothermal nanocarrier due to its capacity for controlled drug release and its response to dual stimuli (pH and NIR). Figure 6 depicts CLSM images of HeLa cells incubated with Fe3O4@TiO2-FTTC microspheres for 8 h, showcasing distinct blue fluorescence from DAPI staining for cell nuclei and green fluorescence from labelled FITC.The Fe3O4@TiO2 was magnetically manipulated by the external magnetic field, showing an obvious green fluorescence signal overlapping with the blue fluorescence signal.The Fe3O4@TiO2 group without magnetic field loading exhibited a weak FITC fluorescence signal at 8 h co-incubation.The results presented in Figure 6 demonstrate that the application of a magnetic field enhances the cellular uptake of the Fe3O4@TiO2.The cytotoxicity assessment of Fe3O4@TiO2 was conducted via a CCK-8 assay, involving various concentrations and times of co-culture with HeLa cells.As shown in Figure 7a, even at the highest concentration, the cell viability was more than 90%.This observation signifies the notable biocompatibility of Fe3O4@TiO2, suggesting its potential as a safe and efficient drug delivery platform.
Photothermal therapy experiments were performed on HeLa cells to assess the effec- The cytotoxicity assessment of Fe 3 O 4 @TiO 2 was conducted via a CCK-8 assay, involving various concentrations and times of co-culture with HeLa cells.As shown in Figure 7a, even at the highest concentration, the cell viability was more than 90%.This observation signifies the notable biocompatibility of Fe 3 O 4 @TiO 2 , suggesting its potential as a safe and efficient drug delivery platform.
+ PDT + CDT + chemotherapy group (Fe3O4@TiO2/DOX + UV + H2O2 + 808 nm), their survival rate notably decreased to 7.8%.This rate was lower than that of the PDT group (Fe3O4@TiO2/DOX + UV), CDT group (Fe3O4@TiO2/DOX + H2O2), and PTT group (Fe3O4@TiO2/DOX + 808 nm).Therefore, the construction of Fe3O4@TiO2/DOX microspheres is capable of achieving the combination treatment of PTT, PDT, CDT, and chemotherapy.As depicted in Figure 7c, the cytotoxic effect of the Fe3O4@TiO2/DOX + 808 + H2O2 group on HeLa cells closely approximates that of the free DOX group.These findings underscore the potential advantages of multimodal therapeutic strategies in cancer treatment.To verify the effects of the H2O2, UV, and 808 nm laser used in the experiment on cells, cells were treated with 100 µM H2O2, UV, and 808 nm lasers, and their survival rates were measured using a CCK-8 assay (Figure 7d).Here, we used an H2O2 solution, 808 nm laser, and UV laser irradiation to treat tumours in vitro and analysed the onset of immunogenic cell death (ICD).The immunofluorescence staining revealed bright green fluorescence (FL) on the membrane of the HeLa cells treated with Fe3O4@TiO2 + 808 nm, Fe3O4@TiO2 + UV, Fe3O4@TiO2 + H2O2, and Fe3O4@TiO2 + H2O2 + 808 nm + UV laser irradiation, indicating the induction of the ICD effect from the combination therapy (Figure 8a).
To examine the generation of ROS in HeLa cells following different treatments, we conducted DCFH-DA staining assays.Increased green fluorescence was observed in UVirradiated cells, indicating ROS formation (Figure 8b).The H2O2-treated HeLa cells Photothermal therapy experiments were performed on HeLa cells to assess the effectiveness of Fe 3 O 4 @TiO 2 in inducing cellular heat using light.The cell viability decreased to 48.99% after 5 min with a 1.5 W/cm 2 NIR laser (Figure 7b).Thus, Fe 3 O 4 @TiO 2 may have good application prospects for photothermal therapy.Next, we studied the PDT effects of Fe 3 O 4 @TiO 2 on HeLa cells in vitro.After 30 min of ultraviolet (UV) light exposure, the cell viability notably decreased from 81.06% to 52.24%, demonstrating the remarkable performance of the material as a photosensitiser.Given the H 2 O 2 concentration in tumour tissues ranging from 50 to 100 µM [39][40][41], a concentration of 100 µM of H 2 O 2 was chosen for subsequent assays related to CDT.As shown in Figure 7b, HeLa cells with exposure to Fe 3 O 4 @TiO 2 + H 2 O 2 exposited a significant decrease in viability compared to that of the Fe 3 O 4 @TiO 2 group cells.Moreover, a CCK-8 assay was conducted to assess the combined therapeutic efficacy of Fe 3 O 4 @TiO 2 /DOX, capable of combining PTT, PDT, CDT, and chemotherapy to target tumour cells.The in vitro experiments conducted on HeLa cells using Fe 3 O 4 @TiO 2 /DOX demonstrated exceptional efficiency in tumour cell elimination.When HeLa cells treated with Fe 3 O 4 @TiO 2 /DOX (1 mg mL −1 ) were exposed to the combined PTT + PDT + CDT + chemotherapy group (Fe 3 O 4 @TiO 2 /DOX + UV + H 2 O 2 + 808 nm), their survival rate notably decreased to 7.8%.This rate was lower than that of the PDT group (Fe 3 O 4 @TiO 2 /DOX + UV), CDT group (Fe 3 O 4 @TiO 2 /DOX + H 2 O 2 ), and PTT group (Fe 3 O 4 @TiO 2 /DOX + 808 nm).Therefore, the construction of Fe 3 O 4 @TiO 2 /DOX microspheres is capable of achieving the combination treatment of PTT, PDT, CDT, and chemotherapy.As depicted in Figure 7c, the cytotoxic effect of the Fe 3 O 4 @TiO 2 /DOX + 808 + H 2 O 2 group on HeLa cells closely approximates that of the free DOX group.These findings underscore the potential advantages of multimodal therapeutic strategies in cancer treatment.To verify the effects of the H 2 O 2 , UV, and 808 nm laser used in the experiment on cells, cells were treated with 100 µM H 2 O 2 , UV, and 808 nm lasers, and their survival rates were measured using a CCK-8 assay (Figure 7d).
Here, we used an H 2 O 2 solution, 808 nm laser, and UV laser irradiation to treat tumours in vitro and analysed the onset of immunogenic cell death (ICD).The immunofluorescence staining revealed bright green fluorescence (FL) on the membrane of the HeLa cells treated with Fe 3 O @TiO 2 + 808 nm, Fe 3 O 4 @TiO 2 + UV, Fe 3 O 4 @TiO 2 + H 2 O 2 , and Fe 3 O 4 @TiO 2 + H 2 O 2 + 808 nm + UV laser irradiation, indicating the induction of the ICD effect from the combination therapy (Figure 8a).

Conclusions
In this study, we explored a smart Fe3O4@TiO2/DOX drug delivery platform for the PTT/PDT/chemotherapy/CDT combination treatment of cancer.The Fe3O4@TiO2/DOX microspheres demonstrated pH and NIR dual-responsive drug release behaviour in vitro.In addition, it has been proven that Fe3O4@TiO2 has superior biocompatibility.Fe 2+ serves as a catalyst in the Fenton reaction, leading to the generation of •OH from H2O2 to facilitate CDT.Moreover, Fe3O4@TiO2 has been shown to catalyse the conversion of H2O2 into O2 within the cytoplasm, consequently enhancing the efficacy of PDT.In drug targeting systems, magnetically sensitive Fe3O4@TiO2/DOX containing medications can accurately accumulate at the tumour site with the help of an external magnetic field.Therefore, Fe3O4@TiO2/DOX has significant potential for the combination therapy of tumours.
An 808 nm laser possesses excellent tissue penetration, capable of penetrating several millimetres to centimetres into human tissues.UV light can penetrate through the epidermis into the dermis [42].Both wavelengths can be applied to melanoma, taking advantage of their ability to reach the targeted tissues effectively.Additionally, Fe3O4@TiO2/DOX microspheres with a diameter of approximately 1 µm can be employed for interventional therapy in cancer.

Conclusions
In this study, we explored a smart Fe 3 O 4 @TiO 2 /DOX drug delivery platform for the PTT/PDT/chemotherapy/CDT combination treatment of cancer.The Fe 3 O 4 @TiO 2 /DOX microspheres demonstrated pH and NIR dual-responsive drug release behaviour in vitro.In addition, it has been proven that Fe 3 O 4 @TiO 2 has superior biocompatibility.Fe 2+ serves as a catalyst in the Fenton reaction, leading to the generation of •OH from H 2 O 2 to facilitate CDT.Moreover, Fe 3 O 4 @TiO 2 has been shown to catalyse the conversion of H 2 O 2 into O 2 within the cytoplasm, consequently enhancing the efficacy of PDT.In drug targeting systems, magnetically sensitive Fe 3 O 4 @TiO 2 /DOX containing medications can accurately accumulate at the tumour site with the help of an external magnetic field.Therefore, Fe 3 O 4 @TiO 2 /DOX has significant potential for the combination therapy of tumours.
An 808 nm laser possesses excellent tissue penetration, capable of penetrating several millimetres to centimetres into human tissues.UV light can penetrate through the epidermis into the dermis Both wavelengths can be applied to melanoma, taking advantage of their ability to reach the targeted tissues effectively.Additionally, Fe 3 O 4 @TiO 2 /DOX microspheres with a diameter of approximately 1 µm can be employed for interventional therapy in cancer.

Figure 2 .
Figure 2. (a) XRD pattern of comparison between Fe 3 O 4 @TiO 2 and TiO 2 ; EDS mapping pictures of (b) Fe 3 O 4 and (c) Fe 3 O 4 microspheres.Scale bar = 200 nm; (d) zeta potential spectra of Fe 3 O 4 and Fe 3 O 4 @TiO 2 .(e,f) XPS spectra of Fe O 4 @TiO 2 composite; (g-i) XPS spectra of Fe 3 O 4 @TiO 2 composite.We then explored the photothermal performance (PTT) of Fe 3 O 4 @TiO 2 .Figure4adepicts the absorption spectra of Fe 3 O 4 and Fe 3 O 4 @TiO 2 microspheres in the UV-visible range.Both Fe 3 O 4 and Fe 3 O 4 @TiO 2 microspheres exhibited excellent PTT, as shown in Figure4b.The TiO 2 coating did not obviously weaken the photothermal effect of Fe 3 O 4 .The temperature increased to 60 • C and 65 • C after 5 min of irradiation for the Fe 3 O 4 and Fe 3 O 4 @TiO 2 suspension, respectively (Figure4b).In the PTT experiments, it was observed that as the concentration of Fe 3 O 4 @TiO 2 microspheres increased, there was a substantial elevation in the temperature of the solution (Figure4c).Further, the solution temperature rise was found to increase with the power of the laser beam (Figure4d).The photothermal stability of Fe 3 O 4 @TiO 2 was further evaluated through several photothermal cycles.The evaluation after five ON/OFF laser cycles indicated the exceptional photothermal stability of Fe 3 O 4 @TiO 2 , showcasing negligible alterations in its performance (Figure4e).Assessments using infrared thermal imaging displayed temperature increments upon exposure to NIR laser radiation.Figure4fillustrates the gradual temperature increase in Fe 3 O 4 @TiO 2 (1 mg mL −1 , 1 W cm 2 ) when exposed to 808 laser irradiations.In comparison, Fe 3 O 4 demonstrated a temperature change of 35.3 • C, whereas Fe 3 O 4 @TiO 2 showed a temperature change of 29.2 • C under identical NIR irradiation conditions (Figure4g).Moreover, the calculated photothermal conversion efficiencies of Fe 3 O 4 and Fe 3 O 4 @TiO 2 were 65.56% and 54.56%, respectively (Figure4h).

Figure 3 .
Figure 3. (a) ROS generation of the as-synthesised Fe3O4@TiO2; (b,c) the time-dependent degradation of DPBF was investigated under UV light irradiation in the presence and absence of Fe3O4@TiO2; (d) time-dependent degradation of MB in the presence of Fe3O4@TiO2 and H2O2; (e) changes in the dissolved oxygen levels were monitored for a 2 min duration in a pH 7.4 environment, catalysed by Fe3O4@TiO2 in a solution containing H2O2 (500 µM).

Figure 3 .
Figure 3. (a) ROS generation of the as-synthesised Fe 3 O 4 @TiO 2 ; (b,c) the time-dependent degradation of DPBF was investigated under UV light irradiation in the presence and absence of Fe 3 O 4 @TiO 2 ; (d) time-dependent degradation of MB in the presence of Fe 3 O 4 @TiO 2 and H 2 O 2 ; (e) changes in the dissolved oxygen levels were monitored for a 2 min duration in a pH 7.4 environment, catalysed by Fe 3 O 4 @TiO 2 in a solution containing H 2 O 2 (500 µM).The saturation magnetisation (MS) and coercive field (Hc) at room temperature are presented in Figure 4i.Both materials exhibit typical superparamagnetic characteristics.The MS and Hc values for Fe 3 O 4 are 23.1 emu/g and 58.75 Oe, respectively, while those for Fe 3 O 4 @TiO 2 are 4.45 emu/g and 55 Oe (Figure4c).The reduction in magnetic saturation is attributed to the introduction of non-magnetic TiO 2 during synthesis.Additionally, the homogeneously dispersed Fe 3 O 4 @TiO 2 rapidly adhered to the vial walls, resulting in a clear and transparent solution upon the application of an external magnetic field (MF).Subsequently, upon removal of the magnets, the Fe 3 O 4 @TiO 2 microspheres uniformly dispersed again after gentle shaking, demonstrating their excellent water-dispersive capability.This magnetic responsiveness holds promise for potential applications in magnetically targeted tumour therapy.N 2 adsorption-desorption isotherm analysis was carried out to measure the specific surface area and pore size distribution to discover the BET-specific surface area and the BJH pore size distribution for the as-synthesised Fe 3 O 4 @TiO 2 (Figure5a,b).The Fe 3 O 4 @TiO 2 microspheres, with an extensive surface area measuring 84.611 m 2 /g and a pore size distribution within the range from 1 to 2 nm, serve as facilitating factors for promoting and enhancing the loading of drugs.The typical chemotherapeutic drug DOX was selected to evaluate Fe 3 O 4 @TiO 2 as a carrier.Compared to the Fourier infrared absorption spectrum of Fe 3 O 4 @TiO 2 , a red shift was noted in Fe 3 O 4 @TiO 2 /DOX, suggesting the effective incorporation of the chemotherapeutic drug DOX into Fe 3 O 4 @TiO 2 microspheres (Figure5c).The concentration of DOX in the supernatant was calculated based on the fitting curve shown in Figure5d.The LC of DOX in Fe 3 O 4 @TiO 2 is 32.2%.The LE of DOX in Fe 3 O 4 @TiO 2 is 85.6%.

Figure 4 .
Figure 4. Photothermal effects of Fe3O4@TiO2.(a) UV-vis absorption spectra of Fe3O4 and Fe3O4@TiO2 sample; (b) temperature variations for Fe3O4 and Fe3O4@TiO2 samples; (c) temperature responses at different Fe3O4@TiO2 concentrations under 808 nm NIR irradiation; (d) temperaturechanges with varying laser power densities for a 1 mg mL −1 Fe3O4@TiO2 solution; (e) photostability test of Fe3O4@TiO2 solution during five cycles under 808 nm; (f) IR thermal photographs of saline and Fe3O4@TiO2 under NIR laser irradiation; (g) photothermal curves of Fe3O4 and Fe3O4@TiO2; (h) photothermal conversion efficiency of Fe3O4 and Fe3O4@TiO2; (i) magnetic hysteresis loops of the Fe3O4 and Fe3O4@TiO2 microspheres (inset of top shows coercive field values (Hc) of samples and inset of the bo om shows their suspensions before and after magnetic separation using an external magnet.

Figure 4 .
Figure 4. Photothermal effects of Fe 3 O 4 @TiO 2 .(a) UV-vis absorption spectra of Fe 3 O 4 and Fe 3 O 4 @TiO 2 sample; (b) temperature variations for Fe 3 O 4 and Fe 3 O 4 @TiO 2 samples; (c) temperature responses at different Fe 3 O 4 @TiO 2 concentrations under 808 nm NIR irradiation; (d) temperature changes with varying laser power densities for a 1 mg mL −1 Fe 3 O 4 @TiO 2 solution; (e) photostability test of Fe 3 O 4 @TiO 2 solution during five cycles under 808 nm; (f) IR thermal photographs of saline and Fe 3 O 4 @TiO 2 under NIR laser irradiation; (g) photothermal curves of Fe 3 O 4 and Fe 3 O 4 @TiO 2 ; (h) photothermal conversion efficiency of Fe 3 O 4 and Fe 3 O 4 @TiO 2 ; (i) magnetic hysteresis loops of the Fe 3 O 4 and Fe 3 O 4 @TiO 2 microspheres (inset of top shows coercive field values (Hc) of samples and inset of the bottom shows their suspensions before and after magnetic separation using an external magnet.

Nanomaterials 2024 ,
14, x FOR PEER REVIEW 14 of 16 exhibited notably elevated fluorescence intensity compared to the control, a ributed to H2O2 conversion into •OH radicals via Fe 2+ catalysis in the Fenton reaction.Particularly noteworthy was the substantial increase in green fluorescence in the Fe3O4@TiO2 + H2O2 + 808 + UV-treated cells, indicating a remarkable rise in cellular ROS production.This outcome underscores the combined impact of CDT and PDT, which collectively amplified ROS generation significantly beyond what PDT alone could achieve.

Figure 8 .
Figure 8.(a) FL imaging of CRT exposure on the membrane of Hela cells upon indicate treatment.Green: CRT; blue: nucleus.Scale bar: 150 µm; (b) FL imaging of intracellular Fe3O4@TiO2 and ROS (green) in Hela cells after indicated treatments.Scale bar: 100 µm.

Figure 8 .
Figure 8.(a) FL imaging of CRT exposure on the membrane of Hela cells upon indicate treatment.Green: CRT; blue: nucleus.Scale bar: 150 µm; (b) FL imaging of intracellular Fe 3 O 4 @TiO 2 and ROS (green) in Hela cells after indicated treatments.Scale bar: 100 µm.To examine the generation of ROS in HeLa cells following different treatments, we conducted DCFH-DA staining assays.Increased green fluorescence was observed in UV-irradiated cells, indicating ROS formation (Figure 8b).The H 2 O 2 -treated HeLa cells exhibited notably elevated fluorescence intensity compared to the control, attributed to H 2 O 2 conversion into •OH radicals via Fe 2+ catalysis in the Fenton reaction.Particularly noteworthy was the substantial increase in green fluorescence in the Fe 3 O 4 @TiO 2 + H 2 O 2 + 808 + UV-treated cells, indicating a remarkable rise in cellular ROS production.This outcome underscores the combined impact of CDT and PDT, which collectively amplified ROS generation significantly beyond what PDT alone could achieve.

Table 1 .
T Atomic mass of individual elements in samples Fe 3 O 4 and Fe 3 O 4 @TiO 2 .
•OH after reacting with H 2 O 2 .Conversely, under neutral conditions, it displayed catalase-like activity, facilitating the decomposition of H 2 O 2 into harmless H 2 O and O 2

Table 1 .
T Atomic mass of individual elements in samples Fe3O4 and Fe3O4@TiO2.