Construction of Multifunctional Fe₃O₄@MSN@PDA-HA-FA Nanocarriers and Research on Synergistic Tumor Therapy

Abstract

Background: Chemodynamic therapy (CDT) and photothermal therapy (PTT) based on nanomaterials have garnered widespread attention in cancer treatment. However, most single-modal nanotherapeutics suffer from limited therapeutic efficacy. Methods: Herein, a magnetic mesoporous composite nanoparticle, Fe₃O₄@MSN@PDA-HA-FA, is successfully fabricated, with Fe₃O₄ nanoparticles as the magnetic core; mesoporous silica nanoparticles (MSNs) as the mesoporous shell; and dopamine hydrochloride (DA·HCl), hyaluronic acid (HA), and folic acid (FA) as the functional ligands. Results: Notably, this composite serves as both an efficient photothermal converter and a chemodynamic promoter, enhancing hydroxyl radical (·OH) generation and improving PTT efficacy. Under near-infrared (NIR) light irradiation, Fe₃O₄@MSN@PDA-HA-FA exhibits high photothermal conversion and heat transfer efficiencies. The Fe²⁺ ions in Fe₃O₄ enable a Fenton reaction-mediated conversion of endogenous hydrogen peroxide (H₂O₂) into ·OH for CDT. Additionally, the MSNs provide a substantial drug-loading capacity, while the HA and FA provide additional surface functionalities that can modulate the nano-bio interactions and improve colloidal stability. Conclusions: In vitro experiments validate the synergistic therapeutic efficacy of PTT, CDT, and chemotherapy. This study demonstrates that Fe₃O₄@MSN@PDA-HA-FA exhibits antitumor efficacy, laying a promising foundation for its potential clinical translation in cancer treatment.

1. Introduction

Cancer remains one of the leading causes of mortality globally [1]. Conventional clinical treatments, including surgical resection, chemotherapy, and radiotherapy, can temporarily suppress tumor growth and metastasis [2], yet they are associated with severe adverse reactions that significantly undermine patients’ quality of life. Over the past few years, photothermal therapy (PTT) has evolved into an effective therapeutic alternative, which eradicates tumor cells via localized thermal ablation [3,4]. Specifically, in PTT, photothermal nanoparticles accumulate at tumor sites, absorb tissue-penetrating near-infrared (NIR) radiation, and transform it into thermal energy, thereby elevating the local temperature above 45 °C to induce tumor cell apoptosis [5]. Compared with traditional therapies, PTT offers lower systemic toxicity, making it a promising cancer treatment strategy. Representative photothermal materials include graphene, gold nanoparticles, organic small molecules, conjugated polymers (e.g., polypyrrole, indocyanine green), and semiconductor materials such as copper sulfide [6,7]. Among these, polydopamine (PDA) exhibits strong NIR absorption and higher photothermal conversion efficiency than many inorganic metal-based materials. As a biomimetic component of human melanin, PDA also possesses excellent biosafety and biocompatibility [8,9], rendering it a highly attractive candidate for tumor PTT. Nevertheless, the efficient enrichment of PDA-based nanomaterials at tumor sites remains a challenge, limiting their therapeutic performance. Thus, the rational design of tumor microenvironment (TME)-responsive nanomaterials has become a key research direction in current tumor therapy.

Hyaluronic acid (HA) is used in the medical and biomedical fields due to its excellent biocompatibility [10]. HA possesses abundant carboxyl and hydroxyl groups, which enable it to be easily conjugated to PDA [11,12,13,14,15,16,17]. Folic acid (FA) is extensively applied as a commonly ligand, with its capacity to act on diverse cancer cells in contrast to the negligible response in normal tissues [18,19,20,21]. Compared with normal tissues, tumor cells exhibit a distinct intracellular microenvironment characterized by a mildly acidic pH (elevated H⁺ concentration) and high hydrogen peroxide (H₂O₂) levels, resulting from aberrant metabolic activity [22,23,24]. Nanomaterials with peroxidase (POD)-like catalytic activity can decompose H₂O₂ to generate hydroxyl radicals (·OH) within tumor cells, inducing lethal oxidative damage [25,26]. Their tumor-specific biochemical features can be harnessed to drive intracellular catalytic reactions. This has led to the development of chemodynamic Therapy (CDT), a novel antitumor strategy. In CDT, transition metal ions (e.g., Mn, Fe, Co, Cu) catalyze Fenton or Fenton-like reactions [27,28]. These reactions produce ·OH. To construct efficient CDT platforms, nanomaterials with high specific surface areas are desirable for providing abundant active sites and enhanced drug-loading capacity [29]. Mesoporous silica nanoparticles (MSNs), with their ordered mesoporous architecture, substantial surface area, and excellent biocompatibility, have become a widely investigated carrier and multifunctional platform for tumor therapy [30].

In this study, Fe₃O₄ nanoparticles containing Fe²⁺ ions are integrated into a nanotherapeutic platform to achieve efficient nanocatalytic CDT [31]. Fe₃O₄ endows the system with magnetic capability, and are further coated with a mesoporous SiO₂ shell for drug loading [32,33,34]. Meanwhile, the reductive nature of PDA can continuously convert Fe³⁺ back into Fe²⁺ during storage and CDT, further enhancing the Fenton catalytic activity of Fe₃O₄@MSN@PDA (Scheme 1). Additionally, PDA’s strong photothermal conversion ability imparts robust PTT performance to the platform [35,36]. Further modification of PDA with HA and FA enhances the surface functionality of the nanoparticles.

2. Materials and Methods

2.1. Materials

Iron(III) chloride hexahydrate (FeCl₃·6H₂O), sodium acetate (NaAc), calcium fluoride (CaF₂), triethanolamine (TEA), ethylene glycol (EG), ethanol (EtOH), 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB), poly(4-styrene sulfonic acid-co-maleic acid) sodium salt (PSSMA, M_w ≈ 20,000), tetraethoxysilane (TEOS), DA·HCl, phosphate-buffered saline (PBS), hydrochloric acid (HCl), HA (M_w = 30,000–50,000), and FA were purchased from Titan Technology Exploration Platform (Shanghai, China). FS-66 was purchased from Sigma-Aldrich (St. Louis, MO, USA). HT29 cells were obtained from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Blood was provided free of charge by Changhai Hospital (Shanghai, China).

2.2. Preparation of Fe₃O₄

Uniformly shaped Fe₃O₄ nanospheres were successfully prepared via a hydrothermal method. Specifically, FeCl₃·6H₂O (3.7 mM), NaAc (36.6 mM), PSSMA (0.5 g), and CaF₂ (0.4 mM) were dissolved in EG (80 mL). The mixture was vigorously stirred at 25 °C for 3 h to ensure full dissolution of reagents and formation of a homogeneous precursor. Afterwards, it was placed into a 100 mL high-pressure reactor, with the temperature set at 200 °C and a reaction time of 12 h. Upon completion of the reaction, the reactor was cooled to 25 °C to avoid product structural damage from sudden temperature changes. The black Fe₃O₄ nanospheres were centrifuged, washed, and dried sequentially, followed by storage in a 4 °C refrigerator for subsequent characterization and application studies.

2.3. Preparation of Fe₃O₄@MSN

First, ethanol-dispersed Fe₃O₄ nanospheres were added into 50 mL of water containing a NaF (2 mM) solution. Then, TEOS was added dropwise. After the reaction finished, the product was washed to remove the unreacted NaF, excess TEOS, and other impurities. Then, the treated product was redispersed into an ethanol solution and subjected to 15 min of ultrasonic treatment to ensure uniform particle dispersion. Subsequently, 5 mL of TEA, TEOS, and the anionic fluorosurfactant FS-66 (0.1 g) were added sequentially, and the reaction was stirred for 6 h. The residual FS-66 was removed by repeated washing with ethanol and water. Through these steps, the Fe₃O₄@MSN magnetic mesoporous silica nanocomposite was successfully prepared.

2.4. Preparation of Fe₃O₄@MSN@PDA-HA-FA

DA·HCl (1 mM) and Fe₃O₄@MSN (200 mg) were dissolved in Tris-HCl (50 mL, pH 8.5) for 6 h to allow for the oxidative self-polymerization of dopamine and the formation of a PDA coating. HA (100 mg) was then added and stirred for another 6 h (400 rpm) to immobilize the HA onto the PDA layer via noncovalent interactions. Subsequently, FA (50 mg) was added and stirred for 6 h (400 rpm). The resulting Fe₃O₄@MSN@PDA-HA-FA nanoparticles were collected by magnetic separation, washed repeatedly to remove unbound species, and dried for storage.

2.5. Characterization

To test the synthesized nanoparticles, we first used a scanning electron microscope (SEM, JEOL JSM-IT500HR, JEOL, Tokyo, Japan) and transmission electron microscope (TEM, Thermo Talos F200X G2, Thermo Fisher Scientific, Waltham, MA, USA) to observe their morphological characteristics and particle size. Then we used an X-ray energy-dispersive spectrometer (EDS, X-MAX-20mm2, Oxford Instruments, Abingdon, UK) to analyze the elemental composition of the nanoparticles. Finally, the synthesis of the nanoparticles was verified via a Fourier transform infrared spectrometer (FTIR, Thermo Scientific Nicolet iS 50, Thermo Fisher Scientific, Waltham, MA, USA) and an X-ray diffraction (XRD) analyzer (SmartLab 9 kw, Rigaku, Tokyo, Japan). Additionally, the absorbance at a specific absorption wavelength was measured by a UV-vis spectrophotometer (Shimadzu, Tokyo, Japan). A NanoZS ZEN3600 instrument (Malvern Instruments, Malvern, UK) was used to determine the dynamic light scattering (DLS) diameter and zeta potential of the nanoparticles. The mass proportion of each component in the nanoparticles was determined by means of a thermogravimetric analyzer (TGA, Shimadzu, Tokyo, Japan). The nitrogen adsorption–desorption isotherms were measured at 77 K on a TriStar II 3020 instrument (Micromeritics Instrument, Norcross, GA, USA). The samples were degassed under a vacuum prior to measurement. The specific surface area was calculated by the Brunauer–Emmett–Teller (BET) method using the adsorption branch in the relative pressure range P/P₀ = 0.05–0.30. The pore size distribution was obtained by the BJH method. For the magnetic properties, the nanoparticles were characterized using a PPMS-9 system from Quantum Design (Qdusa, San Diego, CA, USA). For the photothermal conversion capability, the nanoparticles were evaluated using a laser system provided by Shanghai Connor Fiber Co., Ltd. (Shanghai, China).

2.6. Fenton Reaction Tests

To evaluate the impact of H₂O₂ concentration, a mixed solution composed of Fe₃O₄@MSN@PDA-HA-FA (300 μg/mL) and TMB (0.8 mM) was initially prepared. Then, H₂O₂ solutions of different concentrations (10 μM, 40 μM, 70 μM, and 100 μM) were added to this mixture separately. Finally, the reaction was allowed to proceed for 5 min, after which the absorbance value at a wavelength of 652 nm was continuously recorded for 10 min. To evaluate the impact of Fe₃O₄@MSN@PDA-HA-FA concentration, a mixed solution composed of H₂O₂ (40 μM) and TMB (0.8 mM) was first prepared. Subsequently, Fe₃O₄@MSN@PDA-HA-FA samples with varying concentrations (50 μg/mL, 100 μg/mL, 200 μg/mL, and 300 μg/mL) were added sequentially to the aforementioned mixed solution. Subsequently, the reaction was allowed to proceed for 5 min, following which the absorbance at 652 nm was measured over a duration of 10 min. Reaction-rate curves at varying H₂O₂ concentrations were constructed according to the Michaelis–Menten model, as shown in Equation (1).

$$\upsilon ={\displaystyle \frac{V_{\mathit{max}}\left[S\right]}{K_{m }+\left[S\right]}}$$

(1)

In addition, the kinetic parameters were also obtained by a Lineweaver–Burk linearization for comparison, as shown in Equation (2).

$${\displaystyle \frac{1}{\upsilon }}={\displaystyle \frac{K_m}{V_{\mathit{max}}}}·{\displaystyle \frac{1}{\left[S\right]}}+{\displaystyle \frac{1}{V_{\mathit{max}}}}$$

(2)

where V_max is the maximum reaction velocity, K_m is the Michaelis constant, [S] stands for the H₂O₂ concentration, and $\upsilon$ is the initial reaction rate.

2.7. Photothermal Property Tests

To evaluate the photothermal stability of the Fe₃O₄@MSN@PDA-HA-FA nanoparticles: Use an 808 nm laser (with a power density of 1 W/cm²) to perform the irradiation. Conduct eight cycles of “on-off” operation (on: 300 s; off: 300 s) during irradiation, and evaluate the stability under intermittent irradiation based on the changes in the photothermal performance across these cycles. For the photothermal tests that depend on the concentration: First, prepare Fe₃O₄@MSN@PDA-HA-FA solutions with four different concentrations, which are 200 μg/mL, 500 μg/mL, 800 μg/mL, and 1000 μg/mL, respectively. Then, dispense 200 μL of each concentration solution into 96-well plates. After that, expose each well to an 808 nm laser (with a power density of 1 W/cm²) for a total of 300 s. For the photothermal tests that depend on power: First, take the Fe₃O₄@MSN@PDA-HA-FA solution with a fixed concentration of 1000 μg/mL. Then, measure out 100 μL of this solution each time. Next, irradiate the 100 μL solution with 808 nm lasers that have different power densities (0.2, 0.5, 0.8, and 1.0 W/cm², respectively). Each irradiation process lasts for 300 s. To determine the photothermal conversion efficiency, irradiate the Fe₃O₄@MSN@PDA-HA-FA dispersion (200 μg/mL, 200 μL) with an 808 nm laser at 1 W/cm² for 5 min. The temperature is monitored using an infrared thermal imaging camera. After irradiation, the laser is turned off and the sample is allowed to cool naturally for 5 min, during which the cooling curve is continuously recorded. A water sample with the same volume is measured under identical conditions for background correction. The following Equation (3) is used to calculate the photothermal conversion efficiency (η):

$$\eta (\%) = {\displaystyle \frac{hs \left(T_{max} - T_{sur}\right) - Q_{dis}}{I (1- {10}^{-A})}}$$

(3)

where h is the heat transfer coefficient, s represents the surface area of the container, I stands for the laser power, and A refers to the absorbance. Meanwhile, T_max is the maximum temperature of the sample (°C), and T_sur is the environmental temperature (°C). Q_dis refers to the heat variation during reagent blanking (J·S⁻¹). Note that a PBS blank curve was not acquired; therefore, background heating was estimated using a water blank (Q_dis), and η should be interpreted as an approximate value.

The product value of $\mathrm{hS}$ can be calculated by the following Equation (4):

$$\mathit{hS} = {\displaystyle \frac{m · C_{H2O}}{{\tau }_s}}$$

(4)

where m is the material mass (g); C_H2O is the specific heat capacity of water in the control group (4.18 J/(g·°C)); and ${\mathsf{\tau }}_s$ is the system time constant, which is calculated as follows using Equation (5):

$$t=-{\tau }_s\mathit{ln}\theta =-{\tau }_s \mathit{ln}{\displaystyle \frac{T-T_{\mathit{sur}}}{T_{\mathit{max}}-T_{\mathit{sur}}}}$$

(5)

Here, t (s) represents the duration of the cooling stage following laser exposure, θ is the thermally driven constant, and T is the temperature (°C).

2.8. In Vitro Drug Loading and Release

The DOX stock solution was diluted to obtain standard DOX solutions at concentrations of 50, 100, 150, and 200 μg/mL. Then, the absorbance of each standard solution at 480 nm was assayed via UV-vis to plot the standard curve based on the relationship between the DOX concentration and absorbance. Fe₃O₄@MSN@PDA-HA-FA nanoparticles were dispersed in ultrapure water. Aqueous DOX solutions of varying concentrations (50, 100, 150, and 200 μg/mL) were added, followed by stirring in the dark for 24 h. The nanoparticles were magnetically separated and the supernatants were collected to quantify the residual DOX using the DOX calibration curve. The drug-loading efficiency (%) and encapsulation efficiency (%) were calculated using Equations (6) and (7) below.

$$\mathit{Encapsulation} \mathit{efficiency} (\%)={\displaystyle \frac{M_{ \mathit{loaded}}}{{M }_{\mathit{added}}}} \times 100\%$$

(6)

$$Drug loading efficiency (\%)={\displaystyle \frac{{M }_{\mathit{loaded}}}{M_{ NP}+M_{ \mathit{loaded}}}} \times 100\%$$

(7)

where M_added is the mass of DOX initially added, M_loaded is the mass of DOX loaded in the nanoparticles, and M_NP is the mass of nanoparticles.

Fe₃O₄@MSN@PDA-HA-FA nanocomposites loaded with DOX were first placed into dialysis bags with a 10 kDa molecular weight cutoff (MWCO). Subsequently, the dialysis bags loaded with the samples were incubated in PBS buffer at 37 °C and 50 °C, and the corresponding drug release profiles were monitored over a duration of 50 h. To simulate the transient local hyperthermia effect induced by photothermal therapy, the sample-loaded dialysis bags immersed in PBS buffer were subjected to laser irradiation (808 nm, 1 W/cm²) to achieve the targeted temperatures of 37 °C, 42 °C, 47 °C, and 50 °C. For each temperature node, the dialysis bags were heated for 20 min and then incubated at the corresponding constant temperature. The concentration of the released drug was subsequently determined. At different times, each sample system was sampled for 1 mL of release medium. And then, the removed volume was replenished with an equal amount of fresh buffer to keep the total system volume constant. Each collected sample was tested for absorbance at 480 nm using a spectrophotometer.

2.9. In Vitro Cytocompatibility and Blood Compatibility

For the culture of L929 cells, a DMEM medium was used. This medium was supplemented with three components: 10% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at a temperature of 37 °C and in an environment containing 5% CO₂. To analyze the viability of the L929 cells, the cell viability was assessed by a CCK-8 assay without interference controls and further validated by a Live/Dead staining assay. First, Fe₃O₄@MSN@PDA-HA-FA samples were prepared with different concentrations. Then, these samples were introduced into the cell culture plates one by one according to their concentrations. After that, we continued culturing the cells for 48 h to assess the cell viability. The cell viability (%) was calculated as shown in Equation (8).

$$\mathit{Cell} \mathit{viability} (\%)={\displaystyle \frac{A_s-A_b}{A_c-A_b}} \times 100\%$$

(8)

Here, A_s, A_c, and A_b denote the OD values of the material-treated group, control group, and blank group, respectively.

First, collect fresh blood and wash it three times with PBS. Then place the material and red blood cell suspension in a 37 °C incubator. After incubation, centrifuge the tube at 3000 rpm for 5 min, collect the supernatant, and measure its absorbance at 540 nm using a spectrophotometer. The hemolysis ratio is calculated using the following Equation (9):

$$\mathit{Hemolysis} \mathit{ratio} (\%) ={\displaystyle \frac{A_s-A_n}{A_p-A_n}} \times 100\%$$

(9)

where A_s, A_p, and A_n represent the absorbance of the sample, the positive control group, and the negative control group, respectively.

2.10. Evaluation of the In Vitro Tumor Synergistic Therapy Effect

For the in vitro synergistic therapy evaluation, HT29 cells were plated in 96-well plates for a viability analysis and Live/Dead staining. The HT29 cells were divided into five groups, including control, CDT, PTT, DOX, and combination (combination therapy) groups. The cells in the CDT and PTT groups were incubated with Fe₃O₄@MSN@PDA-HA-FA nanoparticles. The cells in the DOX and combination therapy groups were incubated with DOX-loaded Fe₃O₄@MSN@PDA-HA-FA nanoparticles and Fe₃O₄@MSN@PDA-HA-FA nanoparticles and DOX-loaded Fe₃O₄@MSN@PDA-HA-FA nanoparticles at a concentration of 300 μg/mL. In addition, the CDT group and the combination group were supplemented with 40 μM H₂O₂; the PTT and combination groups were irradiated with an 808 nm laser at 1 W/cm² for 5 min. The cells were then incubated for an additional 24 h. Cell viability was assessed by a CCK-8 assay, and Live/Dead staining was performed using Calcein AM and propidium iodide for fluorescence imaging.

2.11. Statistical Analysis

Data are presented as mean ± SD. Differences were evaluated by one-way analysis of variance (ANOVA), with significance thresholds of * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; and ns: not significant. The sample size was three per group (n = 3).

3. Results and Discussion

3.1. Synthesis and Characterizations

Scheme 1 depicts the procedure adopted for the preparation of Fe₃O₄@MSN@PDA-HA-FA nanoparticles. First, monodisperse Fe₃O₄ nanospheres were synthesized via a hydrothermal method, and the nanoparticles exhibited a spherical morphology with clear particle boundaries, indicating good monodispersity (Figure S1). After the completion of MSN coating modification, a dendritic mesoporous shell clearly formed around the Fe₃O₄ cores (Figure S2). This phenomenon confirmed the successful construction of the core–shell structure. After stepwise surface modification with PDA, HA, and FA, the Fe₃O₄@MSN@PDA-HA-FA maintained a spherical morphology with an evident coating layer. The TEM images (Figure 1a,b and Figure S3) revealed uniformly sized nanospheres and the presence of a surface coating. The TEM images revealed particle aggregation, which may be attributed to the potential aggregation of the particles in the aqueous media during sample drying for the TEM characterization. Consistently, the SEM imaging (Figure 1c) showed particles with a relatively roughened surface. In addition, verification via EDS (Figure S4) analysis and elemental mapping (Figure 1d) indicated the presence of Fe, Si, and C elements in the nanoparticles, consistent with the designed composition.

The DLS measurements revealed that the average diameter of Fe₃O₄@MSN@PDA-HA-FA in aqueous solution was 227 ± 7 nm, with no significant change in the size distribution after standing for 3 h and 6 h (Figure 2a), indicating good colloidal stability. A pronounced Tyndall effect was observed under laser irradiation (Figure S5), further confirming uniform dispersion in water. For Fe₃O₄, Fe₃O₄@MSN, Fe₃O₄@MSN@PDA, Fe₃O₄@MSN@PDA-HA, and Fe₃O₄@MSN@PDA-HA-FA, their zeta potentials were −51 ± 6 mV, −72 ± 6 mV, 9 ± 1 mV, −32 ± 2 mV, and −46 ± 4 mV in sequence (Figure 2b). The significant zeta potential shift from Fe₃O₄@MSN (−72 ± 6 mV) to Fe₃O₄@MSN@PDA (9 ± 1 mV) confirmed successful PDA deposition. The subsequent decreases after HA and FA modification reflected the introduction of negatively charged functional groups. The additional negative shift after FA modification may also have resulted from FA introduction as well as the removal or redistribution of weakly bound HA during FA modification. TGA was carried out to assess the thermal stability and weight loss characteristics of Fe₃O₄@MSN@PDA-HA-FA (Figure 2c and Figure S6). Four stages of weight loss were observed: (1) below 120 °C (2.32%), attributed to adsorbed water release; (2) 120–250 °C (2.89%), due to the release and decomposition of water and small molecules within the MSN channels; (3) 250–440 °C (7.43%), caused by the collapse of surface PDA, HA, and FA structures; and (4) above 570 °C (1.24%), resulting from the complete thermal decomposition of the outer layer. The saturation magnetization value of Fe₃O₄@MSN@PDA-HA-FA was 32.79 emu·g⁻¹. This value is sufficiently effective for in vitro magnetic separation during sample processing (Figure 2d).

XRD was used to confirm the structure of Fe₃O₄@MSN@PDA-HA-FA (Figure 3a). The diffraction peaks were consistent with Fe₃O₄ (PDF #72-2303), indicating that the crystalline phase of Fe₃O₄ was preserved during silica coating and subsequent surface functionalization. To further assess the FA modification, the UV-vis absorbance of free FA and residual FA in the supernatant after mixing with the nanocomposite was measured. A decreased FA absorption in the supernatant was observed, indicating successful FA immobilization on the material surface (Figure S7). Both the Fe₃O₄@MSN and Fe₃O₄@MSN@PDA-HA-FA showed typical mesoporous features in the N₂ adsorption–desorption isotherms (Figure 3b and Figure S8). The Fe₃O₄@MSN exhibited a BET surface area of 197.31 m²/g with a pore size of 4.90 nm, whereas the PDA-HA-FA functionalization reduced the surface area to 169.68 m²/g and shifted the pore size to 3.43 nm, retaining the mesoporous framework for cargo loading and release (Figure 3c and Figure S9).

3.2. Fenton Reaction Tests

The POD-like activity of Fe₃O₄@MSN@PDA-HA-FA was examined with TMB as the chromogenic substrate (Scheme 1). Fe²⁺ in Fe₃O₄ catalyzes H₂O₂ decomposition to generate ·OH, which oxidizes colorless TMB to blue oxTMB. As shown in Figure 4a, the Fe₃O₄@MSN@PDA-HA-FA + TMB + H₂O₂ system exhibited a distinct absorption peak at 652 nm, consistent with oxTMB, while no significant absorption was observed in the TMB or TMB + H₂O₂ groups. The solution color changed from colorless to blue after the reaction (Figure 4f), visually confirming Fenton reaction activity. Figure 4b shows that at constant Fe₃O₄@MSN@PDA-HA-FA and TMB concentrations, the absorption intensity at 652 nm increased with the H₂O₂ concentration. Similarly, at constant TMB and H₂O₂ concentrations, the absorption intensity rose with the Fe₃O₄@MSN@PDA-HA-FA concentration (Figure 4c). A Michaelis–Menten analysis based on the initial rates yielded K_m = 16.7 μM and V_max = 3.5 × 10⁻¹¹ mol·s⁻¹ (Figure 4d), while a Lineweaver–Burk plot showed linearity (R² = 0.9935, Figure 4e), suggesting high catalytic efficiency under biologically relevant conditions.

3.3. Photothermal Properties

The UV-vis spectroscopy showed that the Fe₃O₄@MSN@PDA-HA-FA exhibited significant absorption between 400 and 850 nm (Figure 5a). To assess the photothermal stability under intermittent irradiation, the sample was subjected to laser irradiation, and no significant attenuation of the photothermal effect was observed (Figure 5b), confirming its reliable and repeatable photothermal performance over multiple irradiation cycles. For 0.5 mg/mL Fe₃O₄@MSN@PDA-HA-FA, the photothermal conversion efficiency and heat transfer time constant were calculated as 26.54% and 201.88 s, respectively (Figure 5c,d). In Figure 5d, the heat transfer time constant is derived from the slope of the red linear fit. The photothermal effect of Fe₃O₄@MSN@PDA-HA-FA at different concentrations (200, 500, 800, and 1000 μg/mL) was investigated under 808 nm laser irradiation (1 W/cm²), with water as the control. The temperature increased with the increasing concentration, reaching a 17 °C rise at 1 mg/mL after 5 min of irradiation (Figure 5e,g). Additionally, the temperature rise rate increased with the laser power density (0.2, 0.5, 0.8, 1.0 W/cm²) for 1 mg/mL Fe₃O₄@MSN@PDA-HA-FA (Figure 5f,h), while the water showed a negligible temperature change. These results confirm effective photothermal conversion efficiency and stability, supporting its potential application in PTT.

3.4. In Vitro Hemocompatibility and Cytocompatibility

The blood compatibility tests indicated that the hemolysis rate of the nanomaterial was below 5%, with no significant hemolytic reaction induced (Figure 6a). The CCK-8 assay demonstrated that even at a high concentration of 2000 μg/mL, the survival rate of L929 cells remained as high as 97% ± 3% (Figure 6b). It is worth noting that nanoparticle-related optical or chemical effects may influence colorimetric assays; therefore, the CCK-8 results are interpreted in combination with the consistent Live/Dead staining observations. The Live/Dead fluorescent staining assay showed that the vast majority of cells exhibited bright green fluorescence, while only a negligible number of cells displayed red fluorescence (Figure 6c–f). This result further confirms that the Fe₃O₄@MSN@PDA-HA-FA composite nanospheres have no obvious cytotoxicity and can effectively maintain the normal physiological activity of cells. Collectively, these experimental data fully validate their excellent biocompatibility.

3.5. In Vitro Drug Loading/Release and Cell Therapy

To quantify the DOX loading and release, a UV-vis calibration curve was first established. A good linear relationship between the DOX concentration (25–200 μg/mL) and absorbance was obtained (y = 0.0138 x + 0.0459, R² = 0.9998, Figure S10). The encapsulation efficiency increased initially (50 to 100 μg/mL) and then decreased at higher concentrations, suggesting saturation of available loading sites (Figure 7a), while the drug-loading efficiency increased with the concentration and tended to plateau at higher concentrations (Figure 7b). Notably, 100 μg/mL DOX yielded the highest encapsulation efficiency (42%) and a drug-loading efficiency of 8%, indicating an optimal balance between drug incorporation and formulation performance. Therefore, 100 μg/mL was selected as the standard drug-feeding concentration for subsequent experiments. The cumulative DOX release at 50 °C was 58% ± 1%, significantly higher than that at 37 °C (Figure 7c). The enhanced DOX release at 50 °C is likely attributable to temperature-accelerated diffusion through the mesoporous framework and weakening of noncovalent drug–carrier interactions. To better evaluate the temperature-responsive release behavior, we performed drug release tests at different temperatures (Figure S11). The release profile showed faster release during the heating periods and slower release during the cooling periods, resulting in a stepwise trend. The in vitro synergistic therapeutic efficacy was evaluated using HT29 cells under TME-mimicking conditions. The cell viability after single-modal therapy (chemotherapy, CDT, and PTT) was 47% ± 7%, 82% ± 4%, and 81% ± 7%, respectively. In contrast, the combination of chemotherapy, CDT, and PTT reduced the cell viability to 4% ± 4% (Figure 7d–i, **** p < 0.0001), confirming the synergistic therapeutic effect of the Fe₃O₄@MSN@PDA-HA-FA.

4. Conclusions

In this study, the multifunctional core–shell composite nanomaterial Fe₃O₄@MSN@PDA-HA-FA was successfully prepared via a rational stepwise synthesis strategy. This nanoplatform integrates the advantages of multiple components, overcoming the limitations of single-modal tumor therapies and achieving synergy among chemotherapy, CDT, and PTT. Structurally, the Fe₃O₄ core has dual functions: it provides magnetism and supplies the Fe²⁺ for the Fenton reactions. The MSN shell features an ordered porous structure, which not only protects the Fe²⁺ from oxidation but also enables efficient loading of chemotherapeutic drugs. The PDA layer can absorb NIR light for photothermal conversion, and the HA and FA introduce additional surface functionalities that can modulate the nano-bio interactions and improve colloidal behavior. Functionally, in the TME, Fe²⁺ catalyzes hydrogen peroxide to generate cytotoxic ·OH for CDT. Under NIR light irradiation, the PDA layer converts light energy into thermal energy for PTT and promotes temperature-responsive release of chemotherapeutic drugs, achieving synergistic efficacy. The in vitro experiments confirm the nanomaterial’s excellent biocompatibility, low hemolysis rate and stable photothermal conversion efficiency. The combined therapy inhibits tumor cells significantly better than single therapies, providing a promising strategy.