Dopamine and Mn(II) Chelate Covalent-Doping Coated Ti(IV)-Nanotheranostics for Magnetic Resonance Imaging Guided Phototherapy in Oral Cancer

Introduction

Oral squamous cell carcinoma (OSCC) is a heterogeneous malignant neoplasm from oral epithelial cells and constitutes over 90% of all malignancies within the oral cavity.¹ In 2020, the global newly diagnosed OSCC cases was reported to be 377,713, with a corresponding mortality of 177,757.² Currently, surgical resection is the primary therapeutic intervention for OSCC, supplemented by chemotherapy and radiotherapy as the principal adjuvant.³ However, the high recurrence rate (20–86%), and cervical lymph node (LN) metastasis often limit surgical resection as a definitive intervention treatment.⁴ Concurrently, chemotherapy and radiotherapy lack of tumor specificity cause severe adverse effects on normal tissues and therapeutic resistance.^5,6 Consequently, the five-year overall survival rate remains around 60%, despite multimodal treatments that significantly improve quality of life.^1,7 This underscores the urgent need for more effective strategies to improve local disease control.

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has emerged as a highly effective non-invasive therapeutic modality for the primary treatment of superficial tumors,⁸ especially OSCC. These therapies utilize the spatiotemporal selectivity of photosensitizer (PS) for precise tumor targeting.⁹ In PDT, PS agents generate reactive oxygen species (ROS) upon activation, inducing localized cellular damage.¹⁰ In PTT, near-infrared (NIR) laser irradiation generates heat in situ to thermally killing cancer cells.¹¹ Nevertheless, the dose-dependent and unpredictable nature of ROS and heat generation can result in collateral damage to surrounding normal tissues, highlighting the urgent need for improving monitoring and control of therapeutic effects.

To address this challenge, magnetic resonance imaging (MRI) is emerging as a key auxiliary imaging modality for phototherapy and personalized nanotheranostics.^12,13 MRI offers the capability to track the real-time distribution and metabolism of nanomedicine, optimize the drug-light interval prior to irradiation, delineate tumor margins for precise targeting, evaluate therapeutic outcomes.¹⁴ By integrating these functionality, MRI facilitates the simultaneous initiation and monitoring of intratumoral PDT and PTT, enabling the development of optimal treatment strategies with reduced side effects.

As for PSs, although traditional organic molecules had exhibited significant efficacy in generating elevated levels of ROS, they are often limited by insufficient photostability and sensitivity constraints.¹⁵ Inorganic materials, particularly titanium dioxide (TiO₂), demonstrate long-term physical and chemical stability but are restricted by their ultraviolet light response range, which limits their applicability in deep-tissue phototherapy.^16,17 To address this challenge, polydopamine (PDA) has emerged as a multifunctional modifier. PDA not only extends TiO₂’s absorption to the near-infrared (NIR) region (700–900 nm) through its strong π-conjugated electronic structure, but also synergistically enhances both PTT and PDT effects.^18–20 Specifically, PDA’s abundant catechol and amine groups enable robust anchoring to TiO₂ surfaces, facilitating stable nanocomposite formation.^21,22 Moreover, PDA’s redox-active quinone/catechol moieties promote electron transfer processes under NIR irradiation, which amplify TiO₂’s photocatalytic activity and ROS generation for enhanced PDT.^23,24 Concurrently, PDA’s exceptional photothermal conversion efficiency (exceeding that of gold nanoparticles by over 40%) and broad NIR absorption enable efficient heat generation for PTT.²⁵ This dual functionality—combining TiO₂’s photocatalytic behavior with PDA’s photothermal and redox properties—positions the nanocomposite as an ideal platform for synergistic PTT/PDT applications.

In our previous studies, we have developed a heteroditopic Mn(II) chelate, Mn-DopaEDTA, featuring an Dopa-derived Mn-EDTA complex with a catechol group.²⁶ This configuration facilitates the formation of Mn-metallostar structures with high relaxivity through catechol-M (M = Fe(III) or Ti(IV)) coordination-driven self-assembly, making it highly effective as an MRI contrast agent. The Fe-Mn metallostar, in particular, demonstrates significant MRI relaxivity enhancement, over twice that of monomeric Mn-DopaEDTA, along with synergistic PTT and PDT effects when exposed to NIR light. Building on this, Mn-DopaEDTA has been utilized to coat superparamagnetic iron oxide (SPIO) nanoparticles via catechol-Fe(III) coordination on the Fe₃O₄ surface, forming SPIO@Mn-DopaEDTA structures.²⁷ These core-shell nanoparticles exhibit excellent water dispersibility and possesses optimized T1/T2 relaxivity properties, acting as effective dual-modal MRI contrast agents.

In this study, we develop an novel nanoplatform intergrates TiO₂ nanoparticles with PDA and Mn-DopaEDTA (MnDopa) to address the limitation of single-modality PTT or PDT. Leveraging the excellent coordination properties of catechol group, we used horseradish peroxidase (HRP) catalysis to co-deposit dopamine and Mn-Dopa onto TiO₂ surfaces, achieving surface chemical modification of TiO₂ with both Mn-Dopa and PDA.

Based on the attractive properties of high relaxivity and NIR optical absorption of the core-shell nanoparticles structure discussed above, herein we report the synthesis and characterization of a new MRI/PTT/PDT nanotheranostic TiO₂@PDA-MnDopa. To explore the performance in MRI, we studied its morphology, composition, relaxation properties and imaging effect in vivo. To evaluate the performance in PTT and PDT, we studied its photoactive characteristics, photo-therapy effect and safety in the HSC3 cell line and tumor-bearing mice. This multifunctional nanocomposite achieves synergistic PTT and PDT effects, combining PDA’s photothermal properties with MnDopa’s MRI imaging capability and TiO₂’s photocatalytic behavior. Together, these components enable a robust, image-guided multimodel phototherapy platform with enhanced efficacy for treating oral cancer.

Materials and Methods

Materials and Apparatus

Titanium(IV) chloride, diethylene glycol (DEG), dopamine, and horseradish peroxidase (HRP) were obtained from InnoChem (Beijing, China). Additionally, 1,3-diphenylisobenzofuran (DPBF) was sourced from Macklin (Shanghai, China). The Cell Counting Kit-8 (CCK-8) and the Calcein/PI Cell Viability Assay Kit were acquired from Beyotime (Shanghai, China). Ultrapure water (Millipore Milli-Q grade, 18.2 MΩ) was utilized throughout the experiments. Manganese concentrations were measured using an inductively coupled plasma mass spectrometry (ICP-MS) system (PerkinElmer Nex-ion 350x, USA). The zeta potential was assessed using dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS90 (UK).

Balb/C male nude mice, aged 4 to 6 weeks, were procured from the Laboratory Animal Center of North Sichuan Medical College (NSMC), Sichuan, China. All experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and received approval from the Ethics Committee of NSMC (Application No. 2023007).

Preparation of TiO₂@PDA-MnDopa

TiO₂ nanoparticles were synthesized following a protocol established by Bianca Geiseler et al. In summary, 0.2 mL of titanium(IV) chloride was introduced to 6 mL of diethylene glycol (DEG) while maintaining vigorous stirring under an inert atmosphere at a temperature of 60°C. A white precipitate formed immediately, after which the suspension was heated to 75°C until the solution became clear. Subsequently, 0.1 mL of double-distilled water (ddH₂O) was injected into the solution, and the mixture was refluxed at 160°C for 6 h. Upon cooling to room temperature, 15 mL of acetone was added to facilitate the precipitation of nanoparticles (NPs). The NP solution was then subjected to centrifugation at 3500 rpm for 30 min and washed multiple times with acetone to eliminate residual surfactants. The TiO₂ nanoparticles were characterized using transmission electron microscopy. The synthetic route for MnDopa was adapted from prior research.²⁷ TiO₂ nanoparticles (100 mg) were dissolved in 20 mL of phosphate-buffered saline (PBS) at a concentration of 50 mm and a pH of 6.5, while dopamine (71.2 mg) and MnDopa (92.2 mg) were dissolved in 5 mL of PBS under the same conditions. Dopamine and MnDopa were then gradually added to the TiO₂ solution while stirring vigorously, resulting in a color change from white to orange, indicative of the formation of a charge transfer complex. After 3 h, 1 mL of horseradish peroxidase (HRP) at a concentration of 4 mg/mL was introduced to the solution, followed by the addition of 0.5 mL of hydrogen peroxide (H₂O₂) at a concentration of 1 M every minutes. This addition caused the solution to turn brown-black rapidly. The reaction mixture was subsequently stirred overnight, and the crude product was purified using size exclusion chromatography.

Characterization of TiO₂@PDA-MnDopa

Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images were acquired utilizing a Tecnai F20 transmission electron microscope. X-ray diffraction (XRD) spectra were recorded using a D8 Focus XRD diffractometer (Bruker, USA). Dynamic light scattering (DLS) measurements and Zeta potential assessments were conducted with a Nano-ZS Zeta-sizer nanosystem (Malvern, UK). X-ray photoelectron spectroscopy (XPS) analyses were performed employing an AXIS Ultra DLD XPS instrument (Kratos, UK). Fourier transform infrared (FT-IR) spectra and ultraviolet-visible (UV-vis) absorbance measurements were obtained using a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, USA) and a Lambda 950 UV-vis spectrophotometer (Perkin Elmer, USA), respectively.

Relaxivity

The magnetic resonance (MR) performance was evaluated using a 0.5 T MesoMR23-060H-1 MR instrument (Niumag, China). Specifically, 1 mL of TiO₂@PDA-MnDopa at varying concentrations (Mn²⁺: 0, 0.025, 0.05, 0.1, 0.2, and 0.4 mM) was placed in separate centrifuge tubes. T1-weighted MR imaging was subsequently conducted, and the relaxation times (T₁ and T₂) were measured. Additionally, the longitudinal (r₁) and transverse (r₂) relaxivities were calculated.

Photothermal Performance

An 808 nm laser was employed to investigate the photothermal properties of TiO₂@PDA-MnDopa, with temperature variations and corresponding images recorded using an infrared camera. (1) A range of concentrations of TiO₂@PDA-MnDopa (0, 50, 100, 150, and 200 μg/mL) were subjected to laser exposure for a duration of 10 min at a power density of 2.0 W/cm². (2) TiO₂@PDA-MnDopa at a concentration of 200 μg/mL was irradiated with the 808 nm laser at varying power densities (1.0, 1.5, 2.0, and 2.5 W/cm²) for 10 min. (3) The photostability of TiO₂@PDA-MnDopa was also assessed, wherein 1 mL of the TiO₂@PDA-MnDopa solution (200 μg/mL) was subjected to irradiation from the 808 nm laser (2.0 W/cm²) with alternating on/off cycles every 10 min for a total of five cycles.

Reactive Oxygen Species (¹O₂) Detection

1,3-Diphenylisobenzofuran (DPBF) was employed to quantify the singlet oxygen generated by TiO₂@PDA-MnDopa. A volume of 20 μL of DPBF (1.0 mg/mL, dissolved in DMSO) was combined with 2 mL of TiO₂@PDA-MnDopa (100 μg/mL in water) and subsequently irradiated with an 808 nm laser (1.0 W/cm²) for a duration of 10 min. The absorption spectra of DPBF were recorded and analyzed before and after irradiation. Additionally, pure DPBF and TiO₂ were subjected to irradiation with the 808 nm laser for 10 min, and the generation of reactive oxygen species (ROS) was also assessed.

Cell Cytotoxicity

HSC3 cells were obtained from the American Type Culture Collection (ATCC). The Cell Counting Kit-8 (CCK-8) assay was employed to evaluate the cytotoxicity of TiO₂@PDA-MnDopa. HSC3 cells were plated in a 96-well plate at a density of 1×10⁴ cells per well and cultured for 12 h in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Following this incubation period, the medium was aspirated, and varying concentrations of TiO₂@PDA-MnDopa (0, 25, 50, 100, 150, and 200 μg/mL) were introduced to the cells. Untreated cells, maintained in the medium alone, served as the control group. After 24 h of incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 1.5 h. The absorbance of the medium containing the CCK-8 reagent was subsequently measured at 450 nm using a microplate reader.

In vitro Phototherapy

The in vitro phototherapy was assessed utilizing the CCK-8 assay. HSC3 cells were incubated with TiO₂@PDA-MnDopa at varying concentrations (0, 25, 50, 100, 150, and 200 μg/mL) for 4 h. Afterward, the medium was replaced with fresh medium, and the cells were irradiated with an 808 nm laser for 10 min at a power density of 2.0 W/cm². Following irradiation, the cells were incubated for an additional 24 h. Cell viability was then evaluated using the CCK-8 assay as previously described. Two control experiments were conducted to further investigate the relative contributions of PTT and PDT to the cytotoxicity. For PDT only: experiments were conducted as described above, but HSC3 cells were irritated with an NIR laser (808 nm, 10 min, 2.0W/cm²) at 4°C to suppress temperature elevation. For PTT only: experiments were conducted as described above, but HSC3 cells were co-incubated with a ROS scavenger (Vitamin C, 0.5 mm) to exclude the PDT effect, followed by NIR (808 nm, 10 min, 2.0W/cm²). Finally, the absorbance of each well was measured with a microplate reader at a wavelength of 450 nm.

In vitro Reactive Oxygen Species Detection

HSC-3 cells were cultured in confocal dishes at a density of 1×10⁵ cells per dish for a 24-h incubation period. Subsequently, 1 mL of TiO₂@PDA-MnDopa at a concentration of 200 μg/mL was introduced, followed by an additional incubation of 4 h. After this, 1 mL of DCFH-DA at a concentration of 10 μM was added for a 20-min incubation. The dishes were then washed three times with PBS, and 1 mL of culture medium was added. Finally, the dishes were subjected to irradiation with an 808 nm laser at an intensity of 1.0 W/cm² for 5 min. The generation of reactive oxygen species (ROS) was assessed by measuring the fluorescence of 2,7-dichlorofluorescein (DCF), which is the oxidation product of DCFH-DA in the presence of ROS. This fluorescence was observed using a TSCSPS II confocal laser fluorescence microscope (Olympus, Japan), as DCF can be rapidly oxidized into a highly fluorescent green compound in ROS.

Live/Dead Staining

To visualize the HSC3 cell state, a live/dead staining experiment was conducted. HSC3 cells were evenly distributed across six confocal dishes and incubated for 24 h. Subsequently, the cells in each dish received distinct treatments: PBS as a control, laser treatment alone, TiO₂@PDA-MnDopa treatment alone, TiO₂@PDA-MnDopa at a concentration of 50 μg/mL combined with laser treatment, and TiO₂@PDA-MnDopa at a concentration of 200 μg/mL combined with laser treatment. Laser irradiation was administered after 4 h of incubation with TiO₂@PDA-MnDopa. Following an overnight incubation, a Calcein/PI Cell Viability Assay Kit was employed to co-stain the cancer cells. After 30 min, fresh DMEM medium was added to the plates in preparation for subsequent confocal imaging.

In vivo Biocompatibility with Tumor-Bearing Nude Mice Model

In the assessment of in vivo biocompatibility, healthy nude mice were administered intravenous injections of 100 μL of pure saline and TiO₂@PDA-MnDopa (20 mg/mL in saline), respectively. Following a two-week period, the mice were euthanized, and the major organs, including the heart, liver, spleen, lungs, and kidneys, were excised for analysis. Hematoxylin and eosin (H&E) staining images were subsequently examined using an optical microscope. For the HSC3 tumor-bearing nude mouse model, HSC3 cells (100 μL) suspended in DMEM medium were subcutaneously injected into the right thigh of the mice. The mice were utilized for experimentation once the tumors reached a volume of 100 mm³.

In vivo Imaging

T1-weighted magnetic resonance imaging (MRI) of HSC3 tumor-bearing nude mice was conducted using a 3.0 Tesla Discovery MR750 system (GE Medical Systems, Milwaukee, WI). An intravenous injection of 100 μL of TiO₂@PDA-MnDopa (20 mg/mL in saline) was administered to the HSC3 tumor-bearing nude mice. Subsequently, T1-weighted MRI was performed at 0 min, 30 min, 60 min, 90 min, 120 min, and 6 h post-injection.

In vivo Anti-Tumor Therapy

Four experimental groups were established, each consisting of five mice. The mice were subjected to one of the following treatments: PBS, laser treatment alone, TiO₂@PDA-MnDopa treatment alone, or a combination of TiO₂@PDA-MnDopa and laser treatment. For the groups receiving either laser treatment alone or TiO₂@PDA-MnDopa combined with laser treatment, laser irradiation was administered 1 h post-injection of PBS (100 μL) or TiO₂@PDA-MnDopa (100 μL, 20 mg/mL) via the tail vein for 10 min. Temperature variations were monitored using a thermal camera. Three days later, a second round of laser irradiation was conducted under the same conditions. Tumor volumes and body weights of the mice were recorded every two days. After 14 days, the tumors were excised for photographic documentation and weighing.

Statistical Analysis

Data are presented as means ± SD, and statistical significance was determined using GraphPad Prism software 8. The differences between the groups were determined using using two-tailed unpaired t-tests. The threshold for statistical significance was set at p < 0.05 with 95% confidence intervals.

Results and Discussion

Synthesis and Characterization of TiO₂ and TiO₂@PDA-MnDopa

TiO₂ nanoparticles with small particle sizes were synthesized using the sol-gel method. The water solubility of these nanoparticles is attributed to the presence of diethylene glycol on their surface. The weakly bound diethylene glycol were readily replaced by the catechol groups of dopamine and MnDopa through a ligand exchange reaction. An HRP-catalyzed reaction was employed to facilitate the co-polymerization of dopamine and MnDopa, covalently incorporating them onto the TiO₂ surface to achieve stable and uniform surface modification. This innovative approach enable the precise intergration of both PDA and MnDopa onto the TiO₂ nanoparticles, forming small-sized core-shell structures with enhanced chemical stability. The resulting nanocomposites combine the photothermal properties of PDA with the MRI contrast enhancement provided by MnDopa, offering a multifunctional platform for theranostic applications.

The structural characteristics of TiO₂ were examined using high-resolution transmission electron microscopy (HR-TEM) (Figure 1A). The HR-TEM analysis revealed that TiO₂ exhibited well-defined crystalline structures. The scanning electron microscopy (SEM) image of TiO₂@PDA-MnDopa demonstrated the clustering of several nanoparticles (Figure 1B). To assess the colloidal stability of the synthesized nanoparticles, measurements of the hydrodynamic diameter and zeta potential were conducted. The average size of TiO₂ was determined to be 5.7±0.7 nm, while that of TiO₂@PDA-MnDopa was found to be 121.0±3.6 nm (Figure 1C and Table 1), indicating effective dispersion and stability due to the surface modification. Furthermore, the surface potential of TiO₂ was measured at 38.1±2.0 mV, possibly due to the existence of positively charged Ti (IV) ion on the surface of TiO₂. However, following the surface modification with PDA and MnDopa, the surface potential decreased significantly −52.4 ±1.4 mV (Figure 1D).

Table 1 Physicochemical Characterization of Size, Poly Dispersibility Index (PDI) and Zeta Potential

Figure 1 Physical and chemical characterization of TiO2 and TiO2@PDA-MnDopa. (A) HR-TEM of TiO2. (Scale bar: 5 nm) (B) SEM of TiO2@PDA-MnDopa. (C) Size of TiO2@PDA-MnDopa. (Scale bar: 200 nm) (D) The zeta potentials of the TiO2 and TiO2@PDA-MnDopa in aqueous solution. (E) XPS spectra of TiO2 and TiO2@PDA-MnDopa. (F) XPS spectra of Mn 2p for the TiO2@PDA-MnDopa. (G) XRD of TiO2 and TiO2@PDA-MnDopa. (H) FT-IR spectra of TiO2 and TiO2@PDA-MnDopa. (I) UV-VIS spectrum TiO2 and TiO2@PDA-MnDopa in aqueous solution.

The elemental composition was confirmed through X-ray photoelectron spectroscopy (XPS). In the full-scan XPS spectrum, four characteristic absorption peaks related to C1s (283.75 eV, 61.22%), O1s (530.38 eV, 28.25%), N1s (398.73 eV, 5.12%), Ti2p (457.58 eV, 4.66%) and Mn2p (639.98 eV, 0.75%) were recognized (Figure 1E). In contrast, the NPs without PDA and MnDopa contained only three peaks: C1s (283.67 eV, 41.05%), O1s (528.66 eV, 41.78%), and Ti2p (457.28 eV, 16.00%). The XPS spectra of C1s, O1s and Ti2p of TiO₂ and TiO₂@PDA-MnDopa show different quantities on species, suggesting that Mn doping induces changes in the local chemical environment of C, O and Ti elements of TiO₂. Moreover, the characteristic peak of Mn2p corresponding to MnO₂ demonstrates that a considerable portion of manganese is doped into the TiO₂ (Figure 1F).

The crystal structures of TiO₂ and TiO₂@PDA-MnDopa were examined using X-ray diffraction (XRD) (Figure 1G). The XRD patterns of TiO₂@PDA-MnDopa and TiO₂ were found to be similar which indicates the presence of an amorphous structure in the PDA and MnDopa without alter the crystal structure of TiO₂. To illustrate the conjugation process, the alterations in the surface functional groups were analyzed using Fourier Transform Infrared (FT-IR) spectroscopy. The FT-IR spectra of both TiO₂ and TiO₂@PDA-MnDopa (Figure 1H) exhibited a characteristics peak at 538 cm⁻¹, which corresponds to the Ti-O-Ti bond within the TiO₂ framework, confirming the presence of the titanium-oxygen network. Additionally, a broad absorption peaks at 3416 cm⁻¹ was observed for both TiO₂ and TiO₂@PDA-MnDopa, attributed to the O-H bond stretching vibration from hydroxyl groups, which were present in both the diethylene glycol (DEG) and PDA structures. Notably, the TiO₂@PDA-MnDopa spectrum showed distinct peaks at 1406 cm⁻¹ and 1601 cm⁻¹, which are assigned to the C-H bending and C=C stretching vibrations of the aromatic benzene ring in the PDA and MnDopa components, respectively. These characteristic peaks confirm the successful covalent doping of PDA and MnDopa onto TiO₂ surface.

Finally, TiO₂@PDA-MnDopa demonstrated NIR absorption in comparison to TiO₂, a feature attributed to the outer PDA shell, which is advantageous for phototherapy (Figure 1I).

Relaxation Property and Photoactive Characteristics

The longitudinal and transverse relaxivity (r₁ and r₂) of MnDopa were measured at 0.5 T as 3.48 mM⁻¹s⁻¹ and 4.72 mM⁻¹s⁻¹ (Table 2), respectively. Notably, the relaxivity of the TiO₂@PDA-MnDopa nanocomplexes was significantly enhanced, with values of r₁ = 18.17 mM⁻¹s⁻¹ and r₂ = 23.97 mM⁻¹s⁻¹, representing a 5.22-fold increase compared to the MnDopa monomer (Figure 2A). This substantial enhancement in relaxivity is attributed to the efficient intergration of MnDopa to the TiO₂ surface, which markedly slows down the rotation of MnDopa in solution. Furthermore, the TiO₂@PDA-MnDopa nanoparticles exhibited high relaxivity under higher magnetic field strengths, maintaining excellent r₁ at both 1.5 T and 3.0 T. At these field strengths, r₁ were measured at 14.76 mM⁻¹s⁻¹ and 13.05 mM⁻¹s⁻¹, respectively (Table 2). This suggests that the TiO₂@PDA-MnDopa retains high MR signal enhancement across a range of clinically relevant magnetic fields. To further validate the T1-weighted imaging (T1WI) capabilities, T1WI of aqueous TiO₂@PDA-MnDopa solutions was conducted using a 3.0 T clinical MRI scanner. The results demonstrated a significant enhancement in MR signal and increased image brightness, directly correlating with the concentration of manganese ions in the solution, thereby confirming the strong T1WI performance of the nanoparticles (Figure 2B).

Table 2 Longitudinal (r1) and Transverse (r1) Relaxivity

Figure 2 The Relaxation and photoactive characteristics of TiO2 and TiO2@PDA-MnDopa. (A) The longitudinal relaxation rates (r1 and r2) of TiO2@PDA-MnDopa at magnetic field strengths 0.5 T. (B) T1-weighted imaging with different Mn(II) ion concentrations. (C) Generation of ROS from TiO2 and TiO2@PDA-MnDopa determined by a change in DPBF absorbance. (D) Photothermal profiles of TiO2@PDA-MnDopa in PBS with different concentrations (50, 100, 150, and 200 μg/mL) under laser irradiation (808 nm, 2 W/cm2). (E) Photothermal profiles of TiO2@PDA-MnDopa in PBS under different power density irradiation (808 nm, 1, 1.5, 2, and 2.5 W/cm2). (F) The heating curve of TiO2@PDA-MnDopa in PBS during 5 cycles of on-and-off laser irradiation (808 nm, 2 W/cm2).

To assess the photodynamic properties of TiO₂@PDA-MnDopa, the generation of ROS was monitored using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen (¹O₂) scavenger. The ability of TiO₂@PDA-MnDopa to generate ROS was assessed by tracking the change in absorption at 410 nm, a characteristic peak for DPBF degradation. As illustrated in Figure 2C, following 10 min of irradiation with an 808 nm laser, there was no significant reduction in absorption for the blank control and TiO₂ samples. In contrast, the absorption of TiO₂@PDA-MnDopa decreased by 21%, indicating that TiO₂@PDA-MnDopa is capable of generating reactive oxygen species, particularly singlet oxygen, under laser irradiation.

In order to evaluate the photothermal efficacy of TiO₂@PDA-MnDopa, solutions with varying concentrations were subjected to irradiation for 10 min using an 808 nm NIR laser (2.0 W/cm²). The results indicated that the temperature of the solutions increased in response to both the irradiation time and the concentration of TiO₂@PDA-MnDopa (Figure 2D). Specifically, at a concentration of 200 µg/mL, the temperature of the solution rose from 30.7°C to 52.5°C, reflecting an increase of 21.8°C. In contrast, pure water under the same irradiation conditions experienced a temperature rise of only 1.7°C, confirming the significant PTT response of TiO₂@PDA-MnDopa.

Further experiment demonstrated that increasing the laser power enhanced the photothermal effect. At a concentration of 200 µg/mL of TiO₂@PDA-MnDopa, the solution temperature increased from 46.7°C to 55°C as the laser power was elevated from 1 W/cm² to 2.5 W/cm² (Figure 2E), demonstrating a power-dependent photothermal response.

In addition, under on-off 808 nm laser irradiation (2.0 W/cm²), the temperature of the TiO₂@PDA-MnDopa solution demonstrated consistent cycling behavior, with temperature rising and cooling processes following a predictable pattern (Figure 2F). This indicates the excellent photothermal stability of TiO₂@PDA-MnDopa.

The photothermal conversion efficiency of TiO₂@PDA-MnDopa was calculated to be approximately 15.91%, based on the temperature rise and fall curve, thereby confirming its robust photothermal conversion capability. Previous studies suggest that cancer cells are effectively eliminated at temperatures above 42°C for durations of 15–60 min, with treatment times significantly reduced (4–6s) when temperatures exceed 50°C.²⁸ These findings suggest that TiO₂@PDA-MnDopa has the potential to serve as an effective photothermal agent for tumor treatment.

In vitro Photo-Therapy

To investigate the capacity of TiO₂@PDA-MnDopa to generate sufficient ROS under laser irradiation, HSC3 cells were incubated with TiO₂@PDA-MnDopa and subsequently exposed to an 808 nm laser at an intensity of 1 W/cm² for 10 min. A separate group of cells treated with H₂O₂ served as a positive control, while additional groups subjected to laser irradiation alone or treated with TiO₂@PDA-MnDopa without irradiation acted as negative controls. The cells were stained with DCFH-DA, a well-established probe for ROS detection, and confocal laser scanning microscopy (CLSM) was employed to visualize intracellular ROS production. As illustrated in Figure 3A, groups exposed to either 50 mm H₂O₂ or TiO₂@PDA-MnDopa in conjunction with NIR irradiation exhibited a pronounced green fluorescence signal, indicative of ROS generation. Conversely, the negative control groups, which included cells treated solely with laser irradiation or TiO₂@PDA-MnDopa without irradiation, did not display significant detectable signals. These findings confirm that ROS can be selectively generated in cells through the uptake of TiO₂@PDA-MnDopa followed by NIR irradiation, suggesting that light-triggered TiO₂@PDA-MnDopa are promising candidates for PDT.

Figure 3 In vitro photo-therapy evaluation of TiO2 and TiO2@PDA-MnDopa. (A) Fluorescence microscope images of cellular ROS generation treated with TiO2@PDA-MnDopa (50 mm). (Scale bar: 40 µm) (B) Relative cell viability of HSC3 cells after incubation with different concentrations of TiO2@PDA-MnDopa for 24 h, followed by irradiation with 808 nm laser (2W/cm2) for 10 min (4 °C was used to suppress the PTT effect and Vitamin C was used to shield the PDT effect). ***P < 0.001, ****P < 0.0001, NS means no significance. Data are presented as means ± SD. (C) Fluorescence microscope images of Calcein AM (green fluorescence, live cells) and Propidium iodide (PI, red fluorescence, dead cells) contained HSC3 cells incubated with PBS, Laser, TiO2@PDA-MnDopa and TiO2@PDA-MnDopa + Laser. (Scale bar: 150 µm).

The phototherapy cytotoxicity of TiO₂@PDA-MnDopa on cancer cells was quantitatively assessed using a CCK-8 assay (Figure 3B). After 24 h of incubation, HSC3 cells treated with TiO₂@PDA-MnDopa without laser irradiation exhibited over 90% viability at a concentration of 200 µg/mL. In contrast, the viability of HSC3 cells decreased to less than 5% when exposed to TiO₂@PDA-MnDopa at the same concentration (200 µg/mL) and subjected to NIR laser irradiation for 10 min at a power density of 2 W/cm². To further clarify the relative contributions of photothermal and photodynamic effects to the cytotoxicity, two control experiments were performed. To investigate the photodynamic effect alone, the environmental temperature was maintained at 4°C using an ice bath to suppress hyperthermia. To study the photothermal effect alone, Vitamin C was added to scavenge singlet oxygen and shield the photodynamic effect (Figure 3B). These control experiments suggested TiO₂@PDA-MnDopa can generate synergistic photodynamic and photothermal cytotoxic effects. Notably, the photothermal effect on HSC3 cells was more potent than the photodynamic effect under the conditions tested.

Additionally, the live/dead staining experiment was conducted to visually assess the states of HSC3 cells following various treatments. As illustrated in Figure 3C, exposure to either laser or NPs alone does not affect the viability of HSC3 cells; however, the combination of NPs with laser irradiation (NPs + Laser) effectively induces cell death. An increase in the concentration of TiO₂@PDA-MnDopa, ranging from 50 to 200 µg/mL, correlates with a significant decline in the viability of HSC3 cells, which aligns with the findings from the CCK-8 assay. These results collectively indicate that TiO₂@PDA-MnDopa exhibit favorable biocompatibility in the absence of light while demonstrating efficacy in inducing cancer cell death upon exposure to 808 nm laser irradiation.

In vivo MRI-Guided Anti-Tumor Photo-Therapy

Inspired by the excellent anti-tumor effect in vitro of TiO₂@PDA-MnDopa, the efficacy of in vivo therapy was further evaluated in mice. Prior to initiating the antitumor experiment, MRI was utilized to determine the optimal time point for laser irradiation. Following the intravenous administration of TiO₂@PDA-MnDopa (0.038 mmoL/kg), the T1-weighted MRI intensity at the tumor site exhibited a gradual increase, reaching a peaking at 1 h post-injection, and remained elevated until approximately 2 h. Notably, the signal intensity at the tumor site diminished after 6 h (Figure 4A). The normalized signal-to-noise ratio (nSNR) and the change in contrast-to-noise ratio (ΔCNR) values of the tumor tissues were quantified to assess the tumor’s visibility and the quality of the MRI signal. The nSNR, which quantifies the clarity of the tumor image relative to background noise, and ∆CNR, which measures the contrast between the tumor and surrounding tissues, provide insights into the extent of TiO₂@PDA-MnDopa uptake by the tumor. Both ratios showed a significant increase after TiO₂@PDA-MnDopa injection, with peak values observed around 1 h post-injection. Specifically, the nSNR reached 1.24 ± 0.03, and ∆CNR was 10.16 ± 1.29, both showing a continued upward trend until approximately 1.5 h post-administration (Figure 4B and C). These results indicate a favorable tumor-targeting capability attributed to the enhanced permeability and retention (EPR) effect. Importantly, these findings help guide MRI-based phototherapy by providing key information on the optimal laser irradiation time and irradiation duration. The peak of the nSNR and ∆CNR indicates the ideal time point for initiating laser irradiation (around 1 h post-injection), ensuring the maximum accumulation of TiO₂@PDA-MnDopa at tumor site.

Figure 4 In vivo MRI and PTT of HSC3 tumor-bearing mice. (A) Representative coronal T1-weighted MR images of mice obtained prior, 30 min, 60 min, 90 min, 120 min, and 6 h after intravenous injection of TiO2@PDA-MnDopa. (B and C) Graphs show the normalized signal-to-noise ratio (nSNR) and contrast-to-noise ratio (ΔCNR, tumour vs muscle) time course in the tumour after injection of TiO2@PDA-MnDopa. (D and E) Photothermal images and temperature evolution of HSC3 tumor-bearing mice injected with PBS and TiO2@PDA-MnDopa under NIR laser irradiation (808 nm, 2 W/cm2) for 10 min at 1-h post-injection.

Thus, infrared thermal imaging was performed under the guidance of MRI after a 1 h post-injection, corresponding to the peak of TiO₂@PDA-MnDopa accumulation at the tumor site (Figure 4D). Following the MRI signal confirmation, the 808 nm laser was applied to the tumor region. The temperature at the tumor site in the NPs + Laser group increased by 17 °C, demonstrating effective photothermal conversion. In contrast, the temperature in the PBS+laser control group rose by only 5 °C, confirming the specific photothermal effect of TiO₂@PDA-MnDopa nanoparticles (Figure 4E).

HSC3 tumor-bearing mice with a tumor volume of approximately 100 mm³ were randomly divided into four groups, with five mice per group (Figure 5A). Throughout the 14-day treatment period, tumor size and body weight were monitored every two days. As shown in Figure 5B, the digital photographs taken on the 14th day post-treatment revealed complete tumor elimination in the TiO₂@PDA-MnDopa + Laser group. Tumor volumes in the control groups increased rapidly over the course of the experiment (Figure 5C–E). In comparison, the TiO₂@PDA-MnDopa and PBS+Laser groups showed a slower tumor growth rate than the control groups. These results agree fairly well with the tumor growth curves, confirming the efficacy of TiO₂@PDA-MnDopa under laser irradiation.

Figure 5 In vivo antitumor activity of different treatment of HSC3 tumor-bearing mice. (A) Scheme of HSC3 tumor-bearing mice development and photo-therapy. (B) Representative photos of HSC3 tumor-bearing mice after different days before (0 d) and after (7 and 14 d) different treatments. Saline, without treatment; NPs, injection of TiO2@PDA-MnDopa only; Laser, irradiation with laser only; TiO2@PDA-MnDopa+Laser, injection of TiO2@PDA-MnDopa and irradiation with a laser. (C) Optical images of the dissected tumors from HSC3 tumor-bearing mice in different treatment groups at day 14 after sacrificing the mice. (D and E) Relative volumes of tumors after the above treatments, normalized to their initial volumes. ****P < 0.0001. Data are presented as means ± SD. (F) Histological section of tumor stained with hematoxylin and eosin after intravenous administration of TiO2@PDA-MnDopa. (Scale bar: 50 µm) (G) Average body weights of mice from each group after various treatments. (H) Survival curves of HSC3 tumor-bearing mice in various groups after NIR-light irradiation treatment.

Additionally, histological analysis using H&E staining of tumor sections showed severe cell damage in the tumors of mice from the TiO₂@PDA-MnDopa + Laser group, whereas only partial destruction of tumor cells was observed in the other treatment groups (Figure 5F). Importantly, there were no significant differences in the body weight of the mice across all groups, indicating that the treatments did not induce any overt toxicity or affect the overall health and development of the animals (Figure 5G).

The survival rate of the mice was also monitored throughout the study. After NIR laser irradiation, the TiO₂@PDA-MnDopa group exhibited the highest survival rate among all experimental groups, while the control groups showed signs of distress and succumbed after approximately 40 days (Figure 5H). These findings demonstrate the potent anti-tumor effect and safety profile of TiO₂@PDA-MnDopa-mediated phototherapy.

Conclusion

In conclusion, we have successfully synthesized a novel TiO₂@PDA-MnDopa nanocomposite that combines exceptional MRI capability with powerful PDT and PTT effects, exhibits an exceptionally strong MR signal and notable PDT and PTT capabilities. Both in vitro cellular assays and in vivo experiments demonstrated that TiO₂@PDA-MnDopa can effectively induce synergistic PDT and PTT effects upon laser irradiation. This innovative phototherapy platform, coupled with MRI guidance, shows significant potential as a multifunctional nanotheranostic agent for precise cancer diagnosis and treatment.

However, it is important to note that the animal model utilized in this study involved ectopic transplanted tumors in tumor-bearing mice, which is primarily constrained by the limitations of the imaging and treatment devices employed. Future work will focus on improving experimental equipment, refining the model to better reflect primary tumor conditions, and extending research to explore the potential of TiO₂@PDA-MnDopa for the diagnosis and treatment of primary oral cancer.