Copper–manganese hybrid nanogel for MRI-guided combined photothermal and chemodynamic tumor theranostics

Abstract

Bridging tumor diagnosis and therapy remains a major challenge, largely due to the clinical separation of imaging and treatment, compounded by the low relaxivity of conventional MRI contrast agents. To address these limitations, we developed a copper–manganese hybrid nanogel (CMNG) via the in situ incorporation of Mn²⁺ ions and CuS nanoparticles within a cross-linked polymeric network. This multifunctional design enables T₁-weighted MRI-guided photothermal–chemodynamic therapy. The nanogel matrix significantly enhances the relaxivity of paramagnetic Mn²⁺ ions (r₁ = 10.81 mM⁻¹ s⁻¹), surpassing that of clinically approved Gd-based agents. Under 808 nm laser irradiation, CMNG exhibits efficient photothermal conversion (η = 23.29%), which synergistically enhances Cu⁺/Mn²⁺-mediated Fenton-like reactions, resulting in elevated hydroxyl radical (˙OH) production for effective tumor ablation and inhibition of tumor progression. This work presents a rational materials design strategy for integrated theranostic platforms. By combining MRI-guided tracking with potent therapeutic efficacy, the CMNG system offers a promising paradigm for precision cancer theranostics.

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1. Introduction

Magnetic resonance imaging (MRI) has become a cornerstone in tumor diagnosis due to its non-invasive nature, superior soft-tissue contrast, and three-dimensional anatomical visualization capabilities.¹ However, the intrinsic sensitivity of MRI in distinguishing malignant from healthy tissues remains limited, necessitating the use of contrast agents (CAs) to enhance diagnostic accuracy.² These agents typically contain paramagnetic ions that promote water molecule exchange within their inner and/or outer coordination spheres, thereby accelerating proton relaxation.³ The effectiveness of a CA is largely determined by its ability to enhance the relaxation rates of surrounding water protons at low concentrations.⁴

T ₁-weighted CAs, primarily based on lanthanide metal chelates, enhance proton spin–lattice relaxation, thereby generating positive MRI signals. Although gadolinium-based contrast agents (Gd CAs) are widely used in clinical practice, increasing concerns over nephrotoxicity and long-term tissue retention have raised safety issues.⁵ These concerns have driven interest in manganese ions (Mn²⁺) as alternative paramagnetic agents. With five unpaired electrons and extended electron relaxation times, Mn²⁺ ions are well-suited for T₁ signal enhancement.⁶ Furthermore, manganese is an essential trace element naturally distributed throughout body tissues.⁷

Due to the limited tumor specificity and functionality of conventional manganese complexes, recent research on manganese-based contrast agents (Mn CAs) has increasingly focused on nanoparticle (NP)-based systems over small-molecule chelates. Nanoparticles (NPs) enable tunable optimization of relaxivity through precise control over their size, composition, and surface functionality, while also facilitating multimodal imaging or the integration of diagnostic and therapeutic capabilities.^8–10 Their extended systemic circulation, coupled with the enhanced permeability and retention (EPR) effect, facilitates preferential accumulation in tumor tissues.^11,12

Nanogels (NGs) are three-dimensional networks formed by cross-linked polymer chains, combining the properties of both nanoparticles and hydrogels.¹³ Their nanoscale size, high surface-to-volume ratio, and enhanced stability make them highly suitable for applications in imaging and drug delivery.^14–16 Many MRI-active nanogels incorporate metal chelates into the gel matrix to improve relaxivity.^17–20 For example, Kimura et al. synthesized a Gd-coordinated gelatin nanogel using DOTA-NHS and DOTA-butylamine as Gd ligands, demonstrating excellent biocompatibility and strong relaxivity (r₁ = 5.86 mM⁻¹ s⁻¹) as a T₁-weighted MRI contrast agent.²¹ Effective MRI contrast agents must be hydrophilic to facilitate rapid water exchange and enhance relaxivity.²² Poly(acrylic acid) nanogels (PAA NGs), rich in carboxyl groups, meet this requirement and form dynamic, non-covalent crosslinks with metal ions.²³

These biocompatible nanogels exhibit broad chelation capabilities with various metal ions (e.g., Mn, Ca, Gd, Cu), highlighting their potential for MRI applications.^24,25 The NG-as-chelator approach eliminates the need for external chelating agents, improving structural stability.²⁶ Jiang et al. developed a nanogel (PCG-Fe/DHA) using a nanovesicle template to co-deliver iron and dihydroartemisinin (DHA). This system, assembled from amphiphilic polyphosphazene and coordinated with tannic acid-modified chitosan oligosaccharides and Fe³⁺, achieved a T₁-weighted MRI signal 2.4 times stronger than free Fe.²⁷ Similarly, Chen et al. prepared Fe₃O₄-incorporated PAA nanogels via in situ hybridization, exhibiting high drug-loading capacity, excellent biocompatibility, and superior T₂-weighted imaging performance at tumor sites (r₂ = 180.3 mM⁻¹ s⁻¹).²⁸ In addition to providing contrast enhancement, nanogels also serve as effective drug delivery vehicles, enabling therapeutic applications. The integration of therapeutic agents and imaging functionalities within hybrid nanogel systems represents a key advancement in theranostics.²⁹ Owing to their ability to conjugate multiple functional moieties, nanogels hold significant potential for MRI-guided therapy and real-time treatment monitoring.^30,31

In cancer therapy, photothermal therapy (PTT) is a widely employed strategy, wherein photothermal agents convert near-infrared (NIR) light into localized heat for tumor ablation.³² Copper sulfide (CuS), a NIR-responsive photothermal agent, efficiently mediates PTT under 808 nm laser irradiation.³³ However, its intrinsic hydrophobicity limits aqueous dispersibility and biocompatibility, necessitating the use of stabilizers to maintain colloidal stability—thereby restricting further functionalization.³⁴ To enable real-time monitoring of PTT efficacy, the integration of imaging agents with photothermal components is essential.³⁵ Furthermore, certain transition metal ions (e.g., Cu⁺, Mn²⁺) can catalyze the decomposition of endogenous hydrogen peroxide (H₂O₂) into hydroxyl radicals (˙OH), inducing oxidative damage to tumor cells via chemodynamic therapy (CDT). Studies demonstrate that phototherapy-based combination strategies achieve enhanced anticancer efficacy compared with phototherapy alone.³⁶ Zhang et al. developed a multifunctional nanogel platform co-loaded with gadolinium (Gd) and CuS for combined MRI/photoacoustic (PA) imaging-guided PTT.³⁷ The system was constructed through a multi-step process: synthesis of cross-linked polyethyleneimine (PEI) nanogels, modification with Gd chelates, surface functionalization with folate (FA) ligands via poly(ethylene glycol) (PEG) spacers and 1,3-propanesultone, and subsequent loading of CuS nanoparticles. This multifunctional construct exhibited high longitudinal relaxivity (r₁ = 11.66 mM⁻¹ s⁻¹), excellent photothermal conversion efficiency (26.7%), and demonstrated effective tumor targeting under MRI/PA imaging guidance.³⁸

In this study, we developed a copper–manganese hybrid nanogel (CMNG) as a multifunctional theranostic platform (Scheme 1). The nanogel was synthesized by first forming a cross-linked poly(acrylic acid) (PAA) network, coordinating Mn²⁺ ions via abundant carboxyl groups, and subsequently generating CuS nanoparticles in situ within the gel matrix. This design enables MRI-guided combinatorial PTT and CDT. The incorporated Mn²⁺ ions provide strong T₁-weighted MRI contrast enhancement, while the embedded CuS nanoparticles ensure efficient photothermal conversion under NIR irradiation. Additionally, CMNG facilitates cascade catalysis through Cu⁺/Cu²⁺ and Mn²⁺, synergistically depleting intracellular glutathione (GSH) and amplifying oxidative stress via Fenton-like reactions. By integrating high-sensitivity imaging with multimodal therapeutic functionalities in a single nanostructure, CMNG addresses the limitations of conventional theranostic agents. This platform enables real-time MRI tracking of tumor accumulation and therapeutic progression, and achieves effective tumor ablation in 4T1 murine models through synergistic mechanisms, offering a promising strategy for precision cancer therapy.

Scheme 1 Schematic illustration of CMNG for CDT and PTT of tumor treatment.

2. Materials and methods

2.1. Materials and cells

Ethylene diacrylate (EDA) and tert-butyl acrylate (tBA) were purchased from Acmec (Shanghai, China). Na₂S·9H₂O and MnCl₂·4H₂O were obtained from Adamas-beta (Shanghai, China). Sodium 4-vinylbenzenesulfonate and anhydrous CuCl₂ were supplied by Energy Chemical (Shanghai, China). Coumarin 6 (C6), potassium persulfate, and sodium sulfide hydrate (Na₂S·9H₂O) were acquired from Macklin Corporation (Shanghai, China). Concentrated nitric acid (HNO₃, 75%) was procured from Guangzhou Chemical Preparation Factory (Guangzhou, China).

4T1 murine mammary carcinoma cells and human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin–streptomycin (P/S; Gibco, USA), and maintained at 37 °C in a humidified 5% CO₂ atmosphere.

2.2. Synthesis of polyacrylic acid nanogels (PAA NG)

Tert-Butyl acrylate (tBA) and ethylenediamine (EDA) were purified by passing through a short column of basic alumina to remove polymerization inhibitors and stored at –20 °C prior to use. Poly(acrylic acid) nanogels (PAA NGs) were synthesized via soap-free emulsion polymerization. In a Schlenk flask, potassium persulfate (30 mg) and sodium 4-vinylbenzenesulfonate (40 mg) were dissolved in 40 mL of a methanol/deionized water mixture (1 : 9, v/v). The solution was purged with argon for 30 minutes to remove dissolved oxygen.

Subsequently, 5 mL of tBA (monomer) and 60 μL of EDA (crosslinker) were added, followed by an additional 5-minute argon purge. The reaction mixture was sealed and maintained at 70 °C in an oil bath for 18 hours. The resulting tert-butyl-protected poly(acrylic acid) (t-PAA) precursor was collected and washed twice with methanol by centrifugation (13 000 rpm, 1 h). For hydrolysis, the t-PAA was dispersed in 30 mL of a formic acid/trifluoroacetic acid mixture (2 : 8, v/v) and stirred at room temperature for 36 hours to cleave tert-butyl ester groups. The product was washed twice with ethanol (13 000 rpm, 40 minutes) to remove acidic residues, followed by sequential dialysis against ethanol (24 h) and ultrapure water (24 h) using dialysis membranes (MWCO: 14 kDa). The final PAA nanogels, with a crosslinking density of 1%, were stored at 4 °C until further use.

2.3. Preparation of MNG, CMNG, CMNG-a, MCNG and C6-CMNG

MNG (Mn²⁺-chelated nanogels): poly(acrylic acid) nanogels (10 mg) were dispersed in 2 mL of ultrapure water in a penicillin vial. A solution of MnCl₂·4H₂O (48 mg in 2 mL ultrapure water) was added dropwise under ultrasonication. The mixture was stirred at room temperature for 24 h to allow Mn²⁺ chelation. The resulting Mn²⁺-chelated nanogels (MNG) were collected by centrifugation (13 000 rpm, 15 min) and washed with ultrapure water. The feeding molar ratio of [Mn²⁺]/[–COOH] was maintained at 2 : 1.

CMNG and CMNG-a (CuS-embedded Mn-chelated nanogels): for CMNG preparation, 2 mg of CuCl₂ was dissolved in ultrapure water, and 10 mg of MNG was dispersed separately in ultrapure water. The CuCl₂ solution was added dropwise to the MNG dispersion under bath sonication, followed by stirring at room temperature for 30 min to chelate Cu²⁺. Then, a solution of Na₂S·9H₂O (4 mg in 1 mL ultrapure water) was added slowly under sonication. The reaction mixture was transferred to a preheated oil bath at 70 °C and stirred for 40 min. After cooling in an ice bath, the product was washed once with ultrapure water by centrifugation (13 000 rpm, 10 min), resuspended in ultrapure water, and dialyzed for 24 h using a dialysis membrane (MWCO: 14 kDa). For CMNG-a, the feeding molar ratio of [Mn²⁺]/[–COOH] was adjusted to 1 : 2.

MCNG (Mn²⁺-chelated CuS nanogels): To prepare MCNG, Cu²⁺ was first chelated to PAA nanogels to form CuS-loaded nanogels (CNG). Subsequently, 10 mg of CNG was treated with MnCl₂·4H₂O ([Mn²⁺]/[–COOH] = 2 : 1) under ultrasonication and stirred for 24 h at room temperature.

C6-CMNG (fluorescently labeled CMNG): For fluorescent labeling, 10 mg of CMNG was dispersed in 1 mL ethanol and mixed with 1 mg of coumarin 6 (C6) dissolved in 1 mL ethanol under ultrasonication. The mixture was stirred for 24 h in the dark. The resulting product was washed twice with ethanol and once with ultrapure water by centrifugation (13 000 rpm, 10 min), then resuspended in 1 mL ultrapure water.

2.4. Quantification and optimization of metal ion loading and CuS formation

To evaluate the metal ion loading capacity of nanogels, CMNG, CMNG-a, and MCNG samples were digested with 75% concentrated HNO₃ at 90 °C until complete clarification. The solutions were evaporated to dryness at 110 °C, and the residues were reconstituted in 5% (v/v) HNO₃, filtered through 0.22 μm membranes, and diluted to a final volume of 10 mL. The concentrations of Mn and Cu ions were quantified by elemental analysis using an Optima 8300 inductively coupled plasma atomic emission spectrometer (ICP-AES; PerkinElmer, USA). To further optimize the in situ formation of CuS within the nanogels, experimental groups C2, C3, and C4 were prepared by varying the Cu²⁺ chelation time (30 min, 1 h, and 2 h, respectively) while maintaining a constant heating time of 20 min at 70 °C, whereas group CMNG was prepared with a Cu²⁺ chelation time of 30 min and a heating time of 40 min at 70 °C. Particle characterization of the resulting nanogels, including hydrodynamic diameter by dynamic light scattering (DLS) and zeta potential, was performed using a Zetasizer Nano ZS-90 system (Malvern Panalytical Ltd, UK). Optical properties were analyzed by UV-vis-NIR spectroscopy using a UV-2600i spectrophotometer (Shimadzu Corporation, Japan).

2.5. Morphology and elemental analysis of nanogels

Nanogel samples were diluted in ultrapure water to a final concentration of 100 μg mL⁻¹. A 10 μL aliquot was deposited onto carbon-coated copper Transmission electron microscopy (TEM) grids and air-dried at room temperature. Size distribution and nanostructural morphology were assessed by TEM using a JEM-F200 instrument (JEOL Ltd, Tokyo, Japan) operated at 120 kV. Elemental composition was analyzed by energy-dispersive X-ray spectroscopy (EDS) in both point-scan and mapping modes. For surface chemical characterization, lyophilized CMNG powder was uniformly dispersed on conductive carbon tape mounted on 2 × 2 cm aluminum substrates and compacted under 5 tons of pressure. XPS analysis was performed using a Thermo Scientific Nexsa spectrometer (Thermo Fisher Scientific, USA). High-resolution spectra of Mn, Cu, and S were deconvoluted with XPSPEAK software (v4.1) to determine elemental valence states.

2.6. Photothermal conversion performance of CMNG

The photothermal performance of CMNG was assessed under 808 nm NIR laser irradiation. Sample temperature was monitored in real-time using a thermocouple probe, while ambient temperature and absorbance at 808 nm were recorded for all experimental groups. CMNG solutions (1 mL) at various concentrations (0, 100, 200, 500, and 800 μg mL⁻¹) were placed in quartz cuvettes and irradiated with an 808 nm laser (1 W cm⁻²) for 10 minutes. A thermocouple was centrally positioned in the solution to record temperature changes at 10-second intervals. Time–temperature curves were generated to evaluate the concentration-dependent photothermal response. In a separate experiment, a 500 μg mL⁻¹ CMNG solution (1 mL) was irradiated at different power densities (0.6, 1.0, 1.6, and 2.0 W cm⁻²) for 10 minutes. Temperature was recorded every 10 seconds to generate power-dependent heating profiles. To evaluate photothermal stability, a 500 μg mL⁻¹ CMNG solution (1 mL) was subjected to four consecutive laser ON/OFF cycles under 808 nm irradiation at a power density of 1 W cm⁻². Each cycle comprised 10 minutes of continuous laser exposure followed by natural cooling to ambient temperature. Temperature measurements were recorded every 10 seconds throughout the experiment, and cyclic time–temperature profiles were plotted to assess photothermal stability.

The heat generated from photon energy by photothermal materials is a key parameter for assessing photothermal conversion performance. The photothermal conversion efficiency (η) was calculated according to the method reported by Li et al.³⁹ The η of CMNG was evaluated using a 500 μg mL⁻¹ solution. A 1 mL aliquot was irradiated with an 808 nm near-infrared laser at a power density of 1 W cm⁻² until thermal equilibrium was reached. Immediately after laser termination, the cooling process was monitored by recording the solution temperature at 10-second intervals. The photothermal conversion efficiency was calculated. In this study, the control + L and CMNG + L groups were treated with solvent or CMNG, respectively, followed by irradiation with an 808 nm laser (1 W cm⁻²) for 10 minutes.

2.7. In vitro chemodynamic study of nanogels

The core mechanism of CDT mediated by CMNG relies on Fenton-like reactions catalyzed by Mn²⁺ and Cu⁺ ions. These catalytic processes convert the excess H₂O₂ in the tumor microenvironment (TME) into highly reactive ˙OH, which cause oxidative damage to biomolecules and trigger apoptosis in tumor cells. The representative reactions are as follows:

The generation of ˙OH was evaluated by monitoring the decolorization of methylene blue (MB), a ˙OH-sensitive dye. A stock solution of MB (1 mg mL⁻¹) was prepared by dissolving weighed MB in ultrapure water. MNG and CMNG nanogels were separately suspended in phosphate-buffered saline (PBS), and test solutions (2 mL) were prepared under the following standardized conditions: nanogel concentration (MNG or CMNG) at 300 μg mL⁻¹, H₂O₂ concentration at 200 μM, and NaHCO₃ at 25 mM. The experimental groups were as follows: (i) H₂O₂ alone, (ii) MNG + H₂O₂, (iii) CMNG + H₂O₂, and (iv) CMNG + H₂O₂ + laser (L). For each group, 10 μL of the MB stock solution was added to the reaction mixture in a quartz cuvette and mixed thoroughly. Samples were incubated at room temperature in the dark for 30 minutes. UV-vis absorption spectra were recorded every 5 minutes using a UV-vis spectrophotometer, and changes in absorbance at 664 nm were used to assess MB degradation. For group (iv), the CMNG solution was pre-irradiated with an 808 nm laser at a power density of 1.6 W cm⁻² for 10 minutes before MB addition, followed by incubation and spectral analysis as described above.

2.8. In vitro MRI evaluation of nanogels

The T₁- and T₂-weighted MRI performance of MNG, CNG, and CMNG nanogels was assessed using a 3.0 T clinical MRI scanner (Magnetom Prisma, Siemens Healthineers, Germany). Longitudinal (r₁) and transverse (r₂) relaxation rates were calculated based on relaxation time measurements. MNG and CMNG samples were prepared at varying Mn²⁺ concentrations (0–0.12 mM), while CNG samples contained Cu²⁺ concentrations equivalent to those in CMNG. For benchmarking, commercially available Magnevist (Gd³⁺; varying concentrations) and superparamagnetic iron oxide nanoparticles (SPIONs; varying Fe concentrations) were included as T₁ and T₂ contrast agents, respectively. All samples were loaded into a 48-well plate and scanned under standardized imaging parameters: T₁-weighted imaging (T₁W): echo time (TE) = 14 ms, repetition time (TR) = 500 ms, flip angle (FA) = 150° T₂-weighted imaging (T₂W): TE = 4000 ms, TR = 500 ms, FA = 180°. Relaxivity values (r₁ and r₂) were obtained by plotting 1/T₁ and 1/T₂ against metal ion concentrations and applying linear regression analysis.

2.9. Cellular uptake

The cellular internalization of C6-labeled CMNG (C6-CMNG) was assessed in 4T1 cells using flow cytometry and confocal laser scanning microscopy. For flow cytometry analysis, 4T1 cells were seeded in 6-well plates at a density of 2.0 × 10⁵ cells per well and incubated overnight. After medium replacement, cells were treated with C6-CMNG (C6 concentration: 5 μg mL⁻¹) and incubated for 1, 2, 4, 6, or 8 h. At each time point, cells were trypsinized, centrifuged (1000 rpm, 3 min), washed twice with PBS, and resuspended in 500 μL PBS for fluorescence analysis by flow cytometry. For confocal microscopy, cells were seeded on glass coverslips in 24-well plates (5.0 × 10⁴ cells per well) and incubated overnight. After treatment with C6-CMNG (5 μg mL⁻¹) for varying time points (1–8 h), cells were stained with LysoTracker Red (50 nM in serum-free medium) at 37 °C with 5% CO₂ for 40 min to visualize lysosomes. Following PBS washes, cells were fixed in 4% paraformaldehyde for 15 min, counterstained with DAPI (10 μg mL⁻¹, 10 min), and mounted using antifade reagent. Intracellular localization and uptake were observed via confocal laser scanning microscopy.

2.10. Biocompatibility

The biocompatibility of nanogels was evaluated through both cytocompatibility and hemocompatibility assays. For cytocompatibility evaluation, human umbilical vein endothelial cells (HUVECs) and 4T1 murine breast cancer cells were seeded into 96-well plates at a density of 5.0 × 10³ cells per well and incubated overnight. The medium was then replaced with 100 μL of fresh medium containing varying concentrations (0–400 μg mL⁻¹) of MNG or CMNG nanogels. After incubation for 24 or 48 h, cells were washed with PBS, followed by the addition of 120 μL of MTT solution (final concentration: 1 mg mL⁻¹ in serum-free medium). Plates were incubated for 4 h at 37 °C, and the resulting formazan crystals were dissolved in 150 μL of DMSO. Absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated using the following formula:where: A_Sample: absorbance of treated cells (with test compounds); A_Control: absorbance of untreated control cells (without test compounds); A_Blank: absorbance of background (medium + MTT reagent, no cells).

Hemolysis assays were performed using mouse erythrocytes. Whole blood was centrifuged at 1500 rpm for 10 min at 4 °C, and the pellet was washed with PBS until the supernatant was clear. Erythrocytes were then resuspended in PBS to a final concentration of 2% (v/v). Aliquots (500 μL) of this erythrocyte suspension were incubated with MNG or CMNG solutions (25–500 μg mL⁻¹), PBS (negative control), or 0.1% Triton X-100 (positive control) at 37 °C for 2 h. After centrifugation at 3000 rpm for 10 min, the absorbance of the supernatant was measured at 431 nm. In addition, erythrocyte morphology was examined microscopically to assess potential morphological changes. The hemolysis rate was calculated as:

where: A_Sample: absorbance of the test sample (blood + material/extract); A_Negative: absorbance of negative control (blood + isotonic solution, e.g., PBS or saline); A_Positive: absorbance of positive control (blood + distilled water).

2.11. Cytotoxicity evaluation

Cytotoxicity of MNG and CMNG nanogels was assessed in 4T1 cells using MTT assays, live/dead staining, and scratch wound healing analysis. For the MTT assay, 4T1 cells were seeded in 96-well plates (5.0 × 10³ cells per well) and incubated overnight. The medium was then replaced with fresh medium containing different concentrations (0–250 μg mL⁻¹) of MNG or CMNG, followed by 6 h incubation. For groups receiving photothermal treatment, cells were irradiated with an 808 nm NIR laser (1 W cm⁻², 3 min) before further incubation up to 24 h, after which cell viability was determined by MTT assay. For fluorescence-based viability assessment, cells were seeded in 24-well plates (5.0 × 10⁴ cells per well), treated with MNG or CMNG (200 μg mL⁻¹) for 6 h, and exposed to NIR irradiation where indicated. Calcein-AM/propidium iodide (PI) staining was performed to visualize live and dead cells under a fluorescence microscope. For the scratch assay, cells were seeded in 24-well plates (1.0 × 10⁵ cells per well) and grown to confluence. A uniform scratch was introduced using a sterile 200 μL pipette tip, followed by PBS washing. Cells were then treated with MNG or CMNG (200 μg mL⁻¹), and laser irradiation was applied at 6 h post-treatment where applicable. Wound closure was monitored at 0, 6, 12, 24, and 48 h, and scratch widths were quantified using ImageJ to evaluate cell migration.

2.12. Photothermal therapy effects at the cellular level

Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨ_m) changes were evaluated in 4T1 cells. For ROS detection, cells were seeded in 6-well plates (2.0 × 10⁵ cells per well) or 24-well plates (5.0 × 10⁴ cells per well) and incubated overnight. Cells were treated with MNG or CMNG (100 μg mL⁻¹) for 6 h, followed by incubation with 10 μM DCFH-DA in serum-free medium for 30 min. Laser-treated groups received 808 nm NIR irradiation (1 W cm⁻², 3 min). For flow cytometric analysis, cells were trypsinized, washed, and resuspended in PBS for quantification of DCF fluorescence. For confocal laser scanning microscopy (CLSM; Nikon A1R, Nikon Instruments, Japan) imaging, cells were counterstained with Hoechst 33342 (10 μg mL⁻¹, 20 min) post-irradiation and imaged to visualize ROS distribution. Mitochondrial depolarization was assessed via JC-1 staining. Cells seeded in 24-well plates (5.0 × 10⁴ cells per well) were treated with MNG or CMNG (100 μg mL⁻¹) for 6 h, with or without NIR irradiation. After treatment, cells were incubated with JC-1 working solution for 20 min, washed with PBS, and fluorescence was analyzed by CLSM to evaluate changes in mitochondrial membrane potential.

2.13. Statistical analysis

All experimental data are presented as mean ± standard deviation (SD). Statistical analyses were performed using OriginPro 9.8 software. Differences among groups were evaluated by one-way analysis of variance (ANOVA), with p-values less than 0.05 considered statistically significant.

3. Results and discussion

3.1. Synthesis and characterization of CMNG

In this study, the precursor t-PAA was synthesized via free radical polymerization initiated by potassium persulfate, followed by hydrolysis of the tert-butyl ester groups to obtain poly(acrylic acid) nanogels (PAA NG). Subsequent sequential chelation of Mn²⁺ and Cu²⁺ ions onto the PAA NG yielded the final copper–manganese nanogel (CMNG). Chelation efficiency was assessed by analyzing elemental content under varying reaction conditions, with particular attention to the influence of PAA's weakly acidic ligation capacity. As shown in Table S1, the chelation sequence had minimal impact on Cu incorporation, with Cu mass fractions remaining around 10% across different samples—likely due to the fixed formation of CuS. In contrast, Mn loading was significantly affected by both the order of chelation and the solution conditions. The inherently acidic PAA NG solution suppressed carboxyl group dissociation, thereby limiting Mn²⁺ coordination and resulting in a relatively low Mn content (1.27%) in CMNG. Neutralization with NaOH markedly enhanced Mn loading to 18.60%; however, this adjustment compromised the colloidal stability of the resulting CMNG-a, leading to precipitation. Notably, the MCNG sample exhibited a higher Mn content than CMNG, likely due to partial doping of Mn²⁺ into the CuS crystal lattice. Considering both dispersion stability and the potential toxicity of excessive Mn²⁺, the CMNG formulation with 1.27% Mn and 9.13% Cu was selected for subsequent investigations.

We further examined the effects of Cu²⁺ chelation time and reaction duration on the optical properties and particle size of the nanogels. As shown in Table S2, prolonged chelation and heating led to increased particle sizes, with chelation time exerting a more pronounced impact than heating duration. UV-vis-NIR spectra (Fig. S1) revealed that longer processing times enhanced the characteristic absorption peaks, with sample C4 exhibiting the highest absorbance. Notably, compared with the baseline condition (C2: 30 min chelation/20 min heating), extending the heating time by 20 minutes (CMNG) resulted in a more significant increase in absorbance than extending the chelation time by 30 minutes (C3), indicating that heating duration plays a more critical role in modulating optical absorption. These differences are attributed to variations in CuS loading, particle size, and crystallinity. We hypothesize that nanogels with stronger absorption exhibit superior optical and photothermal conversion efficiencies. Considering both particle size and absorption intensity, a condition of 30 minutes of chelation followed by 40 minutes of heating was identified as optimal for the in situ synthesis of CuS.

Variations in particle size and surface potential during CMNG synthesis are summarized in Table S3. Compared with the precursor t-PAA (128.6 ± 12.23 nm), CMNG exhibited an increased hydrodynamic diameter (255.3 ± 17.75 nm), attributable to nanogel swelling and CuS nanoparticle loading. The resulting nanogels showed a narrower size distribution (PDI < 0.1) and excellent aqueous dispersibility (Fig. S2). Following deprotection of t-PAA to form PAA NGs, the surface potential decreased (from −28 to −33 mV) due to ionization of carboxyl groups. Subsequent chelation with Mn²⁺ increased the potential (from −33.0 to −22.1 mV) in MNGs, indicating enhanced surface charge (Table S3). Successful CuS formation was confirmed by UV-vis-NIR absorption spectra (Fig. S3), with CMNG exhibiting a characteristic peak at 885 nm. Transmission electron microscopy (TEM) analysis (Fig. 1A and B) revealed uniformly spherical nanogels with distinct structural features. Fig. 1A shows PAA nanogels (NGs) in aqueous solution exhibiting larger apparent size (∼120 nm) and lower electron density due to polymer swelling, whereas Fig. 1B demonstrates CMNGs with uniformly distributed, electron-dense inorganic nanoparticles (∼180 nm), confirming successful in situ hydrothermal synthesis of CuS within the nanogel matrix. The observed size differences align with hydrodynamic diameter trends from dynamic light scattering (DLS) measurements.

Fig. 1 (A) Transmission electron micrograph of PAA NG, scale bar = 50 nm; (B) transmission electron micrograph of CMNG, scale bar = 50 nm; (C) XPS full spectrum and (D) S, (E) Cu, (F) Mn elemental mapping result of CMNG; (G) EDS elemental mapping result of CMNG, scale bar = 100 nm; (H) heat-up curves of CMNG at different concentrations; (I) time–temperature curves of four heat-up/down cycles of CMNG; (J) photothermal effect diagram of CMNG. Laser: 808 nm, 1 W cm⁻².

The XPS survey spectrum of CMNG (Fig. 1C) confirmed the presence of C, O, Mn, S, and Cu elements. The S 2p spectrum in Fig. 1D exhibits a single peak at 126.9 eV, assigned to S²⁻, confirming the presence of sulfur. High-resolution Cu 2p spectra (Fig. 1E) exhibited characteristic doublets at 931.7 eV (Cu⁺ 2p_3/2) and 951.7 eV (Cu⁺ 2p_1/2), along with Cu²⁺ signals at 934.3 eV (2p_3/2) and 954.4 eV (2p_1/2), indicative of non-stoichiometric CuS predominantly composed of Cu⁺. The presence of copper vacancies is known to enhance near-infrared absorption and localized surface plasmon resonance (LSPR) behavior. Mn 2p spectra (Fig. 1F) showed binding energies at 641.4 eV (Mn²⁺ 2p_3/2) and 648.0 eV (Mn²⁺ 2p_1/2), with an additional peak at 653.3 eV suggesting partial oxidation to Mn⁴⁺ during thermal processing.

The metal ion content in CMNG was quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES), while elemental mapping of Mn, Cu, and S was performed using energy-dispersive X-ray spectroscopy (EDS). XPS provided insights into the valence states of metal ions. Quantitative EDS analysis (Fig. S4) confirmed Cu, S, and Mn as the dominant elements, uniformly distributed throughout the nanogel matrix (Fig. 1G). These results demonstrate the successful diffusion of metal ions into the three-dimensional gel network, enabling coordination with internal carboxyl groups. Within this hydrophilic nanoconfined environment, restricted water mobility enhances the interaction between paramagnetic Mn²⁺ ions and surrounding water molecules, thereby facilitating dual T₁–T₂ MRI contrast via geometric confinement effects.

As shown in Fig. 1H, temperature elevations (ΔT) were concentration-dependent: following 10 minutes of irradiation at 1 W cm⁻², CMNG solutions at concentrations of 100, 200, 500, and 800 μg mL⁻¹ exhibited ΔT values of 12.2 °C, 15.1 °C, 17.3 °C, and 19.4 °C, respectively. Laser power was found to exert a more pronounced influence on photothermal response (Fig. S5); for instance, at a fixed concentration of 500 μg mL⁻¹, ΔT increased from 10.7 °C at 0.6 W cm⁻² to 34.1 °C at 2.0 W cm⁻². CMNG also demonstrated excellent photothermal stability over four cycles of laser on/off irradiation, with consistent temperature changes ranging from 17.3 °C to 18.4 °C (Fig. 1I). The photothermal conversion efficiency (η) of CMNG was determined to be 23.29% (Fig. 1J), which is higher than that of commercial gold nanorods (21.0%) and nanoshells (13.0%), and comparable to the 26.7% reported for Gd/CuS-loaded nanogels by Zhang et al.^38,40 These results establish CMNG as a promising photothermal agent, where mild photothermal therapy (mPTT, 40–45 °C) may enhance tumor vascular permeability and facilitate targeted intratumoral accumulation.

As confirmed by XPS analysis (Fig. 1E and F), CMNG contains Cu⁺, Cu²⁺, and Mn²⁺ ions, all of which can catalyze the decomposition of H₂O₂ through Fenton-like reactions to generate hydroxyl radical (˙OH). The ˙OH-generating capacity was quantitatively evaluated via methylene blue (MB) decolorization, in which ˙OH-mediated oxidation leads to a decrease in MB absorbance at 664 nm. As shown in Fig. 2A, MNG facilitated time-dependent ˙OH production from H₂O₂/HCO₃⁻ solutions, resulting in gradual MB degradation. In comparison, CMNG exhibited markedly enhanced ˙OH generation kinetics (Fig. 2B), attributable to the synergistic catalytic activity of both Cu⁺/Cu²⁺ and Mn²⁺. Notably, exposure to 808 nm laser irradiation (1.6 W cm⁻²) further accelerated MB degradation in CMNG suspensions (Fig. 2C and 2D), with maximum absorbance loss observed at 5 minutes and continuing reduction at 10 minutes, indicating photothermally enhanced Fenton-like reactivity that improves CDT efficacy.

Fig. 2 UV-visible absorption spectra of MB solution at different times under (A) MNG, (B) CMNG, and (C) CMNG + L treatments; (D) comparison of UV-visible absorption spectra at the 5th min under different treatments; (E) DCF fluorescence intensity, n = 3, *p < 0.05; (F) quantitative analysis of the 4T1 cells after different treatments; (G) confocal laser scanning microscopy images of mixed staining of ROS (DCF dye) and live cells (Hoechst dye) after different treatments of 4T1 cells, Scale bar = 50 μm. Laser: 808 nm, 1 W cm⁻².

Intracellular reactive oxygen species (ROS) generation was further validated using DCFH-DA fluorescence. Flow cytometry revealed negligible fluorescence in the control and control + L groups (Fig. 2E), confirming that NIR irradiation alone does not induce substantial ROS production. Treatments with MNG and CMNG significantly increased DCF fluorescence, reflecting Mn²⁺/Cu⁺/Cu²⁺-mediated intracellular ROS generation, with CMNG producing the highest signal due to dual-metal catalysis (Fig. 2F). Importantly, the CMNG + L group (treated with CMNG and irradiated with an 808 nm laser at 1 W cm⁻² for 10 min) exhibited further fluorescence enhancement, demonstrating that photothermal activation augments CDT efficacy at the cellular level. Consistent with flow cytometry results, confocal fluorescence imaging (Fig. 2G) showed minimal DCF signal in control groups, while increasingly intense fluorescence was observed in the MNG, CMNG, and CMNG + L groups, confirming efficient and photothermally enhanced ROS generation for effective CDT.

3.2 In vitro magnetic resonance imaging of CMNG

The cross-linked nanogel network forms a highly hydrated porous matrix that spatially confines Mn²⁺ ions, thereby facilitating effective water–ion interactions while simultaneously restricting the diffusion and rotational mobility of water molecules. This geometric confinement effect contributes to enhanced T₂-weighted MRI contrast capabilities. Using a 3.0 T MRI scanner, we systematically evaluated and compared the T₁ and T₂ relaxation performances of MNG and CMNG against commercial contrast agents—Magnevist (for T₁) and superparamagnetic iron oxide (SPIO, for T₂). Relaxation rates (r₁ and r₂) were determined from the linear correlation between ion concentration and relaxation rate (1/T₁ or 1/T₂) (Fig. 3). T₁-weighted images demonstrated concentration-dependent signal enhancement (brightening) for MNG and CMNG, while T₂-weighted images showed corresponding signal attenuation (Fig. 3A and C). MNG exhibited high relaxivities (r₁ = 21.62 mM⁻¹ s⁻¹, r₂ = 49.74 mM⁻¹ s⁻¹), outperforming both Magnevist and SPIO (Fig. 3B and D). Notably, the CMNG doped with CuS also exhibited outstanding relaxivity (r₁ = 10.81 mM⁻¹ s⁻¹, r₂ = 23.74 mM⁻¹ s⁻¹). Compared to the clinically used Gd-based contrast agent Magnevist, CMNG produced significantly brighter T₁-weighted images, demonstrating a 3.3-fold higher r₁ value (Fig. 3A and B). Zhu et al. designed a similarly Mn-doped nanogel composed of mitoxantrone, manganese chloride, and PAA, which also displayed a lower relaxivity (r₁ = 7.17 mM⁻¹ s⁻¹) than CMNG.⁴¹

Fig. 3 (A) T1W MRI images and (B) corresponding longitudinal relaxation rates of MNG, CNG, CMNG and Magneviest; (C) T2W MRI images and (D) corresponding transverse relaxation rates of MNG, CNG, CMNG and SPIO.

3.3. Cell experiments of CMNG

The biocompatibility of MNG and CMNG was evaluated through in vitro cytocompatibility and hemocompatibility assays. As shown in Fig. 4A and B, human umbilical vein endothelial cells (HUVECs) incubated with MNG and CMNG for 24 and 48 hours maintained high viability. After 48 hours of exposure at a concentration of 400 μg mL⁻¹, cell viability remained at 78.16% and 78.83% for MNG and CMNG, respectively, indicating low cytotoxicity. Hemolysis assays (Fig. 4C and D) revealed that MNG induced hemolysis rates of ≤0.24% at concentrations up to 400 μg mL⁻¹, suggesting negligible erythrocyte damage. CMNG exhibited slightly higher hemolysis rates, but remained low at ≤2.03% under equivalent conditions. Morphological examination confirmed that erythrocytes retained their characteristic biconcave disc shape without evident abnormalities in both treatment groups. Collectively, these results demonstrate excellent blood compatibility for both nanogels, supporting their suitability for subsequent in vivo studies and potential clinical applications.

Fig. 4 (A) Cellular activity of MNG and (B) CMNG incubated with HUVEC cells for different times, n = 5; (C) hemolysis rates of MNG, CMNG and (D) erythrocyte morphology, n = 5, scale bar = 500 μm.

The cellular uptake kinetics of C6-labeled CMNG (C6-CMNG) by 4T1 cells were quantitatively and qualitatively evaluated using flow cytometry and confocal laser scanning microscopy (CLSM). Flow cytometry analysis demonstrated a time-dependent increase in fluorescence intensity, reaching a maximum at 8 hours post-incubation (Fig. 5A and B), indicative of active internalization of CMNG by the cells. This uptake enables CMNG to utilize intracellular H₂O₂ for initiating Fenton-like reactions, leading to ROS generation. Consistent with these results, CLSM imaging revealed significant co-localization of C6 fluorescence with lysosomal markers (Fig. 5C), confirming lysosome-mediated endocytosis as the primary pathway for CMNG internalization.

Fig. 5 (A) Flow fluorescence intensity and (B) quantitative analysis of C6-CMNG after incubation with 4T1 cells for different times, n = 3; (C) laser confocal imaging images of C6-CMNG after incubation with 4T1 cells for different times, scale bar = 50 μm.

The cytotoxic efficacy of CDT and combined PTT/CDT against 4T1 cells was assessed using cell viability assays, live/dead staining, and scratch wound healing assays. Treatments with Mn²⁺-mediated CDT, Mn²⁺/Cu²⁺ dual CDT, and PTT/CDT showed progressively enhanced suppression of cellular viability. At a concentration of 400 μg mL⁻¹, cell viabilities in the CMNG (CDT) and CMNG + L (PTT/CDT) groups were reduced to 39.42% and 12.61%, respectively, demonstrating significantly improved cytotoxicity in the combined therapy group (Fig. 6A). Live/dead staining using Calcein AM/PI corroborated these results: control and laser-only groups exhibited predominant green fluorescence, indicating healthy cells; the MNG group showed sporadic red fluorescence, reflecting limited CDT efficacy; CMNG treatment induced markedly increased red fluorescence, consistent with dual CDT effects; and the CMNG + L group displayed predominantly red fluorescence with minimal green signal, indicating maximal cell death (Fig. 6B). These observations are consistent with MTT assay results, establishing the cytotoxicity hierarchy as CDT < dual CDT < PTT/CDT.

Fig. 6 (A) Cellular activity of 4T1 cells treated with MNG, CMNG, CMNG + L, n = 5; (B) calcein AM/PI live-dead staining imaging of 4T1 cells after 8 h of different treatments, Scale bar = 300 μm.

The inhibitory effects of CMNG-mediated combined PTT/CDT on the migratory capacity of 4T1 cells were further evaluated using a scratch wound healing assay, with results presented in Fig. 7. In the control groups, with or without laser irradiation, cells exhibited nearly complete wound closure after 48 hours, demonstrating robust proliferation and migration. Treatment with CMNG alone resulted in a notable suppression of cell migration, with a migration rate of 36.63%, indicating the inhibitory effect of CDT. When combined with laser irradiation, the CMNG + L group showed a further significant reduction in migration rate to 9.98%. These results suggest that CMNG-mediated PTT/CDT effectively inhibits 4T1 tumor cell migration, highlighting its potential as a potent tumor-suppressive agent.

Fig. 7 Scratch imaging of 4T1 cells incubated for different time points under different conditions, laser: 808 nm, 1 W cm⁻², 10 min, scale bar = 500 μm.

Mitochondrial membrane potential (ΔΨ_m) serves as a key indicator of mitochondrial functional and structural integrity. As shown in Fig. 8, control cells exhibited strong red fluorescence from JC-1 aggregates, reflecting intact ΔΨ_m and healthy cellular status. Treatment with MNG and CMNG resulted in a marked decrease in red fluorescence accompanied by an increase in green monomer fluorescence, indicating ΔΨ_m dissipation and initiation of early apoptosis. Notably, CMNG induced a more pronounced green fluorescence signal than MNG, attributable to the enhanced CDT effect mediated by dual metal ions (Cu/Mn). The CMNG + laser group displayed complete collapse of ΔΨ_m under combined PTT and photothermally amplified CDT, evidenced by exclusive green fluorescence without detectable red aggregates. These findings demonstrate that laser-activated CMNG synergistically generates ROS via PTT/CDT, effectively disrupting mitochondrial homeostasis and inducing tumor cell apoptosis more efficiently than CDT alone.

Fig. 8 Changes in mitochondrial membrane potential after different treatments in 4T1 cells, Laser: 808 nm, 1 W cm⁻², 10 min, Scale bar = 50 μm.

4. Conclusions

This study successfully developed a multifunctional CMNG platform, wherein poly(acrylic acid) nanogels serve as a matrix for carboxylate-coordinated Mn²⁺ ions, enabling high-performance T₁-weighted MRI contrast with a relaxivity of r₁ = 10.81 mM⁻¹ s⁻¹, alongside in situ-grown CuS nanoparticles that provide efficient photothermal conversion (η = 23.29%). The integrated system facilitates MRI-guided combinatorial PTT/CDT via Cu⁺/Mn²⁺-mediated cascade reactions that deplete glutathione (GSH), enhance ˙OH generation through Fenton-like processes, and induce a synergistic ROS burst under laser irradiation to disrupt mitochondrial function. In vitro studies confirmed significantly enhanced cytotoxicity in CMNG + L group (12.61% cell viability) relative to CMNG group (39.42%), accompanied by effective inhibition of tumor cell migration. The excellent biocompatibility and multimodal therapeutic efficacy of CMNG underscore its promising potential for translational cancer theranostics.

Author contributions

Yijie Luo: writing - reviewing and editing, data curation. Xiaotong Liang: writing – original draft, data curation. Kai Wang: data curation, visualization. Tuohuan Li: visualization. Jia Hua: Resource. Dalin Wu: supervision. Zhong Cao: supervision, resource, funding acquisition.

Conflicts of interest

There are no conflicts to declare.

Data availability

All relevant data are within the manuscript and its additional files. And detailed data are available from the corresponding author upon reasonable request.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5tb01758k

Acknowledgements

This study was supported by the Natural Science Foundation of the Guangdong Province (2024A1515010364, 2025A1515012035), Shenzhen Science and Technology Program (JCYJ20240813151016021, 2023A002), National Natural Science Foundation of China (52073313, 22075327).

References

1. M. Morrow, J. Waters and E. Morris, Lancet, 2011, 378, 1804–1811.
2. M. Shi, W. Xiong, J. Feng, L. Wu, J. Yang, Y. Lu, X. Lu, Q. Fan, H. Nie, Y. Dai, C. Yan, Y. Tian and Z. Shen, J. Nanobiotechnol., 2024, 22, 162.
3. T. Luo, B. Wang, R. Chen, Q. Qi, R. Wu, S. Xie, H. Chen, J. Han, D. Wu and S. Cao, J. Mater. Chem. B, 2025, 13, 372–398.
4. A. Avasthi, C. Caro, E. Pozo-Torres, M. P. Leal and M. L. García-Martín, Surface-modified Nanobiomaterials for Electrochemical and Biomedicine Applications, ed A. R. Puente-Santiago and D. Rodríguez-Padrón, Springer International Publishing, Cham, 2020, pp. 49–91.
5. N. Iyad, M. S. Ahmad, S. G. Alkhatib and M. Hjouj, Eur. J. Radiol. Open, 2023, 11, 100503.
6. R. R. Zairov, B. S. Akhmadeev, S. V. Fedorenko and A. R. Mustafina, Chem. Eng. J., 2023, 459, 141640.
7. E. Kalisińska and H. Budis, Mammals and Birds as Bioindicators of Trace Element Contaminations in Terrestrial Environments: An Ecotoxicological Assessment of the Northern Hemisphere, ed. E. Kalisińska, Springer International Publishing, Cham, 2019, 213–246.
8. G. J. Soufi, A. Hekmatnia, S. Iravani and R. S. Varma, ACS Appl. Nano Mater., 2022, 5, 10151–10166.
9. J. Dolai, K. Mandal and N. R. Jana, ACS Appl. Nano Mater., 2021, 4, 6471–6496.
10. Q. Guo, H. Wang, H. Hao, C. Zhang, X.-J. Liang and D. Liu, J. Mater. Chem. B, 2025, 13, 9698–9719.
11. D. Kalyane, N. Raval, R. Maheshwari, V. Tambe, K. Kalia and R. K. Tekade, Mater. Sci. Eng. C, 2019, 98, 1252–1276.
12. A. Dasgupta, A. M. Sofias, F. Kiessling and T. Lammers, Nat. Rev. Bioeng., 2024, 2, 714–716.
13. J. Gupta and G. Sharma, Drug Delivery Transl. Res., 2025, 15, 455–482.
14. P. N. Kendre and T. S. Satav, Polym. Bull., 2019, 76, 1595–1617.
15. A. J. Dehchani, A. Jafari and F. Shahi, Polym. Adv. Technol., 2024, 35, e6595.
16. A. Vashist, G. Perez Alvarez, V. Andion Camargo, A. D. Raymond, A. Y. Arias, N. Kolishetti, A. Vashist, P. Manickam, S. Aggarwal and M. Nair, Biomater. Sci., 2024, 12, 6006–6018.
17. F. Carniato, M. Ricci, L. Tei, F. Garello, C. Furlan, E. Terreno, E. Ravera, G. Parigi, C. Luchinat and M. Botta, Small, 2023, 19, 2302868.
18. C. V. Gheran, G. Rigaux, M. Callewaert, A. Berquand, M. Molinari, F. Chuburu, S. N. Voicu and A. Dinischiotu, Nanomaterials, 2018, 8, 201.
19. X. Ma, T. Zhang, W. Qiu, M. Liang, Y. Gao, P. Xue, Y. Kang and Z. Xu, Chem. Eng. J., 2021, 420, 127657.
20. F. Carniato, M. Ricci, L. Tei, F. Garello, C. Furlan, E. Terreno, E. Ravera, G. Parigi, C. Luchinat and M. Botta, Small, 2023, 19, 2302868.
21. A. Kimura, J.-i Jo, F. Yoshida, Z. Hong, Y. Tabata, A. Sumiyoshi, M. Taguchi and I. Aoki, Acta Biomater., 2021, 125, 290–299.
22. J. Hasserodt, J. L. Kolanowski and F. Touti, Angew. Chem., Int. Ed., 2014, 53, 60–73.
23. W. Gao, X. Yu, C. Zhang, H. Du, S. Yang, H. Wang, J. Zhu, Y. Luo and M. Zhang, Acta Biomater., 2024, 187, 328–339.
24. J. Guo, Y. Hou, L. Ye, J. Chen, H. Wang, L. Yang, J. Jiang, Q. Sun, C. Xie and B. J. S. C. C. Hu, Sci. China Chem., 2022, 65, 2260–2273.
25. S. Marasini, H. Yue, S.-L. Ho, J.-A. Park, S. Kim, J.-U. Yang, H. Cha, S. Liu, T. Tegafaw and M. Y. J. A. S. Ahmad, Appl. Sci., 2021, 11, 2596.
26. H. Arkaban, M. Barani, M. R. Akbarizadeh, N. Pal Singh Chauhan, S. Jadoun, M. Dehghani Soltani and P. Zarrintaj, Polymers, 2022, 14, 1259.
27. J. Jiang, Y. Peng and L. Qiu, Chem. Eng. J., 2024, 499, 156019.
28. Y. Chen, J. Nan, Y. Lu, C. Wang, F. Chu and Z. Gu, J. Biomed. Nanotechnol., 2015, 11, 771–779.
29. I. Altinbasak, Y. Alp, R. Sanyal and A. Sanyal, Nanoscale, 2024, 16, 14033–14056.
30. A. J. Sivaram, P. Rajitha, S. Maya, R. Jayakumar and M. Sabitha, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol., 2015, 7, 509–533.
31. C. Jacinto, W. F. Silva, J. Garcia, G. P. Zaragosa, C. N. D. Ilem, T. O. Sales, H. D. A. Santos, B. I. C. Conde, H. P. Barbosa, S. Malik and S. K. Sharma, J. Mater. Chem. B, 2025, 13, 54–102.
32. X. Li, J. F. Lovell, J. Yoon and X. Chen, Nat. Rev. Clin. Oncol., 2020, 17, 657–674.
33. J. Ma, N. Li, J. Wang, Z. Liu, Y. Han and Y. Zeng, Exploration, 2023, 3, 20220161.
34. U. Shamraiz, R. A. Hussain and A. Badshah, J. Solid State Chem., 2016, 238, 25–40.
35. X. Meng, B. Zhang, Y. Yi, H. Cheng, B. Wang, Y. Liu, T. Gong, W. Yang, Y. Yao, H. Wang and W. Bu, Nano Lett., 2020, 20, 2522–2529.
36. J. Yi, L. Liu, W. Gao, J. Zeng, Y. Chen, E. Pang, M. Lan and C. Yu, J. Mater. Chem. B, 2024, 12, 6285–6304.
37. Y. Zhuang, S. Han, Y. Fang, H. Huang and J. Wu, Coord. Chem. Rev., 2022, 455, 214360.
38. C. Zhang, W. Sun, Y. Wang, F. Xu, J. Qu, J. Xia, M. Shen and X. Shi, ACS Appl. Mater. Interfaces, 2020, 12, 9107–9117.
39. J. Li, W. Zhang, W. Ji, J. Wang, N. Wang, W. Wu, Q. Wu, X. Hou, W. Hu and L. Li, J. Mater. Chem. B, 2021, 9, 7909–7926.
40. C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell and B. A. Korgel, Nano Lett., 2011, 11, 2560–2566.
41. X. Zhu, Q. He, S. He, X. Li, X. Huang, J. Wang and X. Wei, Chem. Eng. J., 2024, 485, 149752.

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Footnote

† These authors contributed equally to this study.

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