Synthesis and Application Evaluation of the Novel Nanocluster MnS@Tf for Tumor Management

TL;DR

A new nanocluster, MnS@Tf, was developed to target and treat colorectal cancer by combining imaging and therapy.

Contribution

The novel nanocluster MnS@Tf integrates chemodynamic therapy, MRI, and gas therapy for targeted tumor treatment.

Findings

MnS@Tf releases manganese ions for chemodynamic therapy and magnetic resonance imaging.

The nanocluster effectively targets and kills colorectal cancer cells in vitro and in vivo.

MnS@Tf demonstrated stable tumor-targeting and anti-tumor effects in mice over time.

Abstract

Background: Exploring new management and treatment strategies for inoperable colorectal cancer is key to improving patient prognosis. Nanotechnology combining medical imaging with cancer treatment provides a new solution for the management of advanced cancer. Methods: This study designed and synthesized the dual-modal molecular imaging probe MnS@Tf-125I and evaluated its diagnostic and therapeutic applications in colorectal cancer with high expression of transferrin receptors (TfR) through in vitro and in vivo studies. Results: The MnS@Tf synthesized in this study can release manganese ions for chemodynamic therapy (CDT) and magnetic resonance imaging (MRI) and can be combined with hydrogen sulfide (H2S) for gas therapy in response to the acidic tumor microenvironment. The molecular imaging probe MnS@Tf-125I was labeled with 125I to verify MnS@Tf’s targeting and high affinity for tumors with high expression of TfR through in vitro experiments. In vitro cell experiments demonstrated the killing effect of MnS@Tf on CT26 cells. The results of blood clearance and imaging after intravenous injection of MnS@Tf-125I showed that MnS@Tf could stably exert targeted tumor-killing effects in mice over an extended period. In vivo experiments indicated that MnS@Tf not only effectively initiated T1-weighted MRI but also significantly inhibited tumor growth in CT26-bearing mouse models. Conclusions: The dual-modal molecular imaging probe MnS@Tf synthesized in this study can specifically target tumors with high expression of TfR and has good therapeutic effects both in vitro and in vivo, indicating that the nanocluster has broad application prospects in clinical tumor management.

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1. Introduction

The incidence and age-standardized mortality of colorectal cancer (CRC) in China has been increasing year by year, and the incidence rate has risen to second place [1]. Although treatment strategies for advanced or recurrent metastatic colorectal cancer have evolved over the past 10 years, systemic therapy still has a high rate of treatment failure for the majority of patients [2]. Exploring new management and treatment strategies for unresectable colorectal cancer has become key to improving the prognosis. Nanotechnology integrating medical imaging and cancer treatment offers a new solution for the management of advanced cancer [3,4].

In recent years, manganese-based magnetic resonance contrast agents such as manganese sulfide (MnS), manganese carbonate, or manganese iron alloys have been widely used in the diagnosis and treatment of tumors [5,6]. MnS is metastable and can be degraded in an acidic microenvironment and release manganese ions (Mn^2+^). The development of nanomedicine based on MnS for pH-responsive T1-weighted tumor MRI in tumors [7] has shown promise. In addition, a large number of studies have confirmed that Mn ions can decompose endogenous H_2_O_2_ into reactive oxygen species (ROS) in the acidic tumor microenvironment through chemodynamic therapy (CDT) to kill cancer cells [6,8].

Gas therapy is an emerging therapeutic strategy based on the biological effects of multiple gases, such as nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H_2_S), and hydrogen (H_2_) [9,10]. H_2_S gas has been reported to have significant antiproliferative effects on breast cancer cells and living organisms [11]. However, traditional H_2_S donors, such as Na_2_S and NaHS, are released too quickly to maintain long-term effects [12]. Therefore, the development of new sustainable donors of H_2_S is an urgent need for cancer gas therapy.

Transferrin receptor 1 (TfR1), also known as differentiation cluster 71 (CD71), is a type II transmembrane glycoprotein that binds to transferrin (Tf) [13]. The high expression of TfR1 in malignant tumor cells, coupled with its extracellular accessibility and internalization capacity, made it an ideal tumor-targeting ligand and also a carrier for imaging probes [14]. Nuclear medicine molecular imaging can sensitively provide early molecular and functional information about tumors [15]. Tf-labeled molecular imaging probes can specifically bind to TfR on the surface of tumor cells, resulting in high tumor uptake and target ratio. Evans et al. labeled Tf with positron radionuclide ^89^Zr and performed concurrent PET imaging in small animals, which was able to detect advanced prostate cancer and carcinoma in situ [16]. Multimodal imaging technologies can simultaneously obtain morphological information of tumor lesions and high-sensitivity early molecular level information [15].

In this study, we designed and synthesized a multifunctional nanocluster MnS@Tf which can not only act as a dual-modal imaging probe with magnetic and radioactive properties MnS@Tf-^125^I, but can provide treatment by releasing Mn-mediated CDT and H_2_S to kill tumor cells (Figure 1).

2. Materials and Methods

• Synthesis of MnS@Tf

Transferrin (60 mg) was added to 6 mL of ultra-filtered purified water to dissolve. MnCl_2_ and Na_2_S solutions with concentrations of 37 mM were prepared. Then, 1 mL of MnCl_2_ solution (37 mM) was slowly added to the Tf solution (60 mg/6 mL) and stirred at 4 °C in distillation flask. After 30 min, 1.5 mL of Na_2_S solution (37 mM) was added to the same distillation flask and stirred continuously for 2 h. The multifunctional nanocluster solution was purified and concentrated after centrifugation (Sigma 3-30 KS, Waltham, MA, USA, 7200× g, 15 min, 4 °C). The concentrated MnS@Tf solution was placed in a lyophilizer for continuous lyophilization for 24 h, and MnS@Tf solid was finally synthesized.

• MnS@Tf-^125^I Labeling and Quality Control

Next, 10 µL of ^125^I solution with a concentration of 0.1 mCi/µL was added into MnS@Tf solution (5 mM/80 µL), which was then labelled as MnS@Tf-^125^I after 30 min of reaction. The labeling rate of MnS@Tf-^125^I was determined by thin-layer chromatography (Elysia-raytest, Straubenhardt, Germany).

• Chemodynamic Therapy for MnS@Tf

MnS@Tf solutions containing Mn concentration gradients are formulated: 0, 0.01, 0.05, 0.1, 0.3, and 0.5 mM. We added 1 mL of 25 mM NaHCO_3_ and 10 µL MB solution to different concentrations of MnS@Tf solution and mixed the reactant for 30 min at room temperature. Then, 10 µL of H_2_O_2_ solution with a concentration of 30 mM was added, and the reactions were carried out at 37 °C for another 30 min. The spectrophotometer (Thermo, Waltham, MA, USA) instrument was used to determine the spectrum of the solution in the wavelength range of 500–800 nm. Spectral data was analyzed via the built-in workstation.

• In Vitro Targeting Validation

CT26 cells were cultured in 24-well plates at a density of approximately 5 × 10^5^ cells per well. DMEM medium (containing 10% serum and 1% double antibody) was used to prepare MnS@Tf-^125^I and ^125^I solutions at a concentration of 1 µCi/500 µL. The blocking group was prepared for 50 mg/50 µL of Tf solution. Different groups were set up and added to 24-well plates cultured with CT26 cells. The above 24-well plates were incubated (37 °C, 5% CO_2_) for 5 min, 15 min, 30 min, 60 min, 90 min and 120 min, respectively. After washing with PBS, 500 µL of cold NaOH solution with a concentration of 0.1 M was added to each well for 5 min. All cell solutions were transferred to γ counting tubes to detect the counts per minute (CPM) of the solution by a counter. Line charts were drawn and analyzed using GraphPad Prism 8.

• In Vitro Therapy of MnS@Tf

CT26 cells were seeded into 96-well plates at a density of 5000 cells per well. After 12 h in the incubator, cells were incubated with saline, MnS@Tf, MnCl_2_, and Na_2_S ([Mn]/[S] = 0–200 μM) for 24 and 48 h, respectively. A standard MTT assay was performed to assess cell viability.

CT26 cells were seeded into 24-well plates at a density of 1 × 10^5^ cells per well. After 12 h in the incubator, cells were incubated with saline, MnS@Tf, MnCl_2_, and Na_2_S (concentration 0–2 mM) for 24 h. 2 μM calcein green solution and 4 μM PI solution were added to each group of cells and incubated at 37 °C for 15–30 min. After cell processing was complete, the results were observed under a fluorescence microscope (LEICA, Wetzlar, Germany) with calcein green: Ex/Em = 490/515 nm, PI: Ex/Em = 535/617 nm.

After that, 24-well plates of CT26 cells at a density of 1 × 10^5^ cells per well were incubated with saline, MnS@Tf, MnCl_2_, and Na_2_S (concentration 0.5 mM) for 2 h. Enzymatic digestions were performed using pancreatic enzymes without EDTA. The cells were washed twice with pre-cooled PBS. The cells were then resuspended in 100 μL of 1× Annexin V binding buffer (provided by the kit) and transferred to a flow cytometry tube. FITC-labeled Annexin V (5 μL) was added and incubated at room temperature in the dark for 15 min. After the incubation, 10 μL of PI staining solution was added and was detected in the flow cytometer (Beckman-Coulter, San Jose, CA, USA) within 1 h. Additionally, the following control groups were set up: unstained cells, only Annexin V staining, and only PI staining.

• MRI T1 Relaxation Rate Determination

MnS@Tf was weighed to prepared for multiple Mn-containing concentration gradients: 0, 0.005, 0.01, 0.05, and 0.1 mM with pH = 6.8 and pH = 7.4 HEPES buffers. The solutions were transferred to 96-well removable plates at 200 ul per well for 30 min (release Mn^2+^). The scanning parameters of the BioSpec 7.0T/20 cm small animal MR machine from Bruker company in Germany were set as follows: T1-MSME: repetition time (TR) = 400 ms, echo time (TE) = 8.02 ms, field of view (FOV) = 58 mm × 58 mm, matrix (Matrix) = 256 × 256, layer thickness = 1.0 mm; T1-map: repetition time (TR) = 50, 100, 250, 500, 1000, 1500 ms, echo time (TE) = 8.02 ms, field of view (FOV) = 58 mm× 58 mm, matrix = 192 × 192, and then the whole-body volume coil of 72 mm inner diameter rat was selected to scan and collect the cross-sectional positions of 10 wells by T1-MSME and T1-map sequences. The image data were reconstructed and analyzed using Bruker’s built-in ParaVison 6.0 workstation to calculate the T1 relaxation time of MnS@Tf with different concentration gradients. Taking the Mn concentration as the abscissa and the ordinate as the reciprocal of the corresponding T1 relaxation time (R_1_ = 1/T1), the concentration–relaxation intensity curves of different concentrations of MnS@Tf were plotted.

• In Vivo MRI and SPECT/CT Imaging

Each 7-week-old female BALB/c mouse (weighing approximately 18–20 g) was subcutaneously inoculated with 5 × 10^6^ CT26 cells in the right axillary area. The mice were then housed in the IVC level animal room for 1 week. The tumor diameter was measured using a vernier caliper every day and the data were recorded. When the tumor diameter reached 0.8–1.0 cm, the next experiment could be conducted.

Intravenous injection of 5 mg/200 µL of MnS@Tf or 0.0025 mmol/200 µL of MnCl_2_, before injection and 4 h, 8 h, 24 h, 3 d, 5 d, 7 d after injection (n = 5), the German Bruker BioSpec7.0T/20 cm small animal MR machine was used for MRI T1 sequence imaging, and the CT26 Balb/c tumor-bearing mice were acquired by T1-MSME and T1-map sequence cross-sectional scanning. The image data was reconstructed and analyzed using Bruker’s built-in ParaVison6.0 workstation to calculate the relative signal enhancement (rSE) of the tumor site at different timepoints after injection. CT26 Balb/c tumor-bearing mice were randomly divided into an imaging group and a blockade group, and 1 mCi MnS@Tf-^125^I or 10 mg/300 µL Tf (+ 1 mCi MnS@Tf-^125^I) were injected through the tail vein (n = 5), respectively. The nanoScan SPECT/CT (Bioscan, Washington, DC, USA) was used to perform SPECT/CT imaging at 0.5 h, 4 h, 8 h, 24 h, 3 d, 5 d, and 7 d after injection. The tumor-to-muscle ratio (T/M) was used as a semi-quantitative analysis indicator to evaluate the degree of tracer uptake by the lesion relative to the normal background tissue such as the thigh muscle. A high T/M ratio usually indicates that the tumor has a higher specific binding rate, and it is an important indicator for monitoring tumors [17]. After the image acquisition, the tumor tissue was sampled to measure the radioactivity counts per minute (CPM) to calculate the %ID/g.

• In Vivo Biodistribution and Blood Clearance

Female Balb/c mice were randomly divided into 6 groups (n = 3), and all of them were injected with 200 µL containing 20 μCi (0.74 MBq) MnS@Tf-^125^I through the tail vein. At 0.5 h, 2 h, 4 h, 6 h, 8 h and 24 h after injection, the heart, liver, spleen, lungs, kidneys, pancreas, stomach, intestines, bladder, blood, brain, muscle, bone and thyroid were collected to measure CPM. %ID/g was calculated by the percentage of radioactivity per gram of organ tissue to the injected amount. Five female Balb/c mice were injected with 200 µL containing 20 μCi (0.74 MBq) MnS@Tf-^125^I through the tail vein, and the total weight was weighed 1 min, 15 min, 30 min, 2 h, 4 h, 6 h, 8 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d after injection, and the CPM data and %ID/g were calculated. MnS@Tf-^125^I clearance curve in the blood was plotted with GraphPad Prism 8.

• In Vivo Therapy of MnS@Tf

CT26 tumor-bearing mice were prepared consistent with the in vivo imaging experiments. The tumor sizes of each group of mice were recorded, and the average and standard deviation were calculated. CT26 tumor-bearing mice were randomly divided into 4 groups (n = 5). The mean ± SD of each group before treatment were 1.0 ± 0.4, 1.1 ± 0.2, 1.2 ± 0.3, and 0.9 ± 0.2, respectively. Different groups (20 mg/200 µL MnS@Tf, 0.01 mmol/200 µL MnCl_2_, 0.01 mmol/200 µL Na_2_S, and Saline) were injected through the tail vein, respectively. The long and short diameters of tumor-bearing mice were measured every 2 days during the 14 days following dosing, and the tumor volume V = (long diameter * short diameter^2^)/2 and the relative tumor volume RTV = mean tumor volume after treatment/mean tumor volume at baseline. The survival of tumor-bearing mice continued to be observed until day 14, and data were recorded. Bodyweight change curve and survival of CT26 tumor-bearing mice within 14 days to observe the efficacy of tumor treatment. All the mice were sacrificed on the 14th day after treatment, and then the tumors were removed for histological analysis. The tumors were fixed with 10% formalin solution, then embedded in paraffin and cut into 4-micron-thick sections. Hematoxylin and Eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) were performed, respectively, and observed under a digital microscope or confocal scanning microscope (CLSM).

• Statistical Analysis

GraphPad Prism 8 built-in statistical software is used. Data are presented as the mean ± standard deviation (SD). The independent-samples t-test was employed for intra-group comparisons. One-way ANOVA was utilized for between-group comparisons. p < 0.05 was deemed statistically significant.

3. Results

• Preparation and Characterization of MnS@Tf

MnS@Tf was synthesized via a wet chemical method (Figure 1). The manganese source (MnCl_2_) and Na_2_S were gradually added to the Tf solution to form MnS@Tf. The Mn spectrum of MnS@Tf in X-ray Photoelectron Spectroscopy (XPS) characterization exhibited two satellite peaks centered at 641.2 eV and 652.8 eV, corresponding to the Mn 2p_3/2_ and Mn 2p_1/2_ peaks (Figure 2a). The lattice fringes were distributed on the plane of cubic MnS@Tf, with a Mn interplanar spacing of 0.21 nm (Figure 2b and Figure S1), as confirmed by Transmission Electron Microscopy (TEM) characterization. Dynamic Light-Scattering (DLS) characterization indicated that the diameter of MnS@Tf nanoclusters was approximately 40 nm (Figure 2c), with a Zeta potential of about −26.34 mV (Figure 2d). The energy-dispersive X-ray spectroscopy (EDS) results showed that the synthesized material contained Mn, S, C, N, and O elements, indicating successful chelation between MnS and Tf (Figure 2e and Figure S2). Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) characterization revealed Mn content of 5.27% in MnS@Tf. These results demonstrated that MnS@Tf was successfully synthesized using Tf as a template, exhibiting appropriate particle size, making it suitable for the following experiments.

• In Vitro Targeted Validation

Labeling with radioactive nuclide ^125^I as a probe MnS@Tf-^125^I, the TLC results showed good stability while the immediate labeling rate of MnS@Tf-^125^I was greater than 98% and, after 72 h, greater than 90% in 50% fetal bovine serum and physiological saline (Figure 3a and Figure S3). Because of the results obtained in Figure 3a that showed the good stability of MnS@Tf-^125^I, we wanted to examine the targeting characteristics. Therefore, we did cell uptake and blocking experiments. The cell uptake curves of the targeting group (MnS@Tf-^125^I), the blocking group (MnS@Tf-^125^I + Tf), and the control group (^125^I) were statistically analyzed using GraphPad Prism 8, presented as mean ± SD (Figure 3b). The total radioactivity of the three groups was 1 μCi, and each sample was incubated with CT26 cells for 5, 15, 30, 60, 90, and 120 min, respectively. The data curves of the targeting group were compared with those of the control group and the blocking group, which are statistically significant (p < 0.001). The results of in vitro cell uptake experiments proved the targeting specificity of MnS@Tf-^125^I.

In vitro cell competition binding results show there was no statistically significant difference between MnS@Tf-^125^I and Tf-^125^I groups (p > 0.05), while the half inhibition rate of MnS@Tf-^125^I was 12.07 ± 5.84 nmol/L and Tf-^125^I was 9.24 ± 1.25 nmol/L (Figure 3c). When the concentration of transferrin was constant (Figure 3d), the Kd value of the equilibrium dissociation constant of MnS@Tf-^125^I was 32.74 ± 6.74 nmol/L and Tf-^125^I was 42.20 ± 2.04 nmol/L (p > 0.05). The in vitro results indicated that MnS@Tf has the same specific binding to transferrin receptors and the same high affinity as transferrin. Simultaneously, the Mn element in the reagent had no significant effect on the performance of Tf.

• In Vitro Cell-Killing Effect

To evaluate the chemical kinetic effect of MnS@Tf, the rate of methylene blue (MB) degradation in solutions containing H_2_CO_3_ and H_2_O_2_ was measured at different concentrations of MnS@Tf (Figure 4a). The degradation of MB indicates that MnS@Tf can generate concentration-dependent •OH. Next, in vitro experiments of MnS@Tf were conducted to evaluate treatment effect and cytotoxicity. As shown in Figure 4b,c, when incubated with Na_2_S, MnCl_2_, and MnS@Tf (0.1–2.0 mM), CT26 cell viability of MnS@Tf group was the lowest (p < 0.01), showing concentration-dependent and time-dependent therapeutic effects. The fluorescence intensity of dead cells in the Mns@Tf group was higher than that in the Na_2_S and MnCl_2_ groups with concentrations of 0.5, 1, and 2 mM (Figure 4d and Figure S4). The results of flow cytometry showed that after co-incubation for 1 h at a concentration of 0.5 mM, the apoptotic cells in the MnS@Tf, MnCl_2_, and Na_2_S group accounted for 3.65%, 0.62%, and 0.19%, respectively (Figure 4e). The results of cell flow cytometry further confirmed that the cell-killing effect of MnS@Tf was superior to that of Na_2_S and MnCl_2_.

• Biological Distribution and SPECT/CT Imaging

The blood clearance curve of MnS@Tf-^125^I in Balb/c mice showed that the blood distribution phase lasts 60.06 min and the blood clearance phase lasts 23.78 h (Figure 5a). In order to verify the physiological distribution in mice, we injected 200 μCi of MnsTf-^125^I into the tail veins of Balb/c mice and then conducted multiple timepoint biological distribution measurements (Figure 5b and Figure S5). The results of the biodistribution measurement showed that the concentration was the highest in the blood and the lowest in the brain, and it decreased over time. In addition, there was a higher radioactive distribution in the kidneys and bladder, indicating that the probe was excreted through the urinary system. The biodistribution results over 3–7 days showed that the nanoclusters can stabilize their function in the body for a period of time.

CT26 tumor-bearing mice were injected with MnS@Tf-^125^I via the tail vein and underwent SPECT/CT imaging at multiple timepoints. As shown in Figure 5c, the radioactive concentration was concentrated at the tumor site at 8 h. Organs such as the blood pool, gastrointestinal tract, and bladder were also imaged. After 24 h, the radioactive concentration at the tumor site became obvious, and the radioactive distribution in other parts decreased significantly, except in the bladder. In the blocking group, organs such as the blood pool, gastrointestinal tract, and bladder were imaged within 24 h, and the radioactive uptake at the tumor site was not obvious. After 24 h, the radioactive distribution in other organs decreased significantly, and the radioactive uptake at the tumor site was visible but was maintained at a low level. Further analysis of the radioactive uptake in the tumor area and calculation of the target-to-muscle ratio (tumor/muscle) with the contralateral thigh muscle was conducted (Figure 5d). The T/M ratio of the targeted group gradually increased after 24 h, while the T/M ratio of the blocking group did not show any significant change (p < 0.001).

• T1 Relaxation Rate and In Vivo MR Imaging

MnS@Tf was prepared into solutions with concentrations ranging from 0.005 to 0.1 mM using pH 6.8 and pH 7.4 HEPES buffers for MR T1W1 imaging. The results showed that, compared with the condition of pH 7.4, MnS@Tf measuring pH 6.8 could significantly reduce the T1 signal of the buffer solution, and the image gray level gradually became brighter as the solution concentration increased (Figure 6a). The concentration–relaxation intensity curve showed the T1 relaxation rate r1 = 16.96 mM^−1^s^−1^ at pH = 6.8 and 2.40 mM^−1^s^−1^ at pH = 7.4 (Figure 6b). Therefore, MnS@Tf under acidic conditions better conforms to the basic characteristics of MR imaging contrast agents and has a better positive contrast enhancement effect. CT26 tumor-bearing mice were respectively injected with MnS@Tf and MnCl_2_ via the tail vein and underwent MR imaging at multiple timepoints. As shown in Figure 6d, the signal intensity of the tumor area before injection was close to that of the surrounding muscle tissue. Further analysis of the signal-to-noise ratio (S/N) was conducted (Figure 6c). After injection of MnS@Tf, the signal intensity of the tumor area was the strongest at 8 h and then gradually decreased. The tumor signal intensity of the control group MnCl_2_ was the strongest at 24 h and then gradually decreased.

• In Vivo Treatment

The CT26 tumor-bearing mice were randomly divided into four groups: MnS@Tf, MnCl_2_, Na_2_S, and saline group (Figure 7a). Starting from the sixth day after administration, the tumor volume in the MnS@Tf group was significantly lower than that in the other groups, and the tumor growth trend was not obvious. The tumor volumes in the MnCl_2_ group and Na_2_S group were continuously lower than those in the saline group, but the tumor volume gradually increased over time. The relative volume growth curve of the tumors in the MnS@Tf group showed significant differences from the other groups (p < 0.0001). Although the relative volume growth of the tumors in the MnCl_2_ group and Na_2_S group was continuously lower than that in the control group, there was no statistical difference between these two groups (p > 0.05). The bodyweight of the tumor-bearing mice in each group showed no significant changes during the observation period (p > 0.05).

After the 14-day efficacy observation period, the tumor-bearing mice were sacrificed and the tumor tissues were subjected to H&E staining (Figure 7c) and Tunnel staining (Figure 7e). The percentage of blue-stained cell area of the MnS@Tf group was the lowest, while MnCl_2_ and Na_2_S group also had fewer cells than saline group (Figure 7d). Compared with the other groups, MnS@Tf produced the strongest cytotoxicity in vivo. The results of Tunnel staining sections showed that the percentage of apoptotic cell area in MnS@Tf group was significantly higher than that in the other groups, further confirming that MnS@Tf can kill tumor cells (Figure 7f).

4. Discussion

Multimodal molecular imaging, by integrating different imaging techniques such as PET/CT and SPECT/MRI, can display physiological and biochemical processes at the cellular and molecular levels in vivo [18]. It is mainly used for the early diagnosis and precise assessment of tumors, cardiovascular diseases, and neurological disorders. However, due to the high cost of equipment, complex operation procedures, and the high requirements for professional personnel, multimodal molecular imaging has not yet been widely used in clinical practice and is mainly used to supplement traditional imaging in specific scenarios [19]. The commonly used ^99m^Tc-colloids nanoparticles in clinical practice can be used to identify sentinel lymph nodes (SLNs) in various tumor entities and for imaging lymphatic flow [20]. Additionally, the application of nanoparticle-based diagnosis and treatment technologies mainly focused on two core characteristics: “targeted delivery” and “signal amplification”. On one hand, they acted as diagnostic imaging agents to enhance imaging contrast [20]. On the other hand, they served as drug delivery carriers to enrich drugs in tumors, improving therapeutic efficacy and reducing systemic toxicity [21]. Despite its broad prospects, the clinical translation of nanotechnology still faces multiple challenges such as biological safety, large-scale production, and complex regulatory approval pathways [21]. In summary, multimodal molecular imaging and nanotechnology are at a critical stage of transitioning from the laboratory to clinical practice. In this study, MnS@Tf-^125^I was proposed based on nanoparticles’ huge application potential in clinical practice. Compared with other nanoclusters, the dual-modal molecular imaging probe MnS@Tf-^125^I not only plays a more prominent role in observing the molecular-level information and anatomical structure of tumors, but also releases Mn ions and sulfur ions in the acidic tumor microenvironment, which respectively exert cytotoxic effects on tumor cells through CDT and gas therapy, achieving integrated diagnosis and treatment of cancer (Figure 1). We believed that through future in-depth research and continuous optimization, the clinical translation of MnS@Tf-^125^I will be achieved.

MnS is highly stable and non-degradable, which increases its potential long-term toxicity and hinders its further biomedical applications [6]. Further surface modification is required prior to its application in biomedical fields. In our study, MnS was successfully encapsulated in transferrin using a wet chemical method, which can degrade and release Mn^2+^ in specific environments, improving its solubility and biocompatibility. The r1 relaxation rate of MnS@Tf at pH 6.8 in this study was 16.96 mM^−1^s^−1^, which was more advantageous than MnOx-MSNs (6.6 mM^−1^s^−1^) reported by Hongbo Gao et al. [22], indicating that MnS@Tf can be used to increase the MRI T1 imaging contrast agent’s signal contrast in vivo. Additionally, the relaxation rate of MnS@Tf under acidic conditions was higher than alkaline conditions, further indicating that MnS@Tf released more Mn^2+^ in acidic solutions; this is consistent with findings from previous studies [23,24].

A series of results of in vitro cell experiments in our study confirmed that MnS@Tf featured targeting specificity and affinity to TfR. The in vivo SPECT/CT imaging results showed that MnS@Tf-^125^I exhibited continuous and concentrated radioactive uptake starting from 24 h. The tumor lesions in the blocking group did not show significant radioactive high uptake, indicating that MnS@Tf-^125^I has targeting and specificity for tumor tissue uptake in vivo. TfR was highly expressed in rapidly proliferating cells (such as tumor cells), which is a common pathological feature across species [25,26]. The ligand binding and other functional regions of TfR were highly conserved during evolution [26]. A large number of clinical pathological studies confirmed that TfR was widely and highly expressed in various human cancers, including colorectal cancer and breast cancer, which are associated with poor prognosis [27,28]. Therefore, it has clear clinical predictive values to verify the therapeutic strategy targeting TfR in tumor-bearing mouse models. In fact, multiple therapies targeting TfR (such as nanomedicines and bispecific antibodies) have shown significant efficacy in preclinical mouse models and are being translated to clinical practice [29]. We fully recognize the possible subtle differences between species. In subsequent studies, we plan to verify our findings in human tumor xenograft models or humanized TfR mouse models to further solidify the foundation for the clinical translation of our research conclusions. Additionally, the downregulation of TfR expression caused by treatment is a dynamic and multifactorial process [30,31]. In the future, we will dynamically monitor TfR expression and develop combined treatment strategies (such as those that work in combination with anti-angiogenic drugs or immunotherapy) to overcome or utilize this change.

TfR is expressed on various normal tissues (particularly the liver and spleen), which serves as the biological basis for potential off-target toxicity in targeted therapy and false positives in imaging diagnosis [30]. As shown by the biodistribution and SPECT/CT imaging results of MnS@Tf-^125^I, the distribution in the bladder was higher than that in the liver and spleen, possibly due to its slow degradation into small molecules or ions in the body, which are mainly excreted through urine. This requires further verification. Meanwhile, it was observed that MnS@Tf had a relatively high distribution in the blood system early on in the body. The blood distribution and the clearance half-life also suggested that MnS@Tf-^125^I can remain active in the body for a relatively long time, which not only proved beneficial by continuously killing tumor cells but was also convenient for imaging observation. Although it was beneficial for adequate binding to tumor targets, this method’s long-term safety needs to be carefully evaluated in the future.

In this study, we used a subcutaneous tumor-bearing mouse model and administered MnS@Tf or the control treatment. The tumor diameters were measured regularly for 14 consecutive days to calculate the tumor volume, and the changes in bodyweight were recorded. The significant inhibition of tumor volume directly reflected the anti-tumor activity of MnS@Tf, providing key evidence for its efficacy. The relatively stable bodyweight indicated that MnS@Tf caused low systemic toxicity, preliminarily demonstrating its safety. At the end of the 14-day experiment, the animals were sacrificed and tumor tissues were collected for pathological examination. The results showed extensive apoptosis of tumor cells, further confirming the targeted therapeutic effect and providing preclinical evidence for subsequent clinical translation. Future studies need to explore the optimal dosage and frequency of MnS@Tf and conduct further pathological examinations of major organs to monitor organ toxicity.

5. Conclusions

The multifunctional nanocluster MnS@Tf-^125^I targeting the high expression of TfR in CRC was designed in this study as a dual-modal imaging probe capable of both magnetic and radioactive properties. Moreover, by releasing Mn^2+^ to mediate the CDT effect and the H_2_S-induced killing of tumor cells, the integration of diagnosis and treatment was achieved.