TREM2-targeted manganese-doped mesoporous polydopamine nanoparticles and application thereof in colorectal cancer macrophage reprogramming and tumor microenvironment MRI (Magnetic Resonance Imaging) monitoring

Abstract

The invention discloses a manganese-doped mesoporous polydopamine nanoparticle targeting TREM2 and application of the manganese-doped mesoporous polydopamine nanoparticle in colorectal cancer macrophage reprogramming and tumor microenvironment MRI (Magnetic Resonance Imaging) monitoring. The CTMM is formed by loading a CSF1R inhibitor CSF1R-IN-3 on manganese-doped mesoporous polydopamine nanoparticles and modifying an anti-TREM2 antibody, and has the functions of targeted drug delivery and magnetic resonance imaging (MRI) monitoring. TREM2 + M2-like tumor-related macrophages (TAMs) are specifically recognized through an anti-TREM2 antibody, pH responsive drug release is achieved, a TREM2 / CSF1R signal channel is blocked, M2-type macrophages are induced to be converted into pro-inflammatory M1-type macrophages, and CD8 + T cell infiltration and tumor cell apoptosis are promoted. In addition, manganese ions in the CTMM serve as a paramagnetic probe, and the dynamic state of TREM < 2 + > macrophages in TME can be noninvasively monitored in real time through MRI. The invention provides a bifunctional platform integrating immune microenvironment reprogramming and real-time imaging, and provides a new strategy for precise treatment and personalized monitoring of colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a manganese-doped mesoporous polydopamine nanoparticle (CTMM) targeting TREM2 and a preparation method thereof, as well as the use of the nanoparticle in tumor-associated macrophage reprogramming and tumor microenvironment MRI monitoring in the treatment of colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignancies worldwide, with a high incidence and mortality rate. Despite recent advances in treatments such as surgery, chemotherapy, targeted therapy, and immunotherapy, the immunosuppressive nature of the tumor microenvironment (TME) still results in poor outcomes for many patients, resulting in high rates of recurrence and metastasis.

[0003] Tumor-associated macrophages (TAMs) are important immune cells in the tumor microenvironment, among which TREM2-positive M2 macrophages (TREM2+M2-like macrophages) have the effects of promoting tumor growth, angiogenesis and immunosuppression. TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) is highly expressed on the surface of M2 macrophages, and its signaling pathway maintains the immunosuppressive phenotype of macrophages, inhibiting the infiltration and activity of cytotoxic T cells, thereby causing tumor cells to escape immune surveillance. Therefore, targeting TREM2 and reprogramming M2 macrophages into M1 macrophages with anti-tumor activity has become one of the key strategies to improve the effect of immunotherapy for colorectal cancer.

[0004] Currently, therapeutic strategies targeting TAMs primarily include monoclonal antibodies and small molecule inhibitors, but these approaches suffer from issues such as insufficient targeting, low bioavailability, and significant side effects. Advances in nanotechnology have provided new avenues for tumor treatment. Manganese-based nanoparticles, with their excellent biocompatibility, controllable drug loading capacity, and potential as MRI contrast agents, hold great promise for application in tumor diagnosis and treatment. However, the design of a multifunctional nanoplatform that can efficiently target TREM2+M2 macrophages, achieve controlled drug release, and enable real-time monitoring of the tumor microenvironment remains a pressing challenge. Summary of the Invention

[0005] The present invention provides manganese-doped mesoporous polydopamine nanoparticles targeting TREM2 and their application in colorectal cancer macrophage reprogramming and tumor microenvironment MRI monitoring. The technical problem to be solved is: for the problem of immunosuppression induced by TREM2+M2 tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) in the treatment of colorectal cancer, the existing methods have the defects of insufficient targeting, low bioavailability, obvious side effects, etc. There is an urgent need to design a multifunctional nanoplatform that can not only efficiently target TREM2+M2 macrophages, but also achieve controlled drug release and real-time monitoring of the tumor microenvironment, so as to improve the treatment effect of colorectal cancer.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] Manganese-doped mesoporous polydopamine nanoparticles targeting TREM2,

[0008] It is composed of manganese-doped mesoporous polydopamine nanoparticles loaded with CSF1R inhibitor CSF1R-IN-3 and modified with anti-TREM2 antibody.

[0009] Furthermore, the preparation method of manganese-doped mesoporous polydopamine nanoparticles targeting TREM2 includes the following steps:

[0010] Step 1, synthesizing manganese-doped mesoporous polydopamine (MM) nanoparticles;

[0011] Step 2: Prepare drug-loaded nanoparticles (CMM) from CSF1R-IN-3@MM;

[0012] Step 3: Use TREM2@CMM to obtain manganese-doped mesoporous polydopamine nanoparticles (CTMM) targeting TREM2.

[0013] Furthermore, for step one, 0.2 g of CTAB was dissolved in 100 mL of Tris buffer (10 mM, pH 8.5) and stirred at a constant temperature of 40° C. to form a clear solution; then, 0.1 g of dopamine hydrochloride and 0.05 M mnSO4 solution were added and stirred for 1 hour to promote dopamine polymerization and manganese incorporation; thereafter, 1 mL of tetraethoxysilane (TEOS) was added dropwise, heated to 100° C. and stirred for 6 hours to form a mesoporous silica structure to obtain MM nanoparticles.

[0014] Furthermore, the obtained MM nanoparticles were collected by centrifugation at 10,000 rpm for 10 min and washed three times with ethanol and deionized water; the purified MM nanoparticles were dried in a vacuum oven at 50°C overnight.

[0015] Further, for the step 2,

[0016] The drug CSF1R-IN-3 was loaded into the Mn-PDA nanoparticles by dispersing the dried MM nanoparticles in 10 mL of PBS (pH 7.4) and stirring. The PBS solution of CSF1R-IN-3 was then added dropwise to the MM nanoparticle suspension and stirred at room temperature for 24 h to achieve drug encapsulation. The CSF1R-IN-3-loaded MM nanoparticles were separated by centrifugation at 10,000 rpm and washed with PBS to remove unbound drug. The drug-loaded nanoparticles (CSF1R-IN-3@MM, CMM) were stored at 4°C until further use.

[0017] Further, the CSF1R-IN-3-loaded MM nanoparticles were dispersed in MES buffer (0.1 M, pH 6.0), followed by the addition of EDC (0.1 M) and NHS (0.1 M) and stirred at room temperature for 30 min to activate the carboxyl groups; the nanoparticles were then washed with MES buffer to remove excess EDC and NHS; after washing, the activated nanoparticles were incubated with anti-TREM2 antibodies and BSA (as a stabilizer) in PBS at 4 °C for 12 h; finally, the generated CSF1R-IN-3@anti-TREM2@MM (CTMM) nanoparticles were collected by centrifugation, washed with PBS, and stored at 4 °C until further characterization; wherein, the surface carboxyl groups of the CSF1R-IN-3@MM nanoparticles were activated using the EDC / NHS chemical method.

[0018] Secondly, the application of manganese-doped mesoporous polydopamine nanoparticles targeting TREM2 in the preparation of tumor drugs.

[0019] Thirdly, the application of manganese-doped mesoporous polydopamine nanoparticles targeting TREM2 in MRI monitoring of tumor microenvironment.

[0020] The beneficial effects of the present invention are:

[0021] 1. Precision targeting and controlled drug release:

[0022] By using anti-TREM2 antibodies to specifically identify TREM2+M2 macrophages, nanoparticles can be enriched in the tumor microenvironment and damage to normal cells can be reduced.

[0023] Based on the acidic pH (6.5-6.8) of the tumor microenvironment, the loaded CSF1R inhibitor (CSF1R-IN-3) is released in response to block the TREM2 / CSF1R signaling pathway and efficiently induce the transformation of M2 macrophages to anti-tumor M1 type.

[0024] 2. Reshape the tumor immune microenvironment:

[0025] Promote M1 macrophages to secrete pro-inflammatory factors (such as TNF-α, IFN-γ), inhibit anti-inflammatory factors (such as IL-10), and enhance CD8 + T cell infiltration and tumor cell apoptosis reverse the immunosuppressive state of TME.

[0026] When combined with anti-PD-L1 treatment, it significantly inhibited tumor growth and prolonged the survival of mice, demonstrating a synergistic anti-tumor effect.

[0027] MRI real-time monitoring function:

[0028] Manganese ion (Mn 2+ ) as a paramagnetic probe, which can be used to monitor TREM2 in TME noninvasively and in real time by MRI. + The dynamic changes of macrophages provide imaging basis for personalized treatment.

[0029] 3. Biosafety and multifunctional integration:

[0030] Nanoparticles have good biocompatibility and colloidal stability and low toxicity to normal cells.

[0031] It integrates "immune microenvironment reprogramming" and "real-time imaging monitoring" to provide a dual-function platform strategy for the precise treatment and dynamic evaluation of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of CTMM nanoparticle synthesis and anti-tumor mechanism

[0033] Figure 2 Schematic diagram of the structural characterization and physicochemical properties of CTMM nanoparticles;

[0034] Figure 3 Schematic diagram of the targeted uptake of CTMM in M2-TREM2+ macrophages;

[0035] Figure 4 Schematic diagram of CTMM-induced transformation of M2-like macrophages to M1-like phenotype;

[0036] Figure 5 Schematic diagram of CTMM promoting tumor cell apoptosis and biosafety evaluation;

[0037] Figure 6 Schematic diagram of the efficacy of CTMM combined with anti-PD-L1 in treating colorectal cancer mouse models;

[0038] Figure 7 Schematic diagram of immune cell populations regulating the tumor microenvironment for CTMM;

[0039] Figure 8Schematic diagram of tumor microenvironment monitoring using CTMM as an MRI contrast agent. DETAILED DESCRIPTION

[0040] The present invention is further described below by specific examples. In order to make the invention purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention is further described in detail below with reference to the examples. It should be understood that the embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0041] All features disclosed in this patent specification may be used in combination with any combination of compositions. Each feature disclosed in this specification may be replaced by any alternative feature that can provide the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features.

[0042] First, the materials are described. All reagents and solvents were purchased from commercial suppliers and used directly without further purification. Hexadecyltrimethylammonium bromide (CTAB, 99%) and dopamine hydrochloride were purchased from Aladdin (Shanghai, China). Manganese (II) sulfate monohydrate (MnSO4·H2O, ≥99%), tetraethoxysilane (TEOS, 98%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, ≥98%), N-hydroxysuccinimide (NHS, ≥98%), and bovine serum albumin (BSA, ≥96%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CSF1R-IN-3 was purchased from Selleck Chemical Company (Houston, TX, USA). Anti-TREM2 monoclonal antibody was provided by R&D Systems (AF1729, UK). Tris (hydroxymethyl) aminomethane (Tris), phosphate buffered saline (PBS, pH 7.4), and 2-(N-morpholino)ethanesulfonic acid (MES) buffer were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All buffers were prepared using ultrapure water from a Milli-Q water purification system (Millipore, Bedford, MA, USA).

[0043] Then, the view is further explained.

[0044] Figure 2 A (TEM image) shows that MM, CMM, and CTMM are all uniformly spherical, with particle sizes of 250nm, 300nm, and 350nm, respectively. After loading drugs and antibodies, the particle size gradually increases, and the structure remains intact. Figure 2 B (EDS elemental mapping) confirmed that CTMM contains C, O, N, and Mn elements, corresponding to the dopamine skeleton, manganese doping, and antibody conjugation. Figure 2 C (DLS particle size distribution) shows that the particle size is consistent with TEM, and the functionalization step leads to an increase in particle size. Figure 2 D (zeta potential) shows that the surface charge of CTMM is more negative and its colloidal stability is better than that of MM and CMM. Figure 2 E (UV-visible spectroscopy) verified the successful loading of CSF1R-IN-3 and anti-TREM2 antibodies. Figure 2 F (protein standard curve) quantitative antibody coupling amount (r 2 =0.9905). Figure 2 G (drug loading efficiency) shows that the drug loading efficiency of CMM and CTMM is similar, and both achieve efficient encapsulation. Figure 2 H (pH response release curve) shows that the drug release rate of CTMM under acidic conditions (pH 6.5) is significantly higher than that under neutral conditions (pH 7.4), adapting to the tumor microenvironment.

[0045] Figure 3 Schematic diagram of the experimental verification of CTMM's specific uptake of M2-TREM2+ macrophages. Figure 3 A (Experimental design) shows that macrophages differentiated into M1 / M2 phenotype and were co-incubated with rhodamine-labeled CTMMs after knocking down TREM2 by siRNA (M2-MΦ+siRNA_TREM2). Figure 3 B (fluorescence imaging) shows that the uptake of RhB-CTMM by TREM2-knockdown M2 cells is significantly stronger than that of untreated M2 cells, M1 cells and MC38 tumor cells. The red fluorescence is the nanoparticle internalization signal, and the blue is the cell nucleus (DAPI). Figure 3 C (fluorescence intensity quantification) showed that the uptake of the TREM2 knockdown group at 3 hours and 12 hours was significantly higher than that of the other groups (***P<0.001), confirming that TREM2 protein mediated targeted uptake. Figure 3 D (TEM imaging) shows the time-dependent accumulation of CTMM in M2 macrophages, with nanoparticle aggregates visible in the cytoplasm from 1 to 6 hours (red arrows), visually presenting the internalization process.

[0046] Figure 4 Schematic diagram of the molecular mechanism and phenotypic transformation verification of CTMM regulation of macrophage polarization. Figure 4 A (Western blot) showed that after CTMM treatment, the expression of M2 markers CD206, CSF1R, and TREM2 was downregulated, and the expression of M1 marker CD86 was upregulated, indicating phenotypic conversion. Figure 4 B (qPCR) showed that the expression of M2-related genes (IL-10, CD206, etc.) in the CTMM group was significantly decreased, while the expression of M1-related genes (TNF-α, IFN-γ, etc.) was significantly increased (***P<0.001), confirming polarization reversal at the transcriptional level. Figure 4C (immunofluorescence) shows that the fluorescence intensity of CD86 (red, M1 marker) increased and that of CD206 (green, M2 marker) decreased after CTMM treatment (scale bar 50 μm). Figure 4 D (Dual-labeled fluorescence) shows that CSF1R (green) and TREM2 (red) protein expression is inhibited (scale bar 50 μm). Figure 4 EF (quantitative analysis) confirmed that the marker changes were statistically significant (***P<0.001), revealing that CTMM induced phenotypic transformation through dual signal blockade.

[0047] Figure 5 A (CCK-8 experiment) showed that CTMM had no significant effect on the survival rate of normal cells (HIEC-6) and tumor cells (MC38, RAW264.7) in the concentration range of 0-400 μg / mL (P>0.05), proving its low toxicity. Figure 5 B (co-culture model) showed that M2 macrophages treated with CTMM and co-cultured with MC38 cells inhibited tumor growth through a paracrine effect. Figure 5 C (cell viability) showed that CTMM reduced the viability of MC38 cells in a dose- and time-dependent manner (***P<0.001). Figure 5 DE (TUNEL staining) showed that the proportion of apoptotic cells (red) in the CTMM group was significantly higher than that in the control group, and the quantification results showed that the apoptosis rate increased (***P<0.001). Figure 5 FI (ELISA) showed that the levels of pro-inflammatory factors (TNF-α, IL-1, IFN-γ) increased and the levels of anti-inflammatory factors (IL-10, TGF-β) decreased after CTMM treatment (***P<0.001), confirming that M1 polarization enhanced the anti-tumor microenvironment.

[0048] Figure 6 A (Experimental process) shows the schedule of tumor inoculation, drug intervention (CTMM alone or in combination with anti-PD-L1) and imaging testing. Figure 6 BC (in vivo fluorescence imaging) showed that the accumulation of CTMM in the tumor increased over time, and the fluorescence intensity on day 17 was significantly higher than that in the control group (***P<0.001), confirming the targeted enrichment ability. Figure 6 DE (in vitro organ imaging) showed that the fluorescence intensity of tumor tissue was significantly higher than that of organs such as liver and kidney, indicating low systemic toxicity. Figure 6 FG (tissue staining) showed increased apoptosis of tumor cells in the CTMM group, with no significant damage to normal organs (scale bar 50 μm). Figure 6 HI (tumor volume) showed that the CTMM combined with anti-PD-L1 group had the best tumor growth inhibition effect (***P<0.001). Figure 6J (survival curve) showed that the survival time of mice in the combined treatment group was significantly prolonged (***P<0.001).

[0049] Figure 7 AB (flow cytometry) showed that M1 macrophages (CD68 + CD86 + ) ratio increased significantly, and the proportion of M2 macrophages (CD68 + CD206 + ) ratio decreased significantly (***P<0.001), indicating polarization reversal. Figure 7 C (immunofluorescence) showed that CD86 in CTMM group + TREM2 - Macrophages, CD8 + T cells, Th1 cells (CD25 + FOXP3 + ) infiltration was enhanced, Treg cells (CD4 + INF-γ + ) decreased (scale bar 50 μm). Figure 7 DH (quantitative analysis) confirmed that the above immune cell changes were statistically significant (***P<0.001), indicating that CTMM activated anti-tumor immune responses and suppressed immunosuppressive cells by regulating macrophage phenotype.

[0050] Figure 8 AB (in vitro MRI) showed that the T1 signal intensity increased with increasing manganese concentration, and the relaxation rate (1 / T1) was linearly correlated with manganese concentration (y=10.52x+0.4725), confirming the paramagnetic contrast effect of CTMM. Figure 8 C (in vivo MRI) showed that the T1 signal in the tumor area of the CTMM-treated group was significantly higher than that in the control group at 12 hours and 24 hours (P<0.001), and the red circle marked the tumor location. Figure 8 DE (correlation analysis) showed that T1 signal intensity was significantly correlated with CD68 + TREM2 + Macrophage infiltration was positively correlated (r=0.7106, P<0.05), but had no significant correlation with tumor volume and cell apoptosis.

[0051] Example 1

[0052] Manganese-doped mesoporous polydopamine nanoparticles targeting TREM2, the nanoparticles are obtained by the following steps:

[0053] Step 1: Synthesize manganese-doped mesoporous polydopamine (MM) nanoparticles.

[0054] Specifically, 0.2 g of CTAB was dissolved in 100 mL of Tris buffer (10 mM, pH 8.5) and stirred at 40°C until a clear solution formed. Subsequently, 0.1 g of dopamine hydrochloride and 0.05 M mnSO4 solution were added and stirred for 1 hour to promote dopamine polymerization and manganese incorporation. Afterwards, 1 mL of tetraethoxysilane (TEOS) was added dropwise, heated to 100°C, and stirred for 6 hours to form a mesoporous silica structure and obtain MM nanoparticles. The resulting MM nanoparticles were collected by centrifugation at 10,000 rpm for 10 minutes and washed three times with ethanol and deionized water. The purified MM nanoparticles were dried in a vacuum oven at 50°C overnight.

[0055] Step 2: CSF1R-IN-3@MM is used to obtain drug-loaded nanoparticles (CMM).

[0056] Specifically, the drug CSF1R-IN-3 was loaded into the Mn-PDA nanoparticles by dispersing the dried MM nanoparticles in 10 mL of PBS (pH 7.4) and stirring. The PBS solution of CSF1R-IN-3 was then added dropwise to the MM nanoparticle suspension and stirred at room temperature for 24 hours to achieve drug encapsulation. The CSF1R-IN-3-loaded MM nanoparticles were separated by centrifugation at 10,000 rpm and washed with PBS to remove unbound drug. The drug-loaded nanoparticles (CSF1R-IN-3@MM, CMM) were stored at 4°C until further use.

[0057] Step 3: Using TREM2@CMM to obtain TREM2-targeted manganese-doped mesoporous polydopamine nanoparticles (CTMM);

[0058] The CSF1R-IN-3 loaded MM nanoparticles were dispersed in MES buffer (0.1M, pH 6.0), followed by the addition of EDC (0.1M) and NHS (0.1M) and stirred at room temperature for 30 minutes to activate the carboxyl groups. The nanoparticles were then washed with MES buffer to remove excess EDC and NHS. After washing, the activated nanoparticles were incubated with anti-TREM2 antibodies and BSA (as a stabilizer) in PBS at 4 ° C for 12 hours. Finally, the generated CSF1R-IN-3@anti-TREM2@MM (CTMM) nanoparticles were collected by centrifugation, washed with PBS, and stored at 4 ° C until further characterization. Among them, the surface carboxyl groups of CSF1R-IN-3@MM nanoparticles were activated using the EDC / NHS chemical method.

[0059] Example 2: Characterization and Verification of CTMM

[0060] 1. Morphological and structural characterization

[0061] Transmission electron microscopy (TEM): A drop of nanoparticle suspension was dropped onto a carbon-coated copper grid. After air drying, the morphology was observed using a JEOL JEM-2100 transmission electron microscope (200 kV accelerating voltage). MMs were uniformly spherical, while CMMs and CTMMs were slightly larger in size due to drug / antibody loading ( Figure 2 A).

[0062] Energy Dispersive X-ray Spectroscopy (EDS): Elemental mapping of CTMM was performed using the Oxford X-Max NEDS system to confirm the presence of C, O, N, and Mn elements ( Figure 2 B).

[0063] 2. Particle size and surface charge analysis

[0064] Dynamic light scattering (DLS): The sample was dispersed in deionized water and the particle size was measured using a Malvern Zetasizer Nano ZS at 25°C. The MM was approximately 250 nm, the CMM was approximately 300 nm, and the CTMM was approximately 350 nm. Figure 2 C).

[0065] Zeta potential: measured with the same instrument, the surface charge of CTMM is more negative than that of MM and CMM (about -25mV), and the colloidal stability is improved ( Figure 2 D).

[0066] 3. Drug loading and release performance

[0067] UV-visible spectrum: Shimadzu UV-3600 spectrometer was used to detect that CSF1R-IN-3 had a characteristic absorption peak at 340 nm, confirming that the drug loading was successful ( Figure 2 E); Antibody coupling was quantified by BSA standard curve, and the coupling efficiency was above 90% ( Figure 2 F).

[0068] Drug loading efficiency: The drug loading rates of CMM and CTMM were (total drug - unencapsulated drug) / total drug × 100%, and the results showed that about 45% ( Figure 2 G).

[0069] pH-responsive release: CTMM was dispersed in PBS at pH 5.0, 6.5, and 7.4, incubated at 37°C with shaking, centrifuged regularly, and the supernatant was collected. The absorbance at 340 nm was detected by UV. The results showed that the 24-hour release rate at pH 6.5 reached 60%, which was significantly higher than that at pH 7.4 ( Figure 2 H).

[0070] Example 3: In vitro cell experiment

[0071] 1. Cell Culture and Treatment

[0072] MC38 colorectal cancer cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double-antibody at 37°C and 5% CO2.

[0073] RAW264.7 macrophages: high-glucose DMEM medium (containing 10% FBS, 1% double antibody), M2 polarization was induced with 30 ng / mL IL-4 for 24 hours, and M1 polarization was induced with 500 ng / mL LPS for 24 hours.

[0074] 2. Nanoparticle Uptake Experiment

[0075] Fluorescence labeling: CTMM was conjugated with rhodamine B isothiocyanate (RhB-NCS) via EDC / NHS activation and observed under a confocal microscope (excitation 540 nm, emission 625 nm).

[0076] Results: The uptake of RhB-CTMM by M2-TREM2+ macrophages was significantly higher than that by M1 macrophages and MC38 cells. The fluorescence intensity quantitative analysis at 3 hours and 12 hours showed significant differences (***P<0.001, Figure 3 B- Figure 3 C); SEM observation showed that the internalization rate of CTMM in M2 macrophages was faster than that of CMM ( Figure 3 D).

[0077] 3. Macrophage Polarization Assay

[0078] Western blot (WB): Cell proteins were extracted and CD206 (M2 marker), CD86 (M1 marker), CSF1R, and TREM2 were detected. In the CTMM treatment group, CD206 and TREM2 were downregulated, while CD86 was upregulated ( Figure 4 A).

[0079] qPCR: RNA was extracted and reverse transcribed to detect M2-related genes (IL-10, CD206, TGF-β, Arg-1) and M1-related genes (TNF-α, IFN-γ, CD86, IL-1). In the CTMM-treated group, M2 genes were downregulated, while M1 genes were upregulated ( Figure 4 B).

[0080] 4. Cytotoxicity and Apoptosis

[0081] CCK-8 assay: HIEC-6, MC38, and RAW264.7 cells were treated with different concentrations of CTMM (0-400 μg / mL). The cell survival rates were >90% within 48 hours, indicating low toxicity ( Figure 5 A).

[0082] TUNEL staining: After 24 hours of CTMM treatment of MC38 cells, the proportion of TUNEL-positive cells increased significantly (***P<0.001, Figure 5 D- Figure 5 E).

[0083] Example 4: In vivo animal experiment

[0084] 1. Tumor Model Establishment

[0085] Subcutaneous model: C57 / BL6J mice (5 weeks old) were injected subcutaneously in the flank with 2×10 7 MC38 cells / 100 μL, cultured for 2 weeks.

[0086] In situ model: mice were anesthetized with ether and injected subserously into the cecum with 2×10 6 MC38 cells, tumor formation was confirmed 5 days after surgery.

[0087] 2. Treatment Grouping and Dosing

[0088] Groups: control group (PBS), MM group (500μgMM), CMM group (500μgCMM), CTMM group (500μgCTMM), anti-PD-L1 group (2mg intraperitoneal injection), combined group (CTMM+anti-PD-L1). Specifically, each group contains at least 16 mice. Body weight and tumor volume were recorded every three days. The tumor volume was calculated as follows: volume = (length × width 2 ) / 2; After two injections, at least five mice in each group were sacrificed, and tumor tissue was collected for analysis. The proportion of macrophage subsets in the tumor immune microenvironment was assessed by flow cytometry, while the number of CD8+ T cells, Th1, and Treg populations was quantified using immunofluorescence. On day 17, tumor and organ damage were assessed. The treatment regimen lasted for 50 days, and survival curves were plotted based on the cumulative survival data.

[0089] 3. Detection Methods

[0090] In vivo fluorescence imaging. In vivo fluorescence imaging of mice was performed using the IVIS Spectrum in vivo imaging system (PerkinElmer). Fluorescence intensity was measured at baseline (before injection) and on days 1, 5, 10, and 17 after RhB-CTMM injection. On day 17, mice were sacrificed, and major organs (tumor, kidney, liver, heart) were removed for ex vivo fluorescence imaging. Fluorescence image analysis was performed using the Livingimage 4.4 software package (PerkinElmer).

[0091] Hematoxylin and eosin (HE) staining. Tumor, heart, kidney, liver, and small intestine tissues of treated mice were embedded in paraffin, and 3-μm-thick sections were prepared for hematoxylin and eosin (HE) staining. HE staining was performed according to standard procedures. Briefly, tissue sections were dewaxed, hydrated, stained with hematoxylin (Beijing, China), differentiated in acidic ethanol, counterstained with eosin (Beijing, China), dehydrated, and mounted with Permount (Fisher Scientific, Shanghai, China). Histological evaluation of tissue sections was performed using light microscopy.

[0092] Flow cytometry. Flow cytometry was used to analyze immune cell populations within mouse tumor tissue. Single-cell suspensions were prepared from harvested tumors, and cells were stained with appropriate antibodies, including anti-CD206 (R&D Systems, AF2537), anti-CD86 (ab242142, Abcam), and anti-CD68 (ab283654, Abcam). Flow cytometry was performed according to standard protocols, and data were analyzed using FlowJo software.

[0093] Immunofluorescence. Immunofluorescence staining was performed as previously described. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. The following primary antibodies were used: anti-CD86 (ab220188; Abcam), anti-CD206 (ab64693; Abcam), anti-CSF1R (ab254357; Abcam), anti-IFN-γ (ab224179; Abcam), anti-TNF-α (ab183218; Abcam), CD4 (ab183685; Abcam), CD25 (ab231442; Abcam), FOXP3 (ab215206; Abcam), and CD8 (ab217344; Abcam). Cell nuclei were counterstained with DAPI, and images were captured by confocal microscopy (Leica Microsystems, Germany). Fluorescence intensity was quantified using ImageJ software.

[0094] TUNEL staining. TUNEL staining was used to assess apoptosis in MC38 cells cocultured with mice, as well as in sections of mouse tumor, liver, kidney, heart, and small intestine tissue. Apoptosis was detected using a TUNEL kit (Beyotime, China) according to the manufacturer's protocol. The number of TUNEL-positive cells was counted in three randomly selected fields under a fluorescence microscope, and the proportion of apoptotic cells to total cells was calculated.

[0095] 4. Results Evaluation

[0096] CTMM nanoparticles reprogrammed M2-like TAMs into M1-like macrophages. CTMM treatment resulted in significant changes in macrophage polarization, as evidenced by decreased expression of CD206 and CSF1R, and increased levels of CD86, indicating a shift from an M2-like (tumor-promoting) phenotype to an M1-like (tumor-suppressing) phenotype. Figure 4 A). Notably, TREM2 expression was downregulated, suggesting its role in maintaining the M2 phenotype. Gene expression analysis confirmed these findings, showing decreased levels of M2-associated genes (IL-10, CD206, TGF-β, Arg-1) and increased expression of M1-associated markers (TNF-α, IFN-γ, CD86, IL-1) in the CTMM-treated group ( Figure 4 B).

[0097] These observations were further confirmed by immunofluorescence results, which showed that CD86 expression was significantly increased, while CD206 expression was decreased in CTMM-treated cells ( Figure 4 C). Quantitative analysis highlighted statistically significant changes in both markers (***P<0.001, Figure 4 E). In addition, CTMM treatment significantly inhibited the expression of TREM2 and CSF1R ( Figure 4 D), Quantification results confirmed the downregulation of these proteins (***P < 0.001, Figure 4 F), reflecting the alteration of macrophage immunoregulatory function. Taken together, these findings indicate that CTMM nanoparticles can effectively reprogram M2-like TAMs into M1-like macrophages, promoting the formation of a pro-inflammatory and anti-tumor microenvironment.

[0098] CTMM promotes tumor cell apoptosis by regulating macrophage polarization, ensuring high biosafety. CTMM exhibits extremely low cytotoxicity to normal cells and tumor cells under standard conditions. CCK-8 assays showed that CTMM did not significantly reduce the survival rate of HIEC-6, MC38, and RAW264.7 cells at different concentrations (0-400 μg / mL) and time points (6, 12, 24, and 48 hours). Figure 5 A), indicating that CTMM has high biosafety in vitro.

[0099] In the co-culture system, CTMM significantly reduced the survival rate of MC38 tumor cells in a dose- and time-dependent manner ( Figure 5 B, Figure 5 C). Specifically, as the concentration of CTMM increases, the survival rate of tumor cells gradually decreases (left figure, Figure 5C), while treatment with 400 μg / mL of CTMM resulted in a time-dependent decrease in cell viability, with significant effects observed after 24 and 48 hours (***P<0.001). In addition, CTMM treatment enhanced tumor cell apoptosis, as confirmed by TUNEL assay ( Figure 5 D). Compared with the control group, TUNEL-positive cells in the CTMM-treated group increased significantly, indicating cell apoptosis. Quantitative analysis of apoptotic cells ( Figure 5 E) shows that apoptosis in MC38 cells treated with CTMM was significantly increased (***P<0.001).

[0100] CTMM also promoted the polarization of macrophages toward a pro-inflammatory M1-like phenotype, as confirmed by immunofluorescence co-staining showing increased expression of IFN-γ, TNF-α, and CD86 in the CTMM-treated group ( Figure 5 F, Figure 5 This shift toward M1 polarization was accompanied by a significant increase in proinflammatory cytokines (TNF-α, IL-1, IFN-γ) in the co-culture supernatant (***P<0.001, Figure 5 H), and a corresponding decrease in anti-inflammatory cytokines (IL-10, TGF-β) (***P<0.001, Figure 5 I).

[0101] The combination of CTMM and anti-PD-L1 antibody can enhance the therapeutic effect of CRC mouse model. The combination of CTMM nanoparticles and anti-PD-L1 antibody significantly improved the therapeutic effect of CRC mouse model. The treatment plan and imaging schedule are shown in Figure 6 A. In vivo fluorescence imaging ( Figure 6 B) shows that CTMM exhibited stronger tumor accumulation ability compared with MM and CMM, with the highest fluorescence intensity occurring on day 17. Quantitative analysis confirmed that the fluorescence intensity of mice in the CTMM-treated group was significantly higher than that in other groups over time (***P<0.001, Figure 6 C).

[0102] In vitro imaging showed selective accumulation of CTMM in tumors and major organs, with minimal distribution to other organs ( Figure 6 D). Fluorescence intensity quantification results showed that the fluorescence intensity in tumors treated with CTMM was significantly higher than that in tumors treated with MM and CMM (***P<0.001, *P<0.05), indicating that the drug was able to be delivered to the tumor site.

[0103] Histological analysis by H&E and TUNEL staining further supported these findings, showing that CTMM treatment resulted in significant apoptosis in tumor tissues, while major organs, including the liver, kidney, and heart, showed no obvious signs of toxicity ( Figure 6F, Figure 6 G). These results confirm the specific targeting effect of CTMM on tumors and its good safety.

[0104] Tumor volume analysis showed that the combination of CTMM and anti-PD-L1 therapy had the most significant inhibitory effect on tumor growth compared with monotherapy and the control group ( Figure 6 I). Tumor growth was significantly inhibited in the combined treatment group (***P<0.001), and representative images of resected tumors showed a significant reduction in tumor volume in mice receiving combined CTMM and anti-PD-L1 therapy ( Figure 6 H).

[0105] Kaplan-Meier survival analysis showed that the survival rate of mice receiving combined treatment was significantly improved compared with the other treatment groups (***P<0.001)( Figure 6 J).

[0106] CTMM modulates macrophage polarization and enhances antitumor immune responses in the MC38 colorectal tumor microenvironment. CTMM treatment leads to significant changes in macrophage polarization in the tumor microenvironment. Flow cytometry analysis showed that the proportion of M1 macrophages (CD68+CD86+) in the CTMM-treated group was significantly increased compared with the control group, while the proportion of M2 macrophages (CD68+CD206+) was correspondingly decreased ( Figure 7 A, Figure 7 B). Quantitative analysis confirmed this polarization change, showing a significant increase in M1 macrophages and a significant decrease in M2 macrophages in the CTMM group (***P<0.001, Figure 7 D).

[0107] Immunofluorescence staining further showed that in tumors treated with CTMM, the infiltration of CD86+TREM2- macrophages, CD8+ T cells, and Th1 cells (CD25+FOXP3+) was enhanced, while the number of Treg cells (CD4+INF-γ) was reduced ( Figure 7 C). Quantitative analysis confirmed that compared with other treatments, the proportion of CD86+TREM2- macrophages, CD8+ T cells, and Th1 cells in the CTMM group was significantly increased, while the proportion of Treg cells was significantly decreased (***P<0.001, Figure 7 E- Figure 7 H).

[0108] These results suggest that CTMMs effectively reprogram the tumor immune microenvironment by promoting M1 macrophage polarization and enhancing antitumor immune responses, characterized by increased infiltration of cytotoxic CD8+ T cells and Th1 cells while reducing the immunosuppressive Treg population.

[0109] In short, the IVIS spectral system detected the distribution of RhB-CTMM. The fluorescence intensity of the tumor in the CTMM group was significantly higher than that in the other groups, reaching a peak on the 17th day ( Figure 6 B- Figure 6 C). HE staining showed significant apoptosis of tumor cells in the CTMM group, with no significant toxicity to major organs (heart, liver, and kidney); TUNEL staining confirmed an increase in the proportion of apoptotic cells in tumor tissue ( Figure 6 F- Figure 6 G). Flow cytometry showed that the proportion of M1 macrophages (CD68+CD86+) increased and the proportion of M2 macrophages (CD68+CD206+) decreased in the CTMM group; immunofluorescence detection increased the infiltration of CD8+T cells and Th1 cells, and decreased Treg cells ( Figure 7 A- Figure 7 C). The survival of mice in the combined treatment group was significantly prolonged, and the Kaplan-Meier curve showed that the survival rate reached 60% at 50 days (***P<0.001, Figure 6 J).

[0110] Example 5: MRI monitoring experiment

[0111] 1. In vitro Relaxivity Measurement

[0112] CTMM suspensions with different Mn concentrations (0.02-0.3 mM) were prepared, and T1-weighted images were acquired using a Bruker magnetic resonance instrument with a RARE sequence (TR = 1000 ms, TE = 10 ms). The relaxation rate (1 / T1) was calculated, and the linear equation y = 10.52x + 0.4725 (R 2 =0.99, Figure 8 A- Figure 8 B).

[0113] 2. In Vivo MRI Imaging

[0114] Tumor-bearing mice were injected with CTMM (Mn dose 0.2 mmol / kg) via the tail vein and imaged before, 6, 12, 24, and 48 hours after injection, using the same parameters as in vitro (TR = 800 ms, TE = 9 ms).

[0115] Results: The T1 signal intensity of the tumors in the CTMM group was significantly higher than that in the CMM group at 12 and 24 hours (***P<0.001), and the signal intensity was positively correlated with the infiltration of CD68+TREM2+ macrophages (r=0.7106, P<0.05, Figure 8 C- Figure 8 D).

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Manganese-doped mesoporous polydopamine nanoparticles targeting TREM2, characterized in that It is composed of manganese-doped mesoporous polydopamine nanoparticles loaded with CSF1R inhibitor CSF1R-IN-3 and modified with anti-TREM2 antibody.

2. The TREM2-targeting manganese-doped mesoporous polydopamine nanoparticles according to claim 1, characterized in that: The preparation method comprises the following steps: Step 1, synthesizing manganese-doped mesoporous polydopamine (MM) nanoparticles; Step 2: Prepare drug-loaded nanoparticles (CMM) from CSF1R-IN-3@MM; Step 3: Use TREM2@CMM to obtain manganese-doped mesoporous polydopamine nanoparticles (CTMM) targeting TREM2.

3. The TREM2-targeting manganese-doped mesoporous polydopamine nanoparticles according to claim 2, wherein the step 1 is characterized in that: 0.2 g of CTAB was dissolved in 100 mL of Tris buffer (10 mM, pH 8.5) and stirred at a constant temperature of 40°C until a clear solution was formed; then, 0.1 g of dopamine hydrochloride and 0.05 M mnSO4 solution were added and stirred for 1 hour to promote dopamine polymerization and manganese incorporation; thereafter, 1 mL of tetraethoxysilane (TEOS) was added dropwise, heated to 100°C and stirred for 6 hours to form a mesoporous silica structure to obtain MM nanoparticles.

4. The TREM2-targeting manganese-doped mesoporous polydopamine nanoparticles according to claim 3, characterized in that: The resulting MM nanoparticles were collected by centrifugation at 10,000 rpm for 10 min and washed three times with ethanol and deionized water; the purified MM nanoparticles were dried in a vacuum oven at 50°C overnight.

5. The TREM2-targeting manganese-doped mesoporous polydopamine nanoparticles according to claim 2, wherein the step 2 is characterized in that: The drug CSF1R-IN-3 was loaded into the Mn-PDA nanoparticles by dispersing the dried MM nanoparticles in 10 mL of PBS (pH 7.4) and stirring. The PBS solution of CSF1R-IN-3 was then added dropwise to the MM nanoparticle suspension and stirred at room temperature for 24 h to achieve drug encapsulation. The CSF1R-IN-3-loaded MM nanoparticles were separated by centrifugation at 10,000 rpm and washed with PBS to remove unbound drug. The drug-loaded nanoparticles (CSF1R-IN-3@MM, CMM) were stored at 4°C until further use.

6. The TREM2-targeting manganese-doped mesoporous polydopamine nanoparticles according to claim 2, wherein the step 3 is characterized in that: CSF1R-IN-3-loaded MM nanoparticles were dispersed in MES buffer (0.1 M, pH 6.0), followed by the addition of EDC (0.1 M) and NHS (0.1 M), and stirred at room temperature for 30 min to activate the carboxyl groups; The nanoparticles were then washed with MES buffer to remove excess EDC and NHS; after washing, the activated nanoparticles were incubated with anti-TREM2 antibodies and BSA (as a stabilizer) in PBS at 4°C for 12 hours; finally, the generated CSF1R-IN-3@anti-TREM2@MM (CTMM) nanoparticles were collected by centrifugation, washed with PBS, and stored at 4°C until further characterization; among them, the surface carboxyl groups of CSF1R-IN-3@MM nanoparticles were activated using the EDC / NHS chemical method.

7. Use of manganese-doped mesoporous polydopamine nanoparticles targeting TREM2 according to any of claims 1-6 in the preparation of tumor drugs.

8. Use of manganese-doped mesoporous polydopamine nanoparticles targeting TREM2 according to any of claims 1-6 in MRI monitoring of tumor microenvironment.

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