Exploring the theranostic potential of carbon dots/Fe3 O4 superparticles for tumor-associated macrophage targeting and repolarization in colorectal cancer therapy via JAK/STAT and ERK/MAPK pathways

Exploring the theranostic potential of carbon dots/Fe3 O4 superparticles for tumor-associated macrophage targeting and repolarization in colorectal cancer therapy via JAK/STAT and ERK/MAPK pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the theranostic potential of carbon dots/Fe3 O4 superparticles for tumor-associated macrophage targeting and repolarization in colorectal cancer therapy via JAK/STAT and ERK/MAPK pathways Yingying Miao, Xiaoyu Li, Qingsen Zeng, Kai Zhang, Lin Liu, Bai Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5657271/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Colorectal cancer (CRC) immunotherapy has shown remarkable effects in only a small subset of patients, largely due to the influence of tumor-associated macrophages (TAMs), which play a key role in shaping the tumor immune microenvironment. In vivo dynamic imaging of TAMs is critical for personalized immunotherapy, as it enables the identification of patients likely to benefit from treatment and allows for real-time monitoring of therapeutic efficacy. Additionally, reprogramming the polarization state of TAMs from the pro-tumoral M2 phenotype to the anti-tumoral M1 phenotype represents a promising strategy to enhance immunotherapy outcomes. To address these challenges, we developed mannose-coated carbon dots/ Fe 3 O 4 superparticles (Mannose-DSPE-PEG@ Fe 3 O 4 /CDs) specifically designed to target TAMs. These superparticles combine the NMR-enhanced imaging capabilities of Fe 3 O 4 with the red fluorescence properties of carbon dots, enabling precise and non-invasive TAM imaging. Furthermore, Mannose-DSPE-PEG@ Fe 3 O 4 /CDs effectively reprogram TAMs from the M2 to M1 phenotype via the JAK/STAT and ERK/MAPK pathways, thereby reshaping the tumor immune microenvironment and exerting potent anti-tumor effects. In summary, this study demonstrates the potential of Mannose-DSPE-PEG@ Fe 3 O 4 /CDs as a theranostic nanoplatform for the monitoring and modulation of TAMs, offering a novel strategy for improving immunotherapy outcomes in colorectal cancer. tumor-associate macrophages repolarization carbon dots Fe3O4 theranostic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer is the third most common cancer in the world with increasing incidence and mortality rates globally[ 1 , 2 ]. As a novel cancer treatment, cancer immunotherapies have recently driven a paradigm shift in patient survival across tumors, but they only benefited a minority of patients with colorectal cancer[ 3 – 6 ]. As accumulating evidence has shown, Tumor-associated macrophages (TAMs), as abundant and active infiltrated inflammatory cells in the tumor microenvironment (TME), are the most important components in the tumor immunosuppressive microenvironment, promoting tumor metastasis, and therapy sensitivity for colorectal cancers[ 7 ]. In the tumor-promoting microenvironment of colorectal cancer, the inflammatory M2 TAM phenotype accounts for 50% of the tumor cell mass, whereas the immunoprotective M1 TAM phenotype is rare. M2 phenotype macrophages promote tumor cell proliferation by regulating cytokines, chemokines, proteases, and reactive oxygen species. Furthermore, M2 TAMs enhance angiogenesis by modulating VEGF bioavailability and suppressing protective adaptive immune responses. Additionally, M2-polarized TAMs secrete lactate during glycolysis, and through their metabolic processes, secrete other acidic metabolites, reducing the pH value of the TME. TAMs secrete matrix metalloproteinases (MMPs) that degrade extracellular matrix (ECM), affecting local pH buffering capacity and fostering a tumor acidic microenvironment. Therefore, TAMs can promote tumor microenvironment acidification through multiple pathways, making them key factors in facilitating colorectal cancer invasion, metastasis, chemoresistance, and immune evasion. Given their crucial role, TAMs have emerged as highly promising targets in cancer immunotherapy. Given their essential roles, TAMs have emerged as promising targets for cancer immunotherapy[ 8 ]. Immunotherapeutic strategies to suppress TAMs of the M2 phenotype or to reprogram them against the M1 phenotype of tumors have gained enormous momentum[ 9 ]. However, the success of these therapies relies on quantifying the distribution of TAMs in the TME[ 10 ]. Targeting tumor-associated macrophages (TAMs) for imaging and immunomodulation holds significant potential in the personalized immunotherapy of colorectal cancer. By tracing the distribution of TAMs within the body, it is possible to assess the degree of immune suppression in patients, identify those who are more likely to benefit from immunotherapy, and facilitate precision medicine. Additionally, tracking TAMs allows for the real-time, non-invasive monitoring of dynamic changes in the tumor microenvironment during immunotherapy, providing a basis for the dynamic adjustment of treatment protocols. Polarizing TAMs from the M2 to the M1 phenotype can reverse the immunosuppressive tumor microenvironment, enhance the efficacy of immunotherapies such as immune checkpoint inhibitors, delay the onset of resistance, and reduce the risk of tumor recurrence. Integrating TAM modulation with radiotherapy, anti-angiogenic therapy, and cell therapy can produce a synergistic effect, further enhancing therapeutic outcomes. Therefore, targeting TAMs for imaging and immunomodulation offers a personalized immunotherapy strategy for colorectal cancer patients, with the potential to improve patient outcomes. Nowadays, nanomaterials have been widely applied in various fields among which carbon dots (CDs) received more and more attention[ 11 ]. CDs are the most promising candidates of the carbon family with superior properties like ultra-small size, high aqueous solubility, low cytotoxicity, and inherent photoluminescence which makes them suitable for diverse biomedical applications [ 12 ]. A few reports about inflammation indicate that carbon dots could repolarize M2 macrophages, and changes were assessed by fluorescence intensity.[ 13 – 15 ]. This suggests that carbon dots may be a promising material for targeting TAMs. However, few carbon dots can stabilize near-infrared luminescence, resulting in poor tissue penetrability, which limits their dynamic imaging in vivo[ 12 ].SPIONs are FDA-approved T2 contrast agents used in MRI and have been extensively applied to track immune cells, such as DCs and T cells[ 16 , 17 ]. Zanganeh et al. discovered their off-labeled uses that they can induce a phenotypic transition from M2 towards M1 and inhibit tumor growth. Iron oxides repolarize M2 to M1 and induce the Fenton reaction which can generate ROS and promote the apoptosis of tumor cells[ 18 ]. Therefore, iron oxide nanoparticles have promising clinical applications due to their properties such as superparamagnetism and repolarization. The major drawbacks are that it is not possible to distinguish between dead and live cells, that signal void in MRI does not quantitatively report on the number of cells, and that the label undergoes dilution with cell replication in vivo[ 19 ]. In this study, we propose a novel nanoplatform—Mannose-DSPE-PEG@ Fe 3 O 4 /CDs—designed to precisely assess the M2 TAMs within the TME of colorectal cancer and facilitate their polarization switch. This multifunctional material combines the magnetic properties of Fe 3 O 4 with the near-infrared fluorescence of CDs, and is further modified with Mannose-DSPE-PEG to enhance specificity towards M2 TAMs. Upon targeting and binding to M2 TAMs, this nanoplatform not only allows for accurate spatial localization but also employs near-infrared (NIR) fluorescence imaging to precisely identify viable TAMs, inducing the polarization of M2 TAMs to the M1 phenotype, thus improving the immune microenvironment.(Scheme 1) Results and Discussion Colorectal cancer (CRC) patients derive limited benefit from immunotherapy. Analysis of the TCGA database indicates that TAMs not only play a pivotal role in the tumor microenvironment of CRC but also exhibit significant distribution differences between tumor tissues and adjacent normal tissues (Figure S1 A-C). Specifically, patients with higher TAM abundance, particularly M2-type macrophages, have significantly shorter survival times and reduced overall survival rates (Figure S1 D). Therefore, TAMs represent a critical factor influencing the effectiveness of CRC immunotherapy, making precise imaging of TAMs an effective strategy for evaluating patient benefit from immunotherapy. Furthermore, reprogramming the polarization state of TAMs holds promise as a method to improve the tumor immune microenvironment and enhance the therapeutic efficacy of immunotherapy in CRC patients. Synthesis and Characterization of Mannose-DSPE-PEG@Fe 3 O 4 /CDs The Mannose-DSPE-PEG@ Fe 3 O 4 /CDs superparticles were fabricated by first synthesizing carbon dots with stable red-shifted fluorescence through a solvothermal method involving PEG and near-infrared dye IR 813[ 20 ], followed by the encapsulation of Fe 3 O 4 and carbon dots using amphiphilic di-stearoyl phosphatidylethanolamine, polyethylene glycol, and D-mannose (see the Methods for details). Morphological studies show that the Mannose-DSPE-PEG@ Fe 3 O 4 /CDs nanoparticles are spherical in shape and uniformly dispersed, with a size of approximately 169.9 nm (Fig. 1 A). This enables them to possess a favorable enhanced permeation and retention (EPR) effect while avoiding clearance by the liver Kupffer cell system[ 21 , 22 ]. Additionally, the Mannose-DSPE-PEG@ Fe 3 O 4 /CDs are surrounded by hydrophilic groups, allowing them to disperse well in aqueous media. Compared to neutral nanoparticles, anionic nanoparticles are more easily taken up by macrophages[ 23 ] (Figure S2A). The UV absorption spectrum displayed distinct peaks at 214 nm, 251 nm, 307 nm, 373 nm, and 525 nm, with the peak at 307 nm indicating a high content of hydroxyl groups in the carbon dots. The other absorption peaks are attributed to π-π* and n-π* transitions, while the 525 nm peak may arise from the formation of nitrogen-containing heterocycles resulting from the degradation of IR-813(Fig. 1 B), which are combined with PEG[ 24 ]. Leveraging the exceptional near-infrared emission properties of the prepared carbon dots (Figure S2B), the fluorescence emission spectrum demonstrated distinct maximum peaks at 637 nm and 780 nm for the nanoparticles. These peaks exhibited pH sensitivity; as the pH decreased, the intensity of the 637 nm peak diminished, while the intensity of the 780 nm peak increased (Fig. 1 C). Under acidic conditions (pH approximately 0.4), the composite displayed near-infrared fluorescence at 780 nm (Fig. 1 D), indicating potential for visualizing tumor microenvironments. Fourier Transform Infrared Spectroscopy (FTIR) analysis showed that Mannose-DSPE-PEG@ Fe 3 O 4 /CDs exhibited a weak absorption peak at 1650 cm − 1, confirming the successful encapsulation of carbon dots and Fe 3 O 4 in the nanocomposite (Fig. 1 E). Long-wavelength emissions in the red and near-infrared (NIR) regions are important for in vivo imaging, as they can reduce tissue autofluorescence and enhance contrast[ 25 ]. The synthesized nanoparticles formed assemblies that prevent quenching due to aggregation, improving graphitization and π-conjugation[ 26 ]. The specific relaxivity (r*2) of the nanoparticles was determined to be 197.9 mM − 1 s − 1(Figure S2C), indicating their effectiveness in shortening T2, making them suitable as negative MRI contrast agents[ 27 ](Fig. 1 F). The pH value of the routinely prepared aqueous solution was 5.3(Figure S1 D), suggesting prolonged retention in lysosomes[ 28 ]. The CCK8 assay indicated that only when the co-culture time was extended to 48 hours did the 2.5 mg/mL high concentration group show a slight decrease in cell viability (Figure S3). Compared to the control group, there were no significant changes in various tissues and organs of mice injected with the high concentration(Figure S4). Overall, the results demonstrate that Mannose-DSPE-PEG@ Fe 3 O 4 /CDs were successfully synthesized using a simple method, exhibiting good biocompatibility and imaging potential, providing a basis for targeting tumor-associated macrophages (TAMs). Assessment of Nanoparticle Targeting and Imaging Capabilities Macrophages treated with LPS and IL-4 induced M1 and M2 phenotypes, respectively. M1 macrophages showed high expression of IL-12, TNF-α, and iNOS (Figure S5A), while M2 macrophages expressed IL-10, ARG-1, and CD206(Figure. S5B). In colorectal cancer, 60.39% of TAMs expressed CD206(Figure S4C), confirming their predominant M2 immunosuppressive phenotype[ 29 ]. The results of confocal microscopy and flow cytometry showed that the uptake of targeted nanoparticles by M2 macrophages was significantly better than that of non-targeted nanoparticles (Fig. 2 A, B, and D); The CD206 receptor on the surface of macrophages was blocked[ 30 ], and the results of confocal imaging and flow cytometry showed that after blocking with mannose, the uptake of targeted nanoparticles by M2 macrophages was significantly reduced. This suggests that the internalization of the particles occurs through a mannose receptor-dependent mechanism, highlighting a specific interaction between the nanoparticles and the mannose receptors on the surface of M2 macrophages. (Fig. 2 C and D). MRI and fluorescence imaging of subcutaneous tumor models in C57BL/6 mice and liver metastatic models revealed that the targeted nanoparticles exhibit strong optical and magnetic imaging capabilities. Compared to non-targeting nanoparticles, tumors treated with targeted nanoparticles showed pronounced negative enhancement on T2-weighted MRI (T2WI) and distinct fluorescent signals (Fig. 3 A-C), indicating greater nanoparticle accumulation in targeted tumor tissues. In the non-targeted group, non-specific uptake of nanoparticles was evident in the MRI images of tumors, but this was not reflected in the near-infrared (NIR) images. This suggests that the distribution of non-targeted nanoparticles in situ tumors and metastases primarily relies on the enhanced permeability and retention (EPR) effect. By analyzing both NIR and MRI imaging data from the targeted and non-targeted groups, it can be inferred that the non-targeted nanoparticles predominantly accumulate in the extracellular spaces rather than being internalized by living cells. This observation underscores the importance of dual-modality imaging in distinguishing the localization and distribution of nanoparticles. Further, in vitro fluorescence imaging of mouse tumor tissues and major organs revealed that targeted nanoparticles were predominantly absorbed by tumor tissues, with no abnormal concentrations detected in other major organs such as the heart, liver, spleen, lung, and kidney (Fig. 3 D). This selective uptake by tumor tissues post-targeted modification suggests that the nanoparticles are effectively designed to enhance tumor targeting while minimizing off-target effects. Additionally, the persistence of significant fluorescence signals after 4 hours of circulation highlights the in vivo stability of the synthesized material, confirming its potential for sustained imaging applications. Targeted nanoparticles demonstrated significant enrichment within the tumor, with the red fluorescence region indicating that their distribution in tumor-associated macrophages appears to be more concentrated in the tumor center. This combination of selective tumor targeting, dual-modality imaging capabilities, and stable in vivo behavior makes these targeted nanoparticles a promising tool for the advanced imaging and diagnosis of cancer. Immune Microenvironment Modulation and Antitumor Effects of Mannose-DSPE-PEG@Fe 3 O 4 /CDs. The antitumor effect of nanoparticles was evaluated at the cellular level by conditioned medium experiments in MC38 cells. The results of CCK8 showed that the macrophages treated with nanoparticles showed an obvious anti-tumor effect, especially targeting nanoparticles(Figure 4 A). IL-12 and reactive oxygen species are involved in the response of helper Th1 cells to infection to promote antitumor and antigen presentation, inhibit the growth of cancer, and even promote tumor cell apoptosis, along with increased T effector to T regulatory cell ratios[ 31 ]. This anti-tumor activity similar to that of M1 macrophages suggests its significant repolarization ability[ 18 ], indicating that our synthesized nanoparticles have excellent immune adjuvant effects. Antitumor effects of samples were evaluated using the MC38 colorectal cancer mouse model. When the implanted tumor reached 80cm 3 on the seventh day, the nanoparticles were injected through the tail vein every other day, followed by co-injection. The tumors in the group injected with targeted nanoparticles exhibited a significant inhibition of growth (Fig. 4 B-D). Tunel staining indicated that both targeted nanoparticles and non-targeted nanoparticles inhibited tumor growth to a certain extent, especially targeted nanoparticles (Fig. 4 F). At the same time, the results of immunofluorescence showed that the targeted nanoparticles group not only significantly increased M1 macrophages, but decreased M2 macrophages, and the infiltration level of effector T cells (CD3 + CD8+) was significantly higher than that of the PBS group and non-targeted tumors. This shows that the targeted nanoparticles not only anti-tumor through the direct killing effect of M1 after repolarization, but more importantly, remodel the immune microenvironment of the tumor, providing a good environment for immunotherapy, and have the advantages of being compatible with other immunotherapies[ 32 ]. Fluorescence imaging revealed a significant reduction in the distribution of M2-type tumor-associated macrophages in xenograft tumor-bearing mice following treatment with targeted nanoparticles(Fig. 5 ).Therefore, it has great value in combination with other immunotherapeutic approaches. In vivo antitumor: Antitumor effects of nanoparticles in mice. 5*10 5 MC38 cells were planted in the armpit of each mouse and were randomly divided into three groups. Seven days after the tumor was planted, PBS, 500ug/ml DSPE-PEG-Man@Fe 3 O 4 -CDs and 500ug/ml DSPE-PEG-CDs were injected into the tail vein respectively. DSPE-PEG-Man @Fe 3 O 4 -CDs 200ul, administer once every other day and measure the tumor size. After two weeks of treatment, take the tumor (B), tumor growth curve (C) and weigh the tumor mass(D) of each group; prepare a single cell suspension, Flow cytometric analysis of M1 type (F4/80 + CD86+) and M2 type (F4/80 + CD206+) cells in each group (E); immunofluorescence analysis of various M1 type (CD86+) macrophages, M2 type (CD206+) macrophages Phage cells, effector T cells (CD3 + CD8+) and Tunel staining (F) .*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Mechanistic analysis of Mannose-DSPE-PEG@FeO/CDs in promoting TAMs repolarization To gain a comprehensive understanding of the mechanisms by which targeted nanoparticles reprogram tumor-associated macrophages (TAMs), transcriptional profiling was conducted on TAMs co-cultured with nanoparticles at multiple time points—0h, 6h, 12h, 24h, and 36h—revealing the temporal dynamics of TAMs following nanoparticle stimulation. Following quality control, differential analysis of the transcriptomes at different time points was conducted using the limma package, and k-means clustering divided the data into two groups. Cluster analysis revealed a distinct opposing trend in the gene expression of the two groups, with the included genes associated with the phenotypes and polarization pathways of M1 and M2 macrophages. Notably, genes related to the JAK-STAT pathway were most active at 12h, and by 36h, there was a significant divergence in gene expression related to M1 macrophages compared to M2 (Fig. 6 A and B). GO enrichment analysis of the differential gene sets revealed significant macrophage proliferation, activation, and metabolic reprogramming at 6h. By 12h, macrophages exhibited pronounced antigen presentation and tumor-suppressive capabilities, and by 24h, there was a notable activation of the NF-κB pathway, enhancing the presentation of endogenous antigens to CD8 + T cells via MHC I molecules (Fig. 6 C). This indicates that after nanoparticle stimulation, the functions associated with M1 macrophages gradually enhanced under the regulation of pathways such as NF-κB and JAK-STAT. To deeper understand the mechanisms by which targeted nanoparticles reprogram tumor-associated macrophages (TAMs), real-time PCR was employed to analyze the changes in typical mRNA levels. The results showed a significant increase in M1 markers, including CD86, i-NOS, TNF-α, and IL12 (P < 0.01), and a marked decrease in M2 markers, such as CD206, ARG1, and VEGF (P < 0.01), following 24 hours of co-culture (Fig. 7 A). TAMs' insufficient secretion of IL-12 undermines their antigen-presenting ability, thus inhibiting T cell proliferation and cytotoxic activity, promoting the recruitment of Tregs and Th2 cells, and facilitating tumor immune evasion. In colorectal cancer, hypoxic regions enhance Arg1 expression via HIF-1/2, shaping the immunosuppressive effects of TAMs, attracting additional TAMs, and forming a feedback loop that drives tumor growth[ 33 , 34 ]. Furthermore, M2-type TAMs not only suppress immune cytotoxic functions but also promote angiogenesis via VEGF secretion, thereby accelerating tumor metastasis. Targeted nanoparticles significantly reversed this trend (Fig. 7 A). The iron overload provided by nanoparticles triggers the Fenton reaction, generating substantial ROS, and promoting apoptosis in tumor cells[ 23 ]. Additionally, carbon dots themselves also exhibit a significant re-polarization effect (Fig. 7 A), with broad applications in tracking and re-polarizing tumor-associated macrophages. The role of carbon dots as immunoadjuvants is a significant discovery, potentially due to their superior upconversion photoluminescence, excellent photo-induced electron transfer, and unique electron pool properties that inhibit electron-hole recombination. Compared to other family members, they have a natural advantage in biocompatibility, with significantly lower biotoxicity[ 35 – 37 ]. Hypoxia, a hallmark of malignant tumors, participates in inducing epithelial metabolism and TAM infiltration due to the preferential accumulation of macrophages in hypoxic tumor regions and the retention of relatively immature cell types[ 34 ]. The observed elevation in TNF-α and accumulation of ROS suggest that nanoparticles may regulate M1/M2 expression through a mechanism involving ROS-NF-κB, inducing macrophage polarization toward an anti-tumor phenotype (Fig. 7 B). Apoptotic tumor cells further induce M1 polarization, creating a feedback loop that produces substantial tumor necrosis factor-alpha (TNF-α)[ 38 ]. Additionally, the increase of STAT1 and the decrease of STAT6 indicate that nanoparticles reprogram TAMs by changing the balance of STAT1/STAT6, and M1 macrophages release ROS through the ROS-mediated MAPK pathway to form positive feedback to enhance repolarization(Fig. 5 C)[ 39 ]. The ERK protein expression diagram also shows that targeted nanoparticles can inhibit the endoplasmic reticulum stress pathway (Fig. 7 C). PERK is a key driver of M2 polarization in macrophages, and its high expression promotes immune cell infiltration in the tumor microenvironment, leading to poor prognosis[ 40 ]. PERK expression significantly correlates with TAM infiltration levels, particularly with macrophage mannose receptor 1 (MRC1, also known as CD206). Activation of the ERK/MAPK signaling pathway promotes M2 macrophage polarization, and blocking this pathway can inhibit M2 TAM production and enhance T-cell anti-tumor activity[ 41 ]. This could be related to nanoparticle-induced iron overload, which inhibits the PERK signaling pathway, disrupts endoplasmic reticulum-mitochondria interactions, destabilizes mitochondrial homeostasis, prevents M2 TAMs from generating sufficient energy, and ultimately suppresses their immunosuppressive function. Although we observed the repolarization effects and related gene pathway changes induced by nanoparticles, the precise molecular mechanisms underlying repolarization require further in-depth validation. The repolarization effects of targeted nanoparticles on TAMs were also verified in tumor-bearing mice. Immunofluorescence analysis revealed an increase in M1 macrophages and a decrease in M2 macrophages in tumor sections (Figs. 7 D-E). Flow cytometry indicated a higher proportion of M1 macrophages in the nanoparticle-treated group (Fig. 7 F). These findings demonstrate that targeted nanoparticles can overcome the complex tumor microenvironment, exhibit precise TAM-targeting capabilities, and effectively achieve macrophage repolarization. Structurally, Mannose-DSPE-PEG@ Fe 3 O 4 /CDs, with their acidic properties and hydroxyl-rich carbon dots, ensure effective retention within lysosomes. This structural composition facilitates the Fenton reaction while preventing the neutralization of ROS by glutathione in the cytoplasm[ 28 ]. The synergy among iron, hydroxyl-rich carbon dots, and mannose ensures balanced ROS production, thereby avoiding the tumor-promoting proliferation effects of excessive ROS while maintaining levels that enhance tumor immunity. These structural attributes enable the nanoparticles to precisely modulate the oxidative environment of tumors. Biologically, carbon dots promote the polarization of macrophages towards the M1 phenotype by governing the STAT1/STAT6 balance and releasing ROS through the MAPK pathway. This creates a positive feedback mechanism. Additionally, the carbon dots inhibit the endoplasmic reticulum stress and ERK/MAPK signaling pathways, effectively blocking the immunosuppressive roles of M2-type macrophages and augmenting the anti-tumor activity of T cells. Collectively, these biological processes allow carbon dots to re-polarize TAMs within the complex tumor microenvironment, achieving precise recognition and effective re-polarization. In vitro repolarization: qRT-PCR was used to detect the levels of cytokines in control, M1 type, M2 type, M2 + Man@Fe 3 O 4 -CDs, M2 + Man@CDs, M2 + Man@Fe 3 O 4 groups (A); flow cytometry ROS levels in control, M1, M2, M2 + Man@Fe 3 O 4 -CDs groups were detected by instrument (B); M1, M2, M2 + Man@Fe 3 O 4 -CDs and M2 + Fe 3 O 4 -CDs groups were detected by western blot STAT1, STAT6, p-Erk levels (C);repolarization in vivo: 5*105 MC38 cells were planted in the armpit of each mouse, and randomly divided into three groups. Seven days after the tumor was planted, PBS, 500ug/ml Mannose-DSPE-PEG@Fe 3 O 4 /CDs and 500ug were injected into the tail vein respectively /ml DSPE-PEG- @Fe 3 O 4 -CDs 200ul, administered once every other day and measuring the size of the tumor, after two weeks of treatment, the tumor was taken to prepare a single cell suspension, flow cytometric analysis of each group M1 type (F4/80 + CD86+) (D) and M2 type (F4/80 + CD206+) cell mass (E); immunofluorescence analysis of various M1 type (CD86+) macrophages, M2 type (CD206+) macrophages (F). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Conclusion This study successfully synthesized a photomagnetic bifunctional nanocomposite material, Mannose-DSPE-PEG@ Fe 3 O 4 /CDs, capable of converting red light to near-infrared light while possessing MRI imaging capabilities. The material exhibits high sensitivity to the acidic tumor microenvironment (TME), allowing for the targeted imaging of tumor-associated macrophages (TAMs) in colorectal cancer. This nanocomposite facilitates the repolarization of TAMs through the JAK/STAT and ERK/MAPK signaling pathways, significantly enhancing their antigen-presenting ability and thereby improving the tumor immune microenvironment. Surprisingly, the synthesized carbon dots themselves were found to possess the ability to induce changes in TAM phenotypes, offering a novel perspective for developing more effective immunotherapeutic strategies. In summary, the nanocomposite developed in this study provides a powerful platform for the personalized assessment and treatment of colorectal cancer, paving the way for improved tumor immune microenvironments and advancing the field of precision medicine. The limitations of this study include the fact that, while we have initially confirmed the ability of the synthesized carbon dots to track and induce TAM polarization, the specific mechanisms involved require further investigation. Additionally, the relationship between the photomagnetic bimodal imaging results obtained using this nanomaterial and their corresponding clinical benefits needs to be explored further. Materials and Methods Tumor-Associated Macrophage-Related Immune Infiltration and Survival Analysis in TCGA Colorectal Cancer Utilizing clinical and gene expression data from colorectal cancer patients in The Cancer Genome Atlas (TCGA), we leveraged the R package `immunedeconv` to evaluate the abundance and distribution of tumor-associated macrophages (TAMs) in tumor samples compared to adjacent normal tissue. To determine the statistical significance of differences in TAM abundance between these tissues, we employed comparative statistical methods such as the Mann-Whitney U test and t-test. In the survival analysis phase, we constructed survival objects and applied the Cox proportional hazards model to examine the influence of TAM abundance on patient survival, accounting for covariates that could impact survival, including age and tumor stage. We assessed the model’s concordance and utilized statistical tests such as the likelihood ratio test, Wald test, and Score test to evaluate covariate significance. Additionally, we generated survival curves to visually represent the effect of TAM abundance on patient survival outcomes. Materials IR-813P-toluenesulfonate was purchased from Macklin (China), and polyethylene glycol (PEG 400) was purchased from Aladdin (China). DSPE-PEG2000 and DSPE-PEG-Man were purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. Fe 3 O 4 was synthesized by the College of Chemistry, Jilin University. Lipopolysaccharide (LPS) and recombinant mouse interleukin 4 (IL-4) were provided by PeproTech, USA. Dulbecco's Modified Eagle's Medium (DMEM) was purchased from HyClone (GE Healthcare, USA), and fetal bovine serum (FBS) was purchased from Bioind (Israel). Fluorescence-labeled anti-CD11b, F4/80, IL12, iNOS, TNFa, CD206, IL10, ARG1 were purchased from Biolegend (USA). Q-PCR primers were purchased from Sangon Biotechnology (China). Preparation of carbon dots (CDs) Take 50mg of IR-813 p-toluenesulfonate, dissolve in 10ml of polyethylene glycol (PEG 400), mix well, add the mixed solution into a 100ml reaction kettle, and heat at 180°C, 8 hours; then filter with 0.22um filter membrane, 3500 dialysis membrane for dialysis, then centrifugate 10000rpm*15 minutes, discard the supernatant, and obtain solid carbon dots after drying in the fume hood. Preparation of photo magnetic dual-modal targeting nanoprobe ( Mannose-DSPE-PEG@ Fe 3 O 4 /CDs ) : 5mgCDs, 5mg Fe 3 O 4 , and 40mg Mannose-DSPE-PEG were dissolved in 5ml toluene, mix under ultrasound for 20 minutes, then add the mixture into a flask filled with 50ml of deionized water, the temperature is 60°C, the magnetic stirring speed is 400 rpm, via solvent vaporing and self-assembly, to obtain a solid substance. Dissolve in 10ml of PBS, filter through a 0.44um filter, and store in the dark for future use. Characterization of Mannose-DSPE-PEG@Fe 3 O 4 /CDs The absorption spectra of the nanoparticles were measured with a Shimadzu 3600 UV-Vis-NIR spectrophotometer. The morphology of the nanoparticles was observed with a JEM-2100F transmission electron microscope (TEM) produced by JEOL. The particle size and potential of nanoparticles were measured by the dynamic light scattering (DSL) method. 3.0T nuclear magnetic resonance (MRI) was used to image and measure the nanoparticle's T2 and T2 mapping with different concentrations. Biosafety Assessment of Nanoparticles RAW264.7 and MC38 cells were planted in 96-well plates at 8*10 3 per well, and after 24 hours, DSPE-PEG with concentrations of 0, 10, 50, 100, 200, 500, 1000, and 2500 ug/ml Mannose-DSPE-PEG@ Fe 3 O 4 /CDs nanoparticles treated cells. At 12h, 24h, and 48h respectively, the activity of the cells was measured using the CCK8 kit (Solarbio, China). Untreated cells served as a control group and were considered 100% viable. C57BL/6 mice were injected with 200ul of Mannose-DSPE-PEG@ Fe 3 O 4 /CDs nanoparticles at a concentration of 1000ug/ml and 2500ug/ml respectively in the tail vein. After 24 hours, the mice's hearts, livers, spleens, lungs, and kidneys were taken for HE stains, compared with the normal control group. Macrophage induction typing and marker detection Spread RAW264.7 cells in a 6-well plate, about 15*104 cells per well. After 24 hours, add M1 type (DMEM + 10% fetal bovine serum + 1% double antibody + 100ng/ml LPS) and M2 type (DMEM + 10% fetal bovine serum + 1% double antibody + 20ng/ml IL4) induction solution, 2ml per well, placed in a CO2 incubator and incubated for 24h. After 24 hours, the marker levels of M1 and M2 macrophages were detected by flow cytometry (Beckman, USA), among which the M1 type detected i-NOS, IL-12, and TNFα; the M2 type detected IL-10, CD206, and ARG1. RAW264.7 macrophages were placed in the lower chamber of the Transwell chamber, and MC38 cells were placed in the upper chamber, and co-cultured for 24 hours to create a tumor-associated macrophage model. Flow cytometry was used to detect the M2 cytokines IL-10, CD206, and ARG1 levels. The Targeting Ability of Nanoparticles in vitro : RAW264.7 was induced into M2 macrophages and divided into 3 groups. Group 1: adding 500ug/ml targeted nanoparticles (Mannose-DSPE-PEG@ Fe 3 O 4 /CDs); Group 2: adding the same amount of non-targeted nanoparticles (DSPE-PEG@ Fe 3 O 4 /CDs); Group 3: Block with 500ug/ml mannose for 3 h, wash away, and then add 500ug/ml targeted nanoparticles (Mannose-DSPE-PEG@ Fe 3 O 4 /CDs); Laser confocal imaging and flow cytometry was performed to detect the uptake of nanoparticles. The Targeting Ability of Nanoparticles in vivo : C57BL/6 tumor-bearing mice were injected with 200ul of targeted and non-targeted nanoparticles through the tail vein respectively, at a concentration of 1000ug/ml. After 4 hours, the C57 mouse colorectal cancer subcutaneous xenograft tumor model and liver metastases model were treated respectively. Magnetic resonance imaging (Siemens, 3.0T) and in vitro fluorescence imaging (IVIS Spectrum). MRI scanning parameters are as follows: T2WI: TR 3120.0ms, TE 111ms, FOV read 70mm, Voxel size:0.3*0.3*1.0mm, Slice thickness 1.0mm; T2mapping༚ TR 613.0ms, TE1 16.1, TE2 32.2, TE3 48.3, TE4 64.4, TE5 80.5, FOV read 110mm, Voxel size:0.6*0.6*2.5mm, Slice thickness 2.5mm. Coronal imaging of mice. In vitro fluorescence imaging wavelength selection 710-840nm. Then the materials were collected, and fluorescence imaging was performed on the mouse tumor tissue, heart, liver, spleen, lung, and kidney. Macrophage Reolarization Analysis and Transcriptome Analysis The nanoparticles co-incubated with TAMs were subjected to transcriptome sequencing at different time points (0 hours, 6 hours, 12 hours, 24 hours, and 36 hours). Bioinformatics analysis was conducted using R (version 4.3.3). Differential gene expression analysis, clustering analysis, and enrichment analysis were performed on the sequencing results from each time point. Time-related heatmaps were generated to analyze the temporal dynamic changes in TAMs following nanoparticle stimulation. Total RNA extracted from cells was isolated using TRIzol reagent (Invitrogen life technologies, USA), and RNA concentration and purity were measured using Infinite 200 PRO. CDNA was synthesized from 1 ug of total RNA using the PrimeScript™ RT reagent kit with gDNA Eraser(Takara, Japan). The primers were designed and synthesized by Sangon (China) and the sequence was listed in Supplement Table 1 . Quantitative RT-PCR was carried out in an 8-strip tube using FastStart Universal SYBR Green Master(Roche, Switzerland) on a real-time fluorescence quantitative PCR instrument༈Eppendorf, Germany༉. All results were processed by the double-delta method (2 − Δ Ct). Detection of ROS levels before and after transformation of tumor-associated macrophages (TAMs) RAW264.7 cells were divided into a control group, M1 type group, M2 type group, and M2 type group + 500ug/ml targeted nanoparticle repolarization group for 24h after treatment, and then the reactive oxygen species detection kit (Solarbio, China ) and flow cytometry to detect the ROS levels in the above groups. Assessment repolarization with Western blot assay Cells were collected, washed twice with PBS, lysed in RIPA containing 1% PMSF for 30 min on ice, and centrifuged at 15,000 g for 15 min at 4°C. The resulting supernatant was collected, and its protein concentration was determined using the BCA assay (Beyotime, China). Equal amounts of total and prestained protein ladders were separated by SDS-PAGE and transferred to nitrocellulose membranes on 0.22 µm nitrocellulose membranes. After blocking with 5% skim milk for 3 hours, the membrane was incubated with the primary antibody overnight at 4°C. Then, the membrane was washed and subsequently incubated with secondary immunoglobulin G-horseradish peroxidase, bound, and then the proteins were visualized using an infrared luminescence imaging system (LI-COR Odyssey, USA). Antitumor effects of tumor-associated macrophages (TAMs) before and after transformation in vitro Raw264.7 macrophages were induced into M1 and M2 types respectively, and targeting and non-targeting nanoparticles were added to M2 macrophages to induce their repolarization for 24 hours, and then the complete medium was replaced for culture After 6 hours, the conditioned medium was obtained, and the MC38 colorectal cancer cells were incubated for 24 hours, and then the viability of the MC38 tumor cells was detected with a CCK8 kit. Repolarization ability of nanoparticles and antitumor effects in vivo C57BL/6 mice at 6–8 weeks were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and each mouse was subcutaneously inoculated with 5 × 10 5 MC38 tumor cells in the armpit to create a mouse subcutaneous xenograft tumor model, and each mouse was injected with 2 ×10 5 MC38 tumor cells were used to create a liver metastases model. The treatment started 7 days after the tumor was planted, randomly divided into 3 groups, 5 in each group, and injected PBS, 1000ug/ml Fe 3 O 4 /CDs and Man- Fe 3 O 4 /CDs 200ul into the tail vein respectively and administered once every other day. Mice body weight was treated for 14 days. The calculation formula of tumor volume is tumor volume = length × width 2 /2. At the end of the experiment, all the mice were euthanized, and the tumors of each group were taken, weighed, and photographed. Then the tumor was fixed with 4% paraformaldehyde, and the sections were stained with hematoxylin-eosin (H&E), Tunel staining, immunofluorescence detection of CTL cells, and flow cytometry detection of M1 types (CD11b+, F4/80+, CD86+) and M2 Type (CD11b+, F4/80+, CD206+) macrophage proportion. All animal experiments were performed in accordance with the guidelines approved by the Animal Care and Use Committee of Changchun Weishi Testing Technology Service Co., Ltd. Statistical analyses All data subjected to statistical analyses were obtained from at least three parallel experiments, and the results are expressed as mean ± standard error of mean (SEM). The statistical analysis was performed by Student's t-tests for two groups, and one way ANOVA for multiple groups using GraphPad Prism version 9.00 for MACOS (GraphPad Software, La Jolla, CA, USA). A p-value ≤ 0.05 was considered to be statistically significant. Corresponding Authors Bai Yang − State Key Laboratory of Supramolecular Structureand Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun 130012, P.R. China; Joint Laboratory of Opto-Functional Theranosticsin Medicine and Chemistry, The First Hospital of JilinUniversity, Changchun 130021, P. R. China; orcid.org/0000-0002-3873-075X; Email: [email protected] Butian Zhang − Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130021, P. R.China;Email: [email protected] Declarations Competing interests The authors declare no competing interests. Ethics approval All animal experimental procedures were performed according to protocols approved by the Animal Care and Use Committee of Changchun Weishi Testing Technology Service Co., Ltd. Author details Yingying Miao − Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130021, P. R.China; Xiaoyu Li − State Key Laboratory of Supramolecular Structure and Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun130012, P. R. China; Qingsen Zeng − Postdoctoral Researcher at Seoul National University;State Key Laboratory of Supramolecular Structure and Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun130012, P. R. China; Kai Zhang − State Key Laboratory of Supramolecular Structure and Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun130012, P. R. China; Lin Liu − Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130021, P. R.China; Funding This work was supported by grants from the National Natural Science Foundation of China(82001880) and Natural Science Foundation of Jilin Province (YDZJ202201ZYTS538). Author Contribution YB and ZBT proposed and designed the experiments. ZQS , LXY and KZ contributed to and was responsible for designing chemical experiments, performing chemical tests and analyses (Figures 1), and revision the article about chemical section. LL supports the imaging resources. MYY contributed to and was responsible for performing biological tests , ZBT contributed to and was responsible for data analyses (Figures 2-7), and ZBT compolish writing this paper. Acknowledgements The authors are grateful for the financial support from the National Natural Science Foundation of China (82001880) and Natural Science Foundation of Jilin Province (YDZJ202201ZYTS538). Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions. References Sinicrope FA. Increasing Incidence of Early-Onset Colorectal Cancer. N Engl J Med. 2022;386:1547–58. Brenner H, Kloor M, Pox CP. Colorectal cancer. 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PBX3 promotes. migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway - PubMed [Internet]. [cited 2022 Nov 15]. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx scheme1.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5657271","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391825285,"identity":"60a5f32e-578d-451a-9285-f48f12d24d15","order_by":0,"name":"Yingying Miao","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Miao","suffix":""},{"id":391825286,"identity":"c9b77b4c-6526-476e-93aa-8912b02709ac","order_by":1,"name":"Xiaoyu Li","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Li","suffix":""},{"id":391825287,"identity":"8ae9c8ce-1634-403a-9d8d-fe3f513ba3ae","order_by":2,"name":"Qingsen Zeng","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Qingsen","middleName":"","lastName":"Zeng","suffix":""},{"id":391825288,"identity":"27607d8e-2bdd-4a79-9f04-8e739bc53fec","order_by":3,"name":"Kai Zhang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhang","suffix":""},{"id":391825289,"identity":"bd648f34-bb48-437d-b621-282cab8799f3","order_by":4,"name":"Lin Liu","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Liu","suffix":""},{"id":391825290,"identity":"514d1d0a-d00b-4c37-807b-3ee49d9064a2","order_by":5,"name":"Bai Yang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Bai","middleName":"","lastName":"Yang","suffix":""},{"id":391825291,"identity":"4a1ca139-f8b9-4ff6-8d4e-feb38e1876e1","order_by":6,"name":"Butian Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACCSBmbGCQY2wH8diI12JgzNhMqpbEBmZitcjPbn728OuOP+nNzTwGDB/KDjPwz27Ar4VxzjFzY9kzBrmNQC2MM84dZpC4cwC/FmaJBDNpyTaIFmbetsMMBhIJ+LWwSaR/A2lJZwRp+UuMFh6JHDPJj20GCWAtjMRokZDIKZNmPGNs2NjMVnCw51w6j8QNAlrkZ6Rvk/y5Q07esL1544MfZdZy/DMIaAEBZh4gYdjAwHAA5FLC6oGA8QfIOqKUjoJRMApGwYgEAK7KPMl4QIBYAAAAAElFTkSuQmCC","orcid":"","institution":"Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Butian","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-12-17 01:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5657271/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5657271/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72284511,"identity":"2e2db1d2-5ead-4261-a434-6d4a93a3ff8d","added_by":"auto","created_at":"2024-12-24 16:47:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":467443,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image and DLS of Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs (A); UV−vis absorbance of Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs (B); Fluorescence spectrum of Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs (C); Under acidic conditions, the fluorescence emission spectrum of Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs under different excitation lights(D);Fourier Transform Infrared Spectroscopy (FTIR) of Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs, Mannose-DSPE-PEG ,CDs and \u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e(E); T2WI and T2 Mapping imaging of the nanoparticles (F);\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/dce71c384215ddc8006abc6d.jpg"},{"id":72284514,"identity":"cf25100a-564c-48fc-bc56-a966498ddc69","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":513290,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting nanoparticles in vitro. A: Confocal microscopy image and flow cytometry of M2 macrophages 30 minutes after the addition of targeting nanoparticles. B: Confocal microscope image and flow diagram of M2 macrophages added with non-targeting nanoparticles for 30 minutes: C: Confocal images of M2 macrophages blocked with mannose for 3 hours and then added with targeting nanoparticles for 30 minutes confocal microscopy images and flow cytometry results.\u003c/p\u003e","description":"","filename":"Figure2Targetingnanoparticlesinvitro.png","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/17479379443dd3bcb9425b61.png"},{"id":72284512,"identity":"dda8b40e-bd1f-4347-a819-ed616dd332cb","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7145895,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting of nanoparticles in vivo. C57BL/6 mouse axillary xenograft tumor and liver metastases model were respectively injected with PBS, 500ug/ml DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs and 500ug/ml Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs 200ul through the tail vein, 4 hours after MRI T2 Coronal imaging, T2 mapping coronal imaging and fluorescence imaging (A-C); mouse tumor tissue, heart, liver, spleen, lung, and kidney were taken for fluorescence imaging (D).\u003c/p\u003e","description":"","filename":"Figure3Targetingofnanoparticlesinvivo.png","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/5f419bc63e3e42d77fef8b82.png"},{"id":72284525,"identity":"3875a8d9-48d5-4428-b5d9-93c24047c3e3","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1350362,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro antitumor: The viability of MC38 cells after co-incubating with M1 type, M2 type, M2+Man@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs and M2+\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs conditioned medium for 24 hours by CCK8(A).\u003c/p\u003e\n\u003cp\u003eIn vivo antitumor: Antitumor effects of nanoparticles in mice. 5*10\u003csup\u003e5\u003c/sup\u003e MC38 cells were planted in the armpit of each mouse and were randomly divided into three groups. Seven days after the tumor was planted, PBS, 500ug/ml DSPE-PEG-Man@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs and 500ug/ml DSPE-PEG-CDs were injected into the tail vein respectively. DSPE-PEG-Man @\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs 200ul, administer once every other day and measure the tumor size. After two weeks of treatment, take the tumor (B), tumor growth curve (C) and weigh the tumor mass(D) of each group; prepare a single cell suspension, Flow cytometric analysis of M1 type (F4/80+ CD86+) and M2 type (F4/80+ CD206+) cells in each group (E); immunofluorescence analysis of various M1 type (CD86+) macrophages, M2 type (CD206+) macrophages Phage cells, effector T cells (CD3+ CD8+) and Tunel staining (F) .*p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4antitumor.png","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/db60866b0e3207970e3b7d02.png"},{"id":72284516,"identity":"78223723-0e05-4d96-b603-a7a34860ff81","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298646,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence imaging of xenograft tumor-bearing mice before (A) and after (B) treatment with targeted nanoparticles.\u003c/p\u003e","description":"","filename":"Figure5Fluorescenceimagingofxenografttumorbearingmice.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/45dcd3e2e6900e65545fdf1f.jpg"},{"id":72285833,"identity":"b28531b7-6780-4d88-a24d-4e39da4a3adc","added_by":"auto","created_at":"2024-12-24 16:55:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmaps of gene clusters at various time points using the k-means method, depicting the dynamic changes in macrophage polarization-related genes(A); Heatmaps of macrophage polarization-related genes at various time points(B); Time-specific GO enrichment analysis heatmaps and KEGG bubble plot (C).\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/9d4eca5cfd093582aa4c1341.png"},{"id":72284522,"identity":"6ff86310-e571-48d3-bdeb-0dc22bc5f55a","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6902713,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro and in vivo repolarization of nanoparticles.\u003c/p\u003e\n\u003cp\u003eIn vitro repolarization: qRT-PCR was used to detect the levels of cytokines in control, M1 type, M2 type, M2+Man@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs, M2+Man@CDs, M2+Man@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e groups (A); flow cytometry ROS levels in control, M1, M2, M2+Man@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs groups were detected by instrument (B); M1, M2, M2+Man@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs and M2+\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs groups were detected by western blot STAT1, STAT6, p-Erk levels (C);repolarization in vivo: 5*105 MC38 cells were planted in the armpit of each mouse, and randomly divided into three groups. Seven days after the tumor was planted, PBS, 500ug/ml Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs and 500ug were injected into the tail vein respectively /ml DSPE-PEG- @\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e-CDs 200ul, administered once every other day and measuring the size of the tumor, after two weeks of treatment, the tumor was taken to prepare a single cell suspension, flow cytometric analysis of each group M1 type (F4/80+ CD86+) (D) and M2 type (F4/80+ CD206+) cell mass (E); immunofluorescence analysis of various M1 type (CD86+) macrophages, M2 type (CD206+) macrophages (F). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure7AssessmentofPolarizationEffects.png","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/63a8e09daff5bcecf81c67f8.png"},{"id":72289315,"identity":"41296d88-e5d5-4346-822a-2cb3531a8b16","added_by":"auto","created_at":"2024-12-24 17:19:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21059493,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/d99d19fb-d2c7-48b1-97cb-a56e4b02f52c.pdf"},{"id":72284523,"identity":"bde2d16c-b692-4a7f-9b43-783d601dc216","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17660399,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/a69954b081ff4a788fcdc158.docx"},{"id":72284524,"identity":"0db68502-5c4b-4bb4-9506-c88b943ff3ef","added_by":"auto","created_at":"2024-12-24 16:47:42","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":352588,"visible":true,"origin":"","legend":"","description":"","filename":"scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5657271/v1/6e88197460083ad33bb1db2d.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the theranostic potential of carbon dots/Fe3 O4 superparticles for tumor-associated macrophage targeting and repolarization in colorectal cancer therapy via JAK/STAT and ERK/MAPK pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer is the third most common cancer in the world with increasing incidence and mortality rates globally[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a novel cancer treatment, cancer immunotherapies have recently driven a paradigm shift in patient survival across tumors, but they only benefited a minority of patients with colorectal cancer[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As accumulating evidence has shown, Tumor-associated macrophages (TAMs), as abundant and active infiltrated inflammatory cells in the tumor microenvironment (TME), are the most important components in the tumor immunosuppressive microenvironment, promoting tumor metastasis, and therapy sensitivity for colorectal cancers[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the tumor-promoting microenvironment of colorectal cancer, the inflammatory M2 TAM phenotype accounts for 50% of the tumor cell mass, whereas the immunoprotective M1 TAM phenotype is rare. M2 phenotype macrophages promote tumor cell proliferation by regulating cytokines, chemokines, proteases, and reactive oxygen species. Furthermore, M2 TAMs enhance angiogenesis by modulating VEGF bioavailability and suppressing protective adaptive immune responses. Additionally, M2-polarized TAMs secrete lactate during glycolysis, and through their metabolic processes, secrete other acidic metabolites, reducing the pH value of the TME. TAMs secrete matrix metalloproteinases (MMPs) that degrade extracellular matrix (ECM), affecting local pH buffering capacity and fostering a tumor acidic microenvironment. Therefore, TAMs can promote tumor microenvironment acidification through multiple pathways, making them key factors in facilitating colorectal cancer invasion, metastasis, chemoresistance, and immune evasion. Given their crucial role, TAMs have emerged as highly promising targets in cancer immunotherapy. Given their essential roles, TAMs have emerged as promising targets for cancer immunotherapy[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Immunotherapeutic strategies to suppress TAMs of the M2 phenotype or to reprogram them against the M1 phenotype of tumors have gained enormous momentum[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the success of these therapies relies on quantifying the distribution of TAMs in the TME[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Targeting tumor-associated macrophages (TAMs) for imaging and immunomodulation holds significant potential in the personalized immunotherapy of colorectal cancer. By tracing the distribution of TAMs within the body, it is possible to assess the degree of immune suppression in patients, identify those who are more likely to benefit from immunotherapy, and facilitate precision medicine. Additionally, tracking TAMs allows for the real-time, non-invasive monitoring of dynamic changes in the tumor microenvironment during immunotherapy, providing a basis for the dynamic adjustment of treatment protocols. Polarizing TAMs from the M2 to the M1 phenotype can reverse the immunosuppressive tumor microenvironment, enhance the efficacy of immunotherapies such as immune checkpoint inhibitors, delay the onset of resistance, and reduce the risk of tumor recurrence. Integrating TAM modulation with radiotherapy, anti-angiogenic therapy, and cell therapy can produce a synergistic effect, further enhancing therapeutic outcomes. Therefore, targeting TAMs for imaging and immunomodulation offers a personalized immunotherapy strategy for colorectal cancer patients, with the potential to improve patient outcomes.\u003c/p\u003e \u003cp\u003eNowadays, nanomaterials have been widely applied in various fields among which carbon dots (CDs) received more and more attention[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CDs are the most promising candidates of the carbon family with superior properties like ultra-small size, high aqueous solubility, low cytotoxicity, and inherent photoluminescence which makes them suitable for diverse biomedical applications [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A few reports about inflammation indicate that carbon dots could repolarize M2 macrophages, and changes were assessed by fluorescence intensity.[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This suggests that carbon dots may be a promising material for targeting TAMs. However, few carbon dots can stabilize near-infrared luminescence, resulting in poor tissue penetrability, which limits their dynamic imaging in vivo[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].SPIONs are FDA-approved T2 contrast agents used in MRI and have been extensively applied to track immune cells, such as DCs and T cells[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Zanganeh et al. discovered their off-labeled uses that they can induce a phenotypic transition from M2 towards M1 and inhibit tumor growth. Iron oxides repolarize M2 to M1 and induce the Fenton reaction which can generate ROS and promote the apoptosis of tumor cells[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, iron oxide nanoparticles have promising clinical applications due to their properties such as superparamagnetism and repolarization. The major drawbacks are that it is not possible to distinguish between dead and live cells, that signal void in MRI does not quantitatively report on the number of cells, and that the label undergoes dilution with cell replication in vivo[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we propose a novel nanoplatform\u0026mdash;Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs\u0026mdash;designed to precisely assess the M2 TAMs within the TME of colorectal cancer and facilitate their polarization switch. This multifunctional material combines the magnetic properties of \u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e with the near-infrared fluorescence of CDs, and is further modified with Mannose-DSPE-PEG to enhance specificity towards M2 TAMs. Upon targeting and binding to M2 TAMs, this nanoplatform not only allows for accurate spatial localization but also employs near-infrared (NIR) fluorescence imaging to precisely identify viable TAMs, inducing the polarization of M2 TAMs to the M1 phenotype, thus improving the immune microenvironment.(Scheme 1)\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eColorectal cancer (CRC) patients derive limited benefit from immunotherapy. Analysis of the TCGA database indicates that TAMs not only play a pivotal role in the tumor microenvironment of CRC but also exhibit significant distribution differences between tumor tissues and adjacent normal tissues (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C). Specifically, patients with higher TAM abundance, particularly M2-type macrophages, have significantly shorter survival times and reduced overall survival rates (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Therefore, TAMs represent a critical factor influencing the effectiveness of CRC immunotherapy, making precise imaging of TAMs an effective strategy for evaluating patient benefit from immunotherapy. Furthermore, reprogramming the polarization state of TAMs holds promise as a method to improve the tumor immune microenvironment and enhance the therapeutic efficacy of immunotherapy in CRC patients.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eSynthesis and Characterization of Mannose-DSPE-PEG@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CDs\u003c/h2\u003e\n\u003cp\u003eThe Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs superparticles were fabricated by first synthesizing carbon dots with stable red-shifted fluorescence through a solvothermal method involving PEG and near-infrared dye IR 813[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], followed by the encapsulation of \u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e and carbon dots using amphiphilic di-stearoyl phosphatidylethanolamine, polyethylene glycol, and D-mannose (see the Methods for details).\u003c/p\u003e\n\u003cp\u003eMorphological studies show that the Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs nanoparticles are spherical in shape and uniformly dispersed, with a size of approximately 169.9 nm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). This enables them to possess a favorable enhanced permeation and retention (EPR) effect while avoiding clearance by the liver Kupffer cell system[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, the Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs are surrounded by hydrophilic groups, allowing them to disperse well in aqueous media. Compared to neutral nanoparticles, anionic nanoparticles are more easily taken up by macrophages[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] (Figure S2A). The UV absorption spectrum displayed distinct peaks at 214 nm, 251 nm, 307 nm, 373 nm, and 525 nm, with the peak at 307 nm indicating a high content of hydroxyl groups in the carbon dots. The other absorption peaks are attributed to \u0026pi;-\u0026pi;* and n-\u0026pi;* transitions, while the 525 nm peak may arise from the formation of nitrogen-containing heterocycles resulting from the degradation of IR-813(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), which are combined with PEG[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeveraging the exceptional near-infrared emission properties of the prepared carbon dots (Figure S2B), the fluorescence emission spectrum demonstrated distinct maximum peaks at 637 nm and 780 nm for the nanoparticles. These peaks exhibited pH sensitivity; as the pH decreased, the intensity of the 637 nm peak diminished, while the intensity of the 780 nm peak increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Under acidic conditions (pH approximately 0.4), the composite displayed near-infrared fluorescence at 780 nm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating potential for visualizing tumor microenvironments. Fourier Transform Infrared Spectroscopy (FTIR) analysis showed that Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs exhibited a weak absorption peak at 1650 cm\u0026thinsp;\u0026minus;\u0026thinsp;1, confirming the successful encapsulation of carbon dots and \u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e in the nanocomposite (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\n\u003cp\u003eLong-wavelength emissions in the red and near-infrared (NIR) regions are important for in vivo imaging, as they can reduce tissue autofluorescence and enhance contrast[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. The synthesized nanoparticles formed assemblies that prevent quenching due to aggregation, improving graphitization and \u0026pi;-conjugation[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The specific relaxivity (r*2) of the nanoparticles was determined to be 197.9 mM\u0026thinsp;\u0026minus;\u0026thinsp;1 s\u0026thinsp;\u0026minus;\u0026thinsp;1(Figure S2C), indicating their effectiveness in shortening T2, making them suitable as negative MRI contrast agents[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e](Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). The pH value of the routinely prepared aqueous solution was 5.3(Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD), suggesting prolonged retention in lysosomes[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe CCK8 assay indicated that only when the co-culture time was extended to 48 hours did the 2.5 mg/mL high concentration group show a slight decrease in cell viability (Figure S3). Compared to the control group, there were no significant changes in various tissues and organs of mice injected with the high concentration(Figure S4).\u003c/p\u003e\n\u003cp\u003eOverall, the results demonstrate that Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs were successfully synthesized using a simple method, exhibiting good biocompatibility and imaging potential, providing a basis for targeting tumor-associated macrophages (TAMs).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAssessment of Nanoparticle Targeting and Imaging Capabilities\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eMacrophages treated with LPS and IL-4 induced M1 and M2 phenotypes, respectively. M1 macrophages showed high expression of IL-12, TNF-\u0026alpha;, and iNOS (Figure S5A), while M2 macrophages expressed IL-10, ARG-1, and CD206(Figure. S5B). In colorectal cancer, 60.39% of TAMs expressed CD206(Figure S4C), confirming their predominant M2 immunosuppressive phenotype[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe results of confocal microscopy and flow cytometry showed that the uptake of targeted nanoparticles by M2 macrophages was significantly better than that of non-targeted nanoparticles (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, and D); The CD206 receptor on the surface of macrophages was blocked[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e], and the results of confocal imaging and flow cytometry showed that after blocking with mannose, the uptake of targeted nanoparticles by M2 macrophages was significantly reduced. This suggests that the internalization of the particles occurs through a mannose receptor-dependent mechanism, highlighting a specific interaction between the nanoparticles and the mannose receptors on the surface of M2 macrophages. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and D).\u003c/p\u003e\n\u003cp\u003eMRI and fluorescence imaging of subcutaneous tumor models in C57BL/6 mice and liver metastatic models revealed that the targeted nanoparticles exhibit strong optical and magnetic imaging capabilities. Compared to non-targeting nanoparticles, tumors treated with targeted nanoparticles showed pronounced negative enhancement on T2-weighted MRI (T2WI) and distinct fluorescent signals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-C), indicating greater nanoparticle accumulation in targeted tumor tissues.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the non-targeted group, non-specific uptake of nanoparticles was evident in the MRI images of tumors, but this was not reflected in the near-infrared (NIR) images. This suggests that the distribution of non-targeted nanoparticles in situ tumors and metastases primarily relies on the enhanced permeability and retention (EPR) effect. By analyzing both NIR and MRI imaging data from the targeted and non-targeted groups, it can be inferred that the non-targeted nanoparticles predominantly accumulate in the extracellular spaces rather than being internalized by living cells. This observation underscores the importance of dual-modality imaging in distinguishing the localization and distribution of nanoparticles.\u003c/p\u003e\n\u003cp\u003eFurther, in vitro fluorescence imaging of mouse tumor tissues and major organs revealed that targeted nanoparticles were predominantly absorbed by tumor tissues, with no abnormal concentrations detected in other major organs such as the heart, liver, spleen, lung, and kidney (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). This selective uptake by tumor tissues post-targeted modification suggests that the nanoparticles are effectively designed to enhance tumor targeting while minimizing off-target effects. Additionally, the persistence of significant fluorescence signals after 4 hours of circulation highlights the in vivo stability of the synthesized material, confirming its potential for sustained imaging applications. Targeted nanoparticles demonstrated significant enrichment within the tumor, with the red fluorescence region indicating that their distribution in tumor-associated macrophages appears to be more concentrated in the tumor center. This combination of selective tumor targeting, dual-modality imaging capabilities, and stable in vivo behavior makes these targeted nanoparticles a promising tool for the advanced imaging and diagnosis of cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune Microenvironment Modulation and Antitumor Effects of Mannose-DSPE-PEG@Fe\u003c/strong\u003e \u003csub\u003e \u003cstrong\u003e3\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003eO\u003c/strong\u003e \u003csub\u003e \u003cstrong\u003e4\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003e/CDs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antitumor effect of nanoparticles was evaluated at the cellular level by conditioned medium experiments in MC38 cells. The results of CCK8 showed that the macrophages treated with nanoparticles showed an obvious anti-tumor effect, especially targeting nanoparticles(Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). IL-12 and reactive oxygen species are involved in the response of helper Th1 cells to infection to promote antitumor and antigen presentation, inhibit the growth of cancer, and even promote tumor cell apoptosis, along with increased T effector to T regulatory cell ratios[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. This anti-tumor activity similar to that of M1 macrophages suggests its significant repolarization ability[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], indicating that our synthesized nanoparticles have excellent immune adjuvant effects. Antitumor effects of samples were evaluated using the MC38 colorectal cancer mouse model. When the implanted tumor reached 80cm\u003csup\u003e3\u003c/sup\u003e on the seventh day, the nanoparticles were injected through the tail vein every other day, followed by co-injection. The tumors in the group injected with targeted nanoparticles exhibited a significant inhibition of growth (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Tunel staining indicated that both targeted nanoparticles and non-targeted nanoparticles inhibited tumor growth to a certain extent, especially targeted nanoparticles (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). At the same time, the results of immunofluorescence showed that the targeted nanoparticles group not only significantly increased M1 macrophages, but decreased M2 macrophages, and the infiltration level of effector T cells (CD3\u0026thinsp;+\u0026thinsp;CD8+) was significantly higher than that of the PBS group and non-targeted tumors. This shows that the targeted nanoparticles not only anti-tumor through the direct killing effect of M1 after repolarization, but more importantly, remodel the immune microenvironment of the tumor, providing a good environment for immunotherapy, and have the advantages of being compatible with other immunotherapies[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Fluorescence imaging revealed a significant reduction in the distribution of M2-type tumor-associated macrophages in xenograft tumor-bearing mice following treatment with targeted nanoparticles(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).Therefore, it has great value in combination with other immunotherapeutic approaches.\u003c/p\u003e\n\u003cp\u003eIn vivo antitumor: Antitumor effects of nanoparticles in mice. 5*10\u003csup\u003e5\u003c/sup\u003e MC38 cells were planted in the armpit of each mouse and were randomly divided into three groups. Seven days after the tumor was planted, PBS, 500ug/ml DSPE-PEG-Man@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs and 500ug/ml DSPE-PEG-CDs were injected into the tail vein respectively. DSPE-PEG-Man @Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs 200ul, administer once every other day and measure the tumor size. After two weeks of treatment, take the tumor (B), tumor growth curve (C) and weigh the tumor mass(D) of each group; prepare a single cell suspension, Flow cytometric analysis of M1 type (F4/80\u0026thinsp;+\u0026thinsp;CD86+) and M2 type (F4/80\u0026thinsp;+\u0026thinsp;CD206+) cells in each group (E); immunofluorescence analysis of various M1 type (CD86+) macrophages, M2 type (CD206+) macrophages Phage cells, effector T cells (CD3\u0026thinsp;+\u0026thinsp;CD8+) and Tunel staining (F) .*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eMechanistic analysis of Mannose-DSPE-PEG@FeO/CDs in promoting TAMs repolarization\u003c/h3\u003e\n\u003cp\u003eTo gain a comprehensive understanding of the mechanisms by which targeted nanoparticles reprogram tumor-associated macrophages (TAMs), transcriptional profiling was conducted on TAMs co-cultured with nanoparticles at multiple time points\u0026mdash;0h, 6h, 12h, 24h, and 36h\u0026mdash;revealing the temporal dynamics of TAMs following nanoparticle stimulation. Following quality control, differential analysis of the transcriptomes at different time points was conducted using the limma package, and k-means clustering divided the data into two groups. Cluster analysis revealed a distinct opposing trend in the gene expression of the two groups, with the included genes associated with the phenotypes and polarization pathways of M1 and M2 macrophages. Notably, genes related to the JAK-STAT pathway were most active at 12h, and by 36h, there was a significant divergence in gene expression related to M1 macrophages compared to M2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). GO enrichment analysis of the differential gene sets revealed significant macrophage proliferation, activation, and metabolic reprogramming at 6h. By 12h, macrophages exhibited pronounced antigen presentation and tumor-suppressive capabilities, and by 24h, there was a notable activation of the NF-\u0026kappa;B pathway, enhancing the presentation of endogenous antigens to CD8\u0026thinsp;+\u0026thinsp;T cells via MHC I molecules (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). This indicates that after nanoparticle stimulation, the functions associated with M1 macrophages gradually enhanced under the regulation of pathways such as NF-\u0026kappa;B and JAK-STAT.\u003c/p\u003e\n\u003cp\u003eTo deeper understand the mechanisms by which targeted nanoparticles reprogram tumor-associated macrophages (TAMs), real-time PCR was employed to analyze the changes in typical mRNA levels. The results showed a significant increase in M1 markers, including CD86, i-NOS, TNF-\u0026alpha;, and IL12 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a marked decrease in M2 markers, such as CD206, ARG1, and VEGF (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), following 24 hours of co-culture (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). TAMs' insufficient secretion of IL-12 undermines their antigen-presenting ability, thus inhibiting T cell proliferation and cytotoxic activity, promoting the recruitment of Tregs and Th2 cells, and facilitating tumor immune evasion. In colorectal cancer, hypoxic regions enhance Arg1 expression via HIF-1/2, shaping the immunosuppressive effects of TAMs, attracting additional TAMs, and forming a feedback loop that drives tumor growth[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, M2-type TAMs not only suppress immune cytotoxic functions but also promote angiogenesis via VEGF secretion, thereby accelerating tumor metastasis. Targeted nanoparticles significantly reversed this trend (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). The iron overload provided by nanoparticles triggers the Fenton reaction, generating substantial ROS, and promoting apoptosis in tumor cells[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, carbon dots themselves also exhibit a significant re-polarization effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), with broad applications in tracking and re-polarizing tumor-associated macrophages. The role of carbon dots as immunoadjuvants is a significant discovery, potentially due to their superior upconversion photoluminescence, excellent photo-induced electron transfer, and unique electron pool properties that inhibit electron-hole recombination. Compared to other family members, they have a natural advantage in biocompatibility, with significantly lower biotoxicity[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHypoxia, a hallmark of malignant tumors, participates in inducing epithelial metabolism and TAM infiltration due to the preferential accumulation of macrophages in hypoxic tumor regions and the retention of relatively immature cell types[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. The observed elevation in TNF-\u0026alpha; and accumulation of ROS suggest that nanoparticles may regulate M1/M2 expression through a mechanism involving ROS-NF-\u0026kappa;B, inducing macrophage polarization toward an anti-tumor phenotype (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Apoptotic tumor cells further induce M1 polarization, creating a feedback loop that produces substantial tumor necrosis factor-alpha (TNF-\u0026alpha;)[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, the increase of STAT1 and the decrease of STAT6 indicate that nanoparticles reprogram TAMs by changing the balance of STAT1/STAT6, and M1 macrophages release ROS through the ROS-mediated MAPK pathway to form positive feedback to enhance repolarization(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC)[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe ERK protein expression diagram also shows that targeted nanoparticles can inhibit the endoplasmic reticulum stress pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). PERK is a key driver of M2 polarization in macrophages, and its high expression promotes immune cell infiltration in the tumor microenvironment, leading to poor prognosis[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. PERK expression significantly correlates with TAM infiltration levels, particularly with macrophage mannose receptor 1 (MRC1, also known as CD206). Activation of the ERK/MAPK signaling pathway promotes M2 macrophage polarization, and blocking this pathway can inhibit M2 TAM production and enhance T-cell anti-tumor activity[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. This could be related to nanoparticle-induced iron overload, which inhibits the PERK signaling pathway, disrupts endoplasmic reticulum-mitochondria interactions, destabilizes mitochondrial homeostasis, prevents M2 TAMs from generating sufficient energy, and ultimately suppresses their immunosuppressive function. Although we observed the repolarization effects and related gene pathway changes induced by nanoparticles, the precise molecular mechanisms underlying repolarization require further in-depth validation.\u003c/p\u003e\n\u003cp\u003eThe repolarization effects of targeted nanoparticles on TAMs were also verified in tumor-bearing mice. Immunofluorescence analysis revealed an increase in M1 macrophages and a decrease in M2 macrophages in tumor sections (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD-E). Flow cytometry indicated a higher proportion of M1 macrophages in the nanoparticle-treated group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF). These findings demonstrate that targeted nanoparticles can overcome the complex tumor microenvironment, exhibit precise TAM-targeting capabilities, and effectively achieve macrophage repolarization.\u003c/p\u003e\n\u003cp\u003eStructurally, Mannose-DSPE-PEG@\u003cstrong\u003eFe\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e/CDs, with their acidic properties and hydroxyl-rich carbon dots, ensure effective retention within lysosomes. This structural composition facilitates the Fenton reaction while preventing the neutralization of ROS by glutathione in the cytoplasm[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The synergy among iron, hydroxyl-rich carbon dots, and mannose ensures balanced ROS production, thereby avoiding the tumor-promoting proliferation effects of excessive ROS while maintaining levels that enhance tumor immunity. These structural attributes enable the nanoparticles to precisely modulate the oxidative environment of tumors.\u003c/p\u003e\n\u003cp\u003eBiologically, carbon dots promote the polarization of macrophages towards the M1 phenotype by governing the STAT1/STAT6 balance and releasing ROS through the MAPK pathway. This creates a positive feedback mechanism. Additionally, the carbon dots inhibit the endoplasmic reticulum stress and ERK/MAPK signaling pathways, effectively blocking the immunosuppressive roles of M2-type macrophages and augmenting the anti-tumor activity of T cells. Collectively, these biological processes allow carbon dots to re-polarize TAMs within the complex tumor microenvironment, achieving precise recognition and effective re-polarization.\u003c/p\u003e\n\u003cp\u003eIn vitro repolarization: qRT-PCR was used to detect the levels of cytokines in control, M1 type, M2 type, M2\u0026thinsp;+\u0026thinsp;Man@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs, M2\u0026thinsp;+\u0026thinsp;Man@CDs, M2\u0026thinsp;+\u0026thinsp;Man@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e groups (A); flow cytometry ROS levels in control, M1, M2, M2\u0026thinsp;+\u0026thinsp;Man@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs groups were detected by instrument (B); M1, M2, M2\u0026thinsp;+\u0026thinsp;Man@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs and M2\u0026thinsp;+\u0026thinsp;Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs groups were detected by western blot STAT1, STAT6, p-Erk levels (C);repolarization in vivo: 5*105 MC38 cells were planted in the armpit of each mouse, and randomly divided into three groups. Seven days after the tumor was planted, PBS, 500ug/ml Mannose-DSPE-PEG@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CDs and 500ug were injected into the tail vein respectively /ml DSPE-PEG- @Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-CDs 200ul, administered once every other day and measuring the size of the tumor, after two weeks of treatment, the tumor was taken to prepare a single cell suspension, flow cytometric analysis of each group M1 type (F4/80\u0026thinsp;+\u0026thinsp;CD86+) (D) and M2 type (F4/80\u0026thinsp;+\u0026thinsp;CD206+) cell mass (E); immunofluorescence analysis of various M1 type (CD86+) macrophages, M2 type (CD206+) macrophages (F). *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully synthesized a photomagnetic bifunctional nanocomposite material, Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs, capable of converting red light to near-infrared light while possessing MRI imaging capabilities. The material exhibits high sensitivity to the acidic tumor microenvironment (TME), allowing for the targeted imaging of tumor-associated macrophages (TAMs) in colorectal cancer. This nanocomposite facilitates the repolarization of TAMs through the JAK/STAT and ERK/MAPK signaling pathways, significantly enhancing their antigen-presenting ability and thereby improving the tumor immune microenvironment. Surprisingly, the synthesized carbon dots themselves were found to possess the ability to induce changes in TAM phenotypes, offering a novel perspective for developing more effective immunotherapeutic strategies. In summary, the nanocomposite developed in this study provides a powerful platform for the personalized assessment and treatment of colorectal cancer, paving the way for improved tumor immune microenvironments and advancing the field of precision medicine.\u003c/p\u003e \u003cp\u003eThe limitations of this study include the fact that, while we have initially confirmed the ability of the synthesized carbon dots to track and induce TAM polarization, the specific mechanisms involved require further investigation. Additionally, the relationship between the photomagnetic bimodal imaging results obtained using this nanomaterial and their corresponding clinical benefits needs to be explored further.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eTumor-Associated Macrophage-Related Immune Infiltration and Survival Analysis in TCGA Colorectal Cancer\u003c/strong\u003e \u003cp\u003eUtilizing clinical and gene expression data from colorectal cancer patients in The Cancer Genome Atlas (TCGA), we leveraged the R package `immunedeconv` to evaluate the abundance and distribution of tumor-associated macrophages (TAMs) in tumor samples compared to adjacent normal tissue. To determine the statistical significance of differences in TAM abundance between these tissues, we employed comparative statistical methods such as the Mann-Whitney U test and t-test.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eIn the survival analysis phase, we constructed survival objects and applied the Cox proportional hazards model to examine the influence of TAM abundance on patient survival, accounting for covariates that could impact survival, including age and tumor stage. We assessed the model\u0026rsquo;s concordance and utilized statistical tests such as the likelihood ratio test, Wald test, and Score test to evaluate covariate significance. Additionally, we generated survival curves to visually represent the effect of TAM abundance on patient survival outcomes.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMaterials\u003c/strong\u003e \u003cp\u003eIR-813P-toluenesulfonate was purchased from Macklin (China), and polyethylene glycol (PEG 400) was purchased from Aladdin (China). DSPE-PEG2000 and DSPE-PEG-Man were purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. \u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e was synthesized by the College of Chemistry, Jilin University. Lipopolysaccharide (LPS) and recombinant mouse interleukin 4 (IL-4) were provided by PeproTech, USA. Dulbecco's Modified Eagle's Medium (DMEM) was purchased from HyClone (GE Healthcare, USA), and fetal bovine serum (FBS) was purchased from Bioind (Israel). Fluorescence-labeled anti-CD11b, F4/80, IL12, iNOS, TNFa, CD206, IL10, ARG1 were purchased from Biolegend (USA). Q-PCR primers were purchased from Sangon Biotechnology (China).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePreparation of carbon dots (CDs)\u003c/strong\u003e \u003cp\u003eTake 50mg of IR-813 p-toluenesulfonate, dissolve in 10ml of polyethylene glycol (PEG 400), mix well, add the mixed solution into a 100ml reaction kettle, and heat at 180\u0026deg;C, 8 hours; then filter with 0.22um filter membrane, 3500 dialysis membrane for dialysis, then centrifugate 10000rpm*15 minutes, discard the supernatant, and obtain solid carbon dots after drying in the fume hood.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of photo magnetic dual-modal targeting nanoprobe (\u003c/b\u003eMannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs\u003cb\u003e)\u003c/b\u003e: 5mgCDs, 5mg\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e, and 40mg Mannose-DSPE-PEG were dissolved in 5ml toluene, mix under ultrasound for 20 minutes, then add the mixture into a flask filled with 50ml of deionized water, the temperature is 60\u0026deg;C, the magnetic stirring speed is 400 rpm, via solvent vaporing and self-assembly, to obtain a solid substance. Dissolve in 10ml of PBS, filter through a 0.44um filter, and store in the dark for future use.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCharacterization of Mannose-DSPE-PEG@Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/CDs\u003c/strong\u003e \u003cp\u003eThe absorption spectra of the nanoparticles were measured with a Shimadzu 3600 UV-Vis-NIR spectrophotometer. The morphology of the nanoparticles was observed with a JEM-2100F transmission electron microscope (TEM) produced by JEOL. The particle size and potential of nanoparticles were measured by the dynamic light scattering (DSL) method. 3.0T nuclear magnetic resonance (MRI) was used to image and measure the nanoparticle's T2 and T2 mapping with different concentrations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBiosafety Assessment of Nanoparticles\u003c/strong\u003e \u003cp\u003eRAW264.7 and MC38 cells were planted in 96-well plates at 8*10\u003csup\u003e3\u003c/sup\u003e per well, and after 24 hours, DSPE-PEG with concentrations of 0, 10, 50, 100, 200, 500, 1000, and 2500 ug/ml Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs nanoparticles treated cells. At 12h, 24h, and 48h respectively, the activity of the cells was measured using the CCK8 kit (Solarbio, China). Untreated cells served as a control group and were considered 100% viable. C57BL/6 mice were injected with 200ul of Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs nanoparticles at a concentration of 1000ug/ml and 2500ug/ml respectively in the tail vein. After 24 hours, the mice's hearts, livers, spleens, lungs, and kidneys were taken for HE stains, compared with the normal control group.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMacrophage induction typing and marker detection\u003c/strong\u003e \u003cp\u003eSpread RAW264.7 cells in a 6-well plate, about 15*104 cells per well. After 24 hours, add M1 type (DMEM\u0026thinsp;+\u0026thinsp;10% fetal bovine serum\u0026thinsp;+\u0026thinsp;1% double antibody\u0026thinsp;+\u0026thinsp;100ng/ml LPS) and M2 type (DMEM\u0026thinsp;+\u0026thinsp;10% fetal bovine serum\u0026thinsp;+\u0026thinsp;1% double antibody\u0026thinsp;+\u0026thinsp;20ng/ml IL4) induction solution, 2ml per well, placed in a CO2 incubator and incubated for 24h. After 24 hours, the marker levels of M1 and M2 macrophages were detected by flow cytometry (Beckman, USA), among which the M1 type detected i-NOS, IL-12, and TNFα; the M2 type detected IL-10, CD206, and ARG1. RAW264.7 macrophages were placed in the lower chamber of the Transwell chamber, and MC38 cells were placed in the upper chamber, and co-cultured for 24 hours to create a tumor-associated macrophage model. Flow cytometry was used to detect the M2 cytokines IL-10, CD206, and ARG1 levels.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Targeting Ability of Nanoparticles in vitro\u003c/b\u003e: RAW264.7 was induced into M2 macrophages and divided into 3 groups. Group 1: adding 500ug/ml targeted nanoparticles (Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs); Group 2: adding the same amount of non-targeted nanoparticles (DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs); Group 3: Block with 500ug/ml mannose for 3 h, wash away, and then add 500ug/ml targeted nanoparticles (Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs); Laser confocal imaging and flow cytometry was performed to detect the uptake of nanoparticles.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Targeting Ability of Nanoparticles in vivo\u003c/b\u003e: C57BL/6 tumor-bearing mice were injected with 200ul of targeted and non-targeted nanoparticles through the tail vein respectively, at a concentration of 1000ug/ml. After 4 hours, the C57 mouse colorectal cancer subcutaneous xenograft tumor model and liver metastases model were treated respectively. Magnetic resonance imaging (Siemens, 3.0T) and in vitro fluorescence imaging (IVIS Spectrum). MRI scanning parameters are as follows: T2WI: TR 3120.0ms, TE 111ms, FOV read 70mm, Voxel size:0.3*0.3*1.0mm, Slice thickness 1.0mm; T2mapping༚ TR 613.0ms, TE1 16.1, TE2 32.2, TE3 48.3, TE4 64.4, TE5 80.5, FOV read 110mm, Voxel size:0.6*0.6*2.5mm, Slice thickness 2.5mm. Coronal imaging of mice. In vitro fluorescence imaging wavelength selection 710-840nm. Then the materials were collected, and fluorescence imaging was performed on the mouse tumor tissue, heart, liver, spleen, lung, and kidney.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMacrophage Reolarization Analysis and Transcriptome Analysis\u003c/strong\u003e \u003cp\u003eThe nanoparticles co-incubated with TAMs were subjected to transcriptome sequencing at different time points (0 hours, 6 hours, 12 hours, 24 hours, and 36 hours). Bioinformatics analysis was conducted using R (version 4.3.3). Differential gene expression analysis, clustering analysis, and enrichment analysis were performed on the sequencing results from each time point. Time-related heatmaps were generated to analyze the temporal dynamic changes in TAMs following nanoparticle stimulation. Total RNA extracted from cells was isolated using TRIzol reagent (Invitrogen life technologies, USA), and RNA concentration and purity were measured using Infinite 200 PRO. CDNA was synthesized from 1 ug of total RNA using the PrimeScript\u0026trade; RT reagent kit with gDNA Eraser(Takara, Japan). The primers were designed and synthesized by Sangon (China) and the sequence was listed in \u003cb\u003eSupplement Table\u0026nbsp;1\u003c/b\u003e. Quantitative RT-PCR was carried out in an 8-strip tube using FastStart Universal SYBR Green Master(Roche, Switzerland) on a real-time fluorescence quantitative PCR instrument༈Eppendorf, Germany༉. All results were processed by the double-delta method (2\u0026thinsp;\u0026minus;\u0026thinsp;\u003cb\u003eΔ\u003c/b\u003eCt).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDetection of ROS levels before and after transformation of tumor-associated macrophages (TAMs)\u003c/strong\u003e \u003cp\u003eRAW264.7 cells were divided into a control group, M1 type group, M2 type group, and M2 type group\u0026thinsp;+\u0026thinsp;500ug/ml targeted nanoparticle repolarization group for 24h after treatment, and then the reactive oxygen species detection kit (Solarbio, China ) and flow cytometry to detect the ROS levels in the above groups.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAssessment repolarization with Western blot assay\u003c/strong\u003e \u003cp\u003eCells were collected, washed twice with PBS, lysed in RIPA containing 1% PMSF for 30 min on ice, and centrifuged at 15,000 g for 15 min at 4\u0026deg;C. The resulting supernatant was collected, and its protein concentration was determined using the BCA assay (Beyotime, China). Equal amounts of total and prestained protein ladders were separated by SDS-PAGE and transferred to nitrocellulose membranes on 0.22 \u0026micro;m nitrocellulose membranes. After blocking with 5% skim milk for 3 hours, the membrane was incubated with the primary antibody overnight at 4\u0026deg;C. Then, the membrane was washed and subsequently incubated with secondary immunoglobulin G-horseradish peroxidase, bound, and then the proteins were visualized using an infrared luminescence imaging system (LI-COR Odyssey, USA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAntitumor effects of tumor-associated macrophages (TAMs) before and after transformation in vitro\u003c/strong\u003e \u003cp\u003eRaw264.7 macrophages were induced into M1 and M2 types respectively, and targeting and non-targeting nanoparticles were added to M2 macrophages to induce their repolarization for 24 hours, and then the complete medium was replaced for culture After 6 hours, the conditioned medium was obtained, and the MC38 colorectal cancer cells were incubated for 24 hours, and then the viability of the MC38 tumor cells was detected with a CCK8 kit.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRepolarization ability of nanoparticles and antitumor effects in vivo\u003c/strong\u003e \u003cp\u003eC57BL/6 mice at 6\u0026ndash;8 weeks were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and each mouse was subcutaneously inoculated with 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e MC38 tumor cells in the armpit to create a mouse subcutaneous xenograft tumor model, and each mouse was injected with 2 \u0026times;10\u003csup\u003e5\u003c/sup\u003e MC38 tumor cells were used to create a liver metastases model. The treatment started 7 days after the tumor was planted, randomly divided into 3 groups, 5 in each group, and injected PBS, 1000ug/ml \u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs and Man-\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs 200ul into the tail vein respectively and administered once every other day. Mice body weight was treated for 14 days. The calculation formula of tumor volume is tumor volume\u0026thinsp;=\u0026thinsp;length \u0026times; width \u003csup\u003e2\u003c/sup\u003e/2. At the end of the experiment, all the mice were euthanized, and the tumors of each group were taken, weighed, and photographed. Then the tumor was fixed with 4% paraformaldehyde, and the sections were stained with hematoxylin-eosin (H\u0026amp;E), Tunel staining, immunofluorescence detection of CTL cells, and flow cytometry detection of M1 types (CD11b+, F4/80+, CD86+) and M2 Type (CD11b+, F4/80+, CD206+) macrophage proportion. All animal experiments were performed in accordance with the guidelines approved by the Animal Care and Use Committee of Changchun Weishi Testing Technology Service Co., Ltd.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll data subjected to statistical analyses were obtained from at least three parallel experiments, and the results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). The statistical analysis was performed by Student's t-tests for two groups, and one way ANOVA for multiple groups using GraphPad Prism version 9.00 for MACOS (GraphPad Software, La Jolla, CA, USA). A p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorresponding Authors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBai Yang\u0026thinsp;\u0026minus;\u0026thinsp;State Key Laboratory of Supramolecular Structureand Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun 130012, P.R. China; Joint Laboratory of Opto-Functional Theranosticsin Medicine and Chemistry, The First Hospital of JilinUniversity, Changchun 130021, P. R. China; orcid.org/0000-0002-3873-075X; Email: [email protected]\u003c/p\u003e \u003cp\u003eButian Zhang\u0026thinsp;\u0026minus;\u0026thinsp;Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130021, P. R.China;Email: [email protected]\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003e All animal experimental procedures were performed according to protocols approved by the Animal Care and Use Committee of Changchun Weishi Testing Technology Service Co., Ltd.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor details\u003c/h2\u003e \u003cp\u003eYingying Miao\u0026thinsp;\u0026minus;\u0026thinsp;Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130021, P. R.China;\u003c/p\u003e \u003cp\u003eXiaoyu Li\u0026thinsp;\u0026minus;\u0026thinsp;State Key Laboratory of Supramolecular Structure and Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun130012, P. R. China;\u003c/p\u003e \u003cp\u003eQingsen Zeng\u0026thinsp;\u0026minus;\u0026thinsp;Postdoctoral Researcher at Seoul National University;State Key Laboratory of Supramolecular Structure and Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun130012, P. R. China;\u003c/p\u003e \u003cp\u003eKai Zhang\u0026thinsp;\u0026minus;\u0026thinsp;State Key Laboratory of Supramolecular Structure and Materials, Center for Supramolecular Chemical Biology, College of Chemistry, Jilin University, Changchun130012, P. R. China;\u003c/p\u003e \u003cp\u003eLin Liu\u0026thinsp;\u0026minus;\u0026thinsp;Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130021, P. R.China;\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China(82001880) and Natural Science Foundation of Jilin Province (YDZJ202201ZYTS538).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYB and ZBT proposed and designed the experiments. ZQS , LXY and KZ contributed to and was responsible for designing chemical experiments, performing chemical tests and analyses (Figures 1), and revision the article about chemical section. LL supports the imaging resources. MYY contributed to and was responsible for performing biological tests , ZBT contributed to and was responsible for data analyses (Figures 2-7), and ZBT compolish writing this paper.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors are grateful for the financial support from the National Natural Science Foundation of China (82001880) and Natural Science Foundation of Jilin Province (YDZJ202201ZYTS538).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSinicrope FA. Increasing Incidence of Early-Onset Colorectal Cancer. 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Cancer Discov. 2021;11:714\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePBX3 promotes. migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway - PubMed [Internet]. [cited 2022 Nov 15].\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tumor-associate macrophages, repolarization, carbon dots, Fe3O4, theranostic","lastPublishedDoi":"10.21203/rs.3.rs-5657271/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5657271/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) immunotherapy has shown remarkable effects in only a small subset of patients, largely due to the influence of tumor-associated macrophages (TAMs), which play a key role in shaping the tumor immune microenvironment. In vivo dynamic imaging of TAMs is critical for personalized immunotherapy, as it enables the identification of patients likely to benefit from treatment and allows for real-time monitoring of therapeutic efficacy. Additionally, reprogramming the polarization state of TAMs from the pro-tumoral M2 phenotype to the anti-tumoral M1 phenotype represents a promising strategy to enhance immunotherapy outcomes. To address these challenges, we developed mannose-coated carbon dots/\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e superparticles (Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs) specifically designed to target TAMs. These superparticles combine the NMR-enhanced imaging capabilities of \u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e with the red fluorescence properties of carbon dots, enabling precise and non-invasive TAM imaging. Furthermore, Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs effectively reprogram TAMs from the M2 to M1 phenotype via the JAK/STAT and ERK/MAPK pathways, thereby reshaping the tumor immune microenvironment and exerting potent anti-tumor effects. In summary, this study demonstrates the potential of Mannose-DSPE-PEG@\u003cb\u003eFe\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e/CDs as a theranostic nanoplatform for the monitoring and modulation of TAMs, offering a novel strategy for improving immunotherapy outcomes in colorectal cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Exploring the theranostic potential of carbon dots/Fe3 O4 superparticles for tumor-associated macrophage targeting and repolarization in colorectal cancer therapy via JAK/STAT and ERK/MAPK pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-24 16:47:36","doi":"10.21203/rs.3.rs-5657271/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e5619a3-e803-46c3-8834-476168ea5ef8","owner":[],"postedDate":"December 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-24T16:47:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-24 16:47:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5657271","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5657271","identity":"rs-5657271","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}