Functional roles of magnetic nanoparticles for the identification of metastatic lymph nodes in cancer patients

Staging lymph nodes (LN) is crucial in diagnosing and treating cancer metastasis. Biotechnologies for the specific localization of metastatic lymph nodes (MLNs) have attracted significant attention to efficiently define tumor metastases. Bioimaging modalities, particularly magnetic nanoparticles (MNPs) such as iron oxide nanoparticles, have emerged as promising tools in cancer bioimaging, with great potential for use in the preoperative and intraoperative tracking of MLNs. As radiation-free magnetic resonance imaging (MRI) probes, MNPs can serve as alternative MRI contrast agents, offering improved accuracy and biological safety for nodal staging in cancer patients. Although MNPs’ application is still in its initial stages, exploring their underlying mechanisms can enhance the sensitivity and multifunctionality of lymph node mapping. This review focuses on the feasibility and current application status of MNPs for imaging metastatic nodules in preclinical and clinical development. Furthermore, exploring novel and promising MNP-based strategies with controllable characteristics could lead to a more precise treatment of metastatic cancer patients.


Introduction
Cancer cells could escape the primary site through complex biological mechanisms and localize to other body parts, forming metastatic tumors [1].Carcinoma metastasis is responsible for cancer development, prognosis, and therapeutic response [2][3][4].Furthermore, metastasis represents one of the leading causes of cancer-related morbidity and mortality [5,6].Therefore, exploring the underlying biochemical events involved in regulating the metastatic process would be essential and meaningful to identify potential targets for the inhibition and prevention of metastasis.In particular, the definition of metastatic events in the early stages can influence the clinical management of cancer patients.
It is well known that lymph node metastasis (LNM) is an important dissemination event during metastasis.Among these, the sentinel lymph nodes (SLN), the closest lymph nodes (LN) that drain the tumor area, can serve as a host of metastatic cancer cells [7].SLN mapping has recently been used to detect early lymphatic metastases in cancer patients [8].Traditionally, the main assessment methods for LNM are several radiomicbased technologies, including magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography/computed tomography (PET/ CT).However, these detection approaches have some significant disadvantages and limitations.MRI cannot accurately distinguish fibrosis and tumor infiltration, ultimately triggering false positive results for cancer diagnosis and stage [9].CT scans may lead to the potential harm of exposure to ionizing radiation and the risk of overdiagnosis in patients with an incidental finding [10].The challenges for the use of PET/CT have been raised about not only the costs and time consumption but also the misinterpretation in some noncancerous tissues with high radiotracer uptake [11].
It should be noted that although clinical imaging can efficiently determine tumor metastasis, these imaging-based techniques are challenging to detect micrometastases, which occur early in the progression of malignancies [12].When the findings of the imaging modalities reveal the presence of suspicious metastatic nodules, pathological tests are necessary to clarify their origin and location.An SLN biopsy should be performed for patients without nodules to confirm whether cancer cells have spread to the nearby LNs (also called regional lymph nodes, RLNs) or other organs [13].However, pathological examination is unavoidably associated with surgical procedures that could carry the risk of harm to patients.Specifically, even after a sentinel node biopsy, some patients have been reported to have lymphedema, a pathological condition characterized by a localized accumulation of excessive interstitial fluids [14,15].Therefore, exploring novel noninvasive and effective strategies to better detect LNM risk in cancer patients is always of great concern.
Magnetic nanoparticles (MNPs) have been utilized to visualize the lymphatic spread of cancer cells, which is of great significance for cancer staging and treatment.The use of MNPs for human disease diagnosis was first introduced in the 1990s, serving as an agent for liver imaging, and revealed good biological safety [16,17].After that, since MNPs can be detected and manipulated by remote magnetic fields, their clinical application for LNM will substantially impact cancer bioimaging and early diagnosis [18,19].To date, iron oxide nanoparticle (IONP)based formulations, one of the most advanced MNPs, have been used mainly as MRI contrast agents (CAs) for LN mapping [20,21].FDA-approved ferumoxytol, a carboxymethyl dextran-based IONP, helps detect locoregional LN in prostate cancer patients [22].With the help of 1.5 Tesla MRI scanners, intravenous injection of ferrimagnetic or ferromagnetic IONP-labeled macrophages can specifically target LN and its metastases in a nude mouse model (BALB/c) [23,24].Zhou et al. constructed a dye-conjugated near-infrared (NIR) IONP, IONP-NIR830, and found that IONP-NIR830 administration could display strong T2*-weighted magnetic resonance and NIR images for preoperative and intraoperative tracking of SLN [25].Although the application of MNPs is still in its early stages, exploring the underlying mechanisms of MNPs has excellent potential for improving the sensitivity and multifunctionality of LNM mapping.In particular, developing novel MNP-based probes for the precise assessment of LN status will significantly influence the decision-making on operative approaches and therapeutic strategies in cancer patients.
Here, we provide an updated and comprehensive review of MNPs, especially IONPs, in molecular imaging of metastatic lymph nodes (MLNs) in preclinical (Table 1) and clinical (Table 2) development.Furthermore, detailed biological characteristics and application status of different MNPs were discussed, highlighting their essential contribution to diagnosing LNM.

Superparamagnetic iron oxide nanoparticles
The first IONPs, which have a size of 40-150 nm, are termed standard superparamagnetic iron oxide nanoparticles (SPIONs) [26].The superparamagnetic phenomenon was first described in the 1950s by Néel [27], who observed the magnetic properties of small ferromagnetic or ferrimagnetic particles at the nanoscale in the external magnetic field.Typically, SPION has a core of iron oxide (Fe 3 O 4 or Fe 2 O 3 ) coated with optional aggregation preventing monomer or polymer to enhance stability.These coated materials can also be functionalized with targeting molecules or ligands to target the corresponding cells or tissues [28].Vu-Quang et al. investigated the characteristics of mannan-coated SPIONs, which could be targeted to deliver immune cells by receptor-mediated endocytosis.They discovered that normal macrophages and dendritic cells could preferentially phagocytose mannan-SPION very early after intravenous injection [29].
In subsequent studies, the unique properties, such as magnetic responsiveness and high relaxivity, make SPI-ONs useful for their function as attractive MRI CAs in biomedical applications [30].SPION-based MRI offers promising programs for early MLN detection while avoiding the shortcomings of radioactive labels in conventional imaging techniques [31][32][33].The combination of SPION-MRI and handheld magnetometers provides a non-radioactive strategy for the preoperative [34] and intraoperative [35] locations of SLN.Preoperative injection of SPIONs in breast cancer improves detection rate and nodal yields [36].The reliability of SLN detection by SPION-MRI incorporated with the SentiMag magnetometer has also been verified by Minamiya et al. in non-small cell lung cancer [37].As shown in Fig. 1, the underlying mechanism is that IONPs could only be phagocytosed by reticuloendothelial cells in normal lymphoid tissues, resulting in the darkening of normal LNs in T2* weighted images.Inversely, due to the lack of reticuloendothelial cells, the tumor tissues or LNs invaded by tumor cells absorb the nanoparticles that show a high signal in the lymphoid tissues on MRI [38,39].Consequently, a recent study by Johnson et al. [40] confirmed that SPIONs are mainly distributed around metastatic nodules after subcutaneous injection, but are not seen within areas containing metastases.The systematic review and meta-analysis summarizing approximately 20 existing studies demonstrated that the dose, injection site, and nodal metastasis burden had no significant effect on the detection rate of LNM by SPION-based CAs.Despite this, injection of SPIONs 24 h before surgery could improve the detection rate [36].
To approve the potential use of SPIONs for magnetic resonance lymphoscintigraphy, Winter et al. [41] reported a study on the clinical validation of SPIONs for SLN identification in penile cancers.In this case, inguinal SLN could be reliably visualized by MRI after peritumoral injection of SPIONs.For patients with submucosal esophageal cancer, endoscopic injection of SPIONs into the peritumor tissues is a proper method to estimate whether to perform a three-field lymphadenectomy with neck LN dissection [42].More importantly, these patients did not present significant side effects attributable to SPION injection.Based on the statements of Motomura et al. [43], SPION-enhanced MRI imaging showed usefulness in confirming metastatic sentinel nodes located by CT scanning.The accuracy, sensitivity, and specificity of SPION-guided imaging for detecting metastases in SLNs were 89.2%, 84.0%, and 90.9%, respectively.Subsequently, this group evaluated the correlation between SPION-enhanced MRI lymphography and pathologic size to detect metastatic SLNs [44].They showed that for metastatic nodes with pathologic > 2 mm, the high signal intensity indicated by SPION-MRI imaging showed a good correlation with their pathological size.Given the feasible and safe properties, SPION-based magnetic procedures could propose entirely radiation-free methods for the preoperative and intraoperative location of SLNs in cancer patients [45].Therefore, SPION-guided MRI methods are helpful for the visualization of LN mapping, guiding clinical diagnosis and treatment decisions.
It is worth mentioning here that the combination use of SPIONs and specifically targeted SPION conjugates (e.g., cell surface molecule-targeted tags) is available to depict macro-and early micrometastases within the lymphatic system.High levels of tenascin-C could be observed in cervical cancer tissues with node metastasis but not in non-metastatic patients and normal cervix tissues [46].Synthesized SPION-anti-tenascin-C, a magnetic probe oriented specifically to tenascin-C, possessed the non-invasive ability to capture specific MLNs with high expression of tenascin-C in preoperative cervical cancer patients [47].Wang et al. developed a novel SPIOs@A-T imaging probe to target breast cancer and ATP responsiveness to the tumor microenvironment.Mice injected with SPIOs@A-T presented high-intensity fluorescence molecular imaging and magnetic particle imaging with high sensitivity and specificity in the MLN group [48].Recently, Sienna+, a dark brown aqueous suspension of SPIONs, has been utilized in conjunction with the handheld magnetometer SentiMag to non-radioactively locate SLNs.This magnetic nanoparticle tracer, Sienna+/Senti-Mag, could quantitatively and accurately confirm nodal staging, providing a feasible and safe alternative to SLN Successfully targeting E-selectin, the important factor for cancer metastasis [82] SPION-ICG Swine gallbladder cancer model The SPION-ICG dual tracer is feasible and sensitive for the SLNs detection [89] mapping in colorectal cancer patients [13].The result of the consistency examination showed good concordance between the Sienna+/SentiMag probe and the standard radioisotope-guided dynamic SLN mapping [49].In addition, the Sienna+/SentiMag technique enhances clinical outcomes and experience for cancer patients by reducing patient contact time, avoiding radiation exposure, and operating cost-effectively [50,51].
These emerging imaging modalities have extended the applications of SPIONs in molecular oncological diagnostics.However, the questions regarding the pharmacokinetics and safety profiles of SPIONs and conjugates must be well addressed before the approval of these nanoparticles for clinical application in cancer patients.

Ultrasmall superparamagnetic iron oxide
The smaller SPIONs (median diameter less than 50 nm) are called ultrasmall superparamagnetic iron oxides (USPIOs).The smaller particle size of USPIOs leads to their specific lymphotropic behavior [52].After administration, USPIO particles could be phagocytosed by nodal macrophages and then directly drained to normal nodes.After localization to LNs, USPIOs generate a susceptibility artifact on gradient echo MRI sequences, resulting in loss of T2*-related signal [53,54].In contrast, high signal intensity is observed on T2*-weighted images in metastatic LNs lacking macrophages [55].According to this guideline for USPIO-enhanced MRI, LN without blackening or hyperintense in surrounding tissues could be diagnosed as metastatic nodules.The LNs were considered non-metastatic if they displayed low signal intensity [56].Consequently, a recent study by Shen et al. [57] evaluated the changes in the T2*-weighted image after USPIO enhancement.The results did not show a reduced signal intensity for metastatic nodules.
The preferential application of USPIO as MRI CAs in cancer patients will help improve the ability of MRI to detect LN and their metastases early and contribute to better preoperative staging.Compared to pathological morphological findings, using MRI + USPIO results  in greater diagnostic specificity to identify LN status [53].USPIO-based MRI could also provide clues for the surgical removal of suspicious LNs not included during dissection [58].The maximum lymphographic effect could be observed after 24 h of USPIO administration [59,60].In general, the application of USPIO-enhanced MRI improves the detection rates of LNM, particularly for nodules smaller than 5 mm [61].Similar studies revealed that, in patients with bladder or prostate cancer, USPIO-enhanced MRI allows for definite metastases in normal-shaped or smaller LNs [62][63][64].For LN staging in patients with oesophageal cancer, administration with USPIO-enhanced MRI could accurately identify distant and regional nodules, such as celiac and mediastinal LN [65].Furthermore, compared to routine breath-hold MRI, the MRI + USPIO method is more feasible and superior for LNM visualization in esophageal cancer patients [66].A recent in vivo experiment suggested no negative adverse effects in dog oral cancer models treated with USPIO lymphotropic nanoparticles [67].Despite their ability to diagnose LNM, USPIOs have not yet been approved for clinical application [68].
Several USPIO-based technologies, such as ferumoxtran-10, are being evaluated clinically and preclinically [26,69].Ferumoxtran-10, composed of USPIO, is a promising lymphotropic CA that could diagnose more positive metastatic LNs than traditional imaging techniques, such as PET/CT [70,71].Using ferumoxtran-10-enhanced MRI, Tatsumi et al. reported high accuracy in detecting LNM in 194 nodes from 17 gastric cancer patients, with a sensitivity of 100%, specificity of 92.6%, positive predictive value of 85.5%, negative predictive value of 100%, and overall predictive accuracy of 94.8% [72].Tokuhara et al. demonstrated that the positive predictive value and accuracy for nodal staging using ferumoxtran-10 enhanced MRI in 519 nodes of 31 gastric cancer patients were 90.1% and 97.1%, respectively, significantly higher than conventional criteria, which have a positive predictive value and accuracy of 76.8% and 93.3% [73].In addition to some tolerable side effects, such as allergic skin changes or back pain, no other side effects were observed in patients receiving ferumoxtran-10 [74,75].However, current reports are insufficient to support the functional roles of ferumoxtran-10 in all tumor types.
Instead, several novel USPIO-contained CAs, such as P904 [76] and PJY10 [77], and CS015 [78], have been well-developed to show better performance than ferumoxtran-10 in LNM MR lymphography in oncological fields.CS015, a polyacrylic acid (PAA) coated USPIO, enhances the long-term stability of USPIO in various solutions [79].After subcutaneous and intravenous injections, the CS015 particles could be effectively swallowed by macrophages residing in LN, even at a very low working concentration [78].Birkhäuser et al. evaluated the recognizable effects of USPIO-MRI combined with diffusion-weighted MRI (USPIO-DW-MRI) for metastatic LNs in patients with bladder and prostate cancer.The combined USPIO-DW-MRI demonstrated the ability to rapidly detect small metastatic LN preoperatively [80].The median read time of USPIO-DW-MRI in each patient was about 13 min, significantly shorter than that of the traditional method [81].Another strategy has been reported by the Liu group [82], where USPIO-PEG-sLeX molecular probes were constructed based on the conjugation between poly(ethylene glycol) (PEG) coated USPIO and Sialyl Lewis X (sLeX), the high-affinity ligand for the selectin family.The following in vivo models showed that USPIO-PEG-sLeX successfully targeted E-selectin associated with cancer metastasis and enabled early detection of metastasis in nasopharyngeal carcinoma.These findings would help provide promising platforms for the design of novel USPIO-based nanoparticles for LN mapping.For further clarification, studies should be conducted to determine nanoparticle imaging properties, diagnostic roles, and therapeutic values in human cancers.

Other emerging magnetic nanoparticles
Newly designed MNPs with unique physicochemical and pharmacodynamic characteristics have been synthesized and demonstrated excellent diagnostic potential for lymph node mapping (Fig. 2; Table 3).For example, IONPs coated with folic acid (FA-IONPs) have been designed for LN imaging.Intracellular distribution analysis revealed the specific uptake of FA-IONPs by metastatic LNs that express the prostate-specific membrane antigen (PSMA).This folate transporter leads to the targeted detection of LNM by MRI in prostate cancer [83].The combination of MagTrace and MagSeed, two non-radioactive magnetic techniques, showed the effectiveness and safety of the location of SLN in patients with breast cancer [84].Recently, a clinical trial [NCT05569707, https://clinicaltrials.gov/] has been registered to evaluate the clinical application of handheld magnetic probes, Magtrace, in combination with Sen-tiMag and differential magnetometry, in patients with melanoma.As alternative MRI agents, these emerging nanoparticles could be attractive for accurately identifying metastatic LN, providing a surgical roadmap for resectioning suspicious nodules [85].Indeed, the visualization of LNs marked with nanoparticles would provide the foundation for future research to evaluate the underlying effects of MNPs for cancer research and treatment.
Due to the complement of different imaging modalities, nano agents with multimodal imaging capability exhibit high sensitivity and resolution for nodal staging (Fig. 3).The use of dual-modal nanoparticles for fluorescent imaging and MRI of metastatic LNs is gradually attracting attention.Podoplanin and the lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) are explicitly expressed in lymphatic endothelial cells (LECs), serving as particular lymphoid biomarkers [86].Wu et al. have integrated podoplanin/LYVE-1 antibodies on the surface of Fe 3 O 4 -α-ketoglutarate chitosan nanocarriers to form the Fe 3 O 4 @KCTS-LEC double antibody nanoprobe [87].Biochemical and functional assays suggested that these double antibody probes could select fluorescence/magnetic resonance dual imaging for LECs, allowing a precise diagnosis of lymphatic metastases.After tail vein injection, hematoxylin-eosin staining did not show prominent necrotic or inflammatory characteristics in the primary tissues of the mouse xenograft model of colon cancer, demonstrating its high biosafety.Fu et al. [88] prepared manganese porphyrin/indocyanine green (ICG) nanoparticles, F127-ICG/Mn, which efficiently integrated the F127-stabilized ICG and tetra(4carboxyphenyl)porphyrin-Mn(III) (TCPP(Mn)).With the help of F127-ICG/Mn, the visualization of green-stained SLNs was evident, which could be seen with the naked eye.More importantly, micro-and macro-SLN metastasis could be successfully distinguished by fluorescence molecular imaging and magnetic particle imaging.During laparoscopic surgery, these dual-tracer SPION-ICG consisting of MNPs and ICG are more sensitive to  detecting SLNs than MNPs or ICG alone [89].Similarly, the dual-tracer strategy, ICG, and magnetic FerroTrace mixture could be used to identify reliable SLNs specifically and sensitively [90].Furthermore, the clinical trial [NCT05092750] on FerroTrace's SLN mapping has been registered in the ClinicalTrials.govdatabase (https://clinicaltrials.gov/), although no patients have been recruited.Kuwahata's group [91] adopted a different approach to studying LN status.The combination of alternating current (AC) and direct current (DC) provides dual-tracer electromagnetic fields [92], thereby leading to more accurate imaging of SLNs.Consequently, Kuwahata et al. [91] developed a laparoscopic magnetic probe, an AC-DC probe.Gastric SLNs labeled with the AC-DC probe were detected within AC and DC magnetization.In laparoscopic surgery, SLN detection rates were not affected by the shine-through effect at the injection site.
In addition, Gadolinium (Gd)-based CAs (Gd-CAs) appear as one of the most advanced developments in the image-guided mapping of LNM during cancers.IONP conjugated with gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) was helpful in optimally estimating the metastatic potential of tumor-draining LN cells in the B16-F10 footpad melanoma model [93].Administration of Gd-DTPA decreases the threshold size for nodule detection [94], indicating high sensitivity.Meanwhile, subcutaneous injection of generation-6 Gd dendrimer-based CAs (Gd-G6) could effectively characterize micro and macrometastasis within the lymphatic system [95].After labeling with NIR Cy5.5fluorophore, Gd-G6 provided real-time intraoperative guidance for surgical SLN resection under MR and NIR imaging conditions [96].Another two signal-enhanced and highresolution Gd-based MRI agents, Gd 2 O 3 @PCD [97] and Gd 3+ •DOTA⊂SNPs [98] were successfully developed for the biological application of in vivo imaging.These injections of these two agents exhibited a crucial capacity for staging and diagnosing LNs and metastatic spread in tumor mice.With the rapid development of technologies, researchers have developed several multimodal Gd-based contrast imaging agents for detecting micro-metastatic LNs, such as GdIOP@ZDS nanoparticles [99].Yang et al. [100] synthesized the Gd-loaded, polyethyleneimineentrapped gold nano agents (Gd-Au PENPs) with attractive dual-modal CT/magnetic resonance lymphography properties.Functioning as an indirect CT/MRI CA, the Gd-Au PENPs possessed the ability to locate lingual SLN in rabbit VX2 tongue cancer models.Dong et al. [101] established a naked eye and NIR fluorescent nanoprobe inkMNP-PEG-H2 by connecting cuttlefish melanin with NIR fluorescent dye H2.When combined with Gd ions, inkMNP-PEG-H2 showed a high signal-to-noise ratio, accumulation, and sensitivity in lymphatic imaging.The embedding of the NIR dye ZW800 in nanoencapsulation labeled with Gd 3+ and radionuclide 64 Cu made it possible to facilitate optical, MRI, and PET NIR imaging simultaneously [102].These triple-modal MSN probes have several excellent features, such as long intracellular retention time and high stability, contributing to long-term multimodal imaging for metastatic SLNs.

Magnetic nanoparticle-guided imaging and therapeutic effects
Other projects were developed simultaneously for metastatic node imaging and cancer treatment (Fig. 3; Table 4).MNP-based molecular imaging probes provide excellent opportunities for early metastatic diagnosis and cancer treatment [103].Gold-shelled magnetic IONPs were coated with PEG to obtain stable and superior IONC@Au-PEG in physiological solutions [104].In the 4T1 tumor metastatic model, the IONC@Au-PEG nanocomposites could be transferred to SLNs via lymphatic pathways for imaging of cancer metastasis.Given that Au granules have an absorption peak in the NIR range, IONC@Au-PEG also functioned as a potential photothermal reagent possessing pronounced cytotoxic effects against primary tumors and SLNs.Through integrating magnetic Fe 3 O 4 , radioactive 99m Tc, and the NIR dye IR-1061, a research group from Soochow University successfully fabricated a new-style nanotheranostic agent, FIP-99m Tc [105].The FIP-99m Tc nano agent, including MRI, PET/CT, and NIR fluorescence imaging, displayed Meanwhile, GO-IONP-mediated photothermal therapy exhibited an excellent ability to photothermally ablate RLN and cancer cells [108].Applications of these nanotheranostic agents could provide promising strategies for the imaging and treatment of primary tumors and their metastatic nodules in general.In particular, the design of multimodality imaging-based tumor biomarker-targeted nanocomposites holds great promise, as it can offer enhanced specificity for cancer theranostics in clinical management.

Implications and future directions
As demonstrated by the reports highlighted in this review, the appearance of MNPs has already revolutionized our understanding of LN mapping.The development of MNPs, including IONPs, has proposed a promising functional platform that displays the capability to detect tumor metastasis early.As a helpful platform, these MNPs could also carry several different ligands for cancer imaging and treatment [109].The incorporation of multiple components into magnetic nanomaterials allows the delivery of cargo to manage disease progression sensitively.In addition, the targeted transport of drugs or probes to disease sites can potentially weaken adverse effects [110].The successful synthesis of a radio-labeled folate-mediated delivery system allowed for the integration of SPION and the chemotherapeutic drug docetaxel.This delivery system demonstrated remarkable distribution efficiency within folate receptor-positive tumor cells [111].A Fe 3 O 4 -based magnetocaloric nanocarrier based on nanoparticles was designed to carry 4-amino-2-pentylselenoquinazoline and exhibited high specificity and selectivity against various cancer cells [112].Kim et al. [113] constructed a thioketal-linked amphiphilic nanoassembly (MTS) to encapsulate hydrophobic manganese oxide (HMO).After intravenous injection, these MTS@ HMO nanoparticles could be effectively absorbed by tumor cells, consequently inducing significant hydrogen peroxide scavenging and improving radiotherapy-mediated tumor elimination.Thus, we proposed that these magnetic systems could be developed to elucidate the potential activities of anti-cancer agents.However, some critical issues should be carefully addressed before magnetic nanomaterials are approved as theranostic compounds for clinical application.
As a rule, MNPs have been considered innocuous and biocompatible methods for biological applications in vivo.During the medication period, MNPs are relatively safe and well tolerated.Apart from some mild or moderate events, such as headache, back pain, vasodilatation, and urticaria, no serious adverse effects have been observed after injection of ferumoxtran-10 at the diagnostic dose (2.6 mg Fe/kg body weight) [56].However, in rats injected with very high dose levels of ferumoxtran-10 (126-400 mg Fe/kg), Bourrinet et al. observed treatmentrelated toxic effects, such as breathing difficulties, swollen snout, darkening of the body areas, etc. [114].These unexpected risks to chronic health might be due to iron-initiated oxidative stress, which ultimately damages cellular macromolecules and organelles [115].In bronchial epithelial cells, treatment with Fe 3 O 4 nanoparticles could generate lipid peroxidation and oxidative damage by promoting intracellular glutathione consumption and decreasing glutathione-S-transferase activity [116].Under oxidative stress, iron-induced lipid peroxidation is closely related to the cellular production of reactive oxygen species (ROS) potentially harmful to living organisms [117].
Similarly, Gamal et al. found that MNPs could alter the activity of antioxidant enzymes and stimulate ROS production, ultimately leading to damage due to oxidative stress in the rat testes [118].However, coating MNPs with chemical modification might alleviate their toxic impacts.Cellular experimental evidence revealed the minor effects of PEG-modified SPIONs on the biological behaviors of human fibroblasts [119].Due to their prominent stability and negligible cytotoxicity, mannan-coated SPIONs have great potential for specific tracking of cancer cells in SLNs [29].Polycaprolactone/chitosan-covered iron oxide prolonged the survival time of tumor-bearing mice without any toxic responses [120].Therefore, these tests have indicated that the coating materials can effectively protect MNPs, enhancing their stability and demonstrating distinguished biocompatibility.Furthermore, the exploration and development of stabilized micelles open the possibility of IONPs as alternative MRI CAs for bioimaging [121].
Although MNP-based approaches have strong potential for the early diagnosis of MLN in cancer patients, their distribution in body fluids or cells has yet to be clarified.Surface properties have an important influence on the distribution, elimination, and bioavailability of MNPs [122].Among these, the surface charge of MNPs is a critical element in controlling the final intracellular distribution [123].The surface charge of the MNPs was mainly analyzed using the zeta potential [124].Commercial MNPs frequently possess an intrinsic negative charge [125].To explain this phenomenon, Al-Obaidy and colleagues [126] evaluated the physiochemical characteristics of SPIONs in vitro and in vivo.They found that the negative zeta-potential charge of SPIONs effectively prevented the formation of aggregates, resulting in suitable polydispersity ratios.IONPs with a surface charge of -31 mV exhibit good colloidal stability under physiological conditions [127].However, compared to negatively charged MNPs, cultured cells more easily internalized the positively charged nanoparticles.Using quantitative methods, Calatayud et al. [128] demonstrated that almost 100% of positive MNPs were absorbed by SH-SY5Y human neuroblastoma cell lines, while the uptake rates of negative MNPs remained less than 50%.Such differences might be due to electrostatic interactions between the negative charge of the cell outer membrane and the positive charge of the nanoparticles [129].Further investigations are required regarding the surface charge, which will become a pivotal issue in the biomedical fields of MNPs.

Conclusions
The discovery of cancer cell dissemination in SLN has established lymphatic vessels as one of the frequent pathways for cancer cell metastasis.Furthermore, the state of the lymph nodes is recognized as a crucial prognostic and therapeutic factor for cancer patients.Many studies have emphasized the biological significance of LN diagnosis in carcinogenesis and have highlighted the positive impact of LN resection in treating cancer patients with LN infiltration.As a result, there is an urgent need to explore useful technical methods for the preoperative and intraoperative confirmation of macro and micro-metastatic LNs.The emergence of nanomedicine technologies has introduced novel and promising nanoprobes, such as MNPs, for molecular imaging of metastatic LNs.

Fig. 1
Fig. 1 The application of MNPs-based strategies for MRI imaging of tumor lymphatic metastasis.The successful development of MNPs-based strategies in bioimaging recently made it possible to utilize MNPs as the MRI probes for MLNs tracking in cancer patients.After injection, MNPs could be preferentially phagocytosed by cells in normal lymphoid tissues, resulting in the darkening signal on T2*-weighted images.Inversely, high signal intensity on T2*-weighted images is observed for the metastatic LNs which do not take up the nanoparticles

Fig. 2
Fig. 2 Schematic illustrations indicating the different types of MNPs in LN mapping.These MNPs with different physicochemical and pharmacodynamic characteristics have been successfully synthesized, and displayed the excellent diagnostic potential for identification of metastatic LNs in cancer patients

Fig. 3
Fig. 3 The magnetic nanoagents with multifunctional capability exhibit high sensitivity for nodal staging and cancer treatment.With the rapid development of technologies, several multimodal MNPs-based contrast imaging agents have been developed for the LNs detection and cancer theranostics

Table 1
The pre-clinical evaluation of main magnetic nanoparticles in the molecular imaging of metastatic lymph nodes

Table 2
The clinical trials of main magnetic nanoparticles in the molecular imaging of metastatic lymph nodes

Table 3
Other emerging magnetic nanoparticles for imaging of metastatic lymph nodes

Table 4
Magnetic nanoparticles for the metastatic node imaging and cancer treatment