Gold nanoparticles (AuNPs) are considered valuable nanomaterials for the design of radiolabeled nanoprobes for single-photon emission computed tomography (SPECT) imaging. Radiolabeled and functionalized AuNPs could improve lymphatic mapping by enhancing the radioactive signaling of individual particles in the sentinel node.In this study, an alternative method for functionalizing commercial AuNps with mannose is described. The chemical derivatization and biofunctionalization of AuNPs were performed with lipoic acid and mannose, respectively.Several levels of mannose were tested; the thiolate hydrazinonicotinamide-glycine-glycine-cysteine (HYNIC) molecule was also used for 99m Tc radiolabeling. Physicochemical characterization of this system includes U-V spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, and transmission electron microscopy. The most stable nanoprobe, in terms of the aggregation, radiolabeling efficiency, and purity, was tested in a sentinel lymph node model in a rat by microSPECT/computed tomography (CT) imaging. The SPECT images revealed that 99m Tc-radiolabeled AuNPs functionalized with mannose can track and accumulate in lymph nodes in a similar way to the commercial 99m Tc-Sulfur colloid, commonly used in clinical practice for sentinel lymph node detection. These promising results support the idea that 99m Tc-AuNPs-mannose could be used as a SPECT contrast agent for lymphatic mapping.Functionalized AuNPs are ideal contrast agents for hybrid imaging techniques, for instance, single-photon emission computed tomography (SPECT) with computed tomography (CT) [3]. Radiolabeled AuNPs have been widely explored with SPECT imaging due to its high sensitivity, unlimited tissue penetration, and clinic translational capability. For example, 125 I, 111 In, 131 I, and 99m Tc radionuclides have been attached to functionalized AuNPs [4][5][6], while the dual deposition of radionuclides (e.g., 125 I and 111 In) has been used to prepare multimodal probes for SPECT bioimaging [7]. In particular, SPECT/CT mapping has demonstrated a high potential to improve preoperative sentinel lymph node (SLN) localization and the reduction of false negatives compared to either CT or ultrasound scans [8,9]. The SLN denotes the first node in the lymphatic chain draining a primary tumor and provides critical diagnostic and prognostic information for patients, since it can help to identify those with signs of metastasis. In fact, there is a growing interest in the development of new and improved SPECT contrast agents for SLN detection [10].New contrast agents should improve SLN imaging by enhancing the radioactive signaling of individual particles in the tracking of the lymphatic net and/or increase the radiotracer delivery and retention in the sentinel node [10]. Functionalized nanoparticles are relevant in SLN imaging and targeting not only because these particles can be synthesized at well-defined sizes, but also because different radionuclides can be attached to their surface, which is prepared w...