Melanin-Based Nanoparticles for Lymph Node Tattooing: Experimental, Histopathological and Ultrastructural Study

Abstract

In breast cancer, Targeted Axillary Dissection (TAD) allows for the selective excision of the sentinel lymph node (SLN) during primary tumor surgery. TAD consists of the resection of labelled SLNs prior to neoadjuvant chemotherapy (NACT). Numerous clinical and preclinical studies have explored the use of carbon-based colloids for SLN tattooing prior to NACT. However, carbon vectors show varying degrees of inflammatory reactions and, in about one fifth of cases, carbon particles migrate via the lymphatic pathway to other nodes, causing the SLN to mismatch the tattooed node. To overcome these limitations, in this study, we explored the use of melanin as a staining endogenous pigment. We synthesized and characterized melanin-loaded polymeric nanoparticles (Mel-NPs) and used them to tattoo lymph nodes in pig animal models given the similarity in the size of the human and pig nodes. Mel-NPs tattooed lymph nodes showed high identification rates, reaching 83.3% positive identification 16 weeks after tattooing. We did not observe any reduction in the identification as time increased, implying that the colloid is stable in the lymph node tissue. In addition, we performed histological and ultrastructural studies to characterize the biological behavior of the tag. We observed foreign-body-like granulomatous inflammatory responses associated with Mel-NPs, characterized by the formation of multinucleated giant cells. In addition, electron microscopy studies showed that uptake is mainly performed by macrophages, and that macrophages undergo cellular damage associated with particle uptake.

Keywords: breast cancer; lymph node; melanin; nanoparticles; surgery; tattoo.

Figure 1

Schematic representation of the synthesis of melanin-loaded PLGA nanoparticles by the water-in-oil-in-water (w/o/w) method.

Figure 2

Determination of the ease of identification and the location of the particles. (a) Ease of identification scale. (b) Examples of location of the nanoparticles.

Figure 3

Morphology of the NPs during synthesis optimization, SEM micrographs are depicted together with their respective diameter distribution histograms. (a) Optimization of PLGA concentration (5, 10, 15 and 20 mg/mL). (b) Optimization of melanin concentration (0.15, 0.3, 0.5 and 1 mg/mL) using PLGA at 10 mg/mL.

Figure 4

Characterization of the Mel-NPs. (a) Transmission electron micrographs of the particles, where melanin is clearly distinguished by its higher electron density. (b) Thermogravimetric curves. (c) UV–Vis absorbance spectrum of melanin, with a maximum of absorbance at 193 nm. (d) Curve of the release kinetics of the encapsulated melanin from the Mel-NPs in distilled water. (e) Cell viability in the human breast adenocarcinoma (MDA-MB-231), macrophages (J774), and fibroblasts (NHDF-Ad) cell lines. Cytotoxicity is determined by assigning 100% cell viability to untreated control cells.

Figure 5

Identification rate and ease of identification of Mel-NPs tattooed lymph nodes. (a) Photographs of lymph nodes tattooed with Mel-NPs vectors (20 mg/mL) at different times. (b) Identification rates of lymph nodes tattooed with Mel-NPs vectors (20 mg/mL) in the short- and long-term studies. (c) Ease of identification of lymph nodes tattooed with Mel-NPs vectors (20 mg/mL) in the short and long-term studies. (d) Identification rates of lymph nodes tattooed with Mel-NPs vectors using 20 and 10 mg/mL colloids in the long-term studies. (e) Ease of identification of lymph nodes tattooed with Mel-NPs vectors using 20 and 10 mg/mL colloids in the long-term studies.

Figure 6

Histopathological findings of tattooed lymph node of Mel-NPs, where Mel-NPs appear brown and are easily distinguished from H&E staining. (a) The distribution of Mel-NPs shows a quite homogeneous morphology characterized by small granules. A foreign body reaction associated with Mel-NPs was observed, characterized by the presence of multinucleated giant cells (arrows). NP-loaded macrophages are also depicted. (b) Foci of necrosis associated with a foreign body reaction were observed in the ‘short-term studies’.

Figure 7

Location of Mel-NPs (10 and 20 mg/mL) in the adjacent tissue, the pericapsular region, and the inner lymph node region.

Figure 8

Ultrastructural findings in the lymph nodes tattooed with Mel-NPs at 20 mg/mL. (a) Activated macrophages with numerous lamellipodia and filopodia for the phagocytosis of Mel-NPs. (b) Macrophage with Mel-NPs in its cytoplasm. (c) Detail of Mel-NPs within the phagosome of the macrophage in the ‘short-term studies’. (d) Detail of Mel-NPs within the phagosome in the ‘long-term studies’, where the particles are more electron-dense and smaller in size. (e) A macrophage lysed by the action of the Mel-NPs. As observed, large amounts of non-encapsulated and potentially crystallized melanin appear that break the membranes and lead to cell death.