Different Biocompatibility and Radioprotective Activity of Squid Melanin Nanoparticles on Human Stromal Cells

Abstract

Squid ink melanin nanoparticles (NPs) have recently been demonstrated to have a number of bioactivities; however, their biocompatibility has been poorly investigated. In this study, we aimed to evaluate the effects of this NP on stromal cells, including human fibroblasts (hFBs), human umbilical vein endothelial cells (hUVECs), and human umbilical cord-derived mesenchymal stem cells (UCMSCs), and on the development of zebrafish embryos under normal X-ray irradiation conditions. The NPs showed high biocompatibility with low cytotoxicity, no cell senescence induction, and no effect on cell migration in hFBs or cell differentiation in UCMSCs. Nonetheless, this compound prevented cell movement in UCMSCs and significantly suppressed tube formation in hUVECs at a dose of 25 μg/mL. The NPs successfully penetrated the hUVECs but not the other two stromal cell types. The expression levels of functional genes involved in angiogenesis, apoptosis, antioxidant activity, and radiation sensitivity were altered in NPs subjected to hUVECs but were not affected in hFBs and UCMSCs. Melanin NPs significantly rescued cell viability and gene expression in irradiated hFBs and UCMSCs but not in hUVECs. In vivo treatments of zebrafish embryos showed that melanin NPs were nontoxic whether alone or under X-ray irradiation. These findings suggested that nanosized squid ink melanin had biocompatibility with selective stromal cells and was safe for early development.

Figure 1

Synthesis of melanin NPs from the extracted melanin of a squid ink. (A) Extracted melanin under SEM at a magnification of 30,000×. (B) Melanin NPs under SEM at a magnification of 50,000×. (C) FTIR spectra of melanin NPs. (D) Zeta potential of melanin NPs.

Figure 2

In vitro cytotoxicity of melanin on hFBs, and UCMSCs. (A) Images of cell culture in the presence of melanin powder at different concentrations. (B) Percentage of viable cells in the presence of melanin at concentrations ranging from 0 to 125 μg/mL after 24, 48, and 72 h of incubation.

Figure 3

In vitro biocompatibility of melanin NPs on hUVECs, hFBs, and UCMSCs. (A) Percentage of viable cells in the presence of melanin NPs at concentrations ranging from 0 to 125 μg/mL after 24, 48, and 72 h of incubation. (B) Morphology of cell nuclei in the presence of NPs at 50 μg/mL after 72h of incubation. (C) Senescence rates (%) in three cell types. (D) Effect of melanin NPs on cell senescence in hFBs, hUVECs, and UCMSCs. (E) Effect of melanin NPs on the cell differentiation of UCMSCs. Data are presented as the mean ± SD, n = 3, ***p < 0.001.

Figure 4

Effect of melanin NPs on the migration of hFBs, hUVECs, and UCMSCs. (A) Images of the wounded site on the monolayer culture of cells incubated with nanomelanin at different doses and times. (B) Quantitative wound closure rates in hFBs, hUVECs, and UCMSCs were recorded every 4 h up to 72 h of measurement. Data were repeated in triplicate and presented as the mean ± SD, n = 3. **p < 0.01 vs control. Scale bar: 100 μm.

Figure 5

Penetration of melanin NPs in hFBs, hUVECs, and UCMSCs. (A) Presence of melanin NPs in the cell population at a dose of 75 μg/mL after 72 h of incubation. (B) Uptake of melanin NPs in hUVECs at two doses of 25 and 75 μg/mL at 24, 48, and 72 h of incubation.

Figure 6

Effects of melanin NPs on the tube formation of hUVECs. (A) Tube formation in hUVECs in the presence of nanomelanin at two doses: 25 μg/mL and 50 μg/mL. (B) Quantification of tube formation in hUVECs. (C) Effect of nanomelanin on tube formation in hUVECs exposed to X-rays at two doses of 3 and 7 Gy, with or without nanomelanin at 25 μg/mL. Data are presented as the mean ± SD, n = 3. **p < 0.01; ***p < 0.001. Scale bar: 100 μm.

Figure 7

Effect of irradiation and nanomelanin on the expression of functional genes. (A) Expression of tested genes in hFBs under treatment conditions of nanomelanin and radiation. (B) Expression of tested genes in hUVECs under nanomelanin and radiation treatment conditions. (C) Expression of tested genes in UCMSCs under treatment conditions of nanomelanin and radiation. (D) Summarization of the effects of melanin NPs on the expression of functional genes. Data are presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.0001.

Figure 8

Protective effect of nanomelanin against irradiation on cell viability. (A) Changes in the morphology and density of cells associated with irradiation with or without nanomelanin. (B) Cell viability of hFBs, hUVECs, and UCMSCs was significantly recovered in the presence of nanomelanin at different irradiation doses. (C) Scavenging ROS of nanomelanin in the ROS assay. DCF: Dichlorofluorescein. Data are presented as the means ± SDs, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001. Scale bar: 100 μm.

Figure 9

Nanomelanin exposure was safe for zebrafish embryo development. (A) Visible morphological defect was observed only at a dose as high as 200 mg/L, with heart edema being the main phenotype. (B) Dose–response curves of lethal and morphological effects. (C) Nanomelanin at 25 mg/L and irradiation until 15 Gy did not affect zebrafish embryonic development.