**4. Conclusions**

The one-minute microwave-assisted synthesis of fucoidan-coated gold nanoparticles (AuNPs) with controllable, high stability, and antitumoral activity is the first and foremost contribution of the present study, where fucoidan-AuNPs were synthesized by using a fucoidan-enriched fraction extracted from *F. vesiculosus*, as simultaneous reducing and capping agent. The resulting monodispersed and spherical fucoidan-AuNPs present very small diameters, namely 5.8 ± 0.9 nm, 10.4 ± 1.4 nm, and 13.4 ± 3.0 nm that depend on the initial concentrations of fucoidan: 0.5%, 0.1%, and 0.05% (w/v), respectively, together with excellent colloidal stability in acidic and basic solutions, ultra-purified water and culture media. The second innovative input of this study lies in the antitumoral activity of the fucoidan-AuNPs against three human tumor cell lines, namely MNT-1 (pigmented human melanoma), HepG2 (human hepatocyte carcinoma), and MG-63 (human osteosarcoma) cells. Moreover, the use of flow cytometry in combination with dark field imaging confirmed the cellular uptake of fucoidan-AuNPs by the MG-63 cell line, which demonstrates the antitumoral activity of these nanomaterials, and thus their potential for cancer therapy.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1996-1944/13/5/1076/ s1. Figure S1: Digital photographs of fucoidan-AuNPs colloid over time in the di fferent media. Figure S2: Hyperspectral images of MG-63 cells incubated ( **A**) without and (**B**) with fucoidan-AuNPs, measured for the same sample and illumination conditions of Figure 8C,D of the manuscript. The color scale is based on the intensity of the spectra of each pixel at 460 nm. ( **C**) Spectra measured in collection areas of 20 × 20 pixels of (A) (red line) and (B) (blue line). Figure S3: Spectra measured in several single pixels of the hyperspectral image measured for the same sample and illumination conditions of the Figure 8A of the manuscript, before ( **A**) and after ( **C**) correction. (**B**) Spectrum measured in an area of 20 × 20 pixels, of the Figure 8A of the manuscript used to perform the correction. The inset in (A) shows the normalized spectra for better comparison. Figure S4: Spectra measured in several single pixels of the hyperspectral image shown in Figure 8B of the manuscript, ( **A**) before and ( **C**) after correction using the spectrum shown in Figure S3B. The normalized spectra before correction are shown in (**B**) for better comparison.

**Author Contributions:** Conceptualization, R.J.B.P., S.A.O.S., and C.S.R.F.; investigation, R.J.B.P., D.B., C.V., A.M.P.B., A.C.M., and F.C.; resources, R.A.S.F., H.O., M.H.A., S.A.O.S., and C.S.R.F.; writing—original draft preparation, C.S.R.F.; writing—review and editing, R.J.B.P., D.B., C.V., A.M.P.B., R.A.S.F., A.C.M., F.C., H.O., M.H.A., S.A.O.S., and C.S.R.F.; supervision, C.S.R.F.; funding acquisition, R.A.S.F., H.O., S.A.O.S., and C.S.R.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, and CESAM, UID/AMB/50017/2019, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. The research contract of R.J.B. Pinto is funded by national funds (OE), through FCT–Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. The research contract of S.A.O. Santos is funded by the project AgroForWealth (CENTRO-01-0145-FEDER-000001). FCT is also acknowledged for the research contracts under Scientific Employment Stimulus to C. Vilela (CEECIND/00263/2018), H. Oliveira (CEECIND/04050/2017) and C.S.R. Freire (CEECIND/00464/2017). A.M.P. Botas acknowledges SusPhotoSolutions (CENTRO-01-0145-FEDER-000005).

**Conflicts of Interest:** The authors declare no conflict of interest.
