*2.1. Materials*

*Fucus vesiculosus* was collected (January 2014) in Mindelo beach (41◦1838'N, 8◦,4342'W), Portugal. The biomass was washed with water to remove salts, epiphytes, and/or microorganisms and dried at 25 ◦C until reaching a total moisture content of 12–14%. Algae samples were transformed into flakes (1–2 mm) with a knife mill (Retsch SM100, Haan, Germany), packed and stored in airtight bags at the ALGAplus warehouse. The milled algae samples were then washed with a solvent mixture (1 g per 20 mL) of chloroform and methanol (2:1 v/v) under stirring for 20 min, centrifuged at 2500 rpm during 20 min, and dried at 40 ◦C in a vacuum drying oven.

Gold(III) chloride trihydrate (≥99.9% trace metals basis), dimethyl sulfoxide (DMSO), paraformaldehyde, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate buffer saline (PBS, pH 7.4), Dulbeccos modified Eagles medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin and amphotericin B were supplied by Gibco® (Life Technologies, Grand Island, NY, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%) was purchased from Sigma-Aldrich, and <sup>4</sup>,6-diamidino-2-phenylindole (DAPI)-containing Vectashield mounting medium was acquired from Vector Labs. Ultra-purified water (Type 1, 18.2 MΩ·cm at 25 ◦C) was obtained by a Simplicity® Water Purification System (Merck, Darmstadt, Germany). Human osteosarcoma cell line MG-63 was a kind gift by INEB, University of Porto, Portugal. The HepG2 cell line, a liver hepatocellular carcinoma cell line, was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) and supplied by Sigma-Aldrich. MNT-1 cells were kindly provided by Doctor Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

#### *2.2. Microwave-Assisted Extraction (MAE) of Fucoidan from F. Vesiculosus*

The MAE of fucoidan-rich fraction from *F. vesiculosus* followed the methodology described by Rodriguez-Jasso et al. [24]. About 0.4 g of macroalgae sample was suspended in water, with a solid-liquid ratio of 1:25 (w/v). The extraction was performed in a Monowave 300 (Anton Paar, Graz, Austria) equipment, at 172 °C, for 1 min. The samples were cooled on ice and then centrifuged at 4000 rpm during 5 min). The aqueous extract was mixed with a 1% (w/v) CaCl2 aqueous solution, in a solid-liquid ratio of 1:1 (v/v), and maintained overnight at 4 °C, to precipitate alginate. This was separated by filtration, and ethanol was added to the filtrate (1:2, v/v) and maintained at 4 °C for 8 h. The fucoidan-rich fraction (MWF) was obtained after centrifugation (4000 rpm, 5 min) and dried at room temperature.

#### *2.3. Microwave-Assisted Synthesis of Fucoidan-AuNPs*

The microwave (MW)-assisted synthesis of fucoidan-AuNPs was carried out on a Monowave 300 equipment (Anton Paar, firmware version 2.0). Total of 145 μL of HAuCl4.3H2O solution (17.2 mM) was added to microwave vials with 5 mL of the three distinct concentrations of the fucoidan-rich fraction (0.5, 0.1, and 0.05% w/v). The mixtures were heated at 120 °C for 1 min. After the reaction, the obtained fucoidan-AuNPs were centrifuged for 1 h at 15,000 rpm and 4 °C, sonicated and washed three times with ultra-purified water, and finally stored at 4 °C until usage.

#### *2.4. Structural Characterization of the Fucoidan-Enriched Fraction and Fucoidan-AuNPs*

The sulfate content of the fucoidan-enriched fraction was determined by elemental analysis. The fucoidan-rich fraction was grounded with a mortar and analyzed (about 2–3 mg) in a Leco TruSpec 630-200-200 CHNS elemental analyzer (LECO Corporation, St. Joseph, MI, USA), in order to assess the carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) contents. The sulfate content of the sample was

calculated by the following equation: sulfate content (%) = 3.22 × S (%), where S (%) is the S content, as proposed by several authors [25,26].

Total sugars were determined by the phenol-sulfuric acid method, following the methodology described by DuBois et al. [27] where D(+)-glucose was used as standard. An aqueous solution of the fucoidan-rich fraction (0.05% w/v) was prepared and diluted as necessary. The UV absorbance measurements were performed in a Shimadzu UV-1800 spectrophotometer (Shimadzu Corp., Kyoto, Japan) at λ = 490 nm. Triplicate measurements were carried out.

Optical spectra of the fucoidan-AuNPs were recorded by a Thermo Scientific Evolution 220 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) using 100 scans min−<sup>1</sup> with a bandwidth of 2 nm and an integration time of 0.3 s.

The Fourier transform infrared (FTIR) spectra of fucoidan-enriched fraction extracted from *F. vesiculosus* and fucoidan-Au colloidal in the form of KBr pellet were collected by a Mattson 7000 spectrometer using 256 scans at a resolution of 4 cm<sup>−</sup><sup>1</sup> and with a signal gain of 1.

Transmission electron microscopy (TEM) images were obtained by a Hitachi H-9000 (Hitachi High-Technologies Corporation, Tokyo, Japan) operating at 300 kV. Scanning transmission electron microscopy (STEM) images were acquired by a field-emission gun (FEG) SEM Hitachi SU70 microscope operated at 15 kV. Samples for microscopy analysis were prepared by placing a washed colloid drop directly onto a carbon-coated copper grid and then allowing the solvent to evaporate. The average diameter of the NPs was determined by measuring over 100 NPs for each STEM image with the Fiji image processing software.

#### *2.5. Colloidal Stability of Fucoidan-AuNPs*

The colloidal stability of the fucoidan-AuNPs was evaluated in five di fferent mediums, namely ultra-purified water, HCl (0.01 M, pH 2.1), NaOH (0.01 M, pH 12.0), PBS (pH 7.4), and DMEM. Typically, 100 μL of the fucoidan-AuNPs 0.1% (w/v) was added to the vials already with 2.9 mL of the distinct mediums. The colloidal suspensions were placed under mechanical stirring during 48 h at room temperature, and the UV-Vis spectra at specific times (0, 6, 24, and 48 h) were recorded. All assays were performed in triplicate. After 48 h, the NPs in each medium were centrifuged for 15 min at 15,000 rpm (4 °C), sonicated and washed three times with ultra-purified water and analyzed by STEM as previously described.
