**1. Introduction**

Cancer, i.e., the abnormal growth and proliferation of cells, is one of the leading causes of mortality and morbidity worldwide. According to the World Health Organization (WHO), cancer was responsible for 9.9 million deaths in 2018 [1], and the number of cases is anticipated to increase by about 70% over the next two decades. Each cancer type involves a specific treatment procedure that embraces one or more modalities, namely surgery (to remove the tumor), radiotherapy, or chemotherapy. Despite the high cure rates observed when cancer is detected early and if appropriated treatment is provided, most of the presently employed therapies, particularly conventional chemotherapy, are associated with severe side e ffects, including hair loss, nausea and vomiting, pain, anemia, fertility issues, edema, among many others [2], that strongly a ffect the patient's quality of life. In recent decades, the design of more e ffective alternatives that allows a targeting action, with almost no impact on healthy tissues or organs, has received considerable attention [3]. Of these, nanosystems that combine a therapeutic effect and imaging properties, or that promote intertwined diagnosis and therapy, the so-called nanotheranostics, has opened new avenues for cancer-conquering [4–6].

Metal nanoparticles (NPs) are a class of nanomaterials with a panoply of biomedical and therapeutic applications, including cancer therapy and imaging of tumors [7,8]. In particular, gold (Au) nanoparticles owing to their unique physical and optical properties have attracted enormous interest in this realm during the past decades [9–12]. The colloidal stability of NPs in biological environments and their interactions with cells is strongly influenced by their surface properties, and thus distinct coating strategies of NPs, using small molecules, polymers, or lipids, have been described [13]. Moreover, this methodology could also target the reduction of the toxicity of NPs and improvement of biological functionalities, typically associated with the use of active biomolecules [13]. The synthesis of AuNPs using biomacromolecules, including alginate, starch, cellulose, chitosan, gelatin, collagen and fucoidan, among others, as reducing and stabilizing agents is a well-documented strategy to achieve these goals [14] and, at the same time, overcome the environmental e ffects of the conventional methodologies that commonly involve the use of harmful reducing agents.

Fucoidan is a natural occurring sulfated marine polysaccharide extracted from brown seaweeds that presents various biological properties, including antiangiogenic, antitumoral, and anti-inflammatory properties [15,16], and because of that has been widely investigated for the development of nanomaterials for biomedical applications [17]. However, only a few number of papers reported the combination of fucoidan and AuNPs for cancer treatment. One of the first studies in this topic involved the synthesis of AuNPs (44 nm average size) using sodium borohydride as the reducing agen<sup>t</sup> and a fucoidan-mimetic glycopolymer as the capping agen<sup>t</sup> [18]. The obtained AuNPs displayed excellent colloidal stability and selective cytotoxicity to human colon cancer cell line (HCT116). Afterward, Manivasagan et al. [19] produced biocompatible AuNPs (82 nm average size) by using naturally occurring fucoidan as the reducing and capping agent, avoiding the use of harmful reducing agents. This study also demonstrated the applicability of these NPs as a carrier for doxorubicin (DOX) and photoacoustic imaging of breast cancer tumors. In a follow-up study, this research team explored similar fucoidan-AuNPs for dual-chemo-photothermal treatment of eye tumors [20]. In another study, size-controlled fucoidan-AuNPs (15-80 nm) were produced by varying the concentration of fucoidan during the synthesis step [21]. These NPs showed anticancer e ffect against human oral squamous cell carcinoma (HSC3), and its surface modification and conjugation with DOX also improved their e ffect.

These studies clearly demonstrate the prospective of the partnership between AuNPs and fucoidan in cancer treatment. However, to achieve high antitumoral activities, viz., less than 20% cell viability, the conjugation with other chemotherapeutics (DOX), or the use of high dosages (up to 50 μg mL−<sup>1</sup> of NPs) was typically required. Moreover, some methodologies are somewhat time-consuming and laborious, e.g., up to 2 h for the synthesis of the AuNPs and 24 h for the conjugation with DOX. Additionally, the number of investigated tumor cell lines is limited, and the imaging properties of fucoidan-AuNPs have been only marginally explored. Thus, some essential traits still need to be tackled envisioning their scale-up production and broad application, viz., the establishment of fast and straightforward procedures for the synthesis of fucoidan-Au nanosystems with controllable size and morphology and improved antitumoral activity against di fferent tumor cell lines.

As a developing heating tool, microwave irradiation has been shown to considerably reduce the reaction times and provide a uniform bulk heating that allows the synthesis of various nanomaterials, including AuNPs [22,23], with defined structures and narrow size distributions. However, to the best of our knowledge, this methodology has never been sightseen as a simple, time-saving approach to fabricate fucoidan-AuNPs for application in cancer therapy.

In this line, in the present study, we report for the first time a one-minute microwave-assisted synthesis of fucoidan-coated AuNPs with controllable size and high antitumoral activity. This is a pioneering achievement with respect to previous methodologies to produce Au-fucoidan NPs. The fucoidan-AuNPs were synthesized, using a fucoidan-enriched fraction extracted from *F. vesiculosus*, and characterized in terms of structure, colloidal stability, antitumoral activity against di fferent cell lines (MNT-1, HepG2, and MG-63), and cellular uptake by flow cytometry and dark field imaging of NPs. The antitumoral activity of Au-fucoidan NPs against these cell lines and their imaging properties by dark-field is also reported here for the first time.

#### **2. Materials and Methods**
