**1. Introduction**

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide [1]. Cytotoxic drugs-based chemotherapy is the main treatment in cancer management, in addition to surgery, radiation, and biological therapies [2]. 5-Fluorouracil (5-FU) is widely used in CRC chemotherapy, as also other different drugs employed to interfere with cell replication by acting as nucleoside analogs and leading to S-phase arrest, or by damaging deoxyribonucleic acid (DNA) [3]. Unfortunately, owing to the short half-life (5–14 min), poor membrane permeability and rapid metabolism in the body, continuous administration of high doses of 5-FU is required to maintain the minimum therapeutic serum concentration; this is usually associated with numerous side effects and severe toxicity [4]. Nanostructured carriers have been proposed as an alternative therapeutic approach, since they can be properly surface-functionalized and consequently reach a specific target on pathological cells. Therefore, targeted drug delivery nanosystems can ensure the specific release of drugs in tumor areas, at lower and more efficient doses than conventional treatments, thus maximizing the bioavailability of anticancer agents and their uptake into the cancer tissue, while reducing side effects and drug distribution to healthy tissues. Drug-loaded nanovectors have also been demonstrated to be able to overcome the canonical drug-resistance pathways, e.g., membrane pumps, for cellular internalization that usually occurs when using small therapeutic molecules [5,6]. Furthermore, drug delivery nanocarriers can be protected by using pH-dependent polymer coatings or pH-dependent encapsulation systems, to prevent their degradation in the gastric tract [7,8].

Liposomes (LPs) are lipid-based nanostructures with a hydrophilic core and a lipophilic shell, where different types of drugs can be encapsulated [9,10]. The small size of LPs is fundamental to promote extravasation and accumulation phenomena in tumor sites. In the literature, several examples of liposomes have been proposed for CRC treatment, by intravenous, oral or rectal administration [11,12]. The coupling of the LPs surface with selected antibodies, as ligands able to recognize and bind specific plasma membrane proteins that are overexpressed in cancer tissues, allows immuno-LPs to be created, taking a further step forward in the development of efficient targeted drug delivery nanosystems [13,14].

The FZD proteins (FZDs) are the main cell surface receptors for the WNT family of ligands and include ten members (FZD1–FZD10). FZDs are involved in the regulation of several cell processes, which occur not only in normal development of different body systems, but also in various cancers. Indeed, numerous studies proved that FZDs play a crucial role in different cancer functions, thus promoting proliferation, migration, invasion, angiogenesis, and also chemoresistance. Furthermore, modulation of expression levels for specific FZD depending on up- or downregulation of the corresponding messenger RNA (mRNA) was observed in different cancers tissues [15–20]. Among the FZD proteins, FZD10 was suggested to be one of the most promising receptors for the development of targeted therapy of CRC. Indeed, a study by S. Nagayama et al. evaluated the immunohistochemical expression patterns of FZD-10 in tissue samples of patients affected by CRCs. Interestingly, their study indicated that FZD10 was expressed only in cancer cells and it was absent in adjacent normal cells [21,22]. Furthermore, we have reported a study performed on tissues of three different cancers, namely CRC, melanoma and gastric cancer, demonstrating a strong correlation between the expression levels of FZD10 and different tumor stages. In the colon, a significant increase of FZD10 expression in membrane and cytoplasm and, concomitantly, a significant reduction in nuclei expression were observed, passing from nondysplastic to malignant tissue [22,23]. Based on these premises, FZD10 can be effectively exploited as a novel candidate target with a good potential in the development of selective therapeutic approaches for CRC.

The aim of the study will be to create stealth LPs functionalized with FZD 10 antibody and loaded with 5-FU (anti-FZD10/5-FU/LPs), towards the development of efficacious nanostructured formulations for the selective delivery and sustained release of anticancer drug to CRC sites by exploiting active targeting. A deep in vitro investigation on their preclinical therapeutic efficacy in CRC treatment will be performed.
