Cancer remains one of the most significant and worrying causes of death in humans. Several biochemical and physical factors make the growth of the neovasculature, besides accelerated, irregular, increasing the permeability and retention of antineoplastic drugs (Enhanced Permeability and Retention - EPR effect) [[1], [2], [3]]. The type of tumor vasculature and its permeability are not well characterized in the literature, although largely correlated [4]. It is considered a limit for successful drug applications with diameters up to 200 nm, depending on the type of tumor [5,6]. Another difficulty in treating cancer is the absence of a drug with selectivity. In contrast, chemotherapeutic and anti-cancer agents also usually affect healthy cells and accumulate in important organs, causing side effects. In this sense, biomolecule vectorization becomes a promising strategy for the effective and selective delivery of drugs to the target, thus enhancing its action [3,7].

Regarding receptor-target molecules, other factors should be considered before applications: the expression and internalization potential of the receptor, the desired targeting outcome, the specific characteristics of the tumor itself, and the choice of drug for treatment. Receptor expression can be correlated with its incidence on the surface of target cells, preferably avoiding a high degree of heterogeneity in antigen expression [8]. Considering the uptake of these targetable molecules, in some cases, compounds with a particular affinity for their receptors may undergo internalization during or after the binding process with their target receptor [8,9]. This result may be broadly advantageous for vehicular or drug delivery systems in which the delivery or internalization of the active drug is highly desirable. The type of binder is one of the most important factors to consider when it comes to the application [[8], [9], [10]]. There are binders that, besides being cheaper and easier to work with, present at significant levels in the human organism: folate (FA) [11,12], biotin (BT) [[13], [14], [15]], and spermine (SN) [16,17]. Although there may be competitive sites with molecules, using these binders eliminates the researcher's concern regarding the compounds' toxicity and the human body's acceptance of the developed drug [13].

FA, e.g., is a very promising molecule for this type of strategy, and for this reason, many studies involving this molecule have been reported [18,19]. It is a vitamin that belongs to the B complex, essential for the biosynthesis of nucleotide bases. Its deficiency has been associated with an increased risk of cancer (in adults) and gestational problems such as poor embryo development. Furthermore, FA is consumed in high amounts by proliferating cells, such as embryonic and tumor cells, thus, FA receptors are often overexpressed in many types of cancers [18,20,21].

The BT, also known as vitamin B7, is an example of an essential micronutrient for cell function and growth. Similar to FA, BT promotes tumor-targeting properties and is often used as suitable tags in targeted anti-tumor drug delivery systems [13,22]. For this reason, rapidly dividing cells (cancer cells) require high demand for those types of vitamins. Thus, several tumor cells lines express large amounts of specific BT and FA receptors, which facilitate their entrance into cancer cells by endocytosis [22]. Accordingly, several research groups have tried biotin-conjugates and folate-conjugates as cancer cell-specific drug delivery systems [23,24]. In addition, the literature reported the use of BT in mixed micelles, which consist of Pluronic® F127­linked to BT, as well as BT-PEGylated poly(amidoamine) dendrimer. These formulations have demonstrated active targeting capabilities and a promising avenue for exploring the therapeutic potential of drugs against cancer cell lines [25,26].

SN is another interesting compound with similar characteristics but does not belong to the class of vitamins B. This polyamine is a natural antioxidant and anti-inflammatory agent. Its anti-inflammatory activities are perhaps understood as an apparent or synergistic effect of an anti-tumor action [27,28]. Some polyamines are essential for cell growth, mainly irregularly growing cells [29]. SN can decrease local vascular permeability and alter DNA replication/transcription by interacting, thus interfering with protein synthesis, especially kinase activity [30,31]. Additionally, some tumor lines show uptake of polyamine systems, increasing the interest in creating a system in conjunction with this class of compounds as an anticancer agent [27,28].

The search for a suitable carrier system for stabilizing anticancer drugs with the maintenance of their properties desirable for the application that can perform effective delivery without metabolic and biocompatible damages lead us to the recently liposomal vesicles of the L-α-1,2-Dipalmitoyl-sn-3-Glycerol-Phosphatidylcholine (DPPC) coated with the copolymer F127 Pluronic®, which was used to carry the photosensitizing drug hypericin (HY) for photodynamic therapy applications [32,33]. Unfortunately, although very promising in several aspects, this formulation system still has some deficiencies in its configuration, such as the lack of specificity for tumor cells, a problem that should be solved using some binders such as FA, BT, and SN.

Thus, in order to further improve the therapeutic action of HY and increase the selectivity for tumor cells, this drug delivery system had another improvement with the synthetic addiction of the biomolecules FA, BT, and SN as binders in the Pluronic® part, and obtained a new and unpublished model of nanoformulations: the LF127-FA, LF127-BT, and LF127-SN nanoplatforms. Furthermore, all these systems were used to stabilize and carry the photosensitive drug HY. Thus, in this work, we demonstrated the superior therapeutic and theranostic potential of this HY biofunctionalized systems by in vitro assays in B16-F10 (melanoma), Caco-2 (colorectal adenocarcinoma), HaCat (immortalized keratinocyte) and L-929 (fibroblasts) cell lines. In addition, we also seek to mimic in vitro and in vivo biodistribution assays with bovine serum albumin (BSA protein) and hairless mice (Protocol N° 8,843,231,019), respectively, which showed evidence of the influence of these binders on the HY bio-modified nanoformulations.