*Structure of BP*

Phosphorus represents about 1% of the total human body mass, being found specifically in bones. The chemical structure of phosphorus allows it to create links with any other atom by hybridization of its orbitals, obtaining an sp<sup>3</sup> form.

The BP presents an anisotropic reticular structure, which generates two atomic layers with two di fferent interatomic bonds. In this regard, under high pressure, the crystalline structure of BP can be arranged into two di fferent configurations. The structure, under controlled thermal conditions, can shift from an orthorhombic to a rhomboid form at a pressure of 5.5 GPa. Another transition can be obtained after the application of a pressure of 10 GPa, i.e., the rhomboid configuration shifts to a cubic one. Among the di fferent BP tridimensional conformations, the BPQD (BP Quantum Dose) has been one of the most investigated in the recent literature; it has an orthorhombic crystalline structure, which, based on the type of synthesis process, makes it possible to obtain di fferent isoforms that are capable of changing according to mechanical stresses and the intended use. For example, BPQDs maintain a certain structural stability when used in applications aimed at drug-delivery and

bio-imaging. In this context, BPQD has demonstrated that its high stability makes it possible to improve its properties related to light absorption. Moreover, BPQD is able to fluoresce in a way that makes it useful in the diagnosis of cancer. The cubic crystalline configuration, on the other hand, is obtained at high pressures, and allows a redistribution to occur of the electronic density that favors phosphorene conductivity [6]: In this conformation, the use of the BP in medical devices can be considered, in which the sensitivity of electronic elements is essential [4,5]. Given the possibility of obtaining di fferent structural configurations, future studies could concentrate on the definition of the factors that influence the aforementioned transition, in order to obtain adequate characteristics for the chosen field of application, such as biosensors, polymeric sca ffolds and smart drug-delivery systems [10].

Some features make the BP unique, compared to graphene and other biomaterials. In fact, BP is among the most stable biomaterials at room temperature and under normal pressures. It can degrade easily and generate rapid fluorescence in some conditions [11]. Although its use for electronic sectors is not recommended, BP is a perfect fit for the medical sector, mainly thanks to its high biocompatibility and biomimetic biodegradability that reveal its potential in, e.g., bone repairing. Moreover, the excellent optical properties of BP are ensured by a good absorption coe fficient in visible, infrared (IR) and ultraviolet (UV) light. In contrast to graphene and other 2D materials, this allows BP to be used for colorimetric and fluorescent detectors, as well as for biosensing devices. For example, Zhao et al. have shown that the BP makes it possible to detect precise analytes (such as immunoglobulin IgG, myoglobin Mb) and inorganic ions, highlighting the photodynamic and electrochemical properties that make it unique [12]. A physical peculiarity, which is present also in other 2D materials such as graphene or borophene, is related to BP's anisotropic properties; interestingly, there are several techniques by which to synthesize BP, depending on the final application [13] (Figure 1).

**Figure 1.** Process of degradation of polymeric scaffold (PLGA: poly lactic-co-glycolic acid), mixed with BPQDs in a physiological environment. Adapted from Reference [13].

An important evaluation regarding the advantages of BP, compared to graphene and other 2D materials, such as MoS2 and WS2, is related to its lower cytotoxicity, higher in vivo biodegradability, and its tendency to release few nanoparticles in the human body [14]. The toxicity of 2D materials can be influenced by physical parameters, such as size, distribution, concentration, and shape, but also by the time of their exposure to biological tissues. In particular, the toxicity of graphene-based nanomaterials has been mainly linked to several oxidative pathways, and to biological damage involving cell membranes, resulting from the direct interaction between graphene and cells. In this light, studies were carried out on hMSC, human erythrocytes, skin fibroblasts, and glioblastoma cells. It has been hypothesized that the mechanism underlying the cytotoxic process, which basically depends on the concentration of graphene, is linked to the activity of the Reactive Oxygen Species (ROS) generated by graphene, as well as to the direct interaction between graphene and membrane phospholipids [15]. The in vitro cytotoxicity of MOS2 and WS2 was shown to be much lower than that of graphene, as the cells maintained their vitality even when they were exposed to concentrations as

high as 100μg/mL [16]. Specific studies on the viability of cells in vitro showed unprecedentedly low, or a total absence of, cytotoxicity at 1.0 mg ML of BP [17].

The BP monolayers, due to their zig-zag conformation, do not excel in terms of Young's Modulus and thermal conductivity, compared to graphene. On the other hand, they have higher optical absorption and a lower presence of impurities, which facilitate their use for optical pulse detection devices [12]. Two-dimensional materials such as graphene, have good electronic mobility but a fairly low "ON-OFF" current ratio that could impede their use in sensing applications. BP is highly sensitive to electrical disturbances, a characteristic which facilitates its application for gas detection devices [18]. The optical properties of BP are strongly linked to its band-gap; the band-gap of BP nanosheets can be easily modulated from 0 to about 1.45eV through the application of external stimulus. The control of the band gap is fundamental for the use of BP in photothermic, photodynamic, biosensing, and bioimaging applications [19] (Figure 2).

**Figure 2.** Main applications of Black Phosphorous (BP) compared to graphene in biomedical fields.

### **3. Biological Properties of BP**

In the ongoing search for ideal 2D materials for use in biomedicine, a key factor is biocompatibility. In different studies, graphene and BP have been reported to be biocompatible. In detail, BP showed fewer inflammatory reactions, lower cytotoxicity at high concentrations, and better control of corrosion in vivo. All such properties are required for biomedical applications. Moreover, BP can also be used as a coating for the surface of other materials, thus creating composed bilayered scaffolds which are characterized by high chemical and structural stability, and by a low corrosion curve in biological environments [20]. In contrast, the interaction between BP sheets and molecular oxygen seems to worsen the structural properties of this nanomaterial; in fact, oxygen can link the atoms of phosphorus by covalent bonds, thus increasing the overall degradation rate during their interactions.

Moreover, O2 easily dissociates from the BP surface, thereby creating an oxidized covering. Water could also induce structural changes in BP sheets; in fact, the naïve BP hydrophilicity has a strong impact on the degradation rate [21]. In this regard, we may also consider using the influence of oxygen and water on the BP degradation rate to our advantage, as a high degradation rate may be required in some therapeutic or pharmacological applications.

### **4. BP Synthesis and Production**

To produce nanosheets of 2D materials, we will use two different approaches: the "Top-down" and the "Bottom-up" method. The top-down method consists of the realization of a single layer through different exfoliation techniques (mechanical, liquid, and chemical). On the other hand, the bottom-up method involves specific chemical reactions (chemical wetting, CVD chemical vapor deposition) to compose the layer. Both such techniques have advantages and disadvantages. In the top-down approach, nanosheets can have several sizes, but the chemical residues generated by the various exfoliation techniques remain. The bottom-up technique makes it possible to generate BP layers chemically with more compact structures; moreover, it can be used to combine more materials together. On the other hand, this technique showed a low ability to modulate the width and thickness of the single layer [22]. Now we will describe the main synthesis techniques for BP sheets.
