3.1. Physical, Mechanical, and Chemical Features of bP and Phosphorene (bP-NPs)
bP is a semiconductor with a direct band gap, high carrier mobility, and thermal stability in vacuum at around 400 °C. It exhibits a tunable bandgap that varies with the number of layers, ranging from 0.3 eV for bulk to 2 eV for the monolayer. This range covers the spectrum between graphene and transition metal dichalcogenides, rendering bP-NPs an extremely appealing option as a 2D semiconductor
[52,53,54][34][35][36]. Together with the bandgap, carrier mobility is layer-dependent, and it has been calculated to achieve the values of 10,000–26,000 cm
2/Vs for the monolayer
[55][37]. The well-known in-plane anisotropy generates peculiar physical and mechanical responses distinguishing between AC and ZZ directions.
Anisotropy affects even the optical properties, showing the dichroic behavior of bP-NPs. In optics, dichroic materials absorb light differently based on its polarization. In the case of bP and bP-NPs, only light with a polarization component along the AC direction is absorbed for frequencies close to the bandgap energy
[56][38]. Similar results have been recently experimentally obtained by measuring the photoluminescence behavior of bP-NPs
[57][39].
bP exhibits unique mechanical properties due to its puckered structure. When stretched along the y-direction, it displays a negative Poisson’s ratio (−0.027) for the z-direction. This implies that when stretched along one direction, the material expands along the transverse direction, which is contrary to what happens with most materials, where stretching along one direction usually results in a reduction of lateral dimension
[54,58][36][40].
With reference to their actual and effective employment, it is necessary to point out that due to their peculiar structure, bP and especially bP-NPs are materials that exhibit high reactivity. This makes bP-NPs remarkably responsive to the surrounding environment and potentially suitable to be patterned and/or functionalized for specific performant applications, especially in the optoelectronics fields
[46,48,53][33][35][41]. However, phosphorene reactivity under ambient conditions results in physical and structural changes, leading to degradation
[60,61,62,63,64,65][42][43][44][45][46][47].
3.2. Preparation of bP and bP-NPs
Several synthetic procedures have been developed and tested for the preparation of phosphorene using both bottom-up and top-down approaches. Bottom-up methods directly synthesize bP-NPs from different molecular precursors through chemical reactions. Chemical vapor deposition, i.e., direct vapor deposition of red phosphorus or bulk bP in vacuum or argon, has not produced satisfactory results
[50,67,68][48][49][50]. The precursors’ instability and procedural problems constitute a major limitation to the development of massive production of bP-NPs through bottom-up methods. Top-down methods involve separating the stacked layers of bulk bP to obtain single- or few-layered nanosheets by breaking the Van der Waals bonding. For this method, it is essential to begin with high-purity bulk bP, which can be synthesized using established methodologies. Bridgman first synthesized black phosphorus by heating white phosphorus at 200 °C under high pressure (1.2–1.3 GPa). In 2007, it was found that it could be prepared from red phosphorus at low pressure and 873 K by adding small quantities of gold, tin, and tin(IV) iodide
[54][36]. All these processes can produce good-quality bP but are expensive and low-yielding. To date, the absence of a safe, high-throughput, and scalable route for producing bP remains one of the main limits to phosphorene uses.
To produce bP-NPs, exfoliation is needed. Based on multilevel quantum chemical calculations, the exfoliation energy of bP is around 151 meV per atom (larger than that of graphite, 61 meV), which accounts for the relative difficulty in exfoliating bP. This is associated with a non-negligible electronic density overlap between the layers; indeed, it should be pointed out that there is debate regarding whether the interlayer bonding can be classified as a Van der Waals type.
When a solid, layered material is immersed in a liquid, the interfacial tension is significantly high so that the material–solvent interactions are not able to outweigh the interlayer interactions, and spontaneous exfoliation does not occur. It is indeed necessary to apply external energy to win secondary intra-layer interactions and exfoliate the material. Ultrasounds are used to generate microbubbles, the growth and collapse of which are attributed to the cavitation-induced pressure pulses and acoustic waves consisting of alternate regions of compression and rarefaction
[70][51]. The choice of the solvent is crucial; it should have a surface tension similar to the surface energy of the 2D material to maximize the exfoliation rate and inhibit the restacking of nanosheets. In the case of bP, solvents with surface tensions of 35–40 mJ/m
2 are used, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and N-cyclohexyl-2-pyrrolidone (CHP)
[50,54][36][48]. Although anhydrous organic solvents may produce high-quality flakes, they have high boiling points, making postprocessing and disposal more difficult, and they are also hazardous to human health and the environment. After the exfoliation process, if successful, solvent molecules surround the nanoparticles through solvation, which stabilizes them. These molecules are challenging to remove, and they remain even after subsequent centrifugation and redispersion steps that are essential for a final collection of the products. Therefore, particularly in biomedical fields, this can cause safety issues. For this reason, water-based solutions have also been studied as possible sonication mediums. Since water has a surface tension of about 73 mJ/m
2, and bulk bP is insoluble in water, stabilizing surfactants are needed to produce stable flakes and avoid aggregation. It is also important to use de-oxygenated water to prevent phosphorene from oxidizing.
4. Antimicrobial Photoactivity of bP
After its discovery, bP attracted the interest of researchers mainly due to its possible applications in optoelectronics, photonics, and advanced engineering, and only in the last few years has it also emerged as a possible new 2D material for biomedical applications. bP is highly biodegradable, biocompatible, and safe for use. These properties are essential for the use of bP in medicine [46,48,73,74,75,76,77,78,79][33][41][52][53][54][55][56][57][58]. In living organisms, phosphorus is a crucial element that constitutes approximately 1% of the total body weight. When it degrades, it transforms into harmless phosphate, which exhibits high biocompatibility and low cytotoxicity, preventing its in vivo accumulation. As a 2D material, it intrinsically has a large surface area, making it suitable for the absorption of drug molecules and making it easier to control the kinetics of release [80][59]. It also has a high modulus; thus, it can be used to improve the mechanical strength of biomedical implants.
4.1. Mechanisms of bP Photoactivity
As previously mentioned, bP has a bandgap dependent on the number of layers, which varies from 0.3 eV to 2 eV, from bulk to monolayer. The
3O
2/
1O
2 redox potentials fall within this range
[64][46], and thus, bP-NPs can act as light absorbers, mediating the energy transfer to oxygen molecules in the surrounding environment. Molecular dynamic simulations help to predict the mechanism of singlet oxygen (
1O
2) generation: First, oxygen molecules interact with P lone pairs, leading to the adsorption of O
2, and then, the generation of
1O
2 occurs through charge transfer
[14][60]. The so-generated
1O
2 is very unstable and reacts with target systems in the surroundings. Illumination is needed to excite the transition from the ground state of bP-NPs. The most convenient wavelength range for the photosensitizer activation is between 600 and 800 nm, which is called the “optimal therapeutic window”, since it is therapeutically safe and allows for effective tissue penetration of light while still providing enough energy to allow the transition to the excited singlet state of oxygen
[23][14] without compromising biological tissues.
4.2. Bare bP-NPs
The antimicrobial activities of exfoliated bP nanosheets have been compared to those of bulk bP and other 2D materials such as graphene and transition metal dichalcogenides such as MoS
2, showing a better performance in killing both Gram-negative
Escherichia coli and Gram-positive
Staphylococcus aureus that cause serious infections. Under 808 nm laser irradiation, a value of 99.2% in bacterial killing percentage against both
E. coli and
S. aureus was reached mainly by means of photothermal inactivation with a negligible cytotoxicity towards mammal cells even at high bP-NP concentrations
[84][61]. A few nanograms of bP nanosheets have been shown to be enough for a strong and broad-spectrum antimicrobial activity toward the bacteria
Escherichia coli,
Pseudomonas aeruginosa, MRSA,
Salmonella typhimurium, and
Bacillus cereus as well as the fungal strains
Candida albicans,
Candida auris, and
Cryptococcus neoformans, displaying the effectiveness of bP-NPs as an antibacterial additive in surface coatings, too.
High-resolution microscopy and ATR-FTIR studies have revealed that the physical interaction of the bP-NPs with the microbial membranes, together with the oxidative stress, cause important physical and biochemical damages to the phospholipids and to the amide I and II proteins, whereas this results in slight chemical modifications to polysaccharides and nucleic acids of Gram-positive methicillin-resistant
Staphylococcus aureus (MRSA) and Gram-negative
Pseudomonas aeruginosa and to the fungal species
Candida albicans [86][62].
4.3. bP-NPs-Based Hybrid Materials
bP has also emerged as a suitable nanomaterial that allows for drug delivery and therapeutics due to its high surface area. As it is negatively charged in water with an interlay distance of ~5.24 Å, the encapsulation of small and positively charged molecular drugs within the interlayer spaces is possible mainly through electrostatic interactions. To date, three major strategies have been explored for developing bP-NPs-based hybrid materials: electrostatic interaction, covalent bonding, and noncovalent bonding (e.g., hydrophobic interactions)
[83][63].
5. Conclusions
The advancements in biomedical research and the development of nanotechnology can have great potential for fighting multi-drug-resistant microbial pathogens. Among 2D nanomaterials, bP-NP has shown promising properties for various applications, including antimicrobial phototherapy. It has a high specific surface area, which can enhance its interactions with microbial pathogens. The puckered structure of bP-NPs along with their peculiar anisotropy contribute to unique electronic, mechanical, and photoactivity properties that are potentially exploitable in an antimicrobial system. The thickness-dependent band gap (0.3–2 eV) allows it to absorb a wide range of wavelengths from ultraviolet to NIR. Due to its broad absorption band and unique electronic structure, bP-NP possesses an intrinsic photoactivity that makes it an effective phototherapeutic agent in PTT and PDT against pathogenic bacteria. Furthermore, bP nanomaterials have excellent biocompatibility and biodegradability in vivo, minimizing potential adverse effects on living tissues and facilitating their use in antibacterial activity as well as other biomedical applications. Phosphorene-based nanomaterials can be engineered for targeted drug delivery. This is particularly relevant in antimicrobial phototherapy, where the precise delivery of therapeutic agents to the infection site is crucial for maximizing treatment efficacy and minimizing side effects.
Along with their many discussed advantages in antimicrobial applications, there are still several challenges facing bP-NPs that need to be addressed, such as the need for efficient and low-cost synthesis strategies to facilitate large-scale production, stability optimization for ensuring efficacy in biomedical applications, and systematic studies correlating structural characteristics with the antimicrobial properties. In fact, although numerous current studies and insights on the structure, chemical, and physical characteristics of bare bP-NPs are present, the research on applications of bP-NPs-based hybrids in aPDT and aPTT often lacks clear reports on the structure/chemical composition of the investigated hybrid systems and on the mechanisms involved in the photoactivation phenomena. The modification and embedding of bP-NPs in different substrates, such as other photosensitizers, drugs, and protective agents, to create hybrid systems can complicate the identification and rationalization of individual components’ contributions to photoactivation processes.