Since graphene was isolated by Novoselov et al. 1, two-dimensional (2D) few-layered materials have been attracting increasing attention due to their unique electronic 2, optical 3, catalytic 4, electrochemical 5, and mechanical 6 properties in comparison to their multilayered bulk counterparts. The family of 2D materials is a large group of nanomaterials, such as graphene, transition metal dichalcogenides (molybdenum disulfide), MXene 7, and phosphorene, which was discovered in 2014. Phosphorene shows properties such as anisotropy, high carrier mobility, mechanical flexibility, and a tunable bandgap from 0.3 eV (bulk) to 2.0 eV (single layer) 8. For biomedical applications, its strong absorption in the ultraviolet (UV) and near-infrared (NIR) regions 9,10, biocompatibility, and great biodegradability in the physiological environment are especially important. These properties of phosphorene have attracted special attention for clinical applications in biomedicine such as phototherapy 11,12, drug delivery 13, biosensing 14,15, and theranostics 10,16.
Although studies of biomedical applications of FLBP have made a lot of progress, there are still some problems that need to be solved before its clinical transition. Various factor such as size, concentration and test cell line have a great impact on toxicity and treatment effects, it is very important to find an effective method to fabricate FLBP with uniform size and high production. Another remarkable but still underexplored feature of phosphorene is it potential as a carrier for biological and biomedical molecules. Surface functionalisation or conjugation is commonly used to increase stability, improve drug loading and cellular uptake, actively target cancer cells and tumors. To environmental stability of phosphorene is also concern and specialized coatings are necessary to ensure stable performance of the sensor. In this regard, the number functionalisation with other nanoparticulate system or polymers (poly(ethylene glycol) (PEG), Poly-L-lysine (PLL), Poly(lactic-co-glycolic acid) (PLGA), polysialic acid or glycolic acid) have been developed 17,18. The fabrication of BP nanoplatform with polymers could be efficient route to achieve stability of nanomaterial and enhancement of its properties. On the one hand, the involvement of non-ionic, flexible, hydrophilic and biocompatible PEG polymer allowed to resolve the problem with non-specific absorption on the nanomaterial surface19–23. While, application of ionic PLL polymer in functionalisation of nanosheet surface may be a new strategy to overcome drug resistance related to deficient transport 24,25. The polycationic nature of poly-L-lysine enhanced the interaction with plasmid DNA and ensured the protection of biomolecules from nuclease degradation 26,27. These functionalised strategy allow to applied BP in different application area, considering unique features of nanosheets itself or utilizing advantage of synergistic effect of applied components. Constructing such a nanoplatform not only enables encapsulation of specific recognition ligands, but can result in targeted drug delivery to the tumor cell 28–30 .
Peptides may offer new therapeutic opportunities due to low immunogenicity, excellent tissue penetration and low production cost 31,32. Specific targeting of therapeutic moieties to cancer tissue may be a valuable option, especially when local treatment is not possible. The positively charged amino acid residues have great potential as an active targeting ligand 33–35. Peptides rich in KRK motif could promote membrane fusion through electrostatic interaction with lipid and enable the interaction with negatively charged DNA36. This short sequence was uses because it possesses ability to accumulation both on the surface of tumor vessels and within tumor tissue after intravenous injection 37,38.
Next, peptides which can be selectively delivered to tumors are very attractive biomolecules for the prevention and treatment of cancer. The arginine-glycine-aspartic acid (RGD) sequence plays a crucial role in cellular adhesion of extracellular fibronectins39. RGD motif exhibit a strong affinity and selectivity to alpha v beta 3 integrin on the cell surface and enter the cytoplasm receptor-mediated endocytosis40. Within the present work, RGD sequence was used due to the fact that RGD triad is a typical cell-binding domain in order to effective targeting of endothelial cells in tumor vasculature and suppressing angiogenesis and tumor growth. Many research effort have been paid to the determination of therapeutic efficiency and selective tumor targeting ability of black phosphorus. Unfortunately, studies describe the difference in efficiency of the applied linker in delivery systems are still missing. Drug delivery studies with different structural linkers are often performed in different tumor models with a wide dose range, which makes them difficult to compare. Therefore extensive investigations about BP and its bioconjugates various applications as well as critical evaluation on their toxicity assessment are very crucial before introducing it into practical area.
The novel strategies of FLBP nanosheets functionalisation are presented in the paper allowing for enhancement of its environmental stability against air or aqueous solvents. We applied two different strategies of bioligands grafting studying influence of ionic character, surface conformation and biological activity of poly-L-lysine (PLL) and poly-ethylene glycol (PEG), in order to overcome FLBP nanosheets degradation. To the best of our knowledge, the functionalisation of nanosheet incorporating bioligands – tumor-homing peptides with KRK and RGD motifs as a potential drugs in breast cancer therapy were not reported in the literature up to date. The toxicity investigation of FLBP itself and its modifications were carried out in two model of breast cancer lines MCF-7 (luminal A subtype of breast cancer) and MDA-MB-231 (highly aggressive, triple-negative breast cancer).