Advancements in two-dimensional boron nitride nanostructures: properties, preparation methods, and their biomedical applications

Abstract

Novel boron nitride (BN) nanostructures with varied dimensionalities and unique and extraordinary qualities like exceptional mechanical properties, high thermal conductivity, excellent chemical properties have applications in diverse and promising fields, encompassing healthcare, environment, and energy. Amongst the boron nitride nanostructures, two-dimensional BN sheets have been extensively explored by researchers due to their planar topography, which confers them with distinct qualities in terms of chemical, physical, optical, and electronic properties. 2D boron nanostructures have a significant surface-to-volume ratio that enables their enhanced contact with cells and biomolecules, making them appealing candidates for biological applications. This article aims to completely present the latest advances in the development of two-dimensional boron nanostructures, most importantly nanosheets. The properties and different synthesis processes and modifications used for developing 2D boron nanosheets are summarized. We provide a detailed explanation of the unique features of 2D boron nanosheets that enable their usage in biomedical applications like in the field of drug delivery, cancer theranostics, bioimaging, and biosensing. Finally, the prospects of 2D boron nanosheets and their future challenges are discussed. It is anticipated that this review will encourage more developments in the field of 2D boron nanostructures for potential future applications.

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Gitashree Darabdhara

Dr Gitashree Darabdhara is currently serving as an Assistant Professor in the Department of Chemistry at Jagannath Barooah University, Jorhat, Assam. She earned her PhD in 2019 from the Academy of Scientific and Innovative Research (AcSIR), focusing her dissertation on “Reduced Graphene Oxide Sheets Decorated with Bimetallic Nanoparticles: Synthesis, Characterization and their Applications.” Her doctoral work was recognized with the “Best PhD Thesis Award” in Chemical Sciences by AcSIR for the year 2019. Dr Darabdhara has authored over 20 peer-reviewed research articles, with her highest impact publication reaching an impact factor of 16. Her research is centered on the development of advanced two-dimensional materials, particularly for applications in photocatalysis and biosensing.

Priyakshree Borthakur

Dr Priyakshree Borthakur obtained her MSc in Chemistry from Dibrugarh University, Assam, in 2013 and completed her PhD from the Academy of Scientific and Innovative Research (AcSIR) in 2020. Her doctoral research was carried out under the guidance of Dr Manash R. Das, Senior Principal Scientist at CSIR-NEIST, Jorhat, Assam. In 2021, she began her academic career as an Assistant Professor in the Department of Chemistry at Pragjyotish College, Guwahati. Dr Borthakur has contributed to over 20 peer-reviewed journal articles and has authored five book chapters. Her research primarily focuses on the synthesis of semiconductor metal sulfide nanoparticles on graphene and other two-dimensional materials, exploring their applications in photocatalysis, environmental sensing, and water pollutant detection.

Purna K. Boruah

Dr Purna K. Boruah received his PhD in Chemistry from CSIR–North East Institute of Science and Technology, Jorhat, India, in June 2020 under the supervision of Dr Manash R. Das. In April 2021, he began his academic career as an Assistant Professor at Jorhat Engineering College, India. He was awarded the JSPS Postdoctoral Research Fellowship in 2022, and he joined Kyushu University, Japan, in July 2022. In 2023, Dr Boruah was awarded Skłodowska-Curie Individual Postdoctoral Fellowship (H2023), and he is currently working at CNRS–UCCS, University of Lille, France. His research interests focus on the design and synthesis of advanced composite materials with 2D materials, like graphenes and MXenes, in combination with metal oxides, metal nanoparticles, quantum dots, and porous materials such as metal–organic polyhedra and frameworks. He applies these materials to develop functional systems for sensing, photocatalysis, electrocatalysis, and adsorption. Dr Boruah has co-authored over 65 international peer-reviewed publications, 1 patent and 4 book chapters.

Santimoy Sen

Santimoy Sen is a PhD scholar in the Department of Pharmacology and Toxicology at the National Institute of Pharmaceutical Education and Research (NIPER), Guwahati. As an aspiring scholar, he has completed his MS Pharm. in the Department of Pharmacology and Toxicology at NIPER Ahmedabad. At NIPER Guwahati, his research is focused on developing fluorescent nanoparticles for non-invasive theranostic applications and exploiting different molecular mechanisms involved in the neuroinflammation and neurodegeneration in traumatic spinal cord injuries. His approach encompasses the application of photobiomodulation therapy as a minimally invasive therapy for the abrogation of the pathological conditions involved in traumatic spinal cord injury.

Deepak Bhardwaj Pemmaraju

Dr Deepak Bharadwaj Pemmaraju is an Assistant Professor in the Department of Pharmacology and Toxicology at the National Institute of Pharmaceutical Education and Research (NIPER), Guwahati. Dr Deepak has completed his PhD in the Department of Biomedical Engineering at the Indian Institute of Technology, Hyderabad. At NIPER-G, his research is basically focused on developing nanomaterials for non-invasive theranostic applications in various domains, especially in superficial cancers such as breast and skin cancers, wound healing pathology and neurodegeneration and inflammation. His promising approach encircles but is not limited to light-based theranostic applications, especially photothermal and photo-biomodulatory effects, along with the exploitation of their molecular mechanisms involved in the pathologies.

Manash R. Das

Dr Manash R. Das currently serves as a Senior Principal Scientist in the Materials Sciences Group, Coal, Energy and Materials Sciences Division at CSIR–North East Institute of Science and Technology (CSIR-NEIST), Jorhat, India, and as an Associate Professor at the Academy of Scientific and Innovative Research (AcSIR), India. Dr Das received his MSc degree in Chemistry from Gauhati University, Guwahati, Assam, in 2000, and his PhD from Dibrugarh University, Dibrugarh, Assam, in 2007. He pursued his postdoctoral research from May 2007 to April 2008 at the Institut de Recherche Interdisciplinaire (IRI, USR-CNRS 3078) and the Institut d’Électronique, de Microélectronique et de Nanotechnologie (IEMN, UMR-CNRS 8520) in Lille, France. He also served as the Coordinator of the Academy of Scientific and Innovative Research (AcSIR), India, from 2020 to 2025. His research interests encompass the value addition of the graphite minerals of North East India, 2D nanocomposite materials as efficient nanozymes for sensing applications, and the design and development of graphene, borophene, and other 2D nanomaterials. He also focuses on the fabrication of microfluidic paper-based analytical devices (μPADs) for sensing applications, photothermal therapy using 2D nanomaterials for cancer treatment, photocatalytic degradation of water pollutants, development of boron-based nanomaterials for chemical sensing, and exploration of single-atom catalysts as nanozymes. He has published more than 200 papers in reputed journals.

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1. Introduction

Ever since Geim and Novoselov successfully separated two-dimensional (2D) graphene from three-dimensional (3D) graphite in 2004, interest in other such promising and adaptable 2D nanomaterials has increased. 2D materials are free-standing ultrathin layered materials with a planar structure, which imparts them with several beneficial properties such as free flow of electrons, superior mechanical qualities, a high surface-to-volume ratio, and optical transparency, making them materials of choice for several prospective applications.¹ 2D nanomaterials with very high surface-to-volume ratios possess a large quantity of surface atoms, and hence, a large number of surface anchoring sites are present, which leads to enhanced interaction with biological moieties, thus expanding their horizon in various biomedical applications.²

Similar to graphite and graphene, boron nitride (BN) possesses a honeycomb-like structure that is made up of alternating boron (B) and nitrogen (N) atoms. Due to their distinctive characteristics, BN nanostructures have recently drawn a lot of attention. There are four distinct crystal forms of BN, which are layered hexagonal, cubic, rare wurtzite and rhombohedral structures.³ Amongst these crystalline forms, 2D hexagonal BN (h-BN) poses to be the stable form with sp² covalent bonding between B and N similar to graphene. Unlike graphene, B and N's covalent bonding in h-BN exhibits less covalency due to the difference in their electronegativity values leading to higher ionicity, wide band gap (∼5.5 eV) and unique physico-chemical properties.⁴ In recent years, these alluring properties have driven the attention of the scientists to develop BN in different nanostructures such as nanotubes,⁵ nanosheets,⁶ nanoribbons,⁷ nanoparticles,⁸ and nanofibers.⁹

h-BN is the most widely researched form of BN. h-BN layered nanostructures have become an important material of choice because of their extraordinary properties like low dielectric constant, high temperature stability, hardness, and high electric insulation. This has paved the way for their application in different fields including bio-medicine, electronics, catalysis, and solid-state neutron detection.¹⁰ Owing to BN's heteroatom bonding structure, the B–N bond is partly ionic with the electrons localizing closer to N in case of σ-bond due to higher electronegativity of N compared to B. However, in the case of π-bonding that involves empty p orbitals of B and fully occupied orbitals of N, the N electrons are delocalized to a much lesser extent than π–π electrons in C–C bonds, and thus, h-BN offers diverse electrical, optical and chemical properties as compared to graphite and graphene.¹¹ The term “white graphene” is frequently used to describe h-BN since it does not absorb light in the visible spectrum (390–700 nm).¹² Theoretical and experimental studies have revealed that h-BN is biocompatible in nature, and hence, a number of researchers have explored the application of h-BN in several biomedical areas including cancer therapy,¹³ wound healing,¹⁴ and biosensing.¹⁵ As a boron source, h-BN has been widely explored for therapeutic effects, considering the slow release of boron and its long-term effects. Studies on h-BN have shown that it can effectively induce apoptosis in prostate cancer cells, which is dependent on ROS and reducing metastasis by altering the intracellular cytoskeleton because it is totally biocompatible with healthy cells.¹⁶ h-BN has been reported to possess good preferential endocytosis by cells, which promotes its application in drug delivery and bioimaging. However, due to its hydrophobicity, it becomes necessary to functionalize h-BN with other groups such as hydroxyl (–OH), alkoxy (–OR), amine (–NH₂), alkyl (–R), and acyl (–COR). This was reflected in the work of Zhang et al. where hydroxy BN nanosheets decorated with Pd nanoparticles (NPs) were used to load and transport doxorubicin (DOX). The release of this antitumor drug in the tumor microenvironment was pH and NIR dependent. In vivo studies indicated tumor growth inhibition as well as excellent efficacy during subcutaneous injection.¹⁷ Additionally, DOX was delivered using hydroxylated h-BN nanosheets that are water soluble with a loading efficiency of 300 weight percent.¹⁸ Sharker's group produced dopamine- and h-BN-loaded indocyanine green (ICG) using hyaluronic acid as DOX-conjugated (hBNI and hBNI/d-HA-DOX, respectively) systems for photothermal treatment (PTT) and targeted medication delivery for breast cancer.¹⁹ Ag NP-decorated polydopamine-coated h-BN was also used as a wound healing stimulatory agent. The hBN@pdopa-AgNP agent promotes wound closure by decreasing the ROS production and reorganizing tube formation in the cells.²⁰

An h-BN nanosheet-based aptamer sensor was developed by Adeel and his coworkers in which the nanosheets were decorated with Au NPs to achieve a AuNP/BN-nanosheet-based biosensor for the selective detection of myoglobin.²¹ BN nanosheets were also found to have the property of protecting metals from corrosion in a biological environment.²²

According to studies, BN nanostructures, particularly h-BN, have a large specific surface area, minimal cytotoxicity, and good biocompatibility with various cell types. As a result, they are a viable option for drug administration, implants, and biosensors. Additionally, as BN is stable and chemically inert under physiological conditions, it can be used for extended in vivo applications without degrading quickly. It can also be used in therapeutic hyperthermia (such as tumor ablation) due to its high thermal conductivity. Furthermore, it has been demonstrated that BN nanostructures have antibacterial properties, which may be useful for materials used in wound healing or implant coatings. However, there are also certain limitations associated with 2D h-BN nanostructures. Due to its hydrophobic nature and difficulty dispersing in aqueous solutions, pristine BN cannot be used directly in biological environments; instead, it needs to be surface functionalized or chemically modified for biomedical compatibility. Chemical functionalization is frequently required to improve the drug-loading efficiency or biocompatibility. For clinical-grade materials, this increases the costs and makes the fabrication process more difficult. Since BN is a relatively new substance in the biomedical field, its lack of established clinical evaluation methodologies may cause regulatory approval to be delayed.²³

Although h-BN nanomaterials have not yet entered clinical trials, their preclinical success in tissue engineering, bioimaging, and drug delivery offers a solid basis for future translation; however, standardized toxicological evaluations, regulatory approvals, and scalable manufacturing processes are necessary to proceed.

The h-BN nanosheets offering a large surface area provide scope for their interaction with the cells, thus providing a unique bridge between nano- and micro-scale systems. Although numerous studies have already been reported related to the biomedical applications of BN nanosheets, research on BN nanosheet toxicity has not yet been explored at length. Given the rapid development in the field, the scientific community needs a thorough and timely summary of the recent advancements focusing on h-BN nanostructures, despite the fact that the field has made tremendous progress in 2D h-BN nanostructures for different biomedical applications, as demonstrated by the relevant publications. A few notable research studies only addressed the development of h-BN in a single biomedical application, such as medication delivery²⁴ and bone tissue engineering.²⁵ A number of the published volumes provide a detailed overview of h-BN nanostructures and concentrate on their individual applications in tissue engineering, wound healing, targeted drug delivery, nanomedicine, and biosensing.^{13–15,19,26} A large number of reviews include the current development of several types of 2D nanomaterials for biomedical applications rather than just h-BN, thus missing out the important biomedical applications of h-BN.^27–29 The development of h-BN nanostructures is the subject of numerous publications, including their synthesis, history, characteristics, environmental and energy applications, and electrocatalysis.^30,31 However, not many studies are reported on the development of h-BN from its synthesis and properties to its various biological uses. There have been no published reviews that offer a comprehensive overview of the structural details, properties, and diverse biomedical applications of h-BN. These applications include drug delivery, tissue engineering, bioimaging, cancer therapy, electrochemical and colorimetric biosensing, treatments for neurological disorders, and wound healing. Therefore, it is essential to give a comprehensive and in-depth summary of their advancements in this field in order to guide future translational research from basic to clinical, which is where the current work's originality lies.

The current development in the synthesis and biomedical applications of 2D BN nanostructures is the main topic of this article. Primarily, we focus on the properties of BN nanosheets, different methods for its synthesis and how the BN nanosheets can be utilized for different biomedical applications including drug delivery, therapeutics, wound healing, and biosensing. This article will essentially provide a roadmap for the futuristic design of BN nanostructures in forthcoming investigations for medical and related applications.

2. Structure and properties of BN nanosheets

2.1. Structure

The 2D BN nanosheets with sp² hybridization-based covalent bonds are composed of layers of BN with an even number of alternate layers of B and N atoms. The monolayers in the 2D nanostructure are connected through weak van der Waals forces of attraction with an interlayer spacing of 0.333 nm and a B–N bond distance of 0.1446 nm. With a = 2.504 Å and c = 6.661 Å, the crystalline properties of BN are comparable to those of graphite.³² Because of the difference in electronegativity and the location of the electrons at the N atoms, the B–N bond has partial ionic characteristics, and the adjacent layers interact with one another in a lip-to-lip manner. By reducing the amount of bonds dangling at the edges or tips, this event causes a metastable energy minimum, which lessens the “frustration” effect and stabilizes the multilayer structure.^33–35Fig. 1(a) illustrates the crystalline image of the layered BN nanosheets connected through van der Waals forces. The TEM images of layered sheets shown in Fig. 1(b) display folds and partial exfoliation. The crystalline character is indicated through the electron diffraction pattern in Fig. 1(c), and (d) shows the edge portion of the nanosheet, which implies a thickness of around 2 nm. Fig. 1(e) displays the HRTEM image of a BN nanosheet displaying patterns of lattice dots spaced 0.25 nm apart, indicating the separation between two neighboring hexagonal rings of BN.

Fig. 1 (a) Crystal structure of layered BN nanosheets [Reproduced from ref. 34 with authorization. Copyright 2020, Elsevier]. (b) TEM images representing the BN nanosheets made using a ball milling method. (c) Corresponding SAED pattern. (d) and (e) High-magnification TEM images of BN nanosheets [Reproduced from ref. 35 with authorization. Copyright 2014, Nature].

2.2. Properties of BN nanosheets

2.2.1. Electronic and optical properties. Over the last ten years, extensive studies on the optical characteristics of BN nanosheets have been performed. The band gap in pristine BN nanosheets has been found to be ∼6.0 eV because of the strong sublattice asymmetry, which qualifies it as an insulating substance. The insulating property of the BN nanosheet encourages its application as a protective encapsulating shield. However, by doping other materials, this insulating property of BN nanosheets can be altered, making it suitable for various purposes such as photocatalysis and other energy storage applications.³⁶

The Raman spectra of h-BN show a single, strong peak at 1373 cm⁻¹ because of E_2g from atomic motions. E_2g has two frequencies, one low at 49–52.5 cm⁻¹ and the other high at 1363–1373 cm⁻¹ due to sliding of atoms traveling in a plane and adjacent planes, respectively.³⁷ However, the low frequency characteristic is often not seen due to high Rayleigh background. Bond length has a significant impact on these Raman properties, and phonon softening or hardening results in red or blue shifts in the Raman peaks, respectively. Henceforth, a shifting into the red region of the Raman spectra of the BN nanosheets can be attributed also to the reduction or strengthening of the B–N bonds in the temperature due to high concentration of the laser. The Fourier transform infra-red (FTIR) spectroscopy for pure h-BN is located at 800–811 cm⁻¹ and 1350–1520 cm⁻¹ for B–N bending vibrations and stretching vibrations, respectively.³⁸

2.2.2. Thermal properties. Previous studies on h-BN have concluded that it possesses remarkable conductivity and high specific heat.³⁹ They are suitable contenders for various high-temperature applications because of their outstanding ability to conduct heat and high thermal as well as chemical stabilities. However, as the layered structure in the h-BN is anisotropic in nature, the thermal conductivities are also different for both the basal plane and the plane along the c-axis with values ranging from 390 to 2000 W m⁻¹ K⁻¹ and 2–30 W m⁻¹ K⁻¹, respectively.⁴⁰ h-BN also possesses more abundant isotopes leading to strong phonon-isotope scattering. However, the inherent phonon–phonon scattering lowers the conductivity with a bigger reduction at higher temperatures because of the lattice anharmonicity in the h-BN structure.⁴¹ There are numerous possible causes of phonon scattering, such as defects, dislocations, isotopic impurities, and grain boundaries. However, low-temperature thermal conductivity is not accountable to the point defects. Recent research has shown that as the number of layers is reduced, the ability to conduct heat increases as a result of the reduction in interlayer phonon scattering. As a result, h-BN monolayers exhibit greater thermal conductivities than bulk h-BN.⁴²

Approximately 600 W m⁻¹ K⁻¹ is the thermal conductivity of h-BN, and their thermal expansion coefficient ranges from 3.78 × 10⁻² cm⁻¹ K⁻¹ to 3.15 × 10⁻² cm⁻¹ K⁻¹. It is also stable under 1000 °C in air and 1400 °C in vacuum. Having such superior thermal characteristics, this material finds promising applications in batteries with long-term durability and dependability.⁴³

2.2.3. Mechanical properties. Over the past ten years, enough study has been done to determine the mechanical properties of h-BN materials.⁴⁴ Theoretically, Young's modulus value of h-BN was reported to be ∼1 TPa at a tensile strength of ∼100 GPa, making it suitable for several applications.^45,46 According to experimental research, h-BN's Young's modulus value is independent of the layer thickness, while the same is not applicable for graphene and its Young's modulus value has been found to decrease from 1.026 TPa to 0.942 TPa from 1 L to 9 L, respectively. Moreover, the braking strength of h-BN has been found to be constant with the number of layers attributed to less interlayer slippage.^47,48

The value of Young's modulus of h-BN was found to depend on the temperature and its value is found to decline in TPa from 0.96 to 0.65 with the increase in temperature starting from 0 K to 2000 K, respectively, and this starts to show more clearly at zig-zag edges in comparison to the armchair ones. This change in the values can be attributed to enhanced atomic vibrations and deformations at higher temperatures.^49,50

2.2.4. Wetting properties. The wetting property of h-BN is interesting. The hydrophilicity of the h-BN nanosheets switches between hydrophilic and superhydrophobic due to its specific inter-facial behaviors.²⁸ The contact angle varies between 50° and 150°. Because of the chemical inertness of the BN, water's pH value also has no impact on the wetting properties of this material. This adjustable property of contact angle in h-BN is employed for the preparation of functionalized materials applicable in different membrane technologies including water-repelling and self-cleaning applications.^37,51

2.2.5. Chemical properties. h-BN is highly chemically stable towards different reagents and environment, and hence, finds application in high-temperature manufacturing, corrosion-resistant films, etc.^52,53 It is also highly non-reactive towards acids and alkalis. The high chemical inertness is because of the lack of surface-active sites, slack bonds and the presence of o-bonds. However, this inertness can be removed through chemical functionalization or oxidation of the BN sheets with high oxidizing agents such as HCl, HBr, and H₂SO₄, making them Lewis active acids capable of withdrawing electrons from the acid–base interactions' key atoms in the acids. Earth fluorides of both alkaline and alkali metals can also be used to etch the h-BN sheets at high temperatures and pressures.⁵⁴

h-BN also possesses an elevated specific surface area of 1190 m² g⁻¹, and thus, possesses large numbers of surface-active sites, which make it an excellent material for adsorption as well as other applications.⁵⁵

3. Synthesis of 2D-BN nanosheets

Owing to the wide range of applications of the h-BN nanosheets, researchers have concentrated on different techniques and methods through which 2D h-BN nanosheets can be developed with a large surface area and activity. The study reported by Zhu and his co-workers has stated that the surface area of bulk BN is low in comparison to that of the nanosheets with values of 97 m² g⁻¹ for the bulk and 278 m² g⁻¹ for the nanosheets, respectively.⁵⁶ Additionally, the h-BN nanosheets are said to be very pure, crystalline, and thermally stable in nature.⁵⁷ Two widely used methods for h-BN nanosheets synthesis include top-down and bottom-up approaches. The top-down approach involves exfoliation of bulk h-BN by mechanical or chemical exfoliation, whereas bottom-up techniques involve the use of chemical or physical forces at the nanoscale level to build up a larger system. The bottom-up approach involves hydrothermal/solvothermal methods, pyrolysis, and chemical vapor deposition (CVD).^58,59

3.1. Top-down approach

3.1.1. Mechanical exfoliation. The separation of graphene through scotch tape method invented in 2004 paved the way for its application in developing other 2D nanomaterials including h-BN.^60,61 The presence of weak van der Waals forces between the h-BN sheets is weakened and destroyed by mechanical exfoliation using the adhesive tapes. In a work by Pacilé et al., layers of h-BN were removed from a 300 nm-thick SiO₂ substrate using adhesive tapes. For their experiment, they used powdered h-BN of grade AC6004 instead of bulk raw BN.⁵⁹ However, this method is limited to fine separation of the layers due to considerable contact between the BN basal planes, and hence, secondary treatments including ion etching⁶² and high-current density beam⁶³ are required. Instead of direct peeling, the ball milling method with gentle shear force was adopted to fabricate high-quality h-BN nanosheets with a higher yield. Ball milling involves mechanical exfoliation of the bulk BN and comprises various forces such as compression, shear and vibration forces that assist in breaking larger particles into smaller ones. High-quality BN nanosheets were produced by Li's group adopting a mechanical ball milling process under a N₂ environment.⁶⁴ To produce BN nanosheets of superior quality with a high yield and high efficiency, they modified the milling parameters to generate shear forces. Moreover, the near-edge X-ray absorption fine structure measurements showed no degradation to the BN nanosheets' in-plane structure. However, the shear force created during milling may cause minor structure destruction, and hence, for effective milling, few features need to be considered like (i) it is better to use a ball mill with controlled rolling action, (ii) enhancing the motions with a smaller milling area, and (iii) preventing the welding effect while milling using a suitable liquid solution. In this instance, zirconia balls were used to mill the h-BN powders with surfactants before sonication on a low setting. The transmission electron microscopic (TEM) images displayed in Fig. 2(a) of the BN nanosheets indicate their dimension to be ∼250 nm. The electron diffraction (Fig. 2(b)) of the milled sheets indicated its crystalline nature. The high-resolution TEM image (Fig. 2(c)) indicated the distance between lattices of h-BN to be ∼0.25 nm, which is the distance between the centers of two adjacent hexagonal rings of h-BN (Fig. 2(d)). Fig. 2(e) and (f) show three sheets with approximately three, four, and five layers of BN. Fig. 2(g) shows the TEM images of 22 sheets in total, all of which had less than 10 layers or were thinner than 4 nm.

Fig. 2 (a) TEM image of ball-milled BN sheets. (b) Corresponding electron diffraction pattern. (c) HRTEM image. (d) Basal plane of h-BN's crystal structure. (e) and (f) TEM images of three sheets having three, four, and five BN layers. (g) Distribution of the number of layers for 22 sheets of BN [Reproduced from ref. 64 with authorization. Copyright 2011, RSC].

BN nanosheets in significant quantities were generated by Deepika et al. using benzyl benzoate as the milling agent.³⁵ They investigated several parameters that can influence the production of the BN nanosheets and found that small balls in the diameter range of 0.1–0.2 mm were quite successful in BN nanosheet exfoliation. Additionally, they examined the effect of milling time and found that, for the first two hours, few-layer BN yielded a poor product and increased with milling times between five and ten hours. However, the BN nanosheet size decreased after 10 h of milling as opposed to 5 h, indicating that longer milling times are harmful to BN nanosheets and should be avoided in order to allow for complete exfoliation of the material. Fig. 3(a) illustrates the scanning electron microscopy (SEM) images of the fully exfoliated nanosheets following a 10-hour milling period. The X-ray diffraction (XRD) patterns of the BN nanosheets with different milling times are shown in Fig. 3(b), exhibiting strong diffraction, especially for the (002) and (004) planes. Even after 10 hours of milling, there is no discernible broadening of the (002), (004), or (006) peaks, and the (004) and (006) peaks' relative intensities very slightly decrease. These imply that the BN's in-plane structure is not significantly affected by milling. In contrast, the (100), (101), (102), (110), and (112) peaks nearly vanished after 0.5 hours of milling due to the milled BN's favorable orientation on the substrate as a result of their decreased thickness-to-size ratio. To put it another way, the milling successfully exfoliates h-BN particles to produce h-BN nanosheets.

Fig. 3 (a) SEM images of completely exfoliated h-BN nanosheets subjected to a 10 h milling. (b) XRD patterns of the h-BN nanosheets subjected to different billing times [Reproduced from ref. 35 with authorization. Copyright 2014, Nature].

Numerous studies have demonstrated that high-energy ball milling can break down bulk h-BN into smaller fragments during exfoliation. This behavior was first explained in 2000, when it was found that in the initial stages of ball milling, shear forces could cause h-BN to split along its basal planes, forming partial flakes. However, as the shearing continues, the production of BN nanosheets does not significantly increase; instead, various defects, such as stacking faults, begin to appear in the material.⁶⁵ Furthermore, the symmetry of the crystal lattice can be affected.⁶⁶ This has led scientists to reconsider the ball milling principle and enhance the ball milling procedure in order to produce BN nanosheets with high aspect ratios. Parameters such as selection of solvent buffers, milling speed, ball size, and the proportion of milling balls to raw materials all had a substantial impact on the efficiency of exfoliation and the ultimate output of BN nanosheets. However, the preparation of BN nanosheets by a ball milling process still remains relatively arbitrary due to a lack of quantitative study on these parameters, creating fresh hurdle for the production of BN nanosheets on a wide scale.

3.1.2. Liquid/chemical exfoliation. An alternative method of exfoliation utilizes the volume expansion resulting from the chemical or physical interactions within the h-BN layers. For the most part, chemical exfoliation relies on ultrasonication and the organic solvents' surface tension getting beyond the van der Waals contact between the layers. Sonication-based liquid-phase exfoliation to obtain individual layers from layered materials was first introduced in 2008.⁶⁷ The procedure separates agglomerated species into nanoscale components by using cavitation-induced hydrodynamic forces in a suitable solvent. With the aid of organic solvents' surface tension and ultrasonication, the exfoliation is primarily dependent on the capacity to overcome the interlayered van der Waals interaction. Four essential steps—immersion, insertion, exfoliation, and stabilization—make up the liquid exfoliation of multilayer materials. The adsorption of solvent molecules facilitates immersion and spreading across the surface of the bulk substance. Through the solvent molecules intercalating between the layers, the insertion process weakens the interlayer bonds in between the layers. In the exfoliation step, the sheets are separated and stabilized. The steps involved in sonication-based exfoliation in the liquid phase of the h-BN sheets are shown in Fig. 4.⁶⁸

Fig. 4 Diagram illustrating various stages involved in the liquid exfoliation process of 2D layered materials [Reproduced from ref. 68 with authorization. Copyright 2017, ACS].

Chemical exfoliation assisted by ultrasonication was first introduced by Han et al. for obtaining monolayer h-BN sheets using h-BN particles and 1,2-dichloroethane solution of poly(m-phenyl-enevinylene-co-2,5-dictoxy-p-phenylenevinylene) ultrasonicated for a duration of 1 h. The h-BN sheets of varying numbers, i.e., single, double and triple layers, were obtained in their synthesis process. The loose individual crystals of h-BN are broken up and separated by sonication into mono- or few-layer sheets. The yield and the reduction of damage to the final h-BN sheets' crystal structures both depend on the time of the sonication, which needs to be optimized.⁶⁷ Tian and his coworkers presented an easy way for h-BN exfoliation via the use of a low-power hydrothermal technique with sonication assistance, which is motivated by the traits and workings of the hydrothermal treatment technique and sonication-assisted liquid exfoliation. The solution can be injected into the layers while being subjected to the pressure created by the hydrothermal process. Meanwhile, pressure changes can generate and lead to the collapse of tiny gaps or bubbles of micrometer size in the liquids dissolved in the layers, with h-BN in bulk being exfoliated into nanosheets. This is made possible with the use of the energy of sonication. h-BN was exfoliated using a 60% tertiary butanol aqueous solution, as their surface tensions are similar. Meantime, the solution was simple to remove and did not introduce any lingering impurities into the h-BN nanosheets. More significantly, the technique produces single- or double-layered nanosheets without defects, yielding 1.68 weight% of the original h-BN granules; nevertheless, the result may be increased to 3 wt% through recycling of sediments.⁶⁹ When used as a solvent for h-BN exfoliation, isopropyl alcohol (IPA) yields 50% in the dispersions (0.06 mg mL⁻¹ h-BN nanosheets). The mixed solvent method is applied based on Hansen solubility parameter (HSP) theory by comparing the Ra values of various solvent combinations with the HSP space. For example, Zhou et al. exfoliated h-BN using two weak solvents (ethanol and water) and generated a solution of “milky” h-BN nanosheets with a concentration of 0.075 to 0.003 mg mL⁻¹, which is 37 times the amount of exfoliated material in IPA.^70,71 An eco-friendly ultrasound-assisted exfoliation process using various plant extracts was adopted by Deshmukh and their co-workers for fabricating h-BN nanosheets.⁷² Extracts of different plant materials and h-BN were sonicated in a water bath sonicator operating at 40 kHz for 24 h at 30 °C to obtain the corresponding h-BN nanosheets. The extract of plant molecules undergoes a succession of rarefactions and compressions when ultrasonic vibrations are applied to samples that also contain h-BN. Samples include microbubbles that form and burst due to cyclical variations in pressure. “Acoustic cavitation” refers to the formation, expansion, and deflation of microbubbles. Samples undergo agitation, vibration, shock waves, shear forces, acoustic streaming, and high pressure in addition to these other conditions. Acoustic cavitation, which stresses h-BN sheets as they change across the sp² boron nitride planes of the honeycomb lattice, weakens van der Waals forces separating neighboring layers. The entire schematic process is displayed in Fig. 5.

Fig. 5 Schematic displaying the process of exfoliation of h-BN using an ultrasonication process [Reproduced from ref. 72 with authorization. Copyright 2019, ACS].

Using a UV-visible spectrophotometer, the exfoliated h-BN nanosheets were found to exhibit absorption in the 232–236 nm region (Fig. 6(a)). A wide band at 3432 cm⁻¹, which is equivalent to those of OH or H₂O molecule vibrations, and two pointed peaks at 1385 and 808 cm⁻¹, which are related to the bending vibration of B–N–B and stretching vibration of sp²-bonded B–N, respectively, are visible in the FTIR spectra of h-BN in the bulk and nanosheet form (Fig. 6(b)).^73,74 The presence of these peaks demonstrated that the matrix of materials was retained even following exfoliation. The Raman peak of E_2g (B–N vibration), which is characteristic of h-BN, was detected at around 1367 cm⁻¹ (inset of Fig. 6(c)). According to earlier findings, the E_2g peak shifted to 1417 cm⁻¹, which surpasses that of pristine h-BN (Fig. 6(c)). The patterns observed using XRD, as shown in Fig. 6(d), exhibit diffractions at 2θ = 26.7°, 41.6°, 55.1°, and 75.9°, which correspond to h-BN's crystallographic planes (002), (100), (102), and (110). For the h-BN nanosheets (represented as h-BNNs in the figure), a prominent peak appears at 2θ = 26.5°, corresponding to the (002) plane possessing a 3.341 Å interlayer spacing. The h-BN nanosheets' enlarging (002) plane suggests that it has exfoliated from original h-BN.^75,76

Fig. 6 (a) UV-Visible absorption spectra of pristine h-BN and exfoliated h-BN nanosheets, (b) FTIR spectra of pristine h-BN and exfoliated h-BN nanosheets using different extracts, (c) Raman spectra of exfoliated h-BN nanosheets using different extracts; inset shows Raman spectra of pristine h-BN and (d) XRD patterns of h-BN in bulk and nanosheet forms. Inset shows photo of exfoliated h-BN nanosheets [Reproduced from ref. 72 with authorization. Copyright 2019, ACS].

These methods frequently produce unstable, irregular, and impure h-BN nanosheets that demand time-consuming cleaning and post-treatment procedures.⁴ According to Ma et al., the h-BN nanosheet solution begins to combine in a 12-hour period of dispersion and finishes aggregating following three days.⁷⁷ Consequently, in order to create a stable and uniform dispersion, it is imperative to change the surface of h-BN nanosheets. Additionally, Anderson et al. investigated h-BN nanosheets' consistent dispersion potential after exfoliation in a number of solvent systems under various conditions.⁷⁸ Despite these limitations, chemical methods are generally more straightforward and appropriate for industrial synthesis than mechanical peeling. Several investigations have pointed out that for liquid-phase exfoliation, the choice of solvent has a considerable impact on the final product.⁷¹ Effective exfoliation of h-BN nanosheets and avoiding their aggregation must be balanced by the choice of ideal solvents. The exfoliation of h-BN and the creation of nanosheets are facilitated by solvents with the right polarity, which are useful in lowering the contact force between two neighboring h-BN layers. Therefore, one of the most important aspects of liquid exfoliation remains the identification of a suitable solvent with the right polarity. However, the exfoliation mechanism is yet unknown, which warrants further investigation.

3.1.3. Hydrothermal process. The word hydrothermal indicates that the process employs high temperatures as well as high pressures and is carried out in sealed systems. The solvent evaporates as a result of the reaction being carried out in this method at temperatures higher than the boiling point of the solvent. The bubbles that form in proximity to the h-BN particles burst, which initiates the exfoliation.⁷⁹ This heat driving force encourages intercalation and makes it easier for ions, molecules, and particles to diffuse between the layers. Low-yield h-BN nanosheets with high crystallinity are produced by this technique. The diffusion of Na⁺ or K⁺ ions on the negatively charged sites of h-BN causes curling of the layers followed by the entry of the hydroxide ions between the layers, resulting in an increase in the interlayer distance. Several solvents such as NaOH,⁸⁰ LiOH,⁸¹ mixtures of NaOH and KOH⁸² are used as solvents in this process. However, in solvents such as NMP, DMF, and acetonitrile, the B–N bonds can be broken during the exfoliation causing decreased dimensions of the h-BN nanosheets' sides.⁸³ The schematic illustration of the hydrothermal procedure for creating h-BN nanosheets is shown in Fig. 7(a). The corresponding scanning electron microscopic (SEM) images and TEM images of the exfoliated h-BN nanosheets exfoliated using a KOH solution are displayed in Fig. 7(b) and (c), respectively.

Fig. 7 (a) Schematic showing the hydrothermal process for the synthesis of h-BN nanosheets. (b) and (c) SEM and TEM images of the nanosheets of h-BN [Reproduced from ref. 82 with authorization. Copyright 2019, Elsevier].

The solvent type and heating time are the two most crucial parameters influencing this procedure out of all others. The solvent's boiling point and surface energy when aligned with that of h-BN are also significant factors. Because of its autogenous pressures and moderate operating temperatures (between 100 and 250 °C), the hydrothermal technique is generally considered a scalable and environmentally friendly synthesis method for creating h-BN nanosheets. The use of water as the reaction medium also renders it environmentally safe and non-toxic. However, energy-intensive upstream synthesis may be necessary for the use of some common precursors, such as boric acid, boron oxide, and urea or ammonia. If not disposed appropriately, certain boron compounds can be hazardous in aquatic environments.³⁰

3.1.4. Microwave-aided exfoliation. Microwave-assisted heating techniques have been used for decades to create layered nanomaterials such as sub-micron BN nanosheets. Due to the diverse expansion coefficients and h-BN lattice's varied directions, the localized heat produced by this irradiation technique weakens the bonds and various forces. The microwave method offers a number of benefits, including energy efficiency, high reaction rate, short time of reaction, fast volumetric heating, easy workup and production of homogeneous nanoparticles (NPs). Selection of proper solvents and ions boosts the mechanism and reduces undesirable structural defects. In a study conducted by He and his fellow workers, a microwave heating-assisted method was adopted to obtain h-BN nanosheets through 50 min of microwave heating of a boric acid and urea combination.⁸⁴ The obtained nanosheets possessed rich defect structures, which helped in increased light absorption capability of the material and ultimately exhibited excellent photocatalytic H₂ generation. Fu et al. took advantage of the instant heating initiating from the microwave to develop h-BN nanosheets in an ethanol–sodium hydroxide mixture solution.⁸⁵ The small-sized ions such as Na⁺ and OH⁻ emanating from the mixture solution undergo incorporation inside the layer spacings of 3D h-BN as a result of which the interspace between the layers expands, resulting in peeling off of the h-BN nanosheets from the bulk h-BN. Mild ultrasonication results in further exfoliation of the loosely adhered h-BN nanosheets from bulk h-BN. Fig. 8(A) shows the schematic representation of the microwave-assisted method for nanosheet formation. Fig. 8(B) displays the high-resolution X-ray photoelectron spectroscopy (XPS) spectra. In the nanosheets of h-BN, besides the peak at 190.0 eV related to the B–N bond, a smaller and more noticeable peak appears at 191.7 eV corresponding to the B–O bond, suggesting that hydroxylation was used to exfoliate bulk h-BN. Fig. 8(C) compares the yield of h-BN nanosheets using two different systems, i.e., water–NaOH and ethanol–NaOH system. It should be emphasized that ethanol was employed as the exfoliating solvent rather than water. The primary explanation is that h-BN disperses more evenly in ethanol than it does in water. A more effective intercalation process occurs under microwave irradiation conditions due to the h-BN particle's interaction with Na⁺ and OH⁻ and better dispersion in ethanol. Therefore, using ethanol and NaOH produces a lot of nanosheets than using water and NaOH. The yield of h-BN nanosheets using the ethanol–NaOH system was found to be 1.91 as compared to water–NaOH. The Tyndall effect exhibited by the h-BN nanosheets water sheets is shown in Fig. 8(D). A straight laser path was visible in the BNNS dispersion, suggesting that the BNNS consists of ultrathin sheets. Atomic force microscopy (AFM) images were used to measure the h-BN nanosheet thickness, which was found to be 4.36 nm, as shown in Fig. 8(E).

Fig. 8 (A) Schematic the microwave-assisted exfoliation method for h-BN nanosheet synthesis. (B) XPS spectrum of bulk h-BN and h-BN nanosheets. (C) Yield of h-BN nanosheets in different solvent systems. (D) Tyndall effect exhibited by the h-BN nanosheet suspension. (E) AFM image and (F) SEM image of h-BN nanosheets [Reproduced from ref. 85 with authorization. Copyright 2016, Wiley].

A plethora of other techniques have been developed and documented in the recent literature, including high-pressure micro fluidization in conjunction with chloroform/DMF mixed solvents, rapid quenching and liquid exfoliation, electric field-assisted liquid exfoliation, and magnetic stirring-assisted ultrasonication technique intended for large-scale production.⁸⁶ The main disadvantage of the aforementioned techniques lies in their lower reliability in terms of effectiveness and selectivity. Another major disadvantage is the extraction of h-BN by centrifugation, which undoubtedly leads to the production of the separated material in smaller amounts than the initial bulk h-BN powder precursor. Consequently, it was found that the samples made by liquid-phase exfoliation methods yielded fewer crystalline h-BN nanosheets than the samples made by intermediate-assisted grinding exfoliation methods.⁸⁷

3.2. Bottom-up methodology

3.2.1. Chemical vapor deposition. Solid materials with superior quality and performance are made by the vacuum deposition technology known as chemical vapor deposition (CVD). The CVD method has shown great promise for the synthesis of high-quality, large-area, mono- or few-layer h-BN nanosheets with variable thickness. These methods excel at creating precise 2D atomic groupings. This technique entails constructing superior and efficient nanomaterials on substrates. Either atmospheric pressure (APCVD) or low pressure (LPCVD) can be used for the procedure, depending on the environment of the CVD chamber. It is important to highlight that the APCVD approach is less expensive and requires less resources to prepare h-BN products than LPCVD and ultra-high vacuum systems, despite a sharp decline in product quality. The substrate also significantly influences the shape of h-BN generated by CVD. For instance, Ru(0001)⁸⁸ and Rh(111)⁸⁹ substrates yields meshy h-BN monolayers, while Pd,⁹⁰ Pt,⁹¹ Cu,⁹² Co,⁹³ Ni,⁹⁴ Fe,⁹⁵ and graphite⁹⁶ produces monolayer boron nitride. Several parameters such as input rates, background pressures, and precursor purity affect the crystalline structure, number of layers and lateral size of the h-BN nanosheets. Fig. 9 depicts the schematic diagram of a LPCVD system utilized for h-BN synthesis. In addition to equipment, the substrate is a crucial factor that determines the shape of h-BN generated by CVD.

Fig. 9 Schematic showing the CVD synthesis process [Reproduced from ref. 97 with authorization. Copyright 2013, ACS].

Using NH₃BH₃ as the precursor, Wang et al. deposited h-BN nanosheet layers with an elastic modulus of 200–500 Nm⁻¹ and a band gap of 5.5 eV.⁹⁸ Using the LPCVD process, triangular and monolayer BN films can be deposited on Cu and Pt foils using NH₃BH₃ as the precursor. Upon decreasing the roughness of Cu, large-sized h-BN nanosheets can be deposited via APCVD due to the lowering of the density of nucleation and the increase in the domain sizes for BN.

The factors that mostly influence the final morphology of the precursors are their type, purity, and sublimation temperature, and these powerful variables may be adjusted throughout the CVD process. For instance, studies comparing the common NH₃BH₃ precursor to its derivatives, such as dimeric diborazane (H₃BNH₂BH₂NH₃; DAB) and trimeric triborazane (H₃B(NH₂BH₂)₂·NH₃; TAB), demonstrated that the three precursors' growth rates for the films were not the same, despite the fact that all three were capable of producing continuous films. The most potent antecedent, DAB, could create h-BN films in a few layers faster than NH₃BH₃ and TAB, whereas NH₃BH₃ could produce thinner h-BN films. Additionally, as the h-BN transitioned from NH₃BH₃ to TAB, more nanoparticle contaminants were formed. Trimethylamine borane (TMAB) sublimates at lower temperatures than NH₃BH₃,⁹⁹ however when borazine is used in place of ordinary solid NH₃BH₃, monolayers without harmful h-BN particles develop.¹⁰⁰ As impurities usually lead to an excessive number of nucleation sites, great purity is required as the development covers extensive regions in addition to the meticulous choice of antecedents. However, a straightforward filtering technique can be employed to get rid of harmful h-BN particles if there are only poor-quality precursors available.¹⁰¹

The physical features of products can also be altered by growth time and substrate temperature. According to experimental findings, increasing the growth period causes the domain size to increase.¹⁰² Pakdel et al. established a heat-based CVD approach to produce h-BN nanosheets that are pure, C-doped and arranged vertically using B, MgO, and FeO powders under an NH₃ flow on Si/SiO₂ substrates in a horizontal tube furnace.³⁷ Mostly, the nanosheets were thinner than 5 nm, however, as the SEM photos demonstrate, thicker nanosheets were created by increasing the growth temperature or prolonging the development period, as shown in Fig. 10.

Fig. 10 SEM images displaying the surface morphology of h-BN nanosheet coatings under various growth conditions: (a) uniform BN coating formed at 900 °C with a 30 min growth duration, (b) partially vertically aligned nanosheets produced at 1000 °C for 30 min, (c) nanosheets synthesized at 1100 °C for 30 min, (d) coating grown at 1200 °C for 30 min, (e) BN layer developed at 900 °C with a 60 min growth time, (f) partially vertical nanosheets observed at 1000 °C after 60 min of growth, (g) sample obtained at 1100 °C for 60 min, and (h) nanosheet coating formed at 1200 °C with a 60 min duration. [Reproduced from ref. 37 with authorization. Copyright 2011, ACS].

Flow rate is a crucial CVD factor that influences the number of nucleation sites and the size of current domains.¹⁰³ h-BN thin films with high crystalline quality are converted into irregular films with an amorphous structure when the PTOT for the background pressure of Ar/H₂ in the CVD chamber is increased from low pressure of 2 Torr to atmospheric pressure of 760 Torr.¹⁰³ Higher total pressure (PTOT) actually results in a higher pressure of H₂, accelerates expansion and makes it more difficult for the H₃N–BH₃ precursor to decompose. Even under LPCVD conditions, the high H₃N–BH₃ flux ratio results in a faster growth rate and the emergence of undesired amorphous forms. N₂ gas filling the chamber has additionally shown a strong capacity to change the substrate's structure, notably Ni's. The inability of Ni to dissolve the atoms of nitrogen results in the creation of h-BN molecules with irregular triangle shapes and partially B terminated edges. The undesired structure, however, is changed into a completely triangle form with a N atom at the end when N₂ gas is expelled by preventing the removal of adsorbed N atoms from the surface of Ni(111).

According to recent studies, vertically oriented h-BN nanosheets (v-BNNSs) can also emerge, while the majority of 2D-BN nanostructures produced via CVD form in line with the substrate. This form enables the building of extremely thin h-BN nanosheets (having a thickness that is less than 10 nm), as well as the realization of exceptional hydrophobic properties, thanks to the trapped air between the nanosheets.¹⁰⁴

Although the CVD technology has made substantial progress for h-BN nanosheets synthesis, the need to produce large, single h-BN nanosheets with minimal grain boundaries and high surface smoothness to fully realize the material's potential for future applications still persists. The major issues that still need to be resolved involve lowering of growth temperature, managing layer thickness on nonmetal substrates, and producing good crystal quality without using adequate catalysts. Catalytic substrates such as Cu and Ni are still necessary for the synthesis of large and single h-BN nanosheets. The investigation of techniques for producing large, superior nanosheets on non-catalytic substrates is therefore necessary along with control of the uniformity and quality of the defect sites.⁹⁷

3.2.2. Physical vapor deposition. Physical vapor deposition (PVD) is extensively used for film growth because it avoids the intricate interaction of growth parameters required in CVD. By vaporizing the surface of the source material into ions, molecules, and atoms under vacuum, the PVD technique produces a thin coating on the substrate. PVD growth basically occurs in three stages: (1) particle formation from the material source (evaporation and sublimation); (2) particle transit to the substrate; and (3) film development when the particles are deposited on the substrate.⁹⁷

Deposition methods using magnetron and ion beam sputtering are two widely used and extremely efficient processes enabling the creation of extensive monolayer h-BN.¹⁰⁵ Ar⁺ ions are propelled towards a target of solid boron in a standard magnetron sputtering setup when there is a N₂/Ar environment, which causes B atoms to evaporate.¹⁰⁶ As a result, it is feasible for the boron that has evaporated on the Ru substrate to interact chemically with the already radicalized nitrogen environment. The preheated substrate offers B and N a perfect place to land in order to produce the first h-BN nuclei. When B and N atoms arrive, they bind to the locations of nucleation to create h-BN domains. This indicates that the ultimate morphology and ensuing characteristics of the generated h-BN are frequently influenced by the circumstances of the substrate. A large number of metals including Au, Ni, and Cu have been already explored as substrates. By sputtering deposition using an ion beam (IBSD) technique, Gao et al. developed h-BN on Ni(111) films with a size up to 0.6 mm. The same group extended their work on developing a catalyst-free route to obtain high-quality 2D h-BN layers on a sapphire substrate by the same IBSD technique.¹⁰⁷ The grown h-BN layers were highly non-stoichiometric containing excess B that resulted in more N vacancy defects and low crystallinity. However, this technique can yield high-quality, stoichiometric h-BN layers on sapphire substrates at low temperatures when combined with nitrogen and a surface nitridation process that uses an ion beam instead of an Ar beam. Fig. 11 displays the picture of a developed h-BN layer on a sapphire substrate measuring two inches by a N⁺ sputtering technique combined with surface nitridation, which illustrates highly uniform and transparent topographies. The cross-sectional HRTEM images are displayed in Fig. 11(b) and (c) for sample A (an Ar-ion beam is used to prepare sample A without surface nitridation) and D (a N-ion beam with surface nitridation is used to prepare sample D), respectively. In comparison to sample A that shows amorphous nature, sample D exhibits a well-ordered layered structure with a lattice fringe of 0.35 nm corresponding to the basal plane of h-BN. The h-BN sample was found to consist of 8–9 atomic layers with about 3 nm thickness. The elemental mapping as demonstrated in Fig. 11(a)–(h) confirms the presence of B and N with uniform distribution on sapphire. Sample D prepared combining both the N ion beam and surface nitridation produces highly crystalline h-BN nanosheets.

Fig. 11 Few layers of h-BN produced on sapphire (a) h-BN layers grown on a wafer of sapphire. (b) and (c) Cross-sectional HRTEM images. (d)–(h) Elemental mappings of Al, O, C, B and N elements [Reproduced from ref. 107 with authorization. Copyright 2019, RSC].

The final properties of h-BN nanosheets in techniques for physical vapor deposition are mostly influenced by temperature and processing time. An amorphous phase is found when a Cu substrate is employed at 600 °C in temperature, proving that necessary heat as the crystallization's primary driver was not present. However, increasing the temperature from 600 °C to 1050 °C causes the crystalline structure to emerge from the amorphous phase.¹⁰⁸ However, high temperatures also cause the desorption of active species to predominate over domain growth and cause domain sizes to decline again.

Additionally, the product qualities are effectively controlled by the substrate pretreatment, ion beam density, and ambient conditions. When H₂ gas is introduced into the processing environment, 1 μm irregular polygonal h-BN domains change into 5 μm triangular h-BN domains in contrast to the pure Ar environment. The primary causes of this increase in lateral size are the removal of the smaller h-BN nuclei and the breaking of the edge-attachment barrier. While insufficient ion beam density lacks in supplying enough precursors for grain formation, excessive ion beam density results in high-feeding sputtered atoms and a reduction in domain size. Nevertheless, the h-BN nanosheets produced by this approach are affected by lower scaling, less effective utilization of the target, possibility of nonuniform deposition, charge-up, and ion-induced bombardment damages.^26,30

3.2.3. Pyrolysis. Pyrolysis is another widely adopted technique for fabricating large-scale and high-yield h-BN nanomaterials from precursors containing both B and N. Typically, it begins with the low-temperature evaporation of the precursors, followed by the interaction at high temperatures between the vaporized parts and the eventual production of h-BN. The usual precursors utilized in this process include urea, boric acid, ammonia, and borane.^109,110 According to the postulated mechanism, increasing the temperature below 200 °C causes the ammonia borane to break down and transform into an amorphous crystal.¹¹¹ However, B and N atoms are able to reorganize and transform the transition from the amorphous phase to crystalline h-BN nanosheets at higher temperatures up to 1400 °C. Most commonly used precursors include boric acid and urea. A pyrolysis method adopted by Matheswaran et al. for synthesizing h-BN nanosheets from boric acid and melamine under a N₂ atmosphere at 900 °C is shown in Fig. 12.¹¹² The method yielded h-BN sheets that have a specific surface area of 39.252 m² g⁻¹ with the dimension and volume of pores of 2.205 nm and 0.055 cc g⁻¹, respectively.

Fig. 12 Pyrolysis process adopted for the synthesis of h-BN nanosheets [Reproduced from ref. 112 with authorization. Copyright 2023, Elsevier].

Selecting a proper starting material as the precursor and determining the proper annealing temperature are highly important for developing h-BN nanosheets with the desired morphology and crystallinity. High-index facets of urea are formed in low-boiling-point alcohol solutions in water, particularly when methanol and water are included.¹¹³ As a result, atomic distances increase, enabling the production of extremely thin h-BN sheets that have the greatest recorded specific surface area of 1900 m² g⁻¹. Large and thick h-BN nanosheets with a squamous structure are created when the antecedents are initially air-cooled to 900 °C during annealing and then post-annealed at 1200 °C in NH₃. The porous h-BN nanosheets produced by direct annealing around 1200 °C under an NH₃ flow are thinner and smaller and lack squamous structure. Conversely, annealing is useful for bringing down boron atomic distances, and speeds up development by turning [Zn(Ac)₂] into ZnO. Later, the decomposition of [Zn(Ac)₂] to carbon oxides and hydrocarbons lowers both the dimensions and the thickness of the nanosheets. Apart from boric acid, a large number of other borates as B sources have been studied by various researchers to obtain h-BN nanosheets. When a precursor containing a high B content such as MgB₄O₇ is used, thicker nanosheets are obtained, while for precursors such as Mg₃B₂O₆ thin-layered h-BN nanosheets with high specific surface areas are formed.^114,115 The pyrolysis process is simple and cost-effective, thus making it accessible for both research and industrial applications. It is a scalable process capable of producing materials from gram to kilograms, essential for commercial use. It also allows for a wide range of precursor choices, offering flexibility in synthesis routes. Controlling lateral dimension and crystallinity, reliably producing monolayer or few-layer nanosheets, and efficiently eliminating carbon or remaining impurities are among the difficulties associated with this process.¹¹⁵

3.2.4. Pulsed laser deposition. Pulsed laser deposition (PLD) technique is an additional synthesis method of h-BN. Using this technique, highly uniform and pure nanosheets are developed. The hypothesized process states that before the production of the nanosheets can start, a little portion of the material where the light from the laser is impacted rapidly warms up. The newly formed plasma plume, which is made up of high-energy ions and clusters of boron nitride, is now creating a powerful electric field to aid in the movement of surface organisms. As soon as the accommodated ions start to move across the heated substrate's surface, a thin h-BN layer starts to develop. The subsequent covalent bonding of the entering ions B and N having the edge atoms is supported by the high temperature. Adopting the PLD technique, direct expansion of h-BN films was carried out on non-catalytic sapphire substrates. The h-BN films' crystalline quality is most strongly influenced by the substrate temperature, and the ideal pressure varies with target-substrate distance. The h-BN films produced by the PLD technique were found to be highly crystalline in nature.¹¹⁶Fig. 13(a) depicts the PLD experimental set-up, where the laser was operated with a 500 mJ pulse energy in terms of repetition rate of 5 Hz. The SEM images indicate uniform and continuous nature of the films (Fig. 13(b) and (c)) and the corresponding optical microscopic image appears in the inset of Fig. 13(b).

Fig. 13 (a) Experimental set-up of the PLD technique adopted for synthesizing the h-BN nanosheets. (b) and (c) Corresponding SEM images. The inset in (b) displays the corresponding optical microscopy image. [Reproduced from ref. 116 with authorization. Copyright 2021, Elsevier].

Ultrathin sheets of h-BN were deposited on a lattice-matched highly ordered pyrolytic graphite (HOPG) substrate by the PLD technique.¹¹⁷ The HOPG's lower surface energy compared to sapphire results in a prolonged growth time for incoming ions and development that resembles epitaxial growth. Due to the substrate surface being pre-etched, the H₂ environment rather than a vacuum environment effectively regulates the sputtering phenomenon and strengthens the h-BN nanosheets' purity. Their report was the first of its kind where the PLD technique was adopted to fabricate ultrathin crystalline h-BN films with grain sizes of approximately 5 nm and thickness of 1.5–2 nm. Several other synthesis processes such as plasma etching, electrospinning, and surface segregation method have additionally been investigated for the synthesis of h-BN nanosheets. PLD offers several key advantages for the synthesis of h-BN nanosheets including high-purity, stoichiometric films with precise layer-by-layer thickness control. It is also versatile in terms of substrate compatibility and allows deposition on a wide range of materials, including semiconductors and metals. Additionally, the process is scalable and compatible with industrial manufacturing processes, making it a promising method for the large-scale production of h-BN nanosheets for advanced electronic and optoelectronic applications. Despite the advantages, PLD also presents several challenges such as uniform deposition over large areas, which remains difficult, and it can affect film consistency and device performance. Additionally, controlling the in-plane crystallinity and domain size of the layers is a challenge, impacting the material's electronic and mechanical properties. Furthermore, the complexity and high cost of PLD equipment can hamper large-scale industrial applications.^117,118Table 1 presents the comparison of different synthesis methods with their advantages and limitations.

Table 1 Comparison of different methods for the synthesis of h-BN nanosheets

Method	Advantages	Disadvantages	Yield	Cost	Purity	Ref.
Mechanical exfoliation	Ease of operation, low cost, ability to avoid high temperatures and vacuum systems, large area nanosheets production with good dispersion, stability and high yield.	Potential structural defects, control over thickness, size and quality is challenging.	Moderate	Low	High	119 and 120
Liquid/chemical exfoliation	It's a scalable, low-cost, and relatively simple approach, easy and uniform dispersion of h-BN nanosheets in solutions, which is essential for a number of applications.	Possible solvent toxicity, surface contamination, and challenges in regulating the final nanosheets' thickness	Moderate	Moderate	High	121 and 122
Hydrothermal process	Produces monodisperse, chemically homogeneous, and well-dispersed materials under mild conditions, environment friendly, economical, efficient	Difficulty in precisely controlling the thickness, size and properties of the nanosheets, low exfoliation efficiency, possibility of explosion, tendency to agglomerate	Moderate	Low	High	123 and 124
Microwave aided exfoliation	Quick and inexpensive process, scalability, and the capacity to generate materials of superior quality, environment friendly	Low-scale production due to size limitation of equipment, uneven exfoliation due to non-uniform heating, production of flakes of limited size	Moderate	Low	High	125 and 126
Chemical vapor deposition	Superior quality, controllable growth, and the ability to tailor properties, scalability for large-area production, high-purity materials, and exact control over film thickness and structure	High temperature, expensive equipment, slow growth rate	Low to moderate	High	High	127
Physical vapor deposition	A safer and easier method than CVD, control over film characteristics, and the capacity to deposit on a variety of substrates, production of consistent, high-quality films with exact control over thickness.	PVD is slower than CVD, necessitates sophisticated and costly equipment, and has little success in regulating the characteristics of the deposited h-BN nanosheets	Low	High	High	128
Pyrolysis	Economical, simple, low energy consumption, high efficiency	Precise temperature control required to avoid contaminants, post-treatment limits large-scale production	Low to moderate	Moderate	High	129
Pulsed laser deposition	Capacity to process materials at reduced temperatures, high energy density, and exact control over film composition and quality	Particulate formation, uneven coating thickness, and poor substrate adherence, high energy requirement	Low	High	High	130

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4. Functionalization of h-BN nanosheets

h-BN is a robust material with a wide bandgap, thus possessing insulating electrical and thermal properties. However, these properties can be enhanced to meet the properties of other similar materials including graphene, 2D transition metal dichalcogenides (TMDs), and polymers, by creating heterostructures or nanocomposites. A few techniques for the functionalization of the h-BN nanosheets' surface and characteristics are covered in this section.

4.1. Defect engineering

During the synthesis of 2D h-BN, various defects such as grain boundaries, vacancies, and irregular edges are introduced, which have an impact on the properties of both the material and the devices incorporating it. Due to its lower production energy than the N vacancy, the B vacancy is theoretically more thermodynamically advantageous.¹³¹ These defect sites notably enhance the chemical reactivity of the BN monolayer. Defects could result in discrete, smaller, stacked h-BN sheets, altering the structure and adding novel chemical and physical characteristics. For instance, the gradual transformation of a radiation-induced boron vacancy into a triangular hole was noted by Lei et al.¹³² The creation of chains composed of N and B atoms is the first step in this multi-phase process. The optical and electrical properties of the nanosheets are greatly affected by the structural changes caused by point vacancies and edge effects, which result in a noticeable decrease in the work function and the energy band gap. This offers a chance to explore novel approaches for regulating the type and quantity of flaws in order to customize the electronic configuration of h-BN. Activated hexagonal boron nitride, ABN-LiBH₄ and h-BN were combined to form a lithium-functionalized activated nanocomposite by Muthu et al. for use in hydrogen storage.¹³³ The results indicated that the addition of LiBH₄ can reduce the aggregation of ABN. Research indicates that ABN's capacity to store hydrogen is enhanced by the LiBH₄ modification, increasing from 0.29 wt% for unmodified ABN to 1.67 wt% for ABN-LiBH₄ at 100 °C. Lin et al. demonstrated the structure and behavior of linear 4|8 s defects at h-BN grain boundaries.¹³⁴ These defects shifted in response to continuous electron beam irradiation and high temperatures. Furthermore, compared to the 4|8 s grain, the 5|7 s grain's border bandgap is substantially less. Defect engineering has been shown to be one of the most successful methods for functionalizing h-BN sheets. However, other factors such as temperature control need to be taken into account and adjusted properly.

4.2. Doping

Doping is a useful method for improving the performance by changing the electrical structure and surface atomic microstructure of nanomaterials. The most common dopants for h-BN are carbon, oxygen, and fluorine. Numerous unique characteristics or significant changes over the inherent property of BN can be produced by doping specific heteroatoms. Chemical methods such as using H, –NH₂, –F, and –OH ligands and physical methods such as electrostatic adsorption are frequently used to functionalize h-BN heterostructures and porous architectures. This results in improved solvent dispersibility, adjustable surface affinities, biocompatibility, excellent processability, and a tunable energy bandgap, which are crucial for numerous applications. For instance, addition of carbon to h-BN shifts the bandgap from 5.9 eV to 2.6 eV, which is encouraging for non-metal photocatalysts. In order to produce a ternary B–C–N hybrid that is active in both CO₂ reduction and overall water splitting, Huang et al. modified the bandgap of h-BN by adding the appropriate amount of carbon.¹³⁵ Besides carbon doping, studies have been conducted on h-BN nanosheets doped with oxygen. Weng et al. used boric acid and hexamethylenetetramine in a thermal reaction with ammonia at 1000 °C to fabricate oxygen-doped h-BN nanosheets. A minimal oxygen doping concentration of 23.1 at% results in a lowered bandgap of 2.1 eV for the yellow h-BN catalyst.¹³⁶ In a different work, Xue et al. used the sol–gel process to create ultrathin BN nanosheets with oxygen doping that had two to six atomic layers. The addition of oxygen atoms to the BN nanosheets via the creation of B–O bonds was validated by XPS investigation. These oxygen-doped BN nanosheets have a remarkable potential to store hydrogen in comparison to other BN materials that have been previously reported.¹³⁷ Doping h-BN nanosheets with F atoms has been done experimentally to discover a novel way to manipulate the electrical properties of h-BN.¹³⁸ As h-BN changed from an insulator to a semiconductor, high electrical conductivity was achieved at currents of up to 15.854 mA. In order to provide intriguing gas sorption, fluorescence, and electrochemical energy storage capabilities and applications, co-doping with carbon and oxygen may enhance the surface, optical, and electronic characteristics of h-BN.¹³⁹

4.3. Pyrolysis and hybridization

Pyrolysis can create homogenous heteroatom doping and more material modifications than other modification techniques, where functionalization is often carried out at edge or defect site.¹⁴⁰ Combining several 2D material types can result in structures with a wide variety of heterostructures and superlattices. Multiple bandgaps can be added to the heterojunctions to create quantum wells that meet the requirements for charge flow.¹⁴¹ Additionally, heterostructures encourage the creation of defects, which improves the dispersion of charge carriers and increases the electrochemical activity by using free electrons.¹⁴² Moreover, coupling the metal d_z² orbitals with the N-p_z and B-p_z orbitals of h-BN enables tuning of its electrocatalytic performance through electrical interactions with the supporting metal substrate.¹⁴³ Early studies of graphene and h-BN hybrid structures have shown the likelihood of combining carbon atoms with nitrogen and boron atoms to create a variety of B–C–N stacking designs. These studies created a new pathway for the carbon substitution method of generating h-BN from graphene. By reacting with ammonia gas and B₂O₃ powder, these studies created a novel pathway for generating h-BN from graphene via a carbon substitution mechanism.¹⁴⁴ A graphene/h-BN lateral heterostructure on SiC was produced by Bradford et al. using the substitution process from ammonia and boric acid precursors.¹⁴⁵ The creation of graphene hybrid structures employing h-BN produced tunable epitaxial lateral and upright heterostructures because of similarities in growth temperature and development conditions, such as a low-pressure gas flow, growth temperature (1000 °C), and metallic substrate. Nevertheless, the high reactivity of the metallic substrate (Cu/Ni) with S gas molecules and the change in growth temperature make the creation of TMD/h-BN heterostructures very challenging. On copper substrates, epitaxial graphene/hBN heterostructures were created by CVD growth at 1000 °C using the cation substitution method between ammonia gas and boric acid, as reported by Gong et al. in another study.¹⁴⁶ BN nanosheets developed by this method, owing to their distinctive properties, are used in various applications such as spacers, protective layers in tunneling systems, transistors, deep ultraviolet light emitters, solid-state lubricants, and electrocatalysts in energy conversion technologies.¹⁴⁷

5. Biomedical applications

Exceptional qualities such as hydrophobicity,¹⁴⁸ electronegative surface charge,¹⁴⁹ high water dispersibility,¹⁵⁰ biodegradability,¹⁵¹ and hierarchical porosity¹⁵² make 2D h-BN a potential contender in diverse fields. However, the utilization of h-BN in biomedical field is limited by its poor solubility in aqueous and physiological fluids. Numerous methods, such as wrapping, interacting with guest molecules, or surface functionalization with hydroxyl groups, have been shown to address its low solubility. The possibilities of h-BN in numerous biological fields, including biosensing, tissue engineering, drug delivery, and bioimaging, is covered in this section.

The following section discusses biomedical uses of h-BN nanostructures in terms of their main diagnostic tools (bio-imaging and biosensing) to therapeutic applications (drug delivery, tissue engineering, wound healing, and treatment for cancer and neurological diseases).

5.1. Bioimaging

In biomedical applications, bioimaging is regarded as a developed technology for a better understanding of both straightforward and intricate biological processes in cells, tissues, and the entire body. Additionally, bioimaging can assist in identifying aberrant processes related to cancer and other disorders. Recently, various well-established bioimaging technologies including positron emission tomography (PET), computed tomography, photoluminescence imaging (PLI), fluorescence imaging, and optical imaging have been developed. These technologies have all been used extensively in the diagnosis of cancer.¹⁵³ For the imaging of cancer cells, numerous research teams have made use of the fluorescence characteristics of h-BN. Kumar et al. aimed to produce BN nanoflakes (BNNF) exhibiting strong luminescence peaks in the visible blue region at 411 and 435 nm. It was discovered that 10 or 20 g mL⁻¹ concentrated BN nanoflakes were appropriate for marking cancer cells (MCF-7) and visualizing them with a confocal microscope.¹⁵⁴ It was evident from the confocal images that fluorescent BNNFs at a concentration of 1 g mL⁻¹ are not very effective at attaching to and imaging MCF-7 cells. However, it has been discovered that BNNF doses of 10 g mL⁻¹ and 20 g mL⁻¹ are appropriate for identifying the cancer cells. Another noteworthy aspect that was observed was that, even after a 24 h incubation period, no morphological change in the cancer cells could be seen when fluorescent BNNFs were added. This fact explained that the fluorescent BNNFs had no cytotoxic effect on the cancer cells under test. The cytotoxicity effects of BNNFs were analyzed through the MTT assay. The study depicted that when 10 μg mL⁻¹ of BNNFs were present, 97% and 94% of MCF-7 cells remained alive for the full 24 and 48 h incubation periods, respectively. Cell viability gradually dropped to 80% after 48 h of incubation when the BNNF concentration was increased to 60 μg mL⁻¹, and it dropped slightly by 1% (p < 0.05) after 24 h. Thus, the results showed that BNNFs had very little toxicity and seemed to be ideal for bioimaging applications. Due of its capacity to emit blue light when exposed to UV light, hydroxyl-functionalized hexagonal boron nitride (h-BN–OH) was employed for imaging subcutaneous cancers.¹⁵⁵ KB cells were co-cultured and incubated for 4 h to enable h-BN–OH to concentrate in the cell in order to examine the h-BN–OH's cellular uptake and in vitro imaging capabilities. A confocal laser scanning microscope was used to image the KB cells, confirming a notable cellular uptake of h-BN–OH (as shown in Fig. 14). It was noted that a sizable quantity of h-BN–OH was probably found inside the cells. It is believed that the cancer cells absorb non-specific h-BN–OH via a nonspecific EPR/RES mechanism. Using an MTT colorimetric test, the in vitro cytotoxicity of pure h-BN and h-BN–OH at varying doses was assessed in KB cells after a 24 h incubation period. Even at a greater dose (500 μg mL⁻¹), no discernible toxicity of pristine h-BN or h-BN–OH was found because over 90% of the cells were alive. Different quantities of h-BN–OH (10, 20, 50, and 100 μg mL⁻¹) were added to rat blood and co-incubated for the in vitro hemolysis experiment. Even at a high concentration (100 μg mL⁻¹), only about 4% hemolysis was detected, indicating that h-BN–OH is a blood-compatible substance. Additional in vivo research was required to comprehend the biological and biomolecular interactions and biodistribution, even though the initial in vitro tests showed biocompatibility with blood.

Fig. 14 (a) Confocal images and (b) bio-SEM images of KB cells incubated with h-BN–OH. (c) Cytotoxicity of h-BN and h-BN–OH. (d) Hemolytic effect of h-BN–OH [Reproduced from ref. 155 with authorization. Copyright 2016, RSC].

A popular radioisotope in PET imaging, technetium-99m (^99mTc), has been successfully radiolabeled onto BN-PEG nanoparticles by Liu and his co-workers.¹⁵⁶ This study developed PEG-coated BN (BN-PEG) nanoparticles of size ∼10 nm, but with a poor fluorescence quantum yield of ∼7.8% thus making them unsuitable for detecting signals from biological samples. Fluorescence labeling and imaging were thought to be a common way to investigate BN-PEG in vivo, although nonquantitative and less precise biodistribution results could come from possible quenching and other inherent limitations of fluorescence imaging. The biodistribution and pharmacokinetics investigations based on ^99mTc-labeled BN-PEG, in contrast, offered more accurate and quantitative data regarding the in vivo biocompatibility behaviors of functionalized BN. Thus, BN-PEG nanoparticles with high water solubility were synthesized and labeled with ^99mTcO₄⁻, and the toxicity and biodistribution of BN-PEG in mice were investigated in vivo. ^99mTc-labeled BN-PEG nanoparticles accumulated primarily in the liver and spleen, with negligible retention in the brain, according to in vivo biodistribution experiments conducted in mice. Histological slice observations indicated that it may result in visible tissue lesions in the heart, lungs, liver, and spleen. The discovery of cardiac injury is a new and intriguing finding for comprehending and managing the toxicity of BN nanoparticles in mice; the results are further supported by tests of biochemistry parameters such as Cys-C, CREA, ALT, AST, TB, CRP, and BUN in blood. BN-PEG nanoparticles are therefore dangerous substances in vivo, and their biological toxicity was suggested to be considered seriously.

A related work used ∼^99mTc to radiolabel BNNTs functionalized with glycol chitosan.¹⁵⁷ These radio-labelled nanotubes were injected into mice to examine their in vivo biodistribution behavior. Photon correlation spectroscopy (PCS) was used to assess their size, distribution, and homogeneity, and laser Doppler anemometry was used to measure their zeta potential. Additionally, ex vivo biodistribution experiments and scintigraphic imaging in healthy mice were used to assess the BNNTs. The findings demonstrated that within 24 h, the nanostructures had accumulated in the stomach, spleen, and liver before removal by renal excretion. According to the study, functionalized BNNTs may be used as novel medications or radioisotope nanocarriers in therapeutic processes.

The synthesis of BNNTs radiolabeled with gadolinium and samarium for potential use in noninvasive tumor imaging was demonstrated by da Silva et al. A study of BNNT using X-ray fluorescence spectroscopy revealed that 75.3 wt% samarium and 71.1 wt% gadolinium were present.¹⁵⁸ The samples that were exposed to radiation exhibit significant specific activity. The GdBO₃-BNNTs also have magnetic characteristics. At a concentration of 10 μg mL⁻¹, biocompatibility tests demonstrated great biocompatibility in both SAOS-2 cells and fibroblasts. Their findings demonstrated that the systems under study had a great deal of promise for use in biomedicine and can be employed as non-invasive imaging agents, such as MRI contrast media and scintigraphy radiotracers, which can aid in the simultaneous diagnosis and treatment of numerous tumor types.

5.2. Biosensing

Materials that are nanosized with low dimensions (0D, 1D, or 2D) have a better surface-to-volume ratio or mass than that of 3D technologies. This makes them more effective applications involving chemicals and biosensing.¹⁵⁹ The electrical characteristics of the substance beneath are more strongly impacted by increasing the number of locations where the analyte can bind to the sensor surface. This leads to an elevated level of sensitivity and a lower limit of detection (LOD) for the sensor.¹⁶⁰ This section will present an overview of electrochemical and colorimetric sensing of biomolecules that are relevant to the biomedical field.

5.2.1. Electrochemical sensors. Electrochemical sensors use different techniques such as amperometry and cyclic voltammetry (CV) for detecting changes in faradaic current or electrochemical impedance spectroscopy (EIS) for detecting changes in interfacial impedance. By designing a specific working electrode (WE), that incorporates a biorecognition element such as glucose oxidase (GOx), along with synthetic catalysts like hybrid materials, transition metal catalysts, or two-dimensional materials it is possible to detect specific substances using redox reactions. These processes occur when the analyte interacts with the biorecognition layer, and the electrochemical current as determined by the WE noticeably alter as a result. In EIS techniques, redox mediators can be used to indirectly detect a particular target analyte's capture on the functionalized WE. Following the collection of the target analyte, the redox mediator's accessible surface area shrinks,¹⁶¹ which results in a reduction of the electrochemical current or a rise in the resistance of the interface.¹⁶²

Khan et al. utilized 2D-h-BN as the foundation for an electrochemical sensing platform. They demonstrated this by simultaneously detecting DA and determining uric acid (UA) levels through drop-casting 2D-h-BN onto screen-printed graphite microelectrodes (SPEs).¹⁶³ Dopamine (DA) is a vital neurotransmitter in the brain, while uric acid (UR) primarily functions as an antioxidant in the body. However, high levels of UR are linked to gout, a condition that leads to arthritis. The electrochemical response strongly relies on the interaction of 2D-h-BN with the substance of the electrode underneath and the amount of material deposited. This results in an electrocatalytic response compared to bare carbon electrodes. By optimizing the conditions, they have been able to achieve a suitable peak resolution between DA and UA with competitive electroanalytical outputs. By combining low temperature combustion synthesis (LCS) with carbothermal reduction and nitridation techniques, Li et al. created flake h-BN for the electrochemical detection of uric acid (UA), dopamine (DA), and ascorbic acid (AA). Flake 2D-h-BN was used on glassy carbon electrodes (GCE), which possess numerous defects and active surface groups. As a result, there are wider linear ranges, lower limits of detection, and improved anti-interference capabilities.¹⁶⁴ Khan et al. conducted a study regarding the application of surfactant-exfoliated two-dimensional h-BN nanosheets for electrochemical detection. The nanosheets were evaluated for their ability to detect dopamine even in the presence of typical interferents such as AA and UA. To carry out this study, surfactant-exfoliated 2D-h-BN nanosheets (consisting of 2–4 layers) made with an aqueous medium containing sodium cholate were used.¹⁶⁵ The h-BN sample was coated on screen-printed graphite electrodes (SPEs) via a drop-casting modification process. The working capacity of 2D-h-BN-modified SPEs was carefully analyzed to show how the amount of coverage affected their ability to detect biomolecules. They attempted to detect DA and AA simultaneously, but it did not work (Fig. 15(A)). Only one oxidation peak was detected at approximately +0.32 V, with a peak current of 15.7 μA. However, successful detection of DA and UA was achieved simultaneously. An experiment was conducted to test the electrochemical response of DA and UA (both at a 0.5 mM concentration) in pH 7.4 phosphate buffer solution (PBS) using an unmodified SPE. The results indicated corresponding peaks of oxidation at approximately +0.28 V (with a current of 7.48 μA) and +0.51 V (with a current of 9.36 μA) (Fig. 15(B)). When the SPEs were modified with 150 ng of surfactant-exfoliated 2D-h-BN, the maximal potential of oxidation for DA decreased to +0.22 V while the peak current increased to 16.7 μA (compared to the unmodified SPE). However, this modification had a negative effect on the response towards UA. Although the peak potential remained the same at +0.50 V, the peak current decreased to 7.28 μA, suggesting a reduction in sensitivity. It is clear that the surfactant that was utilized to exfoliate 2D-h-BN interferes with the electrochemical response when attempting to simultaneously detect DA and UA. First, several masses of 2D-h-BN exfoliated with surfactant (ranging from 7.5–300 ng) were fixed onto SPEs. In order to investigate the detection of DA further, differential pulse voltammetry (DPV) was employed, as shown in Fig. 15(C). The limit of detection (LOD) for 2D h-BN exfoliated with surfactant was computed, and the SPE modified with 300 ng of 2D-h-BN showed an LOD of 1.57 μM for DA (Fig. 15(D)).

Fig. 15 (A) Typical CV in a solution containing 0.5 mM of each DA and AA in PBS at a pH of 7.4. The measurements were taken using three types of SPEs: an unmodified SPE (black), an SPE exfoliated with 150 ng of 2D-h-BN (red), and an SPE modified with 40 μg sodium cholate (blue). (B) CV recorded in a solution containing 0.5 mM of each DA and UA in PBS at a pH of 7.4 (scan rate: 100 mV s⁻¹, vs. SCE). (C) Typical DPVs; aliquots of DA were added to a 0.1 mM AA solution (pH 7.4 PBS) at concentrations ranging from 3 to 75 μM. The dashed line in the graph indicates a 0.1 mM AA solution without DA. (D) Graphs showing the peak anodic current for DA oxidation at varying concentrations [Reproduced from ref. 165, RSC].

The 2D h-BN has also been used in electrode configurations along with other nanomaterials as nanocomposites for biomolecule detection. As an illustration, Yola and Atar incorporated molecularly imprinted polymers onto graphene quantum dots (GQDs) with 2D-h-BN and placed them on a GCE to detect serotonin (SER).¹⁶⁶ This study found that their approach, which involved using GQDs and 2D h-BN, resulted in a sensor with superior performance compared to other methods previously mentioned in the literature. The facilitation of charge transfer by GQDs, a decrease in the supporting electrode's mass transfer resistance (GCE), and a synergistic interaction between 2D-h-BN and GQDs were some of the factors that were ascribed to improved output. Upon closer examination of their reported voltammetric responses, it was found that there was a notable difference between the voltammetry of a bare GCE and a modified GCE electrode using 2D-h-BN and GQDs/2D-h-BN when tested with a simple redox probe. It appears that there has been a shift from diffusional effects at the GCE to adsorption/thin-layer type effects at the 2D-h-BN and GQDs/2D-h-BN modified electrodes. The authors suggested that this may have played a role within the enhanced electrochemical reaction, but the additional analysis could have been beneficial. An SER imprinted voltammetric sensor was enhanced by conducting CV in the presence of phenol (80 mM) and SER (20 mM). The linearity range was found to be 1.0 × 10⁻¹²–1.0 × 10⁻⁸ M, while the LOD was found to be 2.0 × 10⁻¹³ M. Nonetheless, the authors were able to effectively use their nanocomposite sensor to accurately measure the SER levels in urine samples.

Additionally, 2D h-BN has been combined with a variety of materials to enhance the biosensor performance. Numerous combinations of 2D h-BN have been made with various carbon allotropes, including graphene, multi-walled carbon nanotubes (MWCNTs), and graphene quantum dots (GQDs).¹⁶⁷ For example, the simultaneous detection of β-agonists such as phenylethanolamine A (PEA), clenbuterol (CLE), ractopamine (RAC), and salbutamol (SAL), which are responsible for metabolic disorders, anxiety, and muscular tremors, is greatly aided by a repeatable electrochemical sensor based on 2D-hBN/f-MWCNTs. The 2D-hBN/f-MWCNTs electrode shows improved performance compared to bare GCE, with increased redox peak current of 1.0 mM [Fe(CN)₆]³⁻ and reduced peak potential difference by 110 mV, making 2D h-BN/MWCNTs (5 : 1)/GCE a promising option for detection. According to CV graphs, the electrocatalytic activity of 2D h-BN and the complementary effects of MWCNTs and h-BN are demonstrated by a decrease in DE_p from 200 mV (bare) to 150 mV (2D h-BN/GCE) and subsequently to 90 mV (2D h-BN/MWCNTs/GCE). Fast electron transport is made possible by the unbound electron pairs in MWCNTs, and the system has a greater specific area thanks to 2D h-BN. According to EIS, the charge transfer resistances (R_ct) for 2D h-BN/GCE, 2D-hBN/f-MWCNTs/GCE, and bare GCE are 135 Ω, 100 Ω, and 75 Ω, respectively. The nature of the diffusion-controlled reaction at the 2D h-BN surface is demonstrated by these findings as well as the diffusion-controlled reactions of 2D-hBN/fMWCNTs/GCE phenylethanolamine A, clenbuterol, ractopamine, and salbutamol. The linear range of the sensor was 1.0 × 10⁻¹² to 1.0 × 10⁻⁸ M.

5.2.2. Colorimetric sensor. A colorimetric biosensor is a sensor that changes color when exposed to a specific biological or chemical analyte. This change in color occurs due to a shift in the optical properties of the materials on the sensor's surface. These biosensors are straightforward and affordable because the color change is easy to detect and quantify. The h-BN surface can be modified to attach specific biological molecules or receptors for sensing purposes.

A study found that Pt/h-BN (Pt/h-BNNS) nanosheets used an artificial nanozyme to identify DA. This enzyme sped up the process of oxidizing the peroxidase substrate TMB, causing it to turn blue in the presence of H₂O₂.¹⁶⁸ A nanocomposite of Pt/h-BNNS can be used to detect dopamine, a significant biomolecule, through the oxidation of TMB. The catalytic activity of Pt/h-BNNSs for TMB oxidation is gradually inhibited by increased amounts of dopamine, which allows for selective sensing of dopamine as low as 0.76 μM. Even when common interfering molecules are present, this detection method remains effective. It has also been successful in detecting DA in real blood serum samples. Ce-decorated h-BN nanosheets (Ce-BNNSs) were also used by Fatemeh and Morteza to demonstrate the colorimetric detection of mRNA.¹⁶⁹ Ce-BNNSs have varying affinities towards DNA that is double-stranded (dsDNA) and single-stranded (ssDNA). When miRNA targets hybridize, the probe detaches from the surface of Ce-BNNSs. By combining nanosheets and hydrogen peroxide (H₂O₂), the substrate o-phenylenediamine (OPD) can be oxidized to produce 2,3-diaminophenazine (oxOPD), which is fluorescent in nature. This new substance emits a maximum wavelength of 562 nm and has a shade of yellow with a distinctive 450 nm absorption peak. Because of this reaction, the assay may, under ideal circumstances, detect miRNA-155 in a linear range of 0.1 to 70 nM, with a detection limit of 50.0 pM. The probe is also successful in detecting miRNA-155 in serum samples.

Xu et al. developed a 2D h-BN/CuS nanocomposite and used it as a colorimetric sensor to detect cholesterol. They used the TMB molecule as a probe for this purpose.¹⁷⁰ The growth of brain and nerve cells depends on cholesterol and its fatty acid esters. The h-BN/CuS serves as a catalyst and transporter, helping the electrons to move from the electron acceptor (H₂O₂) to the electron donor (TMB). Cholesterol oxidase helps to oxidize cholesterol when oxygen is present, producing cholest-4-ene-3-one and H₂O₂ as byproducts. The naked eye can detect a shift in color from colorless to blue, demonstrating the existence of cholesterol. This provides a straightforward method for determining the cholesterol levels. The linear standard curve for detecting cholesterol ranges from 0.01 to 0.1 mM (see Fig. 16(A)). To calculate the LOD, the standard deviation (SD) of the blank sample responses and the slope (S) of the calibration curve are used (refer to Fig. 16(B)). The LOD value was precisely found to be 2.9 mM for cholesterol detection. Table 2 provides information on different types of electrochemical and colorimetric sensors based on h-BN nanostructures.

Fig. 16 (A) UV-Vis spectra obtained for oxidized TMB using BNNS@CuS (pH 3.0, at 35 °C, 2 mM TMB concentration). (B) Corresponding pictures of the colored goods, and the linear standard curve for calculating cholesterol levels [Reproduced from ref. 170 with authorization. Copyright 2017, Elsevier].

Table 2 Electrochemical and colorimetric sensors based on h-BN nanostructures

S. no.	Material	Method	Analyte	Linear range	LOD	Ref.
1.	2D-hBN	Electrochemical	Dopamine	—	0.65 μM	163
2.	Uricase-modified BN nanosheets	Electrochemical	Uric acid	5–3000 μM	0.14 μM	171
3.	Mn2O3@h-BN nanocomposite	Electrochemical	Anti-cancer drug flutamide	0.08–1940 μM	0.008 μM	172
4.	Carbon quantum dot-incorporated hexagonal boron nitride nanosheets (CQDs@HBNNS/UiO-66-NH2/MIP	Electrochemical	Oxaliplatin	1.0–20.0 nM	0.37 nM	173
5.	Gold nanoparticles decorated on boron nitride nanosheets (AuNPs/BNNSs)	Electrochemical	Myoglobin (Mb)	0.1–100 μg mL^−1	34.6 ng mL^−1	21
6.	Functionalized hBN nanosheets	Electrochemical	Carcinoembryonic antigen (CEA)	0.1–500 ng mL^−1	0.017 ng mL^−1	174
7.	2D h-BN	Electrochemical	Cancer antigen 125	5–100 U	1.18 U mL^−1	175
8.	2D h-BCN–carbon cloth	Electrochemical	Dopamine (DA) and uric acid (UA)	10–300 mM (DA), 10–500 mM (UA)	5 mM (DA), 2 mM (UA)	176
9.	Flake h-BN	Electrochemical	Ascorbic acid (AA), dopamine (DA), uric acid (UA)	30–1000 mM (AA), 0.5–150 mM (DA), 1–300 mM (UA)	3.77 mM (AA), 0.02 mM (DA), 0.15 mM (UA)	177
10.	Au NPs/2D h-BN	Electrochemical	H2O2	0.04–50 mM	8.3 mM	178
11.	Nafion/myoglobin/2D-h BN	Electrochemical	Trichloroacetic acid	0.2–30.0 mM	0.05 mM	179
12.	Yttrium oxide/h-BN	Electrochemical	Dopamine	0.008–241 μM	0.0003 μM	180
13.	CuO/2D BN	Electrochemical	L-Cysteine	1–10 μM	0.58 μM	181
14.	h-BN–Pt whisker	Electrochemical	Glucose	0.1–2.7 mM	14.1 μM	182
15.	NiO/BCN	Electrochemical	Nitrofurantoin	0.05–230 mM	10 nM	183
16.	GQDs/2D-hBN nanocomposite	Electrochemical	Serotonin	1.0 × 10^−12 M to 1.0 × 10^−8 M	2.0 × 10^−13 M	166
17.	FeV NPs/2D h-BN	Electrochemical	Chlorpromazine	0.04–2222.1 mM	0.004 mM	184
18.	Bi2O3/h-BN	Electrochemical	Anti-cancer drug flutamide	0.04–87 mM		185
19.	h-BN QDs	Colorimetric	E. coli and Klebsiella pneumoniae	1.8 × 10^6 and 1.5 × 10^6 CFU mL^−1		186
20.	Au NPs/Cu^2+–BNNS	Colorimetric	Carcinoembryonic antigen	—	—	187
21.	h-BN/N–MoS2	Colorimetric	H2O2	1–1000 μM	0.4 μM	188
22.	Ag/Fe3O4@h-BN	Colorimetric	H2O2	—	—	189
23.	CoOOH (boron nitride-loaded cobalt oxyhydroxide nanosheets)	Colorimetric	Ascorbic acid	0.05–80 μM	120 nM	190
24.	Ce-FeONPs	Colorimetric	H2O2	0.08 to 1 mM	0.03 mM	191

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5.3. Drug delivery

One of the main concerns in contemporary medical sciences is the delivery of drugs and bio-macromolecules such as genes, peptides, and proteins to particular cells or tissues. In recent years, a nanoplatform was developed in order to address the drawbacks of the conventional old drug delivery systems, such as oral administration or intravenous injection. Since their surface-to-volume ratio is large and it offers a variety of anchoring points and increases the loading capacity, 2D material-based nanocomposites have drawn attention toward the administration of drugs conveyors in biomedical applications.¹⁹² To further achieve targeted drug administration, to more effectively solubilize pharmaceuticals, and to safeguard them from enzyme degradation, the nanomaterials are created with improved biocompatibility. Owing to the special qualities that include stability, low toxicity and biocompatibility, elevated specific surface area and hierarchical porosity, h-BN is considered as a suitable material for drug delivery. However, further biomedical applications of h-BN nanosheets need particular characteristics, such as small sheet dimension, outstanding hydrophilicity and dispersity.¹⁹³ Consequently, hydroxylation must be used to functionalize h-BN in order to exert its excellent performance in practical applications. By using a thermal substitution procedure, Weng et al. created water-soluble and biocompatible hydroxylated h-BN nanosheets and discovered the DOX's low toxicity and outstanding loading capacity for hydroxylated h-BN.¹⁸ The as-synthesized BN nanosheets are able to load anticancer medication efficiently (e.g., doxorubicin (DOX)). The loading amount of DOX onto hydroxylated h-BN was achieved up to 309 wt% which is much higher than other nanocarrier systems as well as three times their own weight. This is due to the π–π stacking and hydrophobic relations between the h-BN nanosheets and drug molecule. Importantly, the drug-loaded hydroxylated h-BN nanosheets outperform free DOX drug molecules in their capacity to decrease the viability of LNCaP prostate cancer cells. After exposure to the BN materials for 24 h at concentrations as high as 100 μg mL⁻¹, cytotoxicity tests showed that more than 92% of cells were still viable, highlighting their safety for use in biomedical applications. Additionally, the BN carriers exhibited stability at physiological pH (7.4) but effectively released their drug payload in acidic environments (pH 5.0), which is characteristic of tumor microenvironments. This pH-sensitive behavior ensures targeted drug release at the tumor site. These qualities make them viable options for boosting cancer treatment effectiveness while reducing adverse effects. Fig. 17 presents the typical fluorescence spectra of DOX solutions before and after being loaded onto hydroxylated BN.

Fig. 17 (a) Typical fluorescence spectra of DOX solutions before and after loading to the hydroxylated BN in PBS buffer of pH = 7.4 (inset: color changes before and after loading). (b) Hydroxylated BN materials' DOX loading capabilities under various equilibrium conditions [Reproduced from ref. 18 with authorization. Copyright 2014, ACS].

Pure and crystalline h-BN nanosheets produced by the NaCl-template process were functionalized by Cheng et al. for effective camptothecin (CPT) delivery. The OH–h-BN nanosheets demonstrated good stability and water dispersibility as they were being created. The anticancer agent CPT could be successfully loaded and delivered onto the highly biocompatible OH–h-BN nanosheets for both in vitro and in vivo suppression of 4T1 breast cancer.¹⁹⁴ Through hydrophobic interactions, the hydrophobic CPT can be adsorbed on the surfaces of the OH–h-BN nanosheets, with a loading capacity of up to 170 wt%. This is due to BN's chemical inertness, which causes huge hydrophobic surfaces to remain even after hydroxylation. Cell viability stayed at 99% for the OH–h-BN nanosheets at a high concentration of 100 μg mL⁻¹. Cell viability was 76% when free CPT was treated for 24 h at a concentration of 1.0 μg mL⁻¹, but this value dropped to 48% when the cells were loaded into OH–h-BN nanosheet nanocarriers, and then to 17% after 48 h. After 14 days of therapy, an in vivo investigation revealed a 43% substantial inhibition of tumor development. According to their findings, OH–h-BN nanosheets could be employed as efficient nanocarriers for the delivery of CPT.

A cancer treatment that combines photothermal therapy (PTT) and chemotherapy agent was created by Sharker et al. using NIR-responsive h-BN nanosheets with indocyanine green (ICG) embedded in them and constructed on dopamine that was additionally embellished with DOX and coupled with hyaluronic acid. (Fig. 18).¹⁹ The technique exhibited high effectiveness rates in specifically harming cancer cells with a smaller impact on healthy cells, according to their in vitro studies, highlighting the synergistic co-therapeutic capability of h-BN. A temporal study of the h-BN, h-BNI, and h-BNI/d-HA-Dox NPs was conducted to assess their potential for thermal heat generation when exposed to NIR light. The total amounts of h-BNI/d-HA-Dox NPs are represented by a concentration of 0.5 mg mL⁻¹. Here, 28% of (weight) ICG, or 0.36 mg of h-BN from the entire 0.5 mg of h-BNI, is present in the total h-BNI. In addition, 0.13 mg of ICG and 0.01 mg of d-HA-Dox are present in 0.5 mg of h-BNI/d-HADox NPs, with the remaining quantity being empty h-BN. It has been shown that low NIR absorption spectra cause the temperature of h-BNI (25–53 °C) NPs to rise in comparison to h-BNI/d-HA-Dox (25–43 °C) NPs. The low levels of h-BNI and the additional decrease in ICG during the d-HA-Dox functionalization may be the cause of the h-BNI/d-HA-Dox NPs' declining NIR absorbance. This is because, in comparison to bare h-BNI, the addition of d-HA-Dox to the h-BNI/d-HA-Dox NPs causes a decrease in NIR absorption and a decrease in temperature generation. The release of Dox from hBNI/d-HA-Dox nanoparticles was evaluated at pH levels of 5.5, 7.4, and 8.5 where pH 5.5 and 7.4 simulate the tumor and endosomal environments, pH 7.4 also represents blood plasma, and pH 8.5 serves as both a negative control and a condition to assess dopamine adhesion stabilization. A lower pH value maintains a higher release pattern, which results in the faster protonation of carboamide group-deteriorated coupling between Dox and h-BNI/d-HA-Dox matrix. It is quite likely that the Dox release profile is directly influenced by acidic conditions since the conjugated –CO–NH– linkage is particularly vulnerable to acid hydrolysis, which easily dissociates Dox from h-BNI/d-HA particles. The intramolecular hydrolysis of the amide bond was thought to be the release of Dox from this conjugated system in an acidic environment. Despite having comparable photothermal heat generating potentialities, h-BNI and h-BNI/d-HA-Dox had different cellular internalization and in vitro photothermal anticancer effects. With the increase in NP concentrations, the NIR-irradiated malignant MDAMB-231 treated with h-BNI/d-HA-Dox NPs demonstrated a dramatic drop in cell viability, going from 42% to 7%. Simultaneously, depending on the concentration, d-HA-Dox- and h-BNI-treated NPs displayed a gradual decrease in cell viability (76 to 20% and 98 to 21%). Higher h-BNI/d-HA-Dox concentrations lead to greater cell death, whereas treated groups with lesser concentrations had distinct in vitro photothermal chemotherapeutic outcomes.¹⁹

Fig. 18 Diagrammatic representation of the process for creating hBNI/d-HA-Dox and a responsive NIR stimulus to activate as photothermal chemotherapy drugs [Reproduced from ref. 19 with authorization. Copyright 2017, Elsevier].

Pd NP-decorated hydroxylated h-BN nanosheets (Pd@OH-BNNS) were designed as a vehicle for the delivery of drugs which demonstrated a large loading capacity towards the anticancer drug DOX.¹⁷ The OH functional groups on the nanosheets increased the drug-loading capacity and hydrophilicity. The two functions of Pd NPs were to improve drug delivery by localized heating and to facilitate photothermal conversion. The Pd@OH-BNNS nanocomposite exhibited a high drug loading efficiency (DLE) of approximately 72–85% and a drug loading content (DLC) of about 18–25 wt%, depending on the drug-to-carrier ratio. The release of DOX from the surface of Pd@OH-BNNS's was pH-responsive, imitating the tumor microenvironment with a markedly increased release under acidic conditions (pH ∼ 5.0). The increased localized Pd photothermal activity-induced heating was caused by NIR laser light at 808 nm. Compared to less than 30% at physiological pH (7.4) without irradiation, cumulative release under NIR irradiation at pH 5.0 exceeded 70% in a 24 h period. Fluorescence microscopy verified the effective uptake of DOX-loaded Pd@OH-BNNS by HeLa cancer cells in terms of cellular uptake and cytotoxicity. When DOX administration and NIR photothermal therapy were combined, the cytotoxicity assays (MTT or CCK-8) showed a synergistic cell-killing impact, lowering cell viability to less than 20% after a 24 h treatment. They also showed low toxicity of the carrier alone. Importantly, Pd@OH-BNNS/DOX therapy significantly slowed tumor growth without appearing to harm mice.

Red blood cells' (RBCs') cell membranes can be destroyed by BN nanosheets, as R. Zhou's research showed, which causes severe hemolysis. BN nanosheets remove phospholipids and compromise membrane integrity by penetrating the lipid bilayer of RBC, according to hemolysis tests and morphological studies. These results were corroborated by molecular dynamics simulations, which demonstrated that strong van der Waals interactions between BN nanosheets and phospholipids make it thermodynamically advantageous for them to penetrate the cell membrane. In vitro hemolysis experiments, treatment of RBCs with 200 g mL⁻¹ BN nanosheets show that BN nanosheets have a strong hemolytic activity that varies with concentration at a hemolysis rate that can exceed 20%.¹⁹⁵

BN surfaces must undergo chemical alterations in order to acquire active tumor-targeting capabilities. This comprises a few of the compounds or targeting groups, like antibodies and folic acid (FA). In order to increase the BN nanosheets’ dispersibility and drug-loading capability, Feng et al. synthesized them with mesoporous silica (MS) functionalized on their surfaces (BNMS).¹⁹⁶ BNMS-FA complexes were nontoxic up to 100 μg mL⁻¹ and could be preferentially absorbed by MCF-7 and HeLa cells by endocytosis mediated by the folate receptor. DOX exhibited a sustained release pattern at different pH levels and could be loaded onto BNMS-FA complexes with high efficiency through π–π stacking and hydrogen bonding. BNMS-FA/DOX complexes outperformed free DOX, BNNS/DOX, and BNMS/DOX complexes in terms of drug internalization and antitumor effectiveness. It was determined that the BNMS and BNMS-FA complexes had respective DOX loading capacities of 52.6 and 49.2 μg mg⁻¹ of carriers. The DOX loading capacity of both complexes was significantly higher than that of BN nanosheet complexes, which were only 6.9 μg mg⁻¹. At lower pH levels, DOX release from BNMS-FA/DOX complexes was noticeably higher. Only 26.1% of DOX was liberated from BNMS-FA at pH 7.4 after 80 h, compared to 81% at pH 5.0. Given the increased hydrophilicity and diminished noncovalent interaction between DOX and BNMS-FA, the protonation of the amino group in the DOX molecule may be the reason for the increased release rate of DOX at acidic pH. Additionally, the H⁺ in an acidic solution would weaken the hydrogen bonds that hold the –OH group of DOX and the –COOH group of FA together, allowing DOX to be released more easily. More internalization of DOX mediated by the FA receptor is the primary reason for BNMS-FA/DOX complexes showing superior in vitro antitumor effects when compared to free DOX, BNNS/DOX, and BNMS/DOX complexes.

Even though h-BN nanosheets demonstrated properties essential for loading and delivering of small molecule therapeutics, the research of these material-based drug delivery agents is still in its initial stages. Therefore, further studies would need to be performed to widen their applications.

5.4. Tissue engineering

Tissue engineering is a developing field of study that combines knowledge from medical, biology, chemistry, and engineering to create artificial tissue paradigms that may address the global scarcity of organs that have been destroyed by various diseases or accidents.^197,198 Many scientists are using h-BN-based nanomaterials in tissue regeneration research because of their exceptional mechanical, electrical, chemical, and physical capabilities. Due to its low cytotoxicity, outstanding thermal stability, and superior mechanical characteristics (high elastic modulus), 2D h-BN is regarded as an appropriate content for bone/tissue regeneration.¹⁹⁹ By using 3D printing technology, Aki et al. created bone tissue scaffolds.²⁰⁰ The scaffolds were produced using 3D printing, incorporating 12 wt% PVA, 0.25 wt% hBN, and varying concentrations of BC (0.1–0.5 wt%). Easy vascularization and nutrient transfer were made possible by the composite's adaptable, homogenous structure, uniform pore distribution, and appropriate pore size for tissue engineering applications. The tensile strength of the scaffolds decreased upon the addition of bacterial cellulose (BC), while the composition containing 12 wt% PVA, 0.25 wt% h-BN, and 0.5 wt% BC exhibited the highest elongation at break, reaching 93%. A notable enhancement in human osteoblast cell viability was observed on 3D scaffolds with this composition. Cell morphology studies indicated that the scaffolds doped with bacterial cellulose supported effective cell adhesion. For bone cells, the functional scaffolds of PVA augmented with h-BN and BC that are 3D printed are suggested to be a suitable extracellular matrix structure.

Ozbek et al. fabricated poly(e-caprolactone) (PCL)/tri-calcium phosphate (TCP)/h-BN composites by an electrospinning method.²⁰¹ Due to the solutions' adequate viscosity, the outcomes showed that the composites had a continuous shape and were bead-free. The composites were discovered to be highly degradable, and the TCP and h-BN levels enhanced the tensile strength. The SaOS-2 cell culture investigation had demonstrated that the presence of TCP and PCL improved SaOS-2 cell adhesion, while h-BN had the opposite effect. The composites of PCL/TCP/h-BN nanofibrous demonstrated excellent degradation and biocompatibility as well as great promise for tissue engineering of the bones. BN-reinforced gelatin, a 2D biocompatible nanomaterial, was reported by Bechelany and his group.²⁰² The incorporation of exfoliated BN into gelatin electrospun fibers leads to an enhancement in Young's modulus. Because of the densely packed hydroxyapatite layers that are created in SBF during mineralization, the BN ESM and glutaraldehyde cross-linked gelatin is extremely bioactive. Increasing enzyme doses have been used over a 24 h period to show that the various ESMs are degradable in vitro. A collagenase concentration of 50 U ml⁻¹ results in complete breakdown of various ESMs. All things considered, these findings demonstrate that the produced ESMs are enzymatically degradable and stable in aqueous solutions, both of which are critical characteristics for tissue engineering applications. BN-reinforced gelatin ESMs are appropriate for the purpose of bone tissue engineering because of their improved capacity for mineralization, cell adhesion, proliferation, and biocompatibility.

Using the technology of spark plasma sintering (SPS), h-BN–B₂O₃, a composite, was developed, which has superior stability, elevated mechanical strength, and favorable biocompatibility.²⁰³ An intricately connected system with an outstanding compressive strength, comparable to that of natural bone tissues, is produced by the covalent connections between B₂O₃ and h-BN created during the process of SPS. The development of mineralized nodules and osteogenic differentiation were seen to be promoted by h-BN–B₂O₃. At a very low SPS temperature of 250 °C, the composite structure exhibits large surface area (0.97–14.5 m² g⁻¹) and substantial densification (1.6–1.9 g cm⁻³). The composite construction achieved a high compressive strength of 291 MPa and a respectably strong wear resistance. Using molecular dynamics simulation, the creation of strong covalent connections between h-BN and B₂O₃ was proposed and confirmed. The composite has a notable impact on cell proliferation and viability. Its utility as a potential osteogenic agent in bone formation is suggested by the higher mineralized nodule development compared to the control. By combining hyperbranched polyglycerol (HPG)-functionalized h-BN nanosheets with gelatin using a filtration system by a vacuum-induced self-assembly methodology, Yoo et al. created materials imitating nacre.²⁰⁴ By using the electrostatic interactions between the cationic amine on gelatin and the anionic hydroxyl on h-BN nanosheets to form interlayer bridges, h-BN nanosheets were ordered into a physical brick and mortar (B&M) structure. Furthermore, the artificial nacre demonstrated outstanding biocompatibility and biodegradation capabilities; this is the first study on the cell survival of both artificial nacre nanocomposites and h-BN nanosheet-based materials. Cell viability increased in response to higher h-BN nanosheet loadings, surpassing that of the control group (TCP). The resulting nacreous nanocomposite (Fig. 19(a)–(e)) displayed mechanical characteristics akin to those of human cortical bone and a controlled nanoscale organization. Ramasamy et al. combined electrospun poly(L-lactic acid) nanofibers (PLLANFs) with amphiphilic triblock copolymer pluronic F-127 (PL)-functionalized nanofillers (PL-functionalized exfoliated boron nitride nanosheets (PL-EBN) and PL-functionalized carboxylated multiwalled carbon nanotubes (PL-cMWCNTs)) to create a composite scaffold.²⁰⁵ The findings indicated that the scaffolds had improved mechanical strength, wettability, and piezoelectric properties. Strong mechanical integrity, conductivity, and piezoelectric properties were also demonstrated by the composite scaffolds, which might be regarded as crucial bio interface features for promoting cell attachment. As a result, the composite scaffolds were effective in encouraging MC3T3-E1 cells' adhesion and osteogenic differentiation (Fig. 19(f)–(h)). The HA/CS/Gel/h-BN bio-composite coating was created by Tozar et al. and applied by EPD to a titanium alloy's surface.²⁰⁶ The covering may mimic the structure and content of bones and display excellent biocompatibility, robustness to wear and flexibility. h-BN coating's chemical characteristics are sturdy and capable of halting metal corrosion.²⁰⁷

Fig. 19 (a) Representation of the synthesis of 2D boron nitride nanosheet (BNNS)-based nanocomposites. (b) Young's modulus and (c) tensile strength of HPG-g-BNNS/gelatin-based nacreous nanocomposites with different HPG concentrations. Representation of the fissure mechanism of (d) BNNS/gelatin and (e) HPG-g-BNNS/gelatin. (f) Representation of the manufacture of PLLA nanofiber scaffolds using the process of electrospinning. (g) Stress–strain curves of the scaffolds. (h) Nyquist charts derived from EIS measurements of the scaffolds. Images of (i) ALP-stained scaffolds (for the days 7 and 14) and (j) ARS-stained scaffolds (for the days 14 and 21). [Reproduced from ref. 24 with authorization. Copyright 2023, Elsevier].

A. Madhan Kumar et al. created a nanocomposite based on polymer coating on conducting polymer and h-BN nanosheets for 316L stainless steel (SS) implants to boost biocompatibility and resistance to corrosion in vitro. TiO₂ NPs (25 nm) were used to decorate the BN surface, and the resulting BN-TiO₂ nanosheets were then mixed with 3,4-ethylene dioxythiophene (PEDOT) coatings. Improved wettability, in vitro corrosion resistance, and the capacity to promote osteoblast adhesion were all displayed by the nanocomposite coatings based on PEDOT/10BN-TiO₂. The results from surface and structural analysis confirmed the development of BN-TiO₂-distributed nanocomposite coatings and the presence of the BN-TiO₂–PEDOT matrix interaction. The value of the corrosion current density (0.0248 μA cm⁻²) was two orders of magnitude lower for PEDOT/BN-TiO₂ nanocomposite coatings than for pure PEDOT, according to in vitro corrosion analysis of coated 316L SS specimens in simulated body fluids. The PEDOT/BN-TiO₂ coatings demonstrated a water contact angle (WCA) value of roughly 20°, further indicating the beneficial role of BN-TiO₂ in the PEDOT matrix, while the uncoated 316L SS specimen had a WCA value of 84.50°. The outcomes show the potential for orthopedic applications of the created nanocomposite coatings on 316L SS implants.²⁰⁷ h-BN has been used since the turn of the century to enhance the mechanical characteristics of bone tissue engineering biomaterials. In order to create composite materials, Sabino et al. combined BN nanosheets with hydroxyapatite (HA) and poly(p-dioxanone) (PPDX).²⁰⁸ In addition to being an efficient PPDX nucleating agent, BN also improved the material's tensile characteristics, significantly increasing its elastic modulus and preventing it from declining when PPDX hydrolyzed. Using a phosphate buffer solution of pH 7.4, the hydrolysis was carried out in vitro over the course of eight weeks at 37 °C. The elastic modulus of PPDX was essentially unaffected by the application of 5% HA, although it was slightly diminished by 20%. These outcomes most probably stem from the matrix's poor dispersion and slight degradation caused by HA. Compared to pure PPDX, samples containing HA hydrolyzed considerably more quickly. Conversely, the presence of BN significantly increased the elastic modulus of PPDX; an increase of 5% from 35 MPa to 110 MPa and 20% from 35 MPa to 160 MPa was observed. Furthermore, the PPDX/BN samples' elastic modulus value falls with the hydrolytic degradation duration but surpasses that of clean PPDX after 8 weeks of degradation. From an application perspective, the results are intriguing because the trabecular bone's reported elastic modulus values fall between 50 and 100 MPa, suggesting that the BN-filled PPDX could be helpful for bone fixation implants.

The mechanical characteristics of tetraneedle-like ZnO whiskers (T-ZnO_w) and BN nanosheets improved the mechanical strength and modulus of poly(lactic acid) (PLLA) scaffolds as found in a study conducted by Feng and his coworkers.²⁰⁹ Cell adhesion and proliferation were enhanced, and stem cell osteogenic differentiation was boosted. Three-dimensional T-ZnO_w supporting two-dimensional BN nanosheets created a space network structure that enhanced their dispersion characteristics in the PLLA matrix. According to the findings, the scaffolds containing 1 wt% BN nanosheets and 7 wt% T-ZnO_w had compressive strength, modulus, and Vickers hardness that were, respectively, 96.15%, 32.86%, and 357.19%, higher than those of the PLLA scaffolds. Additionally, MG-63 cell adhesion and vitality were enhanced by the combination of BN nanosheets and T-ZnO_w in PLLA scaffolds. More notably, the scaffolds fostered osteogenic differentiation and markedly enhanced proliferation of human bone marrow mesenchymal stem cells (hBMSCs). The possibility for use in bone tissue engineering is provided by the improved mechanical and biological characteristics of the developed PLLA/BNNSs/T-ZnO_w scaffolds.

Additionally, biomass resources constitute a significant supply of materials for tissue engineering. Using collagen taken from rainbow trout skin and reinforced with different concentrations of BN NPs (0, 3%, 6%, 9%, and 12%, in weight) via a de-hydrothermal (DHT)–glutaraldehyde (GA) chemical cross-linking procedure, Najafi et al. created nanocomposite scaffolds.²¹⁰ The two-step cross-linking procedure and the synergistic function of BN NPs led to a discernible enhancement in the mechanical characteristics of collagen-BN scaffolds. Notably, adding 6 wt% BN and using a two-step crosslinking procedure greatly enhanced the collagen scaffold's compressive strength (9.5 times) and elastic modulus (four times). Additionally, the MG-63 cell line's proliferation and dissemination were greatly enhanced by nanocomposite scaffolds, demonstrating their biocompatibility. The findings indicated that adding BN NPs and a two-step cross-linking procedure could improve collagen scaffolds' mechanical and thermal properties while also increasing their high cell viability and proliferation, which would support their potential for use in tissue engineering applications.

5.5. Wound healing therapeutics

h-BNs were studied as an intriguing therapeutic agent for the purpose of treating wounds. The use of borate chemicals, mostly boric acid, in wound healing treatment is on the rise; nevertheless, their brief half-life poses a challenge and restricts its application. Boric acid (BA), one of the products of progressive break down of h-BN, was compared against h-BNs in vitro in order to see how each impacted the healing of wounds. To determine the appropriate dosage, the cytotoxicity and antibacterial activity of the synthesized h-BNs were first assessed. The outcomes of the scratch assay demonstrated that, in comparison to BA, even in low doses, h-BNs accelerated wound closure and enhanced angiogenic activity. According to the cell cycle analysis, BA had virtually little influence on cell proliferation, but h-BNs caused the cells to enter the S-phase rather than halt them in the G2/M phase. Furthermore, it was discovered that h-BNs reduced the amount of reactive oxygen species greater than BA. The h-BNs prevented the cells from going through apoptosis, according to the apoptosis/necrosis assay, while BA had very little influence on the process of cell death. Furthermore, at all tested doses, h-BNs did not alter the F-actin structure or harm the mitochondria at low concentrations. These results imply that h-BNs may have a therapeutic value in the treatment of wound healing. It was described as a multipurpose wound healing promoting agent in another investigation. Here, polydopamine (pdopa), silver nanoparticles (AgNPs), and h-BNs nanoparticles were combined using a mussel-inspired chemistry to create pdopa-coated h-BN (h-BN@pdopa) and AgNP-decorated h-BN@pdopa (h-BN@pdopa-AgNPs). Investigations were conducted on these two nanostructures to track the healing process. AgNPs for reduced inflammation and h-BNs for promoted cell migration and proliferation are the reasons for each substance. First, the biocompatibility and excellent cellular absorption capacity of h-BN@pdopa and h-BN@pdopa-AgNPs were assessed in in vitro tests. Their response to elevated ROS concentrations in damaged cells was also examined. Lastly, their impact on F-actin organization, intracellular tube formation, and cellular migration was observed. The outcomes unequivocally showed that h-BN@pdopa-AgNPs greatly reduced the formation of reactive oxygen species (ROS), encouraged wound closure, and rearranged tube formation in cells.²¹¹

In a different study, Turkez et al. used nanotechnology-based treatments to enable wound healing, ease tissue restoration, and lessen related problems including infections in order to address the growing worry of issues resulting from chronic nonhealing wounds.²¹² In the study, NPs were created by conjugating boron carbide (B4C) and alpha lipoic acid (ALA) with h-BN. This novel method seeks to improve wound healing in human dermal fibroblast (HDFa) cell cultures while investigating the antibacterial capabilities of these nanoparticles against strains of the pathogens Escherichia coli and Staphylococcus aureus. The wound healing capabilities of B4C, h-BN, ALA molecules, B4C-ALA, and hexagonal boron nitride–alpha lipoic acid (h-BN–ALA) were investigated using scratch tests on HDFa cell cultures. It was found that all experimental groups exhibited significantly improved wound gap closure healing properties when administered 50 g mL⁻¹ of NPs. Furthermore, h-BN–ALA nanomaterials showed greater wound healing effectiveness in comparison to other experimental groups. After applying the h-BN–ALA nanoparticle for 24 hours, the wound gaps closed by about 75% in contrast to the negative control, which exhibited a 40% gap closure. Furthermore, compared to non-ALA linked groupings, it was found that ALA conjugation to particles greatly enhanced wound healing.

A compelling advancement in the development of scaffolds composed of multifunctional nanocomposite materials with possible biological applications was provided by the work of Mukheem et al.²¹³ These scaffolds offer a potential strategy for infection prevention and wound healing. They consist of h-BN nanodisks infused into a polyhydroxyalkanoate (PHA)/chitosan (Ch) matrix. This synergy greatly boosts the antibacterial activity of the nanocomposite, particularly against multidrug-resistant E. coli K1 (MDR E. coli K1) and methicillin-resistant S. aureus (MRSA). The remarkable bactericidal activity of the polymeric scaffolds causes them to rapidly eliminate dangerous germs. Above all, these nanocomposites exhibit excellent cytocompatibility and minimal cytotoxicity towards human cells. Their broad-spectrum antibacterial action and cytocompatibility make them safe, effective, and durable antibacterial scaffolds for a range of biological applications when applied in proper dosages. The results of this study show that h-BN-based nanocomposites have the potential to treat bacterial infections and greatly accelerate wound healing, offering a novel and intriguing avenue for future biomedical research. Important insights into the potential use of h-BN NPs in dental and wound healing situations are provided by the work of Kivanc et al.²¹⁴ The antibacterial and antibiofilm capabilities of h-BN NPs against a range of microbes, including Streptococcus mutans ATTC 25175, Candida sp. M25, and S. mutans 3.3, were being studied. Importantly, the minimum inhibitory concentration (MIC) of h-BN NPs for various microorganisms was identified, offering information on how they prevent microbial development during wound healing. The results demonstrate that h-BN NPs have potent antibiofilm action, effectively inhibiting the development of significant microorganisms' biofilms, including those of Candida and S. mutans. This discovery holds significant promise for wound healing applications since biofilm growth can obstruct the body's normal healing mechanism.

Besides the wound healing ability of boron nitrides, its anti-inflammatory property was also conformed. This involved simulation of molecular docking that the BNNT-loaded celecoxib could obstruct more pro-inflammatory cytokines as well as IL-1α and TNF-α receptors, thus alleviating the inflammatory condition as well as cardiovascular disorders.²¹⁵ Moreover, the activity of boron nitride in alleviating inflammation in the pathology of breast cancer as well as cardiovascular disorders is reported, when compared with a standard drug sulfasalazine, using molecular docking studies.²¹⁶ In another study, the alleviation of inflammatory biomarkers after LPS induction in rat brain was observed after application of boron nitride in Sprague-Dawley rats. Although numerous research studies are still in progress, the exploitation of pathophysiological events by the theranostics properties of boron nitride has an impactful role.²¹⁷ In a recent study, quercetin-loaded sodium alginate, collagen, and boron nitride have been used as possible wound dressings equipped by employing the experimental design of Box–Behnken. The publication concluded the efficacy of the prepared dressing and inferred about the anti-oxidant and anti-inflammatory role in the pathology of wound healing.²¹⁸

The biodegradability and ease of dispersion in an aqueous environment of h-BN produced from boric acid (BA) have made it a viable therapeutic option for wound healing.¹⁴ In order to understand the distinct proliferation effect of h-BN in these cell lines, cellular uptake capabilities were measured. Human umbilical vein endothelial cells (HUVECs) had a higher absorption capacity than human dermal fibroblasts (HDF). One of the primary processes of wound healing, cell proliferation, is achieved by promoting angiogenesis, collagen deposition, granulation tissue creation, and epithelialization in the injured area. Since HUVECs and HDFs are key players in these processes, the impact of h-BNs on cell proliferation was examined. HDFs are found in the skin's dermis layer, and they produce collagen and extracellular matrix as part of epithelialization. In order to assess the angiogenesis process in wound healing therapy, the HUVECs were chosen as a model. Thus, after exposure to increasing concentrations of h-BNs or BA (10–500 μg mL⁻¹), the WST-1 colorimetric assay was used to measure the cell proliferation rate of HDFs and HUVECs. The evaluation of cell viability on HDFs following treatment with h-BNs or BA at increasing doses revealed that, up to 100 μg mL⁻¹, neither substance significantly impacted cell viability. The evaluation of cell viability on HUVECs revealed that while 150 and 200 μg mL⁻¹ doses of h-BNs had no discernible effect on cell metabolism, the lowest values (10–100 μg mL⁻¹) boosted cell vitality up to 118% (p < 0.05). Cell viability dropped sharply to 42% at the highest h-BN concentrations (300–500 μg mL⁻¹) (p < 0.05). While 100 and 150 μg mL⁻¹ doses of BA had no discernible effect on cell viability, the cells' vitality rose to 119% (p < 0.05) when treated with 25 and 50 μg mL⁻¹ BA concentrations. Cell viability dropped to 48% at the highest BA doses (p < 0.05). However, the cell viability dropped to 64% in a dose-dependent manner (p < 0.05) when the cells were exposed to higher doses of h-BNs (150–500 μg mL⁻¹). After adding the maximum amounts of BA (200–500 μg mL⁻¹), the cell viability progressively dropped to 72% (p < 0.05). HUVECs were also used in an in vitro scratch test. After 8 h, a comparable pattern of wound closure to that of the control cells was noted. In comparison to the untreated cells, the wound area was fully closed after 24 h when the cells were subjected to 25 μg mL⁻¹ of h-BNs. Compared to 25 μg mL⁻¹ quantities of h-BNs, the healing process was not positively impacted by the greater concentration (200 μg mL⁻¹). Following the development of the scratch, HUVECs were also treated with progressively higher concentrations of BA, and the closure of the wound was tracked. The injured area started to close on its own after 8 h. After 8 and 24 h, wound repair was quicker with a low dose of h-BNs (25 μg mL⁻¹) than with control cells. After 8 h, the cells' scratch breadth was noticeably smaller than the control when exposed to 100 μg mL⁻¹ h-BNs. After 24 h, however, a similar scratch breadth was noted. The closure of the wound was nearly identical to that of the control cells at high h-BN concentrations. Furthermore, after 8 h, the scratch closure was induced more quickly by 25 and 100 μg mL⁻¹ concentrations of h-BNs than by the same concentrations of BA. In contrast, the injured region started to close more quickly in cells treated with 200 μg mL⁻¹ of BA than in cells treated with 200 μg mL⁻¹ of h-BNs. When the h-BNs and BA-doped medium were utilized at concentrations of 25 μg mL⁻¹, the scratch region was fully closed after 24 h. The wound area was nearly closed when 100 μg mL⁻¹ quantities of h-BNs or BA were utilized, but the greatest concentrations of either substance (200 μg mL⁻¹) had no beneficial effect on wound healing. According to the cell viability evaluation, this could once more be the result of the harmful effects of the larger dosages of boron-containing compounds. Because of their antioxidant ability, h-BNs may also aid in wound healing by reducing ROS, much like BA. Additionally, it was discovered that whereas BA had virtually no influence on the mechanism of cell death, h-BNs prevented cells from going through apoptosis. Additionally, it was shown that neither BA nor h-BNs depolarized the mitochondria at low doses or interfered with the production of F-actin at any of the examined concentrations. The findings unequivocally demonstrate that both BA and h-BNs hasten wound healing. However, by serving as a source of controlled release for BA, the gradual degradation of h-BNs provides a solution to the short half-life of BA.

5.6. Cancer therapeutics

Cancer therapeutics are the only means to control and combat cancer-a collection of illnesses that display unregulated growth and dissemination of anomalous cells. The goal of cancer therapeutics is to destroy or slow the growth of cancer cells while minimizing harm to healthy cells. Within this segment, we will provide a synopsis of how 2D-h-BN is being studied for its potential use in photothermal therapy (PTT) for cancer cells. 2D-h-BN can convert near-infrared (NIR) light into heat through a photothermal effect. When exposed to NIR light, h-BN can create localized hyperthermia, which can be used to selectively destroy cancer cells.²¹⁹ The NIR light-responsive heating and drug release has been shown to efficiently damage cancer cells while having a lesser impact on healthy cells. This demonstrates the highly effective synergistic effect that is attainable through the use of h-BN.²²⁰

According to reports, indocyanine green (ICG)-functionalized h-BN (h-BNI) can be used as a photothermal therapeutic agent that is responsive to NIR light. Furthermore, a chemotherapeutic agent called d-HA-Dox, which consists of doxorubicin (Dox) and hyaluronic acid (HA) conjugated together, has been recognized as a targeted treatment for tumors.¹⁹ They have combined dopamine (DA) with h-BNI and d-HA-Dox to create a photothermal chemotherapeutic agent called h-BNI/d-HA-Dox that targets tumors. It was found that h-BNI/d-HA-Dox effectively damages cancer cells while sparing healthy cells. In order to assess the potential for thermal heat generation caused by NIR light, the h-BN, h-BNI, and h-BNI/d-HA-Dox nanoparticles were examined over time. For the total quantity of h-BNI/d-HA-Dox NPs, the concentration is 0.5 mg mL⁻¹; 28% of (weight) ICG, or 0.36 mg of h-BN from the total 0.5 mg of h-BNI, is present in this instance; 0.5 mg of h-BNI/d-HADox nanoparticles simultaneously contain 0.01 mg of d-HA-Dox and 0.13 mg of ICG, with the remaining quantity being empty h-BN. Low NIR absorption spectra have been shown to cause the temperature of h-BNI (25–53 °C) NPs to rise in comparison to h-BNI/d-HA-Dox (25–43 °C) NPs. The low levels of h-BNI and the additional decrease in ICG during the d-HA-Dox functionalization may be the cause of the h-BNI/d-HA-Dox nanoparticles' declining NIR absorbance. Additionally, such elevated temperatures may cause cancer cells to suffer irreparable harm. Dox release from h-BNI/d-HA-Dox NPs was measured at pH 5.5, 7.4, and 8.5, which correspond to the environments of tumors and endosomes, blood plasma, negative control, and dopamine adhesion stabilizer, respectively. As the pH increased from 5.5 to 7.4, the produced h-BNI/d-HA-Dox nanoparticle released Dox gradually; after 5 h, the majority (about 50%) of Dox species were accessible. Only 41% of the collected portion of Dox is released at pH 8.5 after a 24 h evaluation, compared to 92% release at pH 5.5 and 70% availability at pH 7.4. Additionally, the release of Dox at pH 8.5 was seen to saturate after 12 h, owing to sustained dopamine adhesion to the h-BNI flat matrix and resistance of carboamide linkage. All things considered, the integration of d-HA-Dox and h-BNI into a single platform resulted in the effective development of a novel drug delivery system for photothermal treatment of cancer. Zhang and co-workers utilized the straightforward thermal replacement technique to produce hydroxylated h-BN, which acted as the base for incorporating Pd NPs and an anti-cancer medication.¹⁷ The nanoplatform that was created demonstrated remarkable stability for photothermal conversion and was responsive to GSH, pH, and photothermal stimuli for DOX release. Pd@OH-BNNS acted as a possible PTT agent since it mostly inherited the Pd NPs' strong NIR absorption property. Since Pd@OH-BNNS exhibited significant and wide absorption throughout the NIR spectrum, several NIR laser wavelengths were used to initiate PTT and drug release. Furthermore, it was evident that the absorbance of the Pd@OH-BNNS dispersion rose with the mass concentration. The temperature curves of 40 ppm and 80 ppm Pd@OH-BNNS dispersions under laser irradiation of 808 nm and 2 W cm⁻² were measured in order to examine the PTT effect caused by NIR absorption. After 5 min of NIR irradiation, the temperature of the 200 μL Pd@OH-BNNS (80 ppm) aqueous solution increased from 32.0 °C to 59.3 °C. Additionally, the Pd@OH-BNNS solution had a clear concentration-dependent heating effect, which enabled it to sufficiently absorb and transform NIR light into thermal energy. In contrast, no discernible temperature change was seen in a water solution exposed to the NIR laser under the same conditions when nanohybrids were not present. Notably, during five cycles of laser-induced heating, Pd@OH-BNNS demonstrated exceptional photothermal stability per cycle. After 808 nm NIR laser irradiation (2 W cm⁻², 5 min at a time) at pH 7.4, an apparent increased release of DOX was seen. This was attributed to the heat produced by the NIR laser irradiating Pd@OH-BNNS, which encouraged DOX detachment. Research done in vivo and in vitro have revealed the effectiveness of a nanoplatform loaded with DOX and subjected to NIR irradiation in inhibiting cell growth and destroying tumors. S180 tumor-bearing mice were used as the animal model for the in vivo anticancer analysis. An infrared thermal camera tracked the local temperature change at the tumor location in vivo. Under 808 nm laser irradiation (2 W cm⁻² for 3 min), the control group only showed a low-temperature increase that was insufficient to destroy the tumor at the tumor tissue location. However, the temperature at the tumor site after Pd@OH-BNNS injection increased quickly from 28 °C to 57 °C in just 3 min, indicating that Pd@OH-BNNS has a useful photothermal impact for treating cancer in vivo.

The therapeutic agent based on h-BN has shown exceptional efficacy against tumors and can also release drugs in response to photothermal stimulation. This can contribute significantly to the development of new and effective treatments for cancer.

Oh et al. conducted a research study that found that TA-Fe/BNS treatment of KB tumor cells, a compound made of tannic acid and Fe³⁺ coordination complex, exhibited greater magnetic resonance contrast that is T₁-weighted than plain BNS or BNS covered with either Fe or tannic acid. When exposed to NIR irradiation at 808 nm, KB tumor cells treated with TA-Fe/BNS experienced significantly higher rates of cell death compared to those treated with other compounds. When given intravenously, TA-Fe/BNS was capable of building up in tumors, according to in vivo MRI imaging. Using MRI information as a guide, targeted laser treatment successfully eliminated all tumor tissues. Based on these findings, it appears that TA-Fe/BNS has potential for theranostics using MRI.²²¹ The use of thermal imaging revealed that when mice were administered with TA-Fe/BNS and subjected to NIR irradiation (Fig. 20(A)), their tumors experienced a significant increase in temperature, with some reaching as high as 57.2 ± 0.7 °C (Fig. 20(B)). Conversely, the temperature readings of mice receiving TA/BNS, Fe/BNS, or simple BNS treatment were comparable to mice without treatment, as shown in Fig. 20(A) and (B). When NIR irradiation was not applied, there were no notable disparities in tumor growth between the different groups of treatments, as shown in Fig. 20(C) and (D). However, after irradiation with NIR, mice that received TA-Fe/BNS presented a marked antitumor effect, while those that received further BNS preparations did not (Fig. 20(C) and (D)). The TA-Fe/BNS treatment caused development of black scabs at the tumor sites, which detached and revealed tumor ablation 20 days later (Fig. 20(C)). Immunohistochemistry revealed a diminution in cell proliferation and an uptick in the death rate in the TA-Fe/BNS-treated (Fig. 20(E)) and NIR-irradiated mice (Fig. 20(E) and (F)); this group had the maximum quantity of cells undergoing apoptosis in the tumor tissues (Fig. 20(F)).

Fig. 20 TA-Fe/BNS photothermal characteristics were tested in vivo on Balb/c nude mice with KB tumors. Different BNS preparations were injected directly into the tumors, and five minutes later, the tumor sites were exposed to near-infrared radiation. (A) After continuous irradiation for 10 minutes, thermal images were captured to display the capability of TA-Fe/BNS in vivo. (B) Data from thermal images for every group (consisting of 5 members) were analyzed and measured. (C) Photographs show what the tumor sites looked like on the first day of radiation and on the 20th day. (D) Five individuals were administered with the BNS preparation and their tumor volumes were measured every two days. (E) Tumor tissues were taken out and studied using immunostaining for PCNA and carrying out a TUNEL assay. This is shown in the bottom panels. The scale bar is 100 μM. (F) Number of cells undergoing apoptosis in five sections of tumor tissues [Reproduced from ref. 221 with authorization. Copyright 2020, Elsevier].

Of all malignancies, melanoma is the most aggressive type and has the highest death rate (5.0–5.6%). In this aspect, Kalugina et al. created h-BN/n·MB (hexagonal BN/methylene blue) heterostructures based on adsorbed methylene blue (MB) at different concentrations (n) and h-BN NPs to improve the effectiveness of its treatment during local photodynamic therapy (PDT).²²² After exposure to an artificial sunlight source for 30 min, heterostructures with 200 mg of MB/g of h-BN (h-BN/200 MB) produced 3.7 × 10⁻² ± 0.2 × 10⁻³ μM × μg⁻¹ of reactive oxygen species (ROS) and decreased the survival of A-375 melanoma cells by 90% after 48 h. The h-BN/200 MB hybrid material exhibits oxidative and anticancer activity levels that are noticeably higher than those of the MB and h-BN system components alone. It is demonstrated that MB adsorbed on h-BN NPs has improved photooxidative activity and stability. The adsorbed MB showed enhanced biocompatibility with normal fibroblast cells (Wi-38) and exhibited minimal dark phototoxicity as compared to the MB solution. The examination of h-BN/n·MB cytotoxicity in relation to A-375 and Wi-38 cells revealed that the h-BN/200 MB sample possesses the best possible balance between biocompatibility and antitumor activity. The anticancer efficacy of h-BN/n·MB heterostructures decreases, and the biocompatibility is mostly unaffected when the MB concentration is raised above 200 mg g⁻¹ of h-BN. After cultivating A-375 cells for 24 h, the IC50 value of h-BN/200 MB heterostructures was found to be 7.5 μg. The findings demonstrated encouraging potential of h-BN/n-MB heterostructures for melanoma PDT. In a separate study, Liu et al. developed a novel multifunctional CuPc@HG@BN theranostic platform composed of hexagonal boron nitride nanosheets (h-BNNS), conjugated DNA oligonucleotide and copper(II) phthalocyanine (CuPc), wherein the CuPc molecule served two important functions: in situ monitoring of miR-21 by surface-enhanced Raman spectroscopy (SERS) and photodynamic treatment (PDT).²²³ Real-time monitoring of biomarker levels, particularly miR-21, was made possible by the SERS-active reporter molecule, which achieved an ultralow detection limit of roughly 0.7 fM in living cells. The ability of CuPc@HG@BN to produce singlet oxygen (¹O₂) was examined using the singlet oxygen sensor green (SOSG) probe, which reacts with ¹O₂ to produce strong green fluorescence. Strong green fluorescence was seen in MCF-7 cells treated with CuPc@HG@BN and SOSG after 5 min of radiation. After 8 h, significant ¹O₂-induced cell membrane damage was seen, confirming CuPc@HG@BN's superior PDT efficiency. Furthermore, the SERS signal of CuPc@HG@BN was barely affected by the production of ¹O₂. Following treatment with CuPc@HG@BN under irradiation, the rates of early and late apoptosis of MCF-7 cells were 50.03% and 31.31%, respectively. These were noticeably greater than the 1.11% and 0.08% of cells treated with CuPc@HG@BN alone. Using MCF-7 tumor-bearing nude mice as a model system, the in vivo therapeutic efficacy was further evaluated. Mice were administered with Cy3-label CuPc@HG@BN via tail vein in order to measure the biodistribution. At 6 h post-injection, significant Cy3 fluorescence signals were seen at the tumor location of the animals. CuPc@HG@BN were accumulated in the tumor sites and retained for a long enough period of time, as evidenced by the stronger Cy3 signals that were seen in the tumor than in the normal tissues after 12 h and that persisted even after 48 h. The Cu concentration was measured using coupled plasma mass spectrometry (ICP-MS) to assess the pharmacokinetics profile of CuPc@HG@BN in blood, tumor, and normal tissues. The probe's clear buildup in the liver and spleen indicated that the phagocytes in the reticulo-endothelial system had partially intercepted h-BNNS. Conversely, the accumulation of CuPc@HG@BN in the kidney was significantly less than that in the liver and spleen. Even though the late-stage tumor was not entirely eradicated, CuPc@HG@BN and irradiation were nevertheless able to successfully limit tumor growth with few adverse effects. Mice with a tumor that had grown for 25 days were also used to examine the effectiveness of treatment. Only tumors treated with CuPc@HG@BN under irradiation showed a significant tumor regression. Minimal toxicity to the normal tissues was demonstrated by the H&E-stained organ slices from the mice treated with CuPc@HG@BN under irradiation and the insignificant body weight loss within 15 days. All of these findings confirmed effective intracellular transport and the CuPc@HG@BN theranostic probe's maximum anticancer activity in vivo with few adverse effects.

5.7. Neurological disorders

Neurodegenerative diseases are conditions marked by a steady loss of neurons linked to protein depositions, inflammation, apoptosis and changes in the trophic factors, which ultimately lead to changed physical and chemical characteristics of the brain, spinal cord and peripheral organs. Some of the frequently challenging primary neurodegenerative disorders include Parkinson's disease (PD) and Alzheimer's disease (AD). There are a number of reports indicating the neuroprotective role of the different nanomedicine approaches in various in vitro and in vivo disease models.

Despite progress in understanding the processes behind the emergence of neurological diseases and the activities of neurotransmitter drugs, drug delivery to the central nervous system (CNS) remains a formidable problem. The barrier between the blood and brain restricts the availability of drugs to the central nervous system. In addition, the distribution of neuroactive substances throughout the body to promote neuronal regeneration is fraught with inherent issues, such as the toxicity and unstable nature of several bioactive components. The applications of bio-nanotechnology with relation to the CNS are intended to engage with tissues and cells at the subcellular molecular level, namely, at cellular components such as transmembrane proteins, ion channels, and surface receptors. Designing and utilizing bionanotechnologies are made more difficult by the complexity of the CNS's cellular heterogeneity, structure, and functional organization. However, the potential provided by the special qualities of devices and materials with nanoengineering complements additional neurological techniques and offers a significant chance to advance our fundamental knowledge concerning neurophysiology and cellular neurobiology as well as to develop new clinical treatments for neurological disorders.²²⁴

Therefore, applications of nanomaterials have become an upcoming trend, especially the exploitation of metallic nanoparticles in neurodegenerative disorders. There are several metallic nanoparticles that have been used in the management of neurodegenerative disorders under pre-clinical conditions.²²⁴ The application of zinc, silver, gold and more specifically boron has been used as a treatment for both traumatic and non-traumatic neurodegenerative disorders.^225–228 There are enormous difficulties in these nano-formulations, especially development, bio-metabolization, soluble nature, and challenges, in crossing the blood–brain barrier (BBB) and cytotoxicity. In this context, the multipotent properties of boron nitride nanotubes (BNNTs) and hollow boron nanotubes (h-BNs), such as their high surface area, good physical durability, and favorable biocompatibility, make them ideal nanocarrier systems along with low cytotoxicity profile, greater solubility and increased cell uptake and greater capacity to cross the blood–brain barrier. One such forefront approach was the use of poly-L-lysine-coated boron nitride nanotubes (PLL-BNNTs), which were shown to be excellent neural transducers. Using BNNTs as carriers of NPs to transport electrical and mechanical signals inside a biological system on demand was the subject of another research. Once BNNTs have internalized, their piezoelectric behavior allows them to be used as external “wireless” mechanical sources to provide electrical stimulation to a tissue or cell culture. The percentage of differentiated cells in the culture serves as a proxy for the differentiation status. In control cultures, measurements of neurite length and the quantity of neuronal processes per cell were made. Three types of cultures were studied: those that were stimulated with a “wireless” mechanical source, those that were incubated with BNNTs, and those that were stimulated with both. Nerve growth factor (NGF) was added to the culture medium and PC12 cells were stimulated for up to five days. No statistically significant difference was observed between the groups when it came to cellular differentiation: every culture attained above 95% of differentiation without showing any variation from the control. Remarkably, in the cultures that underwent both ultrasonic stimulation and BNNT incubation, the quantity of neural processes within each differentiated cell significantly increased.²²⁹ As PC12 is used as an in vitro model of spinal cord injury (SCI), BNNTs can also be used in multiple disorders related to SCI. However, its use in spinal injury animal models have not yet been performed, and it can prove to be a boon in SCI.

Despite the fact that BN has not been used extensively in neuroscience, a few noteworthy studies have given a comprehensive understanding of its applicability in neurodegenerative illnesses and neuronal pathology. One such study used the mHippoE-14 embryonic mouse hippocampus cell line to examine the biocompatibility and oxidative stress-relieving properties of h-BNs and their breakdown product, boric acid (BA). To determine the viability of the cells at different concentrations of h-BNs, the researchers evaluated ROS generation, processes of cell death and study of the cell cycle. They also applied Dox to induce cell stress, which improved the cells' ability to survive and reduced the production of ROS and apoptosis. The findings point to the potential medicinal uses of h-BNs as biocompatible agents, such as reducing oxidative stress brought on by medications used to treat brain tumors and neurological disorders. Another important report examined the structure and electrical characteristics of boron nitride nanoribbons adsorbed with the neurotransmitters: adrenaline and dopamine systemically.²³⁰ These nanoribbons would be employed as a biological cargo carrier for neurological applications in medicine. Dopamine plays an important role in various neurological conditions. Applications of boron nitrides in conjugation with dopamine and adrenaline can be an effective therapeutic approach in PD, AD, epilepsy, and in episodes of depression.

Another study investigated the neuroprotective effects of boron nitride nanoparticles against MPP+ induced apoptosis in an animal model for Parkinson's disease.²³¹ PD is the progressive degeneration of dopaminergic neurons in substantia nigra. Although there are different nanoparticles that are both metallic and non-metallic, the use of these materials still poses problems and its application is still restricted to pre-clinical studies. This led to a study that was used to determine if h-BNs might reduce the toxicity of 1-methyl-4-phenylpyridinium (MPP+) in an experimental Parkinson's disease model. A diverse range of doses of MPP+ were applied to cultures of human embryonal carcinoma cells with developed pluripotent cells (Ntera-2, NT-2) to create an experimental PD model. Cell viability experiments, which involved the release of MTT and LDH, were used to study the efficacy of h-BNs to protect against MPP+ toxicity. Using tests for total oxidant status (TOS) and total antioxidant capacity (TAC), oxidative changes caused by the administration of h-BNs in PD cell culture models were examined. Using the fluorescence of Hoechst 33258 staining technique, the effects of h-BNs and MPP+ on nuclear integrity were examined. Using a colorimetric test, the activities of the acetylcholinesterase (AChE) enzyme were assessed in response to h-BN administration. An examination of flow cytometry data was employed to investigate the mechanisms of cell death brought on by exposure to MPP+ and h-BNs. Based on experimental data, the use of h-BNs against MPP+ application in the PD model enhanced cell viability. TAS and TOS experiments showed that after administering h-BNs, the antioxidant capacity increased while oxidant levels decreased. Moreover, the administration of h-BNs greatly inhibited MPP+-induced apoptosis, as demonstrated by flow cytometric analysis. To sum up, the results indicated that h-BNs have enormous potential to prevent MPP+ toxicity and can be employed as novel neuroprotective and drug delivery agents in the treatment of Parkinson's disease (PD).²³¹

One of the most recent studies sought to determine, for the first time, how differentiated human SH-SY5Y neuroblastoma cell cultures were shielded against beta amyloid (Aβ_1–42) by varied doses of hexagonal boron nitride nanoparticles (h-BN-NPs).²³² MTT and LDH release tests were used to evaluate the neurotoxicity and therapeutic potential of h-BN-NPs generated by Aβ_1–42 on differentiated SH-SY5Y cells. A review of cellular morphologies, gene expression levels linked to AD, and values of total oxidant status (TOS) and total antioxidant capacity (TAC) was conducted. The percentage of viable cells was significantly lower when exposed to Aβ_1–42, and the amount of TOS was also enhanced. Aβ_1–42 caused necrotic and apoptotic cell death. When exposed to Aβ, the expression levels of the genes APOE, BACE 1, EGFR, NCTSN, and TNF-α greatly increased, while the expression levels of the genes ADAM 10, APH1A, BDNF, PSEN1 and PSENEN significantly reduced. Applications of h-BN-NPs reduced all neurotoxic insults caused by Aβ_1–42. Additionally, the notable increase in the signal for Aβ after 48 hours of exposure to Aβ_1–42 was inhibited by h-BN-NPs. The findings suggested that h-BN-NPs might considerably reduce the neurotoxic harm caused by Aβ. Therefore, h-BN-NPs may represent a new and potential anti-AD agent for use in bio-nano imaging, medication delivery, or successful drug development.²³²

To investigate BNs capacity for neural regeneration, Qian et al. integrated it into a nerve scaffold.²³³ Using piezo-response force microscopy, the authors first confirmed that the 2D BN scaffold had outstanding piezoelectric properties (3.3 to 25.7 pCN⁻¹). In order to create mechanical deformation on the 2D BN scaffold and produce electrical currents, they then implemented a treadmill running routine. A fully regenerated peripheral nerve made up of axons and myelin sheaths was discovered when the morphology of the regenerated nerve tissues was examined by transmission electron microscopy eighteen weeks after implantation. Axon regions, myelin sheath thickness, myelinated axon diameter, and myelinated axon number all increased in the axons inside the 2D BN scaffold. Additionally, there was an improvement in the restoration of locomotor function as assessed by distal compound muscle action potential (DCMAP) and walking track analysis. Because denervation causes muscle atrophy, artificial nerve scaffolds have difficulty restoring motor function. The 2D BN scaffold encouraged the transition of the muscle fiber phenotype from slow to fast while maintaining the endplate function. Since micro vessel rebuilding constituted the foundation for axonal regeneration, the faster axonal regeneration was the cause of this occurrence. As anticipated, the 2D BN scaffold group showed a large rise in CD34, indicative of neo-vessel development. In addition, the scaffold is very biocompatible in vivo because it causes less damage to the main organs.

6. Conclusion and future prospects

In the past few decades, significant work has been performed on developing several potential 2D layered nanomaterials. In this context, 2D h-BN began attracting the attention of the researchers after the gold rush for 2D nanomaterials began with the discovery of graphene. Considering the fascinating chemical and physical characteristics of 2D h-BN, researchers have concentrated on different routes for their synthesis. Herein, we attempt to provide an outline of the developments in this review and progress regarding 2D h-BN, including their remarkable properties, different physical and chemical techniques for their synthesis, and their possibility of utilizing in biological applications such as drug delivery, tissue engineering, bio-imaging, cancer therapeutics, and biosensing. The distinct surface characteristics of h-BN, including its huge active surface locations and a high surface area, hierarchical porosity, biocompatibility, presence of strong quantum confinement, and significant opto-electronic properties, make it a prospective applicant for biomedical uses ranging from fluorescence sensing to bio-imaging. 2D-graphene is an example of a well-studied low-dimensional 2D layered nanosystem, and although the 2D-h-BN nanostructure have intriguing properties and a bright future, research on their essential physical characteristics and possible uses has been relatively slow up to this point. This could be explained by the difficulties in mass-producing materials with excellent crystallinity and high purity, which restrict their industrial applications and obstruct the exploitation of all of their functional potentials.

Amongst the many synthetic methods, such as mechanical and liquid phase exfoliation, hydrothermal/solvothermal procedures, deposition techniques, and CVD with or without a metal catalyst, CVD has been identified as a flexible way to create a variety of BN nanostructures. Nevertheless, the high processing temperature (∼1000 °C or higher) is incompatible with the majority of semiconductor techniques. Additionally, there is a significant chance of degradation from chemical contamination when transferring to other, more valuable substrates, typically through the use of polymers. Furthermore, even when BN foams are generated at about 1000 °C, their quality and crystallinity remain inadequate. It should be investigated further whether careful catalyst tuning may significantly lower the defect density. The viability of scaling up these CVD methods is also greatly influenced by the intricate growth parameters including gas flow rate, substrate homogeneity, and precursors.

Exfoliation techniques by means of mechanical or liquid phase exfoliation processes can produce high-quality h-BN nanosheets on a large scale in high yields. However, the resulting h-BN nanosheets usually suffer from non-uniform size and thickness distributions, which may hinder reproducible device performance. As another low-cost, low-processing temperature and easy upscaling method, bottom-up assembly and top-down hydrothermal/solvothermal approaches have been used for the preparation of BN composites. However, the microstructure, quality and morphology of the resulting architectures are highly dependent on the synthetic parameters such as type and volume of precursor and solvent, reaction temperature and time and polymer additives.

Bandgap engineering is another possibility of modifying the capabilities of 2D-h-BN. Although studies have focused on this view, there are several potential scopes for surface alterations to modify the characteristics of the nanosheets. Consequently, surface alterations offer enormous potential for customizing the characteristics of 2D-h-BN. It is possible to process the nanocrystals in situ, which could be a novel technique to modify their optoelectronic characteristics to suit the needs of a particular application. The effects of surface and edge flaws on the band structure of 2D-h-BN require further investigation.

It is necessary to create new green chemistry-based techniques and synthesis protocols. The prospective uses of h-BN in bioengineering domains such as drug delivery, biosensing, and bioimaging underscore the need for creating procedures that employ non-toxic solvents for their preparation. More biocompatible solvents with high polarity should be investigated because commonly used solvents such as DMF and NMP are harmful.

Although researchers have explored the use of 2D h-BN nanostructures in various biomedical applications, significant challenges remain before their successful clinical translation. One major concern is the insufficient evaluation of the biosafety of these 2D nanomaterials, with current studies failing to establish their full biocompatibility. Most existing toxicological research focuses primarily on short-term toxicity and underlying mechanisms, leaving critical gaps in our understanding of their pharmacokinetics, in vivo transformations, structure–activity/toxicity relationships, reproductive and developmental toxicity, and long-term effects—particularly for materials other than graphene. Therefore, comprehensive investigations into these aspects are essential for advancing the biomedical potential of h-BN nanostructures. Second, it is essential to elucidate the metabolism and biodegradation pathways of BN nanostructures in vivo. While h-BN has demonstrated excellent cytocompatibility and has been widely utilized, its short- and long-term biosafety profiles in living systems remain a concern, particularly when employed as an anticancer nanozyme or drug delivery nanocarrier. Despite its promising applications, current research and available data are insufficient to fully address these critical safety questions. Third, the targeting capabilities of BN-based nanomedicines need significant improvement—an issue common across many nanomedicine platforms. Due to its chemical inertness, h-BN is particularly difficult to modify, posing a challenge for the attachment of targeting ligands essential for site-specific drug delivery or accumulation in target tissues or organs. Currently, most BN nanocarriers rely on passive targeting strategies, which limit cellular uptake and reduce therapeutic efficacy. Therefore, developing effective surface functionalization techniques for h-BN is a crucial first step. In addition, further research is needed to identify efficient targeting ligands compatible with BN materials and to optimize coupling strategies that enable their stable and functional integration. A deeper understanding of the relationship between the chemical structure of BN nanomaterials and their emission characteristics is crucial for advancing their use in bioimaging applications. Bioimaging enables real-time visualization and analysis of physiological processes and pathological changes within living organisms. The development of advanced diagnostic and therapeutic technologies urgently requires modern, efficient fluorescent probes. However, due to the wide bandgap of h-BN, it inherently lacks visible light emission. Therefore, inducing luminescence in BN materials primarily relies on band engineering strategies such as doping, defect introduction, and surface functionalization. Despite these efforts, the structure–luminescence relationships in BN remain poorly understood.

Integrating both diagnostic and therapeutic functionalities into a single BN-based nanoplatform holds great promise for cancer treatment and other diseases. Designing multifunctional BN systems capable of drug delivery, bioimaging, catalytic (nanozyme) activity, and more could significantly enhance therapeutic outcomes. However, this is highly challenging, as each function often demands distinct material architectures, and the parameters governing them are interdependent rather than individually tunable. Overcoming these challenges could represent a major breakthrough in the field. BN nanostructures show considerable promise, and continued research in this area is warranted. This review aims to inspire and guide future investigations into the design and application of BN-based nanomaterials in biomedicine and beyond.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Acknowledgements

The authors express their appreciation towards the Director, CSIR–NEIST, Jorhat, for showing interest in the execution of this project work (Manuscript No. CSIR-NEIST/PUB/2024/081). MRD acknowledges the financial support from the CSIR (OLP 2411 and OLP 2504 A).

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