2D layered nanomaterials for therapeutics delivery

Abstract

Two-dimensional (2D) nanomaterials are ultrathin, layered materials with a high surface-to-volume ratio that can deliver various therapeutics including small-molecule drugs, peptides, and large proteins. Their high surface area allows for high therapeutic loading and sustained therapeutic release over time. Some 2D nanomaterials respond to external stimuli, providing control over triggered or on-demand therapeutic release. 2D nanomaterials explored for biomedical applications include carbon-based (graphene), nanoclays, black phosphorous, layered double hydroxides, metal organic frameworks, covalent organic framework, 2D metal carbides and nitrides, transition metal dichalcogenides, transition metal oxides, polymer nanosheets, and hexagonal boron nitride. Most of these nanomaterials are biocompatible and degrade into nontoxic products, which is advantageous for therapeutic delivery systems. In this article, we will evaluate these nanomaterials for therapeutic delivery. We will highlight some of their unique physical and chemical characteristics, discuss their biological stability, and investigate their ability to deliver various therapeutics. Recent developments in 2D nanomaterials as drug delivery systems will also be discussed.

Introduction

Many U.S. Food and Drug Administration-approved drugs exhibit poor water solubility which leads to low bioavailability and inefficient distribution [1]. Drug delivery systems are designed to enhance the solubility and chemical stability of drug molecules to maintain a therapeutic concentration at the target site [2]. Recent research has shown that two-dimensional (2D) nanomaterials improve the delivery of hydrophobic drugs by increasing drug efficacy, reducing toxicity, and improving bioavailability [2–5]. Their anisotropic charge distribution, large surface area-to-volume ratio, and photodynamic and thermal properties make 2D nanomaterials potentially useful as targeted delivery carriers.

Two-dimensional nanomaterials have a flat structure with lateral dimensions on the nanoscale or microscale, and a thickness of a single to a few atomic layers [5,6]. This atomic thickness creates the highest specific surface area of any type of material, providing a large number of contact points and increasing the propensity for surface interactions with drugs for high loading and controlled release kinetics. These materials can also load drugs via π–π stacking [7,8], which efficiently stabilizes drug molecules and promotes stimuli-responsive release [9]. The variation in energy band gaps, as well as physical, electrical, and chemical properties can differ among each material and allows for tunability in the design of therapeutic delivery systems [10].

In this review, we will focus on 2D nanomaterials and their applications as drug delivery systems that exhibit localized, prolonged, and sustained release of therapeutics. The discussion is limited to the types of 2D nanomaterials most relevant to drug delivery based on biocompatibility, sustained and prolonged delivery ability, and stimuli-responsive ability. This includes carbon-based (graphene), nanoclays, black phosphorous, layered double hydroxides (LDHs), metal organic frameworks (MOFs), covalent organic framework (COFs), 2D metal carbides and nitrides (MXenes), transition metal dichalcogenides (TMDs), transition metal oxides (TMOs), polymer nanosheets, and hexagonal boron nitride (hBN) (Figure 1). The scope of this paper is to highlight the current and emerging state of 2D nanomaterial-based drug delivery systems. We will highlight some of the most promising types of these 2D nanomaterials for therapeutics delivery applications, specifically for regenerative medicine, and cancer therapeutics.

Figure 1.

2D nanomaterials for drug delivery applications. The high surface area of these nanomaterials is used to sequester a range of therapeutics including drug, gene, peptide and proteins for various biomedical applications.

Carbon-based materials (Graphene)

Graphene is the oldest known 2D nanomaterial and has applications in numerous research areas including electronics, sensing, and energy [11–13]. The atomic structure contains layers with strong pi bonds between carbon atoms and weak interlayer binding through Van der Waals forces which lead to extreme mechanical strength [14]. Graphene also has exceptional physiochemical, thermal, and biomedical properties based on its highly reactive structure [15,16]. Graphene oxide (GO) is a modified graphene material with carboxylic acid, epoxide, and hydroxyl groups in the planar structure [14], as well as unmodified sections of hydrophobic graphene, making GO an amphiphilic molecule useful for stabilization of hydrophobic drugs in solution [17]. The amphiphilic nature of the molecule and presence of functional groups are beneficial for drug delivery applications [18]. Graphene oxide can be further modified to obtain reduced graphene oxide (rGO) which has increased electrical properties, but reduced surface charge and hydrophilicity compared to GO [14]. Reduced graphene oxide is a stimuli responsive material [19] and has improved dispersibility [20] compared to GO, both of which can be leveraged to synthesize stable drug carriers that perform a sustained release.

Graphene oxide nanosheets were used for targeted delivery of chemotherapeutic agents such as docetaxel [21]. Docetaxel has poor solubility in water, and current FDA-approved delivery methods utilize surfactants that could cause extreme adverse effects in some cases. GO was used to load docetaxel through π–π stacking for efficient delivery without the use of a surfactant. The 37% loading capacity with GO was increased by a factor of 3.7 relative to reported data for commonly used small-molecule drug delivery systems [21]. Drug release was pH-dependent, as there was a 2.73-fold increase in acidic pH relative to neutral pH over identical time periods. Transferrin was incorporated to promote accumulation of the GO nanosheets in cancer cells that contain an abundance of transferrin receptors. In vivo testing is necessary to determine how targeted delivery will be affected by normal cells that express high levels of transferrin receptors and cause delocalized delivery [21].

Intracellular delivery of therapeutics is especially beneficial because specific cell compartments can be targeted to achieve better therapeutic activity. A recent study investigated the enhancement of cellular uptake and drug release properties of GO via surface modification without limiting biodegradability [22]. Doxorubicin was loaded by intercalation onto G-4 quadruplex DNA strands which were used to line the GO surface. Actuated by NIR irradiation, the photothermal effect of GO would cause the DNA to denature and release the drug. This newly formed single strand binds to hemin on the GO surface and, because of high hydrogen peroxide in the tumor environment, catalyzes a reaction that degrades GO into biocompatible, rapidly cleared products. In vivo testing showed a significant level of tumor growth inhibition, attributed to both the inherent properties of GO and benefits of the surface modification [22]. Graphene-based materials have shown to exhibit high loading ability, biodegradability, tunability, and controlled drug release for small-molecule drugs [23]. Due to this, these materials have also been investigated for their ability to sequester and release larger biomolecules [24].

In another study, a GO-based hydrogel carrier for bone morphogenetic protein 2 (BMP2) was designed for the improvement of BMP2 therapeutic function in cartilage [25]. The initial burst release seen in hydrogels with free BMP2 was greatly hindered upon incorporation of GO, after which there was sustained release for 40 days. The controlled release behavior is especially beneficial in treating osteoarthritis, as improper doses of BMP-2 can cause side effects such as ectopic bone formation or osteolysis. In addition, there was an enhanced therapeutic effect and greater inhibition of osteoarthritis in the gels that included GO [25]. Graphene oxide can also be used to promote vaccine delivery of biomolecules [26]. Graphene oxide has been investigated as a potential nanocarrier for protein-based therapeutic agents to facilitate their interactions with dendritic cells. Graphene oxide strongly interacts with proteins that are more hydrophobic, and those with more lysine residues leads to strong covalent interactions. In vitro testing showed GO enhanced the vaccine antigen presentation to CD8⁺ T cells which elicit a more potent immune response than the CD4⁺ T cells that cause the antibody mediated response [27]. Because of the high surface area, sheet-like structure, and inherent hydrophilicity, GO can efficiently load and deliver proteins via multiple administration routes.

When used as therapeutic carriers, GO nanosheets have a tendency to agglomerate due to interlayer bonding which can reduce the efficiency of drug release [28]. To address this, rGO was synthesized via spray drying and used to form iron oxide–decorated rGO microspheres for synergistic cancer therapy. The microspheres showed a very high loading efficiency (92.15%) of doxorubicin which can be attributed to the high surface area of rGO, strong π–π stacking and hydrogen bonds [28]. Release rates of doxorubicin were analyzed in physiologic pH (7.4) as well as acidic pH (5.4) to simulate microsphere behavior in both healthy and tumor tissue. In the first 10 h, nearly twice as much therapeutic was released in pH = 5.4 compared to pH = 7.4. The cumulative release totals for the microgels followed a similar trend, showing a significantly higher release in acidic pH after 98 h. In addition to pH responsiveness, the photothermal properties of rGO allowed for NIR stimulus to influence microsphere behavior in two ways: accelerated doxorubicin release as well as an increased temperature both related to NIR intensity. In vitro cytotoxicity assays showed high biocompatibility and excellent antitumor properties [28]. Although it is unclear how results from the in vitro assessments would translate to in vivo microsphere performance, the available data show that rGO can be used to form stable, smart delivery systems for small-molecule drugs.

The stimuli-responsiveness of rGO can also be utilized to create drug carriers to deliver agents for treating chronic diseases such as cancer, diabetes, or arthritis that require frequent doses over extended periods of time. Hydrogels modified with rGO were used for on-demand release of insulin over 7 days while maintaining its metabolic activity [29]. Insulin-loading capacity increased with rGO concentration and the hydrogels showed a small burst release of only 10% when submerged in PBS for 1 day. This period was not followed by any further release which indicates the ability of the hydrogels to stabilize loaded cargo and only release when triggered. With daily NIR laser illumination for 1 week there was a repeated release of over 30% of the loaded insulin on each day. The metabolic activity of photothermally released insulin was analyzed by measuring protein kinase B phosphorylation (p-Akt), which occurs as a direct result of insulin binding to HepG2 cells. Specifically, the p-Akt/Akt ratio was measured for native insulin as well as insulin released due to NIR stimulus from the hydrogels. The photothermally released insulin showed a ratio of 6.14 compared to 4.82 by native insulin, indicating a greater activity in the hydrogel-released hormone [29]. The reason is unclear, but this is possibly due to the ability of the rGO to stabilize and increase the bioactivity of insulin. In vitro assays showed a high biocompatibility of the hydrogels as well as the ability to perform NIR-stimulated transdermal delivery of insulin through porcine ear skin [29]. The ability of the rGO-incorporated gels to load, stabilize, and release sustained levels of insulin on demand without signs of cytotoxicity is promising for diabetes treatment and can be expanded to design therapeutic carriers for other chronic diseases.

Nanoclays

Clay materials are naturally occurring, finely grained materials that contain phyllosilicate minerals [30]. Smectites are a type of phyllosilicate with a 2:1 unit structure of two tetrahedral sheets sandwiching one dioctahedral (montmorillonite (MMT)) or trioctahedral (Laponite or saponite) sheet formed from a metal cation. These types of clay minerals are biocompatible, have a large specific surface area, and have high swelling capabilities in the presence of water [31,32]. Laponite and MMT clays have extremely small particle size, large cation exchange capacity, anisotropic charge distribution, and interactions with inorganic and organic substances, imparting physiochemical properties that make them useful for drug delivery applications [33].

The structural and charge characteristics of nanosilicate clays have been utilized to efficiently load and release small molecules [34]. Cisplatin, 4-fluorouracil, and cyclophosphamide were each loaded into Laponite disk–based nanohydrogels to evaluate the loading and release of different chemotherapeutics for breast and ovarian cancer. Laponite disintegrates into ions at pH values lower than 7; therefore, the nanosilicate hydrogels were expected to be stable at physiologic pH and release their cargo near pH = 5.5. This would allow for the hydrogels to release the loaded cargo prior to intracellular degradation by lysosomal enzymes [35]. In an acidic environment (pH = 5.5), the hydrogels increased in size by a factor of 15 and burst into small pieces, releasing the loaded molecules. When compared to the free drug, each drug loaded separately in Laponite-based gels showed high cytotoxic activity in both HeLa cells and MCF-7 cells. Combined into one gel, the mixture showed a positive synergistic cytotoxicity effect with an IC₅₀ value that is 10 times lower than the theoretical value. When injected intravenously into mice, the free Laponite gel showed high biocompatibility. The hydrogels were biodegradable and showed 100% clearance in 24 hours [35]. The use of multilayered systems is common for nanosilicate clays, as the flat structure allows for facile synthesis of versatile layer-by-layer materials with high loading capabilities and efficient surface functionalization [36,37] (Figure 2). MMT/hyaluronic acid (HA)–gentamicin layer-by-layer films were synthesized for on-demand self-defense drug release of antibiotics to prevent bacterial colonization after implantation. The nanofilms showed a high loading dosage of 0.85 mg/cm², which was partly due to the high MMT adsorption to positively charged molecules. The films exhibited enzyme-responsive release, and in vivo testing showed good bactericidal properties due to the layer-by-layer enzymatic degradation [38]. The results of these experiments showed that smectite nanoclays can be used in drug delivery to increase performance and enhance biocompatibility of therapeutic carrier systems.

Figure 2.

Different synthesis methods for therapeutic controlled delivery systems. (a) Layer by layer polymer encapsulation of therapeutics sequester them until particle degradation. (b) Incorporation of bound therapeutic within hydrogel scaffold provides them with NIR and mechanical stimuli release. (c) Interactions between nanoparticles and proteins provide them with protection from mechanical and thermal stress and allow release following degradation.

Drugs often have rapid clearance and short half-lives; therefore, supraphysiological doses are required to achieve their intended therapeutic effect, despite often resulting in adverse effects [39,40]. Nanosilicates can remain in circulation for prolonged periods, promoting increased bioavailability of smaller amounts of loaded therapeutics [41,42]. The high surface area of nanoclays can be used to sequester a range of protein therapeutics for sustained and prolong delivery [43–46]. In a recent study, Laponite-based delivery systems were used to reduce the concentration of delivered recombinant human BMP2 (rhBMP2) and transcription growth factor beta-3 (TGF-β3) while maintaining bioactivity [47]. After an initial burst release of loosely bound protein, sustained release was observed for over 30 days. Laponite nanoclays showed inherent osteoinductive properties, and a synergistic effect on osteogenic differentiation was seen in the rhBMP2-loaded nanoclay. Delivery of TGF-β3 with nanosilicates provided a greater stimulation of specific hMSC differentiation than delivery of TGF-β3 alone, using 10-fold less TGF-β3 [47]. In another study, 2D Laponite particles were incorporated into injectable alginate hydrogel walls to tune protein release and prevent denaturation. Five proteins of varying size and net charge were bound to nanoparticle-walled hydrogels and analyzed against alginate-only gels. Laponite particles were able to eliminate the initial burst release and maintain a controlled release over 4 weeks without affecting bioactivity [48]. Further investigation should be conducted in vivo to determine the effects of cellular infiltration on the gels as well as how the osteogenic properties of Laponite may cause calcification of the hydrogel and disrupt its function.

Black phosphorus (BP)

Black phosphorus (BP), or phosphorene, is a semiconductor phosphorus allotrope with an orthorhombic pleated honeycomb structure [49]. Black phosphorus is structurally similar to graphene but has unique properties that offer advantages over the latter in some biomedical applications such as its tunable band gap and high carrier mobility [49,50]. The anisotropic behavior, high biocompatibility, and nontoxic degradation products make BP an ideal material for use in drug delivery systems [51]. Some of these unique characteristics of BP can be leveraged to develop bioresponsive and on-demand delivery carrier.

Negatively charged BP sheets exhibit strong binding to positively charged small-molecule drugs via electrostatic interactions [52]. These nanosheets have stimuli–responsive properties that can be leveraged for photothermal therapy treatment of cancers. A BP nanosheet–based drug delivery system was used to deliver a chemotherapeutic agent and reverse multidrug resistance seen in tumor cells [53]. Intracellular mutant p53 in cancer cells has anti-apoptotic capability and allows the cells to tolerate chemotherapeutics. Phenethyl isothiocyanate (PEITC) can remove mutant p53, leading to elimination of drug resistance. Here, the BP nanosheets were loaded with PEITC and doxorubicin and investigated [53]. Polydopamine (PDA) and polyethylene glycol-amine were added to enhance binding to PEITC and enhance the solubility of the nanocarriers. Doxorubicin release was investigated, and it was determined that this system had both pH- and NIR-responsive capabilities. In acidic environments, PDA dissociates and the solubility of doxorubicin increases, leading to an accelerated release. NIR stimulus caused the BP nanosheet to generate heat, decreasing electrostatic interactions between the drug and carrier. In vivo assessment of the delivery system showed that the carriers have high biocompatibility and, when used in combination with NIR-stimulation, synergistic anticancer effect is observed [53].

Black phosphorus also has inherent antimicrobial properties [54] that are enhanced when used in combination with another antimicrobial agent [55]. In a recent study, titanium aminobenzenesulfanato (Ti-SA₄) complexes were loaded onto BP nanosheets via simple mixing [56]. The loading efficiency and antibacterial effect of this platform against drug-resistant bacteria were investigated. Strong P–Ti coordination led to a high level of loading at about 43%. BP/Ti-SA₄ has a positive surface potential, which is beneficial for binding to negatively charged bacteria, while its sharp edges penetrate and destroy the bacteria membrane. Significant improvement in therapeutic effect was seen in the hybrid nanocarrier relative to the free BP and Ti-SA₄ [56]. It is known that BP has good biocompatibility and biodegradability, but to expand the applications of BP in small-molecule drug delivery, in vivo assessment is necessary to determine how surface modification can affect these properties.

Many of the current DNA delivery methods are subject to increased risk of off-target toxic effects due to over-dosing [57]. To achieve direct and sustained delivery without excessive release of drugs, BP nanosheets were employed for cytosolic delivery of Cas9 ribonucleoprotein [58]. Cas9 was loaded to BP nanosheets via electrostatic interactions at an efficiency of 98%. Upon cellular uptake via endocytosis, the BP/Cas9 complex performed endosomal escape, and cytoplasmic degradation of the BP led to sustained Cas9 release over 12h. In vivo testing showed promising results, demonstrating that the nanocarrier system showed significant gene silencing activity [58]. These results indicate that BP can support efficient loading and delivery via targeted cellular uptake.

Layered double hydroxides (LDHs)

Layered double hydroxide materials have a layered structure, similar to clays, with two metal hydroxide outer layers sandwiching the inner anionic layer. These materials are responsive to pH and will biodegrade in response to acidic pH, forming biocompatible byproducts. Their positive charge also allows for efficient loading of anionic molecules. These properties are promising for designing site-specific drug delivery systems [59,60].

While current chemotherapy drugs have been shown to prolong patient lifespan, there are issues of toxicity and extreme adverse effects. Layered double hydroxides have low toxicity and, due to their 2D structure, can deliver the required therapeutic dose of drug without causing adverse effects due to over-dosing [59]. Layered double hydroxides were investigated as a nanocarrier delivery system for etoposide (VP16), a common chemotherapy drug with side-effects such as hair loss, gastrointestinal problems, and leukopenia [61]. Use of the LDH-based system increased cellular uptake of the free drug from 61.6% in 1 hours to over 90% in that same timeframe and led to significant reduction in common VP16 side effects, including liver and blood toxicity. Release was also PH-dependent, with a high initial burst release of ~40% and ~25% at acidic pH of 4.8 and 5.8, respectively. The delivery system also greatly increased the bioavailability of the drug, reaching a peak plasma concentration at 24 hours compared to 4 hours for the free drug [61]. LDHs were also used to synthesize a nanofilm for delivery of butyrate, an antibacterial agent, focusing on LDH H₂O₂ responsiveness [62]. The films, when in a high H₂O₂ environment, were able to revert H₂O₂ to OH⁻ which would exchange with the interlayer butyrate. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria were used to measure the antibacterial activity of the films in vivo. Layered double hydroxide samples without butyrate showed some antibacterial properties, with an inhibition rate of close to 100% for S. aureus but only about 70% for E. coli. Samples containing butyrate were able to kill both types of bacteria with inhibition rates of 100%. H₂O₂ controlled release was seen, with alternating burst release and sustained release depending on whether the film was submerged in H₂O₂ or PBS [62]. Layered double hydroxide–based drug delivery enhanced many aspects of the loading, targeting, and sustenance mechanisms of both VP16 and butyrate, indicating that these 2D nanomaterials would be effective delivery vehicles for small-molecule drugs.

To effectively deliver larger molecules such as proteins or peptides, the denaturation of immobilized proteins must be prevented to maintain biocompatibility and performance [63]. Properties of LDH such as the high surface-to-volume ratio, high hydrophilicity, and high biocompatibility are advantageous for protein carriers that will transport immobilized proteins without altering their activity [64]. Mg–Al–Cl-type LDH/aluminum hydroxide (Alhy) nanoplates were formed and tested for their ability to load and release bovine serum albumin (BSA) as a model protein [63]. The carrier showed an extremely high loading capacity of 996 mg/g. This is due to an increase in porosity and tunability of the surface area of LDH sheets with the addition of Alhy. Hydrogen phosphate stimulated protein release, due to the high affinity of LDH films for anionic molecules, causing 52% and 49% desorption for the LDH-Alhy and a commercial LDH, respectively. Chloride did not cause BSA release, showing 1.8% and 1.1% release for the two composites [63]. In vivo experiments are necessary to determine how different physiological pH would affect carrier performance, but the high loading capacity and selective protein release prove that LDH materials have potential for applications in protein delivery.

Metal organic frameworks (MOFs)

MOFs are organic–inorganic hybrid materials composed of metal ion nodes and organic linkers. The links form a cage-like, hollow structure with high porosity and an extremely high surface area beneficial for drug encapsulation and loading. Many combinations of metal and organic linkers can be used to form MOFs with specific properties and functions. Their versatile structure along with their biocompatibility and degradability make MOFs useful within various biomedical fields [65–67].

Curcumin (CCM) is a naturally occurring molecule with many health benefits including anti-inflammatory, anti-oxidative, and anti-HIV properties [68]. The therapeutic benefits of CCM, similar to other small-molecule drugs, are hindered by poor drug solubility and instability [69]. Zeolitic imidazolate framework (ZIF) MOFs were used to design an effective delivery system for CCM. Zeolitic imidazolate frameworks are a type of MOF with high biocompatibility, large pores, and biostability, which promote their use as drug carriers [70]. ZIF-8 showed a high encapsulation efficiency of 83.3%, and the carriers exhibited pH-responsive release with a cumulative release of 88% and high burst release at acidic pH, with only 28% cumulative release and very minor burst release at physiological pH. In vitro assessment of the ZIF-8–encapsulated drug effect on HeLa cells showed that MOF enhanced the cytotoxic effect of CCM [70].

In another study, [Zn₄O(3,5-dimethyl-4-carbox ypyrazolato)₃] (Zn₄O(dmcapz)₃) MOF was used to encapsulate hydrophobic drugs (aminobenzoic acid and benzocaine), a hydrophilic drug (5-fluorouracil), and an amphiphilic drug (caffeine) [71]. The effects of the MOF on the loading, delivery, and stability of each drug were analyzed. Over 60% encapsulation efficiency was seen in the hydrophobic and amphiphilic drugs, but there was only 22% encapsulation efficiency for 5-fluorouracil. There was also very little attenuation of the burst release for the hydrophilic and amphiphilic drug, while a sustained release was shown for the more hydrophobic drugs. The enhanced performance of hydrophobic drugs when encapsulated in MOF is presumably due to the hydrophobic nature of Zn₄O(dmcapz)₃ [71]. The results of these experiments prove that MOF materials are capable of forming effective drug delivery systems for hydrophobic drugs due to their high loading capacity, stimuli-responsive release, and tunable structural properties that promote strong hydrophobic interactions.

Similar to LDH materials, MOFs have the ability to stabilize enzymes, viruses, and antibodies while maintaining their physical and chemical properties [72]. ZIF-8 growth on protein surfaces has proved to be a simple, efficient method of stabilization that is possible for many different molecules [73]. Tobacco mosaic virus (TMV) was investigated as a model protein and encapsulated in ZIF-8 to test the MOF ability to stabilize and enhance the functionality of protein-based vaccines. Results showed that shell formation, stress, and exfoliation did not significantly alter the protein surface. In vivo assessments of the release, host response, and histocompatibility were performed. With multiple subcutaneous injections of the TMV-ZIF-8 carrier and no observed instances of toxicity or tissue damage, it is apparent that the MOF carrier can stabilize the protein without altering its biocompatibility. The delivery system was also able to promote sustained TMV delivery over 14 days, while the free TMV almost fully expended within ~8–9 days [74]. The versatility, biostability, and loading capabilities of MOFs indicate that they could be advantageous in vaccine delivery.

Covalent organic frameworks (COFs)

Covalent organic frameworks are crystalline porous organic polymers with a layered sheet-like structure. Contrast to the irreversible bonds seen in conventional covalent organic polymers, COFs are formed via reversible condensation reactions, providing better crystallinity and biodegradability. Similar to MOFs, there is versatility in the organic compound used to form the material, allowing for diverse properties. Their tunable porosity and pore function as well as the lightweight atomic composition makes COFs slightly advantageous over MOFs due to reduced potential hazard [75]. Although the large surface area, porosity, and high tunability of COF materials are beneficial properties for drug delivery applications, these materials have intrinsically poor targeting, low permeability, and physiological instability [76].

Polymers are commonly integrated with porous materials to form composites with desired properties. Nanocomposites composed of polyethylene-glycol-modified curcumin derivatives and amine-functionalized COFs (PEG-CCM@APTES-COF-1) were developed for In vitro and in vivo assessments of their loading characteristics, drug retention, stability, and release [76]. Amine-functionalized COF by itself is hydrophobic which leads to inefficient loading of hydrophobic doxorubicin. The PEG-CCM@APTES-COF-1 nanocomposites, on the other hand, showed a remarkably high loading capability of 9.71 wt% and high encapsulation efficiency of 90.5%. The carrier system considerably inhibited HeLa cell growth with low doxorubicin concentrations compared to controls that had no significant effect on cell growth. Injection of doxorubicin-loaded nanocarriers showed increased antitumor efficiency via combinational therapy of targeted cellular uptake due to PEG-CCM encapsulation as well as doxorubicin release (Figure 3). The delivery system also demonstrated prolonged blood circulation in mice [76].

Figure 3.

Application of 2D nanomaterials. (a) In vivo testing of doxorubicin-loaded COFs demonstrating pH-responsive therapeutic release. (b) In vivo testing of protein-loaded nanoclays demonstrating localized angiogenesis over time. (c) Cas9 proteins bound to nanosheets enable genome editing following cellular import of the 2D nanomaterials. (d) Antibacterial effects of 2D TMDs result in ROS production within bacteria causing cell lysis.

In another study, a COF-based delivery system was designed by sandwiching an imine-derived COF drug reservoir between biocompatible, porous PCL films [77]. The release of methylene blue (MB), a model drug, as well as oxytocin was analyzed. The ordered structure and smooth pores of the COF allowed for controlled release and hindrance of an initial burst for both molecules. High biocompatibility was achieved in the COF membrane with cell viability higher than 80% [77]. The wide range of materials available to form COFs allows for tunable porosity, which is advantageous for the design of drug delivery carriers with high drug loading. As some of the building block materials of COFs may have inherent toxicity, polymers can be used as modification tools to improve the safety and efficiency of COF-based drug carriers.

Boroxine-linked COFs are able to bind with insulin or proteins via coordination bonds between the electron-deficient boron atom and imidazole or amine groups [78]. As previously stated, he ability to stabilize proteins and inhibit denaturation during transport is essential for therapeutic delivery. Dual-responsive nanocarriers based on boroxine-linked COFs were designed to load and deliver insulin in response to glucose and pH [79]. PEG was used as a hydrophilic component, forming a micelle that traps the insulin inside the carrier until the intended stimuli is received. The incorporation of glucose oxidase endowed glucose–responsive properties, while the COF decomposed in acidic conditions. Insulin release was triggered by either an increase in glucose concentration or a decrease in pH, and there was no conformational change of released insulin in either case. In vitro, the insulin-loaded micelles performed cytosolic delivery to A549 cells and were able to degrade into biocompatible products [79]. The efficiency of the carriers in their response to stimuli and their ability to regulate glucose levels are both promising discoveries for the use of COFs in protein-based delivery systems.

Metal carbides and nitrides (MXenes)

MXenes include transition metal carbides, nitrides, and carbonitrides. M_n+1X_n is the generic formula, the main three being M₂X, M₃X₂, and M₄X₃, where M represents an early transition metal and X represents carbon or nitrogen. The tunable structure of these materials allows for flexibility in design and functionality. In addition to the high drug loading benefits, MXenes have unique characteristics such as enzymatic degradation and efficient cellular uptake making MXene-based drug delivery systems promising [80].

Two-dimensional MXenes have unique properties enabling their applications in photothermal cancer treatment [81,82]. Specifically, MXenes are triggered via NIR exposure, absorbing the emitted light and increasing in temperature, causing hyperthermia within the tumor microenvironment. Some limitations of these materials include physiological instability and inability to control drug release [83]. A possible solution is the incorporation of hydrogels, as these materials are highly biocompatible and commonly used in drug delivery. The known lack of mechanical strength in hydrogels would be restored with incorporation of 2D MXenes. In a recent study, Ti₃C₂ MXenes were integrated into cellulose hydrogels to potentially achieve controlled release and biocompatibility [83]. The composite hydrogels were loaded with doxorubicin and evaluated in vitro and in vivo. The cellulose/MXene gels demonstrated a slow increase in drug concentration over time, indicating NIR-stimulated controlled release. Results of biotoxicity assays proved that both the free and composite hydrogels were highly biocompatible. The photothermal properties of the composite hydrogels were also evaluated in vivo, utilizing gels ground into slurries and injected to tumor sites in mice. Bimodal cancer therapy was achieved with a combination of photothermal treatment and chemotherapeutic release from the hydrogels [83].

In another study, polyacrylamide (PAA_m) hydrogels were modified with Ti₃C₂ nanosheets to increase their mechanical properties and enhance drug release [84]. Using bisacrylamide (BIS)/PAA_m hydrogels as a control, in vitro testing was performed to compare the loading and release of chloramphenicol, a hydrophobic, antimicrobial agent, from the two gels. The nanogels had a greatly increased swelling ratio, higher drug loading capacity, and greater release. This can be attributed to the uniform pore distribution achieved with incorporation of the MXene sheets. While there was a positive correlation between concentration of Ti₃C₂ and inhibition of the burst release, even the highest concentration evaluated (0.4%) did not show a lower initial burst release than 0% [84]. The reason that the initial burst release was higher for gels containing Ti₃C₂ is because these gels exhibited better swelling and release properties, with a higher cumulative release. The higher burst was not due to lack of control but increased overall release of drug relative to the gels with 0% Ti₃C₂. It is possible that higher concentrations, such as 1% or above, would further inhibit the burst release; however, this may cause reduced swelling and hinder drug loading. Overall, incorporation of MXene nanosheets into hydrogels improved their mechanical strength and drug delivery properties, making MXenes a promising vehicle for drug delivery.

Transition metal dichalcogenides (TMDs)

Transition metal dichalcogenides are a class of 2D nanomaterials with a generic formula MX₂, where M represents a group 4–10 transition metal and X represents a chalcogen. The hexagonal metal layer is sandwiched between two chalcogen layers. There are approximately 40 different categories of TMDs, each with unique physical and chemical properties. The cytocompatibility, versatile surface chemistry, and high photothermal conversion properties make TMDs a good candidate for use as a photothermal agent in drug delivery applications [85–88].

Layered MoS₂ nanosheet–based delivery systems were synthesized for synergistic cancer therapy [89]. The nanosheets were first functionalized with DNA (D1/D2) and then loaded with doxorubicin. High loading was seen due to doxorubicin interactions with both the DNA as well as MoS₂ nanosheets. MoS₂/D2 and MoS₂/D1 loaded nanosheets were compared for their anticancer effects. Both systems showed a significant increase in apoptotic cell population relative to free doxorubicin. The MoS₂/D2 carrier showed fivefold increase, whereas the MoS₂/D1 carrier showed a 3.4-fold increase [89]. In a recent study, hydrogels loaded with MoS₂ nanosheet can be designed for on-demand release of doxorubicin by leveraging NIR-responsive characteristics of MoS₂ nanosheet [88]. Interestingly, the wetting characteristics of these MoS₂ nanosheet can be modulated by presence of atomic defects [90,91], a range of hydrophilic as well as hydrophobic drugs can be loaded for on-demand triggered release. High loading capacity, enhanced therapeutic effects, and stimuli-responsive release are promising characteristics for use of MoS₂ nanosheets as small-molecule drug carriers.

TMDs have innate antibacterial properties that can be used in combination with similar agents for synergistic effects. Nanocomposite films with MoS₂ and GO were investigated for their in vitro antibacterial activity [92]. Nanosheets of GO, MoS₂, and GO-MoS₂ were synthesized and equal concentrations of E. coli cells were dispersed onto each nanosheet. The hybrid film showed the greatest antibacterial effect, with 61% of loss of E. coli viability after 2 hours compared to approximately 55% and 45% for the MoS₂ and GO films, respectively. SEM images of bacteria cells post-interaction with the nanofilm show compromised cell integrity, indicating direct physical disruption, oxidative stress, and charge transfer [92]. The behavior of this MoS2-based film suggests that this system is a promising candidate for an antibacterial film.

Transition metal oxides (TMOs)

TMO nanosheets can be obtained via delamination or deposition methods [93] and are highly advantageous because of their high biocompatibility and loading capabilities [94]. Manganese dioxide (MnO₂), specifically, has proved to be beneficial for therapeutic delivery due to its harmless degradation products and stimuli–responsive properties [95].

MnO₂ nanosheet–based carriers were prepared to load and deliver chlorine6 (Ce6) for cancer therapy [96]. The nanocarriers exhibited high loading of Ce6 (351 mg/g), presumably due to strong bonds between Mn atoms in the nanosheets and Mn atoms in Ce6. NIR and pH dependence of the release kinetics was tested by determination of release rates in acidic and neutral pH, with or without NIR stimulation. An attenuation of burst release was seen at pH of 5.0 and 7.4 without NIR laser irradiation, with the cumulative release doubled in acidic pH. With NIR stimulation, there was approximately a 3.5-fold increase in cumulative release and less attenuation of the burst release for both gels [96]. In acidic environments, degradation of MnO2 nanosheets forms Mn²⁺ ions that are nontoxic and can be used as contrast agent for MRI. This property was utilized in another study to design a cancer theranostic platform with curcumin-loaded, hyaluronic acid-functionalized MnO2 nanosheets [97]. A high loading efficiency of 75% was observed, in part due to electrostatic and van der Waals interactions. The nanosheets showed controlled release at neutral pH, with less than 20% cumulative release after 12 hours. In acidic pH, cumulative release increased to over 70% in the same time period. The amount of Mn²⁺ generated by reduction of the nanosheets was an adequate amount to perform MRI imaging, which proves the capability of MnO2 sheets to form effective cancer theranostic delivery carriers.

Polymer nanosheets

Natural and synthetic polymers are extensively researched and have numerous applications in biomedical engineering due to their high biocompatibility [98], high permeability [99,100], tunable properties [101], and degradation rates [102]. Polymer nanosheets (PNS) have been researched as alternatives to graphene and GO due to the cytotoxicity and low biocompatibility of the latter materials [103]. While PNS offers the advantage of biocompatibility, drug delivery applications require specific physiochemical properties to effectively perform targeted and prolonged delivery [102]. Research has thus been focused on utilizing different combinations of polymers to achieve specific properties for intended applications [104,105].

Poly (lactic acid) and poly (lactic-co-glycolic) acid – based nanofilms synthesized via layer-by-layer fabrication were assessed for their ability to perform topical and transdermal delivery. Betamethasone valerate (BV), an anti-inflammatory steroid, was used as the model drug. The nanofilms showed a high adhesiveness, and the skin concentration of BV after application was comparable to commercial ointments. The films were able to incorporate and release therapeutic levels of the model drug without compromising adhesiveness or breathability [106]. In another study, multilayered PCL-based nanosheets were investigated as controlled release vehicles for methyl orange, a model drug. The nanosheets exhibited thickness-dependent release, with a faster release for the thinnest films. Drug release was also pH dependent, showing a doubled release rate at pH of 11 compared to pH of 3 [107]. The simple fabrication methods and tunability of these materials are highly beneficial to drug delivery applications.

Hexagonal boron nitride (hBN)

Hexagonal boron nitride (hBN) is a material composed of equal parts boron and nitride atoms and has a honeycomb lattice structure similar to graphene. Hexagonal boron nitride is one of the most researched forms of boron nitride along with cubic boron nitride. The B and N atoms form a strong covalent bond that creates the cage structure while the layers are held together with van der Waals interactions. In vitro studies have shown size-dependent biocompatibility of these materials and more in vivo work is necessary to determine long term performance [108]. However, multiple studies have reported the use of hBN to deliver cancer therapeutics [109,110]. In one study, palladium nanoparticles were grown on the surface of hydroxy boron nitride nanosheets (Pd@OH-BNNS) to load and deliver doxorubicin. The antitumor drug was efficiently loaded onto the nanosheets with a loading capacity of 32%. This is due to the π–π stacking as well as hydrophobic interactions between the hBN-based nanosheets and doxorubicin. Release was pH- and NIR-dependent, indicating the possibility of photothermal therapy and high drug release in a tumor microenvironment. In vivo testing also showed tumor growth inhibition and high efficacy during subcutaneous injection [111]. While the research of hBN nanosheets as drug delivery agents is still in its infancy, these materials have demonstrated properties that can be utilized to load and deliver small-molecule therapeutics. Further evaluation of their interaction with proteins and larger biomolecules, as well as interactions with cells, would need to be performed to widen their applications.

Conclusion and future directions

Two-dimensional nanomaterials can bind to numerous proteins and small molecules because of their sheet-like structure and large surface area. They also possess unique photothermal and chemical properties and are highly tunable. Stimuli-responsive degradation and high biocompatibility allow 2D materials to be utilized in targeted and sustained drug delivery. This review highlighted recent and promising studies involving 2D nanomaterial-based therapeutic delivery systems (Figure 4, Table 1). Graphene-based materials, GO specifically, has been the most extensively researched material in drug delivery among the various 2D nanomaterials. However, nanoclays, BP, and LDH have also been widely explored in therapeutic delivery due to their unique properties. Nanoclays have an anisotropic charge distribution that allows for simultaneous loading of differently charged molecules. The puckered honeycomb structure of BP increases the number of contact points on the surface and promotes higher drug loading. LDHs are hydrophilic, positively charged molecules that can efficiently bind to and stabilize proteins. MXenes, MOFs, COFs, and TMDs have been mainly applied within fields such as biosensing, photothermal therapy, and medical imaging; however, their photothermal, chemical, and electrical properties may be useful for delivery of chemotherapeutics, antibiotics, protein vaccines, and anti-inflammatory drugs. Although recent investigations have shown promising findings in vitro that indicate 2D nanomaterials could be used as drug delivery vehicles, more extensive in vivo assessments are still necessary to determine how surface modification, degradation, and clearance of these materials would affect their functionality. Overall, 2D nanomaterials are promising means for the development of delivery systems that exhibit efficient, localized, and prolonged delivery of therapeutics.

Figure 4.

2D nanomaterials have shown strong potential for various biomedical applications including cancer treatment, immunomodulation, regenerative medicine, vaccine delivery and antimicrobial.

Table 1.

Examples of drug delivery systems based on different types of 2D nanomaterials.

2D Nanomaterial		Delivery Molecule	Application	In vivo	Reference
Carbon-based nanomaterials	GO	Doxorubicin	Colon cancer	Yes	[22]
	GO	Bone morphogenic protein 2	Osteoarthritis, bone regeneration	Yes	[25]
	GO	Docetaxel	Breast cancer	No	[21]
	GO	Ovalbumin (model protein 43 kDa)	Vaccine delivery	No	[26]
	rGO	Doxorubicin	Cervical cancer	No	[28]
	rGO	Insulin	Diabetes	No	[29]
	rGO	Paclitaxel	Breast cancer	No	[20]
	Graphene	Chlorhexidine	Antibacterial	No	[23]
Nanoclays	Montmorillonite	gentamicin	Antibiotic	Yes	[37]
	Laponite	Cisplatin	Breast cancer, cervical cancer	Yes	[34]
	Laponite	4-fluorouracil	Breast cancer, cervical cancer	Yes	[34]
	Laponite	cyclophosphamide	Breast cancer, cervical cancer	Yes	[34]
	Laponite	rhBMP2	Bone regeneration	No	[42]
	Laponite	TGF-β3	Tissue regeneration	No	[42]
	Laponite	GM-CSF (Model protein (14.2 kDa))	Vaccine delivery	No	[47]
	Laponite	Flt3L (Model protein [18.6 kDa])	Vaccine delivery	No	[47]
	Laponite	IL-15 (model protein [13.3 kDa])	Vaccine delivery	No	[47]
	Laponite	CCL20 (model protein (8.0 kDa))	Vaccine delivery	No	[47]
	Laponite	IL-2 (model protein [17.2 kDa])	Vaccine delivery	No	[47]
Black Phosphorus (BP)		Cas9	Genome editing, gene silencing	Yes	[57]
		Doxorubicin/PEITC	Breast cancer, multidrug resistant cancer	Yes	[52]
		Ti-SA4 complexes	Antibacterial	No	[55]
Layered Double Hydroxides (LDHs)		Etoposide	Non-small cell lung cancer	Yes	[60]
		Butyrate	Antibacterial, breast cancer, liver cancer	Yes	[61]
	Mg–Al–Cl	BSA (model protein (66.5 kDa))	Protein stabilization	No	[62]
Metal Organic Frameworks (MOFs)	ZIF-8	TMV (model protein (300 × 18 nm))	Vaccine delivery	Yes	[74]
	ZIF-8	Curcumin	Anti-inflammatory, anti-HIV	No	[70]
	Zn4O(dmcapz)3	Aminobenzoic acid	UV blocker	No	[71]
	Zn4O(dmcapz)3	Benzocaine	Anesthetic	No	[71]
	Zn4O(dmcapz)3	5-fluorouracil	Antineoplastic agent, anticancer	No	[71]
	Zn4O(dmcapz)3	Caffeine	Liporeductor cosmetic	No	[71]
Covalent Organic Frameworks (COFs)	PEG-CCM@APTES-COF-1	Doxorubicin	Cervical cancer	Yes	[76]
	Boroxine-linked COF	Insulin	Diabetes	Yes	[79]
	Imine-derived COF	Methylene blue (model drug (319.8 g/mol))	Prolonged delivery	No	[77]
	Imine-derived COF	Oxytocin	Uterine contraction stimulant	No	[77]
Metal carbides and nitrides (MXenes)	Ti3C2	Doxorubicin	Astroglioma, glioblastoma,	Yes	[83]
	Ti3C2	Chloramphenicol	liver cancer Antimicrobial	No	[84]
Transition Metal Dichalcogenides (TMDs)	MoS2	Doxorubicin	Breast cancer	No	[88,89]
Transition Metal Oxides (TMOs)	MnO3	Cisplatin	Non-small cell lung cancer	Yes	[95]
	MnO2	Ce6	Prostate cancer	No	[96]
Polymer nanosheets	PLA/PLGA	Betamethasone valerate	Topical and transdermal delivery	Yes	[106]
	PEO/PCL-PEI-b-PCL	Methyl orange (model drug [327.3 g/mol])	Controlled release	No	[107]
Hexagonal boron nitride (hBN) nanosheets		Doxorubicin	Anticancer	Yes	[111]
		5-fluorouracil	Antineoplastic agent, anticancer	No	[109]
		6-mercaptopurine	Anticancer	No	[109]
		6-thioguanine	Anticancer	No	[109]

GO, graphene oxide; 2D, 2 dimensional; PLA, poly(lactic acid); PLGA, poly(lactic-co-glycolic) acid; BSA, bovine serum albumin; COF, covalent organic framework.

Even though recent research has generated promising results, further investigation is necessary to allow for clinical translation of these 2D nanomaterials. Although many studies focus on loading capabilities and release kinetics of the nanocarriers, little work explores their long-term cytotoxicity or clearance from the body. When applied to chemotherapeutic delivery, multiple studies attribute the high nanocarrier accumulation in the tumor to the enhanced permeability and retention effect of nanoparticles which indicates there is room for improvement. To achieve more robust targeting for cancer among other applications, more research investigating the use of targeting ligands is crucial. Other promising methods to enhance the properties of 2D nanomaterials include composite systems with hydrogels or other 2D materials (MXene hydrogels, GO-MoS₂ composite systems). Because of the high variation in chemical properties and surface interactions of these materials, an extensive biosafety analysis of each specific hybrid material would be necessary. The majority of current research has focused on cancer drugs or other small molecules. Further investigation into the interactions of 2D nanomaterials with biomolecules could lead to expanded applications in vaccines, immune engineering and in situ regeneration strategies [112]. The effects of nanomaterial size and structure on cellular uptake mechanisms should also be explored further, as this could lead to improved delivery of drugs through selective channels such as the blood–brain barrier. Although significant progress remains before the clinical application of these materials is possible, 2D nanomaterials have shown great potential in effective small-molecule drug, peptide, and protein delivery.