Two-dimensional graphene analogues for biomedical applications

This journal is©The Royal Society of Chemistry 2014 Chem. Soc. Rev.

Cite this:DOI: 10.1039/c4cs00300d

Two-dimensional graphene analogues forbiomedical applications

Yu Chen,ab Chaoliang Tan,c Hua Zhang*c and Lianzhou Wang*a

The increasing demand of clinical biomedicine and fast development of nanobiotechnology has

substantially promoted the generation of a variety of organic/inorganic nanosystems for biomedical

applications. Biocompatible two-dimensional (2D) graphene analogues (e.g., nanosheets of transition

metal dichalcogenides, transition metal oxides, g-C3N4, Bi2Se3, BN, etc.), which are referred to as

2D-GAs, have emerged as a new unique family of nanomaterials that show unprecedented advantages

and superior performances in biomedicine due to their unique compositional, structural and

physicochemical features. In this review, we summarize the state-of-the-art progress of this dynamically

developed material family with a particular focus on biomedical applications. After the introduction, the

second section of the article summarizes a range of synthetic methods for new types of 2D-GAs as well

as their surface functionalization. The subsequent section provides a snapshot on the use of these

biocompatible 2D-GAs for a broad spectrum of biomedical applications, including therapeutic

(photothermal/photodynamic therapy, chemotherapy and synergistic therapy), diagnostic (fluorescent/

magnetic resonance/computed tomography/photoacoustic imaging) and theranostic (concurrent

diagnostic imaging and therapy) applications, especially on oncology. In addition, we briefly present

the biosensing applications of these 2D-GAs for the detection of biomacromolecules and their in vitro/

in vivo biosafety evaluations. The last section summarizes some critical unresolved issues, possible

challenges/obstacles and also proposes future perspectives related to the rational design and

construction of 2D-GAs for biomedical engineering, which are believed to promote their clinical

translations for benefiting the personalized medicine and human health.

1. Introduction

The fast development of biomedicine and nanobiotechnologyprovides broad efficient strategies as promising alternativestowards disease diagnosis and therapy, especially on oncology.1–6

It is believed that these emerging techniques strongly depend onthe fabricated biomaterial systems at nanoscale with desirablestructures, compositions, morphologies and physicochemicalproperties. It has been demonstrated that the morphology ofnanomaterials did have significant impact on their biologicalperformances such as cellular uptake, biodistribution, excretionand even blood circulation durations.7–10 The most exploredmorphology of diverse nanosystems for biomedical applications isthe spherical nanoparticles (NPs), most probably due to the easypreparation of such type of shape. Moreover, other types of

nanostructures with rich topologies, such as tubes,11,12 wires,13

ellipsoidal,14,15 and cages,16–18 have also been successfullyconstructed as either drug delivery nanosystems or synergisti-cally therapeutic agents for cancer treatment.19 Previousstudies on mesoporous NPs have proved that the large surfacearea of mesopores is highly favorable for loading guest drugmolecules (Fig. 1a).2,20–26 Therefore, it is expected that thenanostructures with high surface area would be appealingcandidates for biomedical applications.

As a newly emerging class of nanomaterials, two-dimensional(2D) nanosheets with planar topography exhibit some uniqueproperties that originate from their ultrathin thickness and 2Dmorphological feature, such as high surface-area-to-mass ratio andspecific physicochemical properties, enabling them very promisingnanoplatforms for biomedical applications (Fig. 1a).27–34 Represen-tatively, biocompatible graphene derivatives, such as grapheneoxide (GO) and reduced GO (rGO), have been recently demon-strated to be attractive candidates for biomedical applications,including the anticancer drug delivery,35–38 gene transporta-tion,39–41 photothermal therapy (PTT),42–44 photodynamic therapy(PDT),45 biosensing46,47 and even tissue engineering,48–50 whichshowed superior performances compared to other conventional

a Nanomaterials Center, School of Chemical Engineering and AIBN,

University of Queensland, Queensland, 4072, Australia. E-mail: [email protected] State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050,

P. R. Chinac School of Materials Science and Engineering, Nanyang Technological University,

639798, Singapore. E-mail: [email protected]

Received 10th September 2014

DOI: 10.1039/c4cs00300d

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nanostructures such as NPs, tubes, wires, and cages. Graphene-based hybrid nanomaterials by integrating graphene with otherfunctional nanomaterials have been also developed for diagnosticimaging such as fluorescent imaging,51–54 magnetic resonanceimaging (MRI),55,56 computed tomography (CT),57–59 and radio-nuclide imaging.60–62 Their corresponding biological effects/behaviors have also been revealed, including cytotoxicities, bio-distribution, excretion, and hemo/histocompatibility.63–69 How-ever, the biomedical engineering and clinical translation ofGO/rGO suffer from many severe problems. For example, thephotothermal conversion efficiency of hydrophilic GO duringphotothermal therapy (PTT) is relatively low due to the exis-tance of abundant structural defects. In addition, the in vivobiodegradation rate of GO/rGO is extremely low; therefore,

the long term accumulation of GO/rGO in the body mightcause some severe biosafety issues.

In addition to graphene nanosheets, the 2D grapheneanalogues, which are referred to as 2D-GAs, with an ultrathin-layered feature, such as transition metal dichalcogenides(referred to as TMDs), transition metal oxides, graphitic carbonnitride (designated as g-C3N4), boron nitride (BN), and Bi2Se3,are also receiving considerable research interest due to theirunique physical, chemical, and electronic properties in the pastfew years.70–77 In this review, the 2D-GAs mainly refer to theinorganic materials with planar topology, ultrathin thickness(single or several atomic layers), which have structural similar-ity with graphene but exhibit distinctive physicochemical/biological properties. Although having similar structural features

Yu Chen

Yu Chen received his bachelordegree in Polymer MaterialSciences and Engineering atNanjing Tech University and PhDdegree at Shanghai Institute ofCeramics, Chinese Academy ofSciences (SICCAS). Since then, hehas worked as a research associatein SICCAS. He is now a researchassociate at the University ofQueensland. His research focuseson the design, synthesis andbiomedical applications of multi-functional inorganic nano-

materials, including drug-delivery systems for chemotherapy,contrast agents for molecular imaging, synergistic agent forultrasound/magnetic hyperthermia and non-virus gene deliveryvehicles.

Chaoliang Tan

Chaoliang Tan received his BEdegree from Hunan University ofScience and Technology in 2009.After he received his MS degreefrom South China NormalUniversity, he moved in 2012 tothe School of Materials Scienceand Engineering of NanyangTechnological University inSingapore where he is a PhDcandidate under the supervisionof Professor Hua Zhang atpresent. His research interestsfocus on the synthesis, assembly

and applications of two-dimensional nanosheets (e.g. graphene andsingle- or few-layer transition metal dichalcogenides) and theircomposites.

Hua Zhang

Hua Zhang obtained his BS andMS degrees at Nanjing Universityin 1992 and 1995, respectively,and completed his PhD with Prof.Zhongfan Liu at Peking University in1998. As a Postdoctoral Fellow, hejoined Prof. Frans C. De Schryver’sgroup at Katholieke UniversiteitLeuven (Belgium) in 1999, andthen moved to Prof. Chad A.Mirkin’s group at NorthwesternUniversity in 2001. After heworked at NanoInk Inc. (USA)and Institute of Bioengineering

and Nanotechnology (Singapore), he joined Nanyang TechnologicalUniversity in July 2006. His current research interests focus onsynthesis of two-dimensional nanomaterials and carbon materials(graphene and carbon nanotubes), and their applications in nano-and bio-sensors, clean energy, and water remediation.

Lianzhou Wang

Lianzhou Wang is currently aProfessor in School of ChemicalEngineering and Research Directorof Nanomaterials Centre, theUniversity of Queensland (UQ),Australia. He received his PhDdegree from Shanghai Institute ofCeramics, Chinese Academy ofSciences in 1999. Before joiningUQ in 2004, he has worked attwo national institutes (NIMSand AIST) of Japan for five years.Wang’s research interests includethe design and application of

nanostructured materials, including two-dimensional layeredcompounds for energy, environment and relevant applications.

Review Article Chem Soc Rev

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with graphene, these 2D-GAs possess much different physico-chemical properties, composition and surface status. In thisregard, these biocompatible 2D-GAs could be ideal alternativesor complementary candidates of graphene-based nanomaterialsfor biomedical applications such as drug delivery, phototheram/photodynamic therapy, diagnostic imaging, and biosensing(Fig. 1b). In this review, the state-of-the-art progress, challengesand perspectives of 2D-GAs in biomedical applications willbe highlighted and discussed. In particular, the very recentprogresses of 2D planar biomaterial nanosystems, includingtheir synthesis strategies, surface engineering and diagnostic/therapeutic performance against cancers, as well as the biosensingapplications and biosafety evaluations will be summarized with afocus on 2D MoS2, WS2, BN, Bi2Se3, g-C3N4 and MnO2. Of note, inthis article, we will exclude the studies on biocompatible 2Dnanosheets of layered double hydroxides (LDHs) for biomedicalpurposes because several excellent reviews concerning this topichave already been published.78,79

2. Synthesis of 2D-GAs

Layered materials have been investigated for decades, whereasthe recent research interest on graphene in single-layer sheetfashion has triggered the increasing research efforts on thisfamily of materials due to some unique physicochemical pro-perties arising from the thickness reduction to single or fewlayers. For instance, the indirect bandgap of bulk MoS2 is1.3 eV, but it can convert to a direct bandgap of 1.8 eV in itssingle-layer form.80 Various kinds of single- or few-layer TMDnanosheets, such as MoS2, WS2, MoSe2, NbSe2, TiS2, ZrS2, TaS2,and WSe2, have been successfully constructed for a range ofapplications such as catalysis, electronic/optoelectronic devices,energy harvesting and storage.70,73,80–88 In addition, many other2D nanomaterials, including transition metal oxides (e.g., TiO2

and MnO2),89–93 g-C3N4,94–96 BN97–99 and Bi2Se3 (ref. 100) are alsoreceiving extensive investigation.

To date, a number of synthetic methods, including mechanicalexfoliation,101–103 liquid-phase exfoliation,104,105 ion-intercalationand exfoliation,106,107 chemical vapor deposition (CVD),108–112

and hydro-/solvo-thermal synthesis,113 have been developed forthe preparation of various classes of 2D-GAs. Generally, thesesynthetic methodologies can be divided into two distinctcategories, i.e. top-down and bottom-up approaches.

2.1 Top-down synthesis

The top-down methods are based on the direct exfoliation oflayered bulk crystals by various driving forces. The typical top-down method is the mechanical-exfoliation approach, whichwas first used to produce graphene sheets (Fig. 2a and b).Similarly, atomically thin sheets of layered 2D-GAs can also befabricated by this procedure from their initial bulk materialsfeatured with stacks of strongly bonded layers with weakinterlayer attractions. By using the micromechanical cleavage,Geim et al. successfully prepared several types of single-layernanosheets such as graphene, NbSe2, Bi2Sr2CaCu2Ox, BN andMoS2.101 Ultrathin 2D-GAs prepared by this approach arepristine and well-crystallized sheets with large size (up to tensof micrometer) deposited on certain substrates (e.g. SiO2/Si),which are suitable for fundamental understanding of theirintrinsic properties. However, this approach is featured withrelatively low throughput; therefore, it cannot be used for thoseapplications (e.g. biomedicine) that require a large amount ofsolution processed 2D nanosheets. Alternatively, low-energy ballmilling was further employed to substitute the initial Scotch tapefor mechanical exfoliation, which could mechanically peel hexa-gonal boron nitride (h-BN) particles into BN nanosheets whileonly little crystallographic damage would be generated withinthe in-plane crystal structures. Importantly, this ball milling

Fig. 1 (a) Schematic illustration of the conventional spherical mesoporous NPs and new 2D planar nanostructures for drug delivery. (b) Summary of thebiocompatible 2D-GAs for biomedical applications.

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method could potentially produce BN nanosheets in high yieldand large scale.102

Chemical exfoliation of layered bulk crystals in liquid is oneof the most developed strategies to synthesize 2D-GAs for bio-medical applications, especially suitable for large-scale productionand easy control of the crucial structural/compositional para-meters of prepared 2D-GAs. The intercalation of bulk TMD crystalsby lithium ions (Li+) has been proven to be effective in thehigh-yield production of single-layer TMD nanosheets. As atypical paradigm, we recently developed an electrochemicalLi-intercalation and exfoliation method to precisely controlthe intercalation process and enhance the product yield(Fig. 3).106 By using this method, single- and few-layer TMDnanosheets such as MoS2, TaS2, TiS2, WS2, ZrS2, NbSe2, WSe2,

Sb2Se3, Bi2Te3, and BN can be prepared in high yield and largescale.106,107 Recently, a H2SO4-assisted liquid-exfoliationapproach was developed to prepare highly dispersed WS2

nanosheets.114 One of the advantages of this intercalationprocess is that it can be processed in air and water comparedwith the Li-intercalation method. As an interesting paradigm,Coleman et al. reported a simple and general dispersion/exfoliation method to prepare several kinds of 2D nanosheetssuch as MoS2, WS2, MoSe2, TaSe2, NbSe2, NiTe2, BN, andBi2Te3.104 This method simply dispersed the commerciallayered bulk powders into the solvents with dispersive, polarand H-bonding components of the cohesive energy densitywithin certain well-defined ranges according to the Hansensolubility parameter theory. Solvents such as N-methyl-pyrrolidone (NMP) and isopropanol (IPA) were demonstratedas the most effective solvents to minimize the energy forexfoliation. Note that the yield of single-layer nanosheets forthis method is relatively low compared to the electrochemicalLi-intercalation and exfoliation method.106,107 Exfoliation byultrasonication in liquids containing various solvents, surfac-tants and polymers can produce the single-layer nanosheetsin large scale depending on the mechanical effect of theultrasound. It should be noted that this exfoliation processcan produce 2D-GAs with specific surface modification, whichcan avoid the re-aggregation of as-formed nanosheets. Impor-tantly, such a surface modification can endow these 2D-GAswith high stability in physiological conditions, which is veryessential for further in vivo biomedical applications. However,the liquid exfoliation can potentially cause large amount of

Fig. 2 (a) Typical synthetic procedure for surface-modified 2D layered nanosheets by the typical top-down exfoliation approach. (b) Schematicillustration of biomedical applications of 2D-GAs against cancers, including drug delivery, photo/photodynamic therapy, diagnostic imaging and otherspecific applications such as biosensing.

Fig. 3 Li-intercalation and exfoliation method for preparation of 2D TMDs.Reprinted with permission from ref. 106. Copyright 2011 Wiley-VCH.


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defects within the nanosheets, the impurities of the product,and single-layer and multilayer 2D-GAs co-exist in the exfoliatingsolution, and it is rather difficult to separate them.

2.2 Bottom-up approach

Aforementioned top-down methods are only applicable to thelayered bulk compounds. Alternatively, the bottom-up approaches,such as CVD growth and wet-chemical synthesis, are also widely usedfor preparation of 2D-GAs. The CVD method can produce high-quality and large-area 2D-GA nanosheets with atomic thickness. Forinstance, single-layer MoS2 films can be deposited onto amorphousSiO2 substrates by the CVD process with MoO3 and S powers asthe reactants at 650 1C.112 The pre-treatment of the substrate withrGO, perylene-3,4,9,10-tetracarboxylic acid tetrapotassium salt andperylene-3,4,9,10-tetracarboxylic dianhydride could facilitate thegrowth of MoS2 nanosheets. Large-area MoS2 atomic layers on aSiO2 substrate could be also prepared by a similar CVD approach.115

By etching the SiO2 substrate, the fabricated MoS2 nanosheets couldbe transferred to arbitrary substrates for further characterizationsand applications. Typically, the CVD method is suitable for large-scale device fabrication,111,112,115 but not suitable for biomedicalapplications, especially for therapeutic purposes, for which smallsheet size is highly desirable.

Hydro-/solvo-thermal synthesis is expected to produce high-quality 2D TMD nanosheets that can be adapted to biomedicalapplications.113,116–118 Previous results showed that single- or few-layer MoS2 nanosheets could be synthesized by the hydrothermaltreatment of molybdic oxide and KSCN in deionized water at453 K.113 However, there is still no appropriate synthetic procedureto prepare nano-sized TMD nanosheets with high dispersity andcontrolled structural parameters by hydro-/solvo-thermal synth-esis, which is essential to satisfy the strict requirement of biome-dicine. An effective bottom-up hydro-/solvo-thermal synthesis of2D-GA nanosheets is still rare; however, it is expected that abun-dant 2D-GA nanosheets with controlled and desirable structuralparameters could be synthesized by this method in the near futurebased on its simple and controllable nature.

2.3 Surface-modification and multifunctionalization

Similar to other nanosystems in biomedicine, the exfoliated thin2D-GAs are not stable in physiological conditions, and thus surfacemodification is essential to endow them with the high dispersityand stability in a physiological environment, site-specific targetingcapability and improved biocompatibility. Generally, the surfaceengineering of 2D-GAs is to modify their surface with certainpolymers through physical adsorption or weak non-covalentchemical bonding. As a typical paradigm, a simple in situsonication-assisted exfoliation approach assisted with poly(acrylicacid) (PAA) was developed to fabricate highly water-soluble WS2

nanosheets by modifying PAA onto the surface of WS2 via therelatively strong coordination interaction between carboxyl groupsand tungsten atoms.119 The enhanced stability of PAA-modifiedWS2 sheets is evidenced by the fact that they could be stored forweeks at ambient temperature. Similarly, the adsorption of bovineserum albumin (BSA) onto the surface of WS2 nanosheets couldalso enhance their dispersity and stability in phosphate buffered

saline (PBS).114 Recently, we successfully grafted the surface of MnO2

nanosheets with polyethylene glycol (PEG) molecules to improve theirstability in physiological condition for concurrent pH-responsivedrug releasing and T1-weighted MRI.120 It should be noted that sucha surface modification would decrease the anchoring points of2D-GAs for therapeutic molecules; thus, the modification degreeshould be carefully conducted and controlled.

The decoration of 2D-GAs with other functional materials canfurther broaden their applications. For instance, the integrationof 2D nanosheets with contrast agents (CAs) can endow themwith concurrent diagnostic imaging and therapeutic perfor-mance (designated as theranostic).121–123 However, such a multi-functionalization strategy is still in infancy compared to thewidely explored GO/rGO because of the synthetic and integratingdifficulties.84,124,125 It is believed that the fast development onsynthesis and applications of 2D-GAs will lead to diverse 2Dnanosheet-based multifunctional nanosystems for biomedicalengineering in the near future.

3. Therapeutic applications of 2D-GAs

The unique 2D planar structure and diverse chemical composi-tions lead to unique properties of 2D-GAs for biomedical applica-tions. Till now, these 2D nanosheets have been explored for avariety of biomedical applications such as drug delivery, photo-thermal/photodynamic therapy, diagnostic imaging, and bio-sensing. Moreover, the biological effect and behavior of these2D nanosheets are also now under extensive exploration. It hasbeen demonstrated that these 2D nanosheets present relativelyhigh biocompatibility and biosafety such as low cytotoxicity andhigh hemo-/histo-compatibility. In this section, we mainly focuson the introduction of these biocompatible 2D nanomaterials forvarious biomedical purposes (Table 1 and Fig. 2b).

3.1 Photothermal therapy

Photothermal therapy (PTT) typically employs laser to generateheat and induces hyperthermia within tumor tissues, causing thedenaturation of proteins, the disruption of cell membrane andcorresponding irreversible damage to cancer cells.126 However,the nonselectivity and high power density of laser therapy candamage both the normal and tumor tissues, causing severe sideeffects.127 The introduction of photothermal agent (PTA) canincrease the selectivity of laser, which means that the heat canonly be generated within the local microenvironment of tumortissue at relatively low power density of laser.128–136 GO and rGOhave been extensively explored as the PTA for in vitro and in vivophotothermal ablation of cancer cells, due to their strong light-absorbance in the near-infrared (NIR) window of wavelength inthe range of 700–1300 nm.42,55,131,132 Biological tissues are trans-parent to this NIR window; thus, the light-penetrating depth ishigh and tissue-induced light-absorption is low, which causes thehigh photothermal conversion efficiency within tumor tissues.

Similar to the high photothermal conversion efficiency of GOor rGO, some 2D-GAs with various chemical compositions havealso been reported as PTA for efficient PTT. Recently, amphiphilic


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chemically exfoliated MoS2 (designated as ceMoS2) was synthe-sized via the Morrison method to break the interlayer van derWaals force in bulk MoS2 through ultrasonication.133 The as-synthesized ceMoS2 exhibited the typical 2D planar morphologywith an average sheet size of 800 nm and thickness of 1.54 nm(n = 40 sheets, Fig. 4a). The mass extinction coefficient of as-synthesized ceMoS2 was calculated to be 29.2 Lg�1 cm�1 by theNIR absorbance (Fig. 4b), which was about 7.8-fold of GOnanosheets (3.6 Lg�1 cm�1) and better than that of rGOnanosheets (24.5 Lg�1 cm�1). The temperature of the aqueoussolution could be rapidly increased to above 40 1C at ceMoS2

concentrations in the range of 38 ppm to 300 ppm (Fig. 4c). Thein vitro photothermal evaluation against HeLa cells (Fig. 4d)showed that the cells could be completely killed afterco-incubation with ceMoS2 and NIR irradiation (l = 800 nm,20 min) because the solution temperature could be quickly

raised to over thermal-ablation thresholds. This report onthe photothermal effect of MoS2 nanosheets provides directevidence that the photothermal conversion efficiency of MoS2

nanosheets was comparable to the hydrophobic rGO, but theyexhibited a hydrophilic nature. In addition, this report onin vitro PTT of cancer cells using 2D-GAs as the PTA clearlyreveals that graphene can be substituted by other 2D-GAnanosheets for efficient PTT. However, the large sheet size(around 800 nm) and lack of surface modification of ceMoS2

restrict their further in vivo PTT against tumors.WS2 is another representative member of the TMD family.

Similar to MoS2, 2D WS2 nanosheets also exhibit promisingphotothermal conversion capability for PTT. 2D WS2 nano-sheets with the sheet size of 50–100 nm were synthesized bythe Morrison method to reveal their PTT efficiency (Fig. 5a).134

After further PEGylation through the W–S bond between lipoic

Table 1 Summary of representative in vivo diagnostic imaging and therapeutic applications of 2D-GAs

Nanosheettype

Surfacemodifications

Sheet size(nm)

Imagingmodality

Therapeuticmodality

Administrationmode Performance Ref.

MoS2 PEG 50 (AFM) — PTT &Chemotherapy

Intravenous &Intratumor

(a) High PTT efficiency(b) Synergistic PTT and chemotherapy outcome

140

MoS2 Chitosan 80 (AFM) CT PTT &Chemotherapy

Intratumor (a) Preliminary excellent CT imaging performance(b) Enhanced synergistic PTT andchemotherapeutic efficiency

141

WS2 BSA 20–100 (TEM) CT PTT & PDT Intratumor (a) Strong CT imaging signals of the injectedsite of tumor(b) Synergistic PTT and PDT outcome

114

WS2 PEG 50–100 (TEM) CT PTT Intravenous &Intratumor

(a) Excellent CT imaging performance(b) Complete tumor eradication by PTT

134

Bi2Se3 PVP 90 (TEM) CT PTT Intratumor (a) Contrast-enhanced tumor CT imaging(b) Complete tumor eradication by PTT

100

MnO2 PEG 80–100 (TEM) MRI Chemotherapy Intratumor (a) pH-responsive intelligent drug releasing(b) pH-responsive MR imaging of tumor tissue

120

MnO PEG 8–70 (TEM) MRI — Intravenous Significantly contrast-enhanced MRI in thewhole body

164

Fig. 4 (a) Atomic force microscopy (AFM) image of ceMoS2 sheets (inset: the diameter and thickness diagrams). (b) Absorbance spectra of ceMoS2 atdifferent concentrations (inset: Beer’s law plot at 800 nm). (c) The temperature increase with the prolonged exposure duration to laser at l = 800 nm(0.8 W cm�2) at elevated concentrations. (d) MTT results to determine HeLa cell viabilities after diverse treatments. Reprinted with permission fromref. 133. Copyright 2013 Wiley-VCH.


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acid-conjugated PEG (LA–PEG) and WS2 nanosheets, theas-synthesized PEGylated WS2 nanosheets (designated as WS2–PEG) could be easily dispersed into saline with high stability.The average thickness of WS2–PEG was determined to be about1.6 nm by AFM characterization (Fig. 5b). According to theUV-vis-NIR spectrum of WS2–PEG nanosheets, the extinctioncoefficient WS2–PEG at 808 nm was calculated to be as high as23.8 Lg�1 cm�1. Importantly, the capability of WS2–PEG for PTTwas systematically revealed in vivo. It was found that the surfacetemperature of the tumor could be quickly raised to about 65 1Cwithin a 5 min-irradiation by a 808 nm laser at the power densityof 0.8 W cm�2 (Fig. 5c), irrespective of the intratumoror intravenous administration of WS2–PEG into mice. In parti-cular, the administration of WS2–PEG combined with laserirradiation could bring with the significantly improved in vivophotothermal-therapeutic efficiency, where the tumors could becompletely eradicated without obvious reoccurrences (Fig. 5d).The survival of mice could also be significantly improvedbecause of this high PTT outcome (Fig. 5e).

Furthermore, it was found that 2D Bi2Se3 nanoplates arealso efficient in absorbing the NIR laser and converting it intoheat for PTT.100 Polyvinylpyrrolidone (PVP)-coated 2D Bi2Se3

nanoplates with the sheet size of about 90 nm were synthesizedwith the outer layer thickness of about 3.9 nm and inner layerthickness of about 21.5 nm. The high photothermal conversionefficiency was shown by a fast temperature increase to 50.2 1Cafter 5 min-irradiation via the 808 nm laser with a powerdensity of 1 W cm�2 at Bi2Se3 concentration of 50 mg mL�1.The co-incubation with Bi2Se3 nanoplates combined with laserirradiation could cause 75% H22 cell death at the concentrationof 50 mg mL�1. After intratumor injection of Bi2Se3 nanoplatesassisted by the 808 nm laser irradiation, the tumors could becompletely eradicated, further demonstrating their high in vivophotothermal efficiency against tumor.

3.2 Drug delivery

One of the outstanding structural characteristics of 2Dnanosheets is their large surface-to-volume ratio, which canprovide numerous anchoring points available for loading guestmolecules (Fig. 1a).137 2D graphene and its derivatives have thestrong absorption ability for anticancer drugs through supra-molecular p–p staking and hydrophobic interaction due to theultrahigh surface area and unique sp2-bonded carbonaceoussurface.53,66 In addition, GO and rGO were demonstrated as

Fig. 5 AFM images of (a) WS2 and (b) WS2–PEG. Inset: photographs of WS2 and WS2–PEG dispersed in saline at the concentration of 0.05 mg mL�1.(c) IR thermal images of tumor-bearing mice after the intratumor (i.t.) and intravenous (i.v.) administration of WS2–PEG after exposure to the irradiation ofthe 808 nm laser at the power density of 0.8 W cm�2. (d) The tumor volume changes and (e) the corresponding survival curves of mice after differenttreatments. Reprinted with permission from ref. 134. Copyright 2013 Wiley-VCH.


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efficient carriers for gene macromolecules or even contrast agents(CAs).53,66,67 Similar to the GO or rGO sheets, the 2D-GAs are alsoexpected to have a similar desirable function for drug deliverybased on the following considerations. First, inorganic 2D-GAsshow relatively high chemical/physiological stability comparedto the organic liposomes and micelles. Therefore, the drugmolecules can be released from the carrier via a sustainedmanner to avoid the explosive release of drugs caused by thebreak-up of traditional organic nanocarriers. Second, the highsurface-to-volume ratio of 2D-GAs endows the carrier with highdrug-loading capacity. Third, the unique chemical composition ofsome members in the 2D-GA family can form specific interactionswith drug molecules, which can endow the carrier with high drug-loading capacity and response to external triggers for on-demanddrug releasing. Last but not least, the multifunctionalities of thecarriers themselves can bring the synergistically therapeutic out-come such as photothermal-/chemo-therapy and/or theranostics(concurrent diagnostic imaging and therapy). The aforementionedfeatures and functions are difficult to achieve by traditionalorganic liposomes or micelles.

Similar to the structure of graphite, graphitic-phase carbonnitride (g-C3N4) is generally regarded as the N-substitutedgraphite via a regular manner. As a new 2D semiconductormaterial, g-C3N4 nanosheets exhibit intriguing performance inphotochemical and electrochemical catalysis.94,139 However,the biomedical applications of g-C3N4 require their specificphysical/chemical/biological properties, i.e., small sheet size,high dispersity, high hydrophilicity and low toxicity. Recently,highly dispersed g-C3N4 nanosheets were synthesized with thehydrodynamic diameter of only 55 nm and thickness of about1.1 nm by chemical oxidation of bulk g-C3N4 followed byultrasonic exfoliation.138 The as-synthesized g-C3N4 could not

only encapsulate/deliver anticancer drugs (doxorubicin, Dox)but could also act as the photosensitizer for photodynamictherapy (Fig. 6). Importantly, the loading amount of Dox ong-C3N4 could reach as high as 18 200 mg g�1, which is muchhigher than traditional mesoporous NPs. The release of Doxfrom g-C3N4 was pH-dependent where the acidity could accele-rate the releasing rate of drugs from the carrier. Furthermore,the Dox-loaded g-C3N4 exhibited comparable cytotoxicitiescompared to free Dox molecules.

Biocompatible transition metal oxide nanosheets can also actas drug delivery nanosystems for chemotherapy. In particular,biocompatible MnO2 nanosheets exhibit a unique break-up natureunder mild acidic environment. We recently constructed a pH-responsive drug-releasing platform based on MnO2 nanosheets.120

The drug molecules (Dox) could be quickly released from MnO2

nanosheets when Dox-loaded MnO2 nanosheets were impregnatedinto an acidic environment (Fig. 7a). The successful loading of Doxonto MnO2 nanosheets were demonstrated by the changes of Zetapotential from �40.6 mV to +22.3 mV (Fig. 7b). The Dox-releasingin neutral solution after 5 h was only 24.8% (Fig. 7c). Based onthe disintegration of MnO2 nanosheets, the releasing rate showeda significant increase in a mild acidic environment where the5 h-releasing amounts could reach 58.9% (pH 6.0) and 94.3%(pH 4.6), respectively. Such an ultrasensitive intelligent drug-releasing performance can concurrently reduce the side-effectsand improve the therapeutic efficiency of anticancer drugs because

Fig. 6 The scheme of 2D g-C3N4 nanosheets for synergistically photo-dynamic therapy and chemotherapy. Reprinted with permission fromref. 138. Copyright 2014 Royal Society of Chemistry.

Fig. 7 (a) The scheme of the break-up nature of 2D MnO2 nanosheets forpH-responsive drug releasing. (b) Zeta potentials of as-prepared MnO2

nanosheets and Dox-loaded MnO2 nanosheets. (c) Cumulative-releasingpercentage of Dox from Dox-loaded MnO2 nanosheets at different pHs.(d) T1 values of the releasing medium after 300 min drug-releasing (top image,MRI-T1 images from left to right: water, buffer solution at pH 7.4, 6.0 and 4.6).Reprinted with permission from ref. 120. Copyright 2014 Wiley-VCH.


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these drugs are mostly released within the acidic microenviron-ment of tumor tissues. Importantly, the disintegration of MnO2

nanosheets could substantially improve the T1-weighted MRIperformance (Fig. 7d), which provides the potential for monitoringthe drug-releasing procedure by T1-weighted MR imaging. Further-more, in vitro cellular evaluations showed that Dox-loaded MnO2

nanosheets could enhance the anticancer outcome and circumventthe multidrug resistance (MDR) of cancer cells by escaping thepumping effect of molecular pumps such as P-glycoprotein.

The 2D nanosheets can be further assembled to form porous3D materials for drug transportation. For instance, a highly watersoluble, porous and biocompatible BN material was constructedfor anticancer drug delivery.98 The 3D porous BN nanostructurewas formed by self-assembling a few-nanometer sized hyroxylatedBN layers along the [0001] direction. It is well known that BN isone of the typical structural analogues of carbon materials, inwhich C atoms are substituted by alternating B and N atoms. Thefabrication of the porous BN materials was based on the thermalsubstitution reaction of C atoms in graphitic carbon nitrides,which could produce porous BN with unprecedently high hydroxy-lation degrees, guaranteeing their high hydrophilicity. Impor-tantly, the as-synthesized porous BN nanostructure exhibitedextremely high drug-loading capacity of as high as 309 wt% (forDox), which is rationalized to the inherent light nature of BNcomponents and extra p–p interactions between the BN frame-work and aromatic Dox molecules. Furthermore, the Dox-loaded

BN showed enhanced in vitro therapeutic efficiency against LNCaPcells compared to free Dox molecules.

3.3 Synergistic therapy

2D nanosheets of TMDs, such as MoS2 and WS2, have beendemonstrated as the efficient therapeutic agents for photothermaltherapy. It is anticipated that anchoring anticancer agents ontothe surface of these 2D nanosheets can bring the synergisticallytherapeutic outcome, i.e., combined chemotherapy and photo-thermal therapy, which is difficult to be achieved by using Au NPsas the photothermal agent. On this ground, highly dispersed 2DMoS2 nanosheets were synthesized by a chemical-exfoliationapproach (Fig. 8a), which was further modified by lipoic acid-conjugated PEG (LA–PEG) to improve their stability in PBS.140 Themass extinction coefficient of as-synthesized MoS2 nanosheetswas determined to be 28.4 Lg�1 cm�1 by UV-vis-NIR absorbancespectra, much higher than that of traditional GO (3.6 Lg�1 cm�1)and better than that of rGO (24.6 Lg�1 cm�1). Similar to g-C3N4,the high surface area of the atomic-thin planar nanostructureendows MoS2–PEG with extraordinarily high drug-loading capa-city, which could reach 239% for Dox, 39% for Ce6 and 118% forSN38, much higher than PEGylated GO with 150%, 25% and 15%loading amount for Dox, Ce6 and SN38, respectively. After intra-venous administration of Dox-loaded MoS2–PEG into 4T1 tumor-bearing mice, a significantly synergistic therapeutic efficiencycould be achieved after further tumor exposure to the 808 nm

Fig. 8 (a) Schematic illustration of the synthetic procedure of the MoS2–PEG and drug-loading process. (b) The scheme of intravenous administration ofDox-loaded MoS2–PEG, and the combined PTT and chemotherapy. (c) IR thermal images of 4T1 tumor-bearing mice after various treatments. (d) Thetemperature-variations of tumor as a function of irradiation duration after exposure to the 808 nm NIR laser. (e) Tumor volume changes as a function oftime after the various treatments. Reprinted with permission from ref. 140. Copyright 2014 Wiley-VCH.


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NIR laser (Fig. 8b). The tumor temperature could be quicklyincreased to 45 1C after laser irradiation (Fig. 8c and d). Theenhanced tumor-suppressing effect could be achieved in thegroup of MoS2–PEG/Dox combined with 808 nm laser irradiation(Fig. 8e) compared to the groups of MoS2–PEG/Dox and MoS2–PEG combined with the NIR exposure, demonstrating the highsynergistically therapeutic outcome.

Similar to 2D MoS2 nanosheets for synergistically concurrentchemotherapy and PTT, a modified oleum treatment exfoliationprocess was developed to synthesize single-layer MoS2 nanosheetsfor efficient NIR-triggered on-demand drug releasing and syner-gistically chemo-photothermal therapy.141 After further surfacemodification with chitosan (CS), the MoS2–CS could load anti-cancer agent Dox for NIR laser-triggered drug releasing. TheON/OFF introduction of 808 NIR laser could quickly raise thetemperature of the releasing media and produce the pulsatiledrug-releasing patterns where the ‘‘ON status’’ of the laser quicklypromoted the releasing of Dox. The releasing percentage couldreach 80% at the power density of 1.4 W cm�2 while only less than20% Dox released without the NIR laser triggering. The intratu-mor administration of Dox-loaded MoS2–CS into pancreatictumor-bearing mice could cause the high accumulation of thenanosheets into tumors. After the irradiation by 808 NIR irradia-tion (0.9 W cm�2), the temperature of tumors raised rapidly(DT = 22.5 1C) to induce the hyperthermia and trigger the Doxreleasing. The maximum tumor growth-inhibition effect could beachieved by such a synergistically chemo-photothermal therapy.

In addition to the encapsulation and delivery of anticancerdrugs for chemotherapy, 2D layered nanosheets can also beused to deliver other therapeutic agents such as photosensiti-zers. Methylene blue (MB) was loaded onto the surface of WS2

nanosheets as the photosensitizer to generate the cytotoxicsinglet oxygen for photodynamic therapy (PDT).114 Combinedwith the photothermal effect of WS2 nanosheets, the local-generated heat could promote the fast release of MB moleculesand significantly restore the generating amounts of singletoxygen. The high synergistically therapeutic effect could be

realized by combining irradiations of 665 nm and 808 nmlasers. These results give the strong evidence that 2D-GAnanosheets can act as the carrier of photosensitizers for PDT.In addition, chemically exfoliated MoS2 nanosheets exhibitedunique antibacterial activity due to their 2D planar structureand high conductivity, which could induce the membrane/oxidative stress and produce reactive oxygen species.142

4. Diagnostic imaging of 2D-GAs

The unique physicochemical property of 2D nanosheets providesan excellent platform for diagnostic imaging, which indicates thatthey can act as the CAs to improve the imaging performanceof diverse imaging modalities. Compared to traditional CAs,these 2D layered nanomaterials exhibit their specific features forcontrast-enhanced imaging, mainly based on their chemicalcomposition, layered structure and physicochemical properties.By combining the concurrent therapeutic and diagnostic perfor-mances, these 2D-GAs can further act as the theranostic agents fordiagnostic imaging and therapy simultaneously.

4.1 Fluorescent imaging

Compared to conventional organic fluorescein, inorganicquantum dots (QDs) exhibited significantly enhanced fluores-cent performance for bio-imaging such as tunable wavelength,enhanced photostability and high quantum yields.143–146

However, traditional inorganic QDs typically contain toxicheavy metal atoms (e.g., Cd-based QDs), which severely restrictfurther clinical translations. Graphene QDs, a newly developedmetal-free fluorescent nanomaterial, showed potential utiliza-tion for fluorescent imaging.147,148 However, these grapheneQDs suffer from a low photo-response and the intrinsic hydro-phobicity. To develop more reliable inorganic fluorescent nano-materials, ultrathin g-C3N4 nanosheets were developed to act asprobes for two-photon fluorescence imaging of cellular nucleusbased on the p-conjugated electronic structure and rigid C–N

Fig. 9 ((a) inset: size distribution) TEM, (b) AFM and (c) height diagram of g-C3N4 QDs. Confocal fluorescent images (one-photon and two-photon) ofHepG2 cells after co-incubation with (d) 1,1-dioctadecyl-3,3,30,3-tetramethylindocarbocyanine perchlorate (Dil), fluorescein diacetate (FDA), g-C3N4

QDs, and (e) Dil, FDA, 40,6-diamidino-2-phenylindole (DAPI), respectively (d1 and e1: one-photon fluorescence of Dil; d2 and e2: one-photonfluorescence of FDA; d3: one-photo fluorescence of g-C3N4 QDs; d4: two-photon fluorescence of g-C3N4 QDs; d5: merged image of d1, d2 and d3;d6: merged image of d1, d2 and d4; e3: one-photon fluorescence of DAPI; e4: two-photo fluorescence of DAPI; e5: merged image of e1, e2 and e3; e6:merged image of e1, e2 and e4). Reprinted with permission from ref. 149. Copyright 2014 Wiley-VCH.


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planes of thin-layered nanostructures.149 Theoretical calcula-tion shows that single-layered g-C3N4 with small sheet size caneasily cause two-photon absorption (TPA). g-C3N4 QDs with thediameter of only a few nanometers (B4 nm, Fig. 9a) and thethickness of a single C–N layer (B0.35 nm, Fig. 9b and c) wereprepared by three steps, including acid treatment, exfoliationand further hydrothermal treatment. The synthesized g-C3N4

QDs could concurrently absorb two near-infrared photons andemit bright fluorescence in the visible-light region, which is thetypical TPA feature. Fig. 9d shows that g-C3N4 QDs could enterthe nuclei of HepG2 cells while g-C3N4 with relatively largesheet size (B30 nm and B100 nm) could not pass throughthe nuclear membrane to penetrate into the nuclei. The two-photon fluorescence-imaging performance (Fig. 9d and e) ofg-C3N4 QDs was compared with the commercial nuclear stainingregent 40,6-diamidino-2-phenylindole (DAPI). It was found thatthe two-photon fluorescence nuclear imaging of g-C3N4 QDsagreed with that of DAPI, but the cost of g-C3N4 QDs was relativelylow. Importantly, the nuclei information could be effectivelylightened up after the two-photon fluorescence imaging ofg-C3N4 QDs, demonstrating their high intracellular and intra-nuclear fluorescent-imaging capabilities.

A one-step degradation of C3N4 nanosheets with the assistanceof catalase was developed to produce fluorescent N-doped carbon(N–C) dots with particle size of 5 nm, which can overcome thedrawbacks of large sheet sizes of C3N4 while the excellent fluores-cent properties could be maintained. The N–C dots exhibited lowcytotoxicity and strong blue fluorescence at the excitation wave-length of l = 405 nm, which can also be used for cell imaging.150

Xie et al. also found that the ultrathin liquid-exfoliated g-C3N4

showed an extremely high photoluminescence quantum yield upto 19.6% for intracellular bioimaging.151 Note that bioimaging ofg-C3N4 nanosheets for intracellular imaging is highly effective.However, their application for further in vivo fluorescent imagingis severely restricted because of their emission of blue fluores-cence, which is not in the range of the widely accepted NIRwindow of wavelengths (700–1300 nm).

4.2 Photoacoustic tomography

Photoacoustic tomography (PAT) has attracted much recent atten-tion for diagnostic imaging due to its specific features of highimaging depth and spatial resolution.152 As a new diagnostic-imaging modality, PAT typically requires the CAs to further improvetheir imaging performance. Nanoprobes with strong NIR-absorbance are generally regarded as the desirable CAs for PAT.Based on the excellent NIR-absorbance performance, Liu et al.employed PEGylated WS2 nanosheets as the CAs for PATimaging.134 Under a PAT imaging system using a 700 nm laser asthe excitation source, strong photoacoustic signals of the 4T1 tumorcould be recorded after either intratumor or intravenous adminis-tration of WS2–PEG while only major blood vasculatures could befound in mice without the administration of WS2–PEG CAs.

4.3 Computed tomography imaging

An element with high atomic number (Z) can be used as the CAfor computed tomography (CT). 2D-GAs composed of elements

of high Z are expected to act as CAs for CT imaging. The use ofWS2–PEG as the CA for CT imaging was successfully demon-strated due to the higher Z of W (Z = 74) compared to theclinically used I (Z = 53).134 The enhanced in vitro CT perfor-mance of WS2–PEG was revealed compared to commercialiodine-based CT CAs (Iopromide). Importantly, WS2–PEG couldaccumulate within tumor tissues to give the apparent contrast-enhanced CT imaging after the intravenous injection of WS2–PEG. Zhao et al. also showed the high CT-imaging performanceof BSA-modified WS2 nanosheets for nude mice bearing HeLatumors after intratumoral administration of the CAs.114 Thecapability of MoS2 nanosheets as the CAs for CT imaging waspreliminarily revealed, showing slightly higher in vitro imagingperformance compared to the commercial Iopromide.141

Bismuth (Z = 83) is regarded as the element with almost thehighest atomic number (Z) among various metal elements withsatisfactory biocompatibility. Different from most explored Bi2S3

NPs for CT imaging,153,154 2D topological insulator bismuthselenide (Bi2Se3) nanoplates possess specific functions of con-current photothermal therapy and CT imaging.100 In addition tothe low cytotoxicity of Bi,155 Se is an essential trace element for thehuman body. Thus, Bi2Se3 nanoplates are expected to show highbiocompatibility. Compared to other metals, Bi shows one of thelargest X-ray attenuation coefficients (Bi: 5.74, Au: 5.16, Pt: 4.99,

Fig. 10 (a) In vitro CT images of Bi2Se3 nanoplates and Iopamidol at variedconcentrations. (b) CT HU value of Bi2Se3 nanoplates and Iopamidol as afunction of Bi and I concentrations. (c) In vivo CT coronal imaging oftumor-bearing mouse after the intratumor administration of Bi2Se3, andcorresponding three-dimensional CT reconstruction images. Reprintedwith permission from ref. 100. Copyright 2013 Nature Publishing Group.


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Ta: 4.3 and I: 1.94 at 100 keV).154 Thus, the in vitro CT images(Fig. 10a) of Bi2Se3 nanoplates displayed the substantially enhancedHU values compared to the commercial Iopamidol at the same Biand I concentrations (Fig. 10a and b). The enhanced CT-imagingperformance of Bi2Se3 nanoplates indicates that the low doses ofCAs can cause the equivalent CT contrasts compared to clinicaliodinate-based CAs, which can concurrently increase CT-imagingaccuracy and the biosafety of CAs. After the in situ injection ofBi2Se3 nanoplates into tumor tissues, a significantly brighter CTsignal could be observed within tumor tissues compared to thesurrounding soft tissues (Fig. 10c), demonstrating the high in vivoCT imaging performance of Bi2Se3. Although the intratumor injec-tion is not a preferred manner, this preliminary in vivo assessmentindeed showed their high performance for in vivo CT imaging. It isanticipated that the in vivo CT-imaging performances after intra-venous administration can be further realized by additional surfacemodifications such as PEGylation or targeting engineering.

4.4 Magnetic resonance imaging

Due to the high spatial resolution, excellent contrast difference ofthree-dimensional soft tissues and non-invasive feature, magnetic

resonance imaging (MRI) has been extensively applied forclinically diagnostic imaging.156,157 Although Gd-based CAs aredemonstrated to be effective in improving the accuracy ofdiagnosis, the US Food and Drug Administration (FDA) haswarned about their potential toxicities related to nephrogenicsystemic fibrosis with impaired kidney function.158 Compara-tively, manganese-based T1-weighted MRI CAs show highpotential for substituting Gd-based MRI CAs because of thenecessary daily uptake of manganese for physiological meta-bolism by the human body.159 In addition, the human body cancontrol the homeostasis of manganese.160 The slow developmentof manganese-based MRI CAs is mainly due to their relativelylow imaging performance (typically r1 o 0.5 mM�1 s�1) com-pared to commercial Gd-based agents (r1 E 3.4 mM�1 s�1).161–163

The high in vivo MRI performance of PEGylated 2D MnO nano-plates (r1 = 5.5 mM�1 s�1) was demonstrated by Hyeon et al.164

They found that the contrast-enhanced T1-weighted MRI couldoccur almost in the whole body by using the intravenous adminis-tration of 8 nm-sized PEGylated MnO nanoplates as CAs. Kidneyexcretion of MnO CAs was also observed due to the smallparticulate size of PEGylated MnO nanoplates.

Fig. 11 (a) TEM image of PEG–MnO2 nanosheets. In vitro dynamic measurement of T1-weighted MR imaging of PEG–MnO2 in (pH = 4.6, b) mild acidicenvironment and (pH = 7.4, c) neutral condition. (d) Axial and (e) coronal T1-weighted MR imaging of 4T1 tumor-bearing nude mice before (d1 and e1) andafter (d2–d9 and e2–e9) injection of PEG–MnO2 nanosheets saline solution within tumor and normal subcutaneous tissue. Quantitative T1-wieghted MRIsignal intensity before and after the injection of PEG–MnO2 nanosheets saline solution (f: axial tumor region in d, g: coronal tumor region and normalsubcutaneous tissue in e). Reprinted with permission from ref. 120. Copyright 2014 Wiley-VCH.


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Very recently, we solved the critical issue of low-MRI perfor-mance of Mn-based CAs based on the unique break-up nature of2D MnO2 nanosheets.120 The highly dispersed MnO2 nanosheetswere fabricated by the typical chemical exfoliation approach. Thesurface was further under PEGylation to improve their stabilityin physiological conditions (Fig. 11a). The in vitro dynamicevaluation of T1-weighted MRI showed that the T1 signal gave asubstantial increase after the incubation of MnO2 nanosheetswithin the mild acidic environment (pH 4.6, Fig. 11b) while theimpregnation of PEG–MnO2 nanosheets under neutral condition(pH 7.4) did not cause the obvious positive-signal enhancement(Fig. 11c). The relaxation rate (r1 value) was only 0.007 mM�1 s�1

in the neutral solution due to the high valence (IV) of Mn, butincreased to 3.4 mM�1 s�1 and 4.0 mM�1 s�1 under the mildacidic environment of pH 6.0 and pH 4.6, respectively. Such anultrasensitive pH-triggered MRI enhancement was due to thegradual disintegration of MnO2 nanosheets. The released MnII

ions from MnO2 nanosheets had the maximum chance to inter-act with water molecules, thus leading to the enhancement ofMRI signals. Importantly, the in vivo evaluation on tumor xeno-graft further demonstrated that the acidic tumor microenviron-ment can trigger the break-up of MnO2 nanosheets to enhancethe T1-weighted MRI while the normal tissues with neutral pHmicroenvironment cannot cause such a change (Fig. 11d–g).165

Such a unique break-up nature of 2D MnO2 nanosheets can alsofacilitate the excretion of nanosheets out of the body due to theextremely small size of leaked MnII ions cleared by kidney.

It is well known that traditional CAs, e.g., Fe3O4, Au andIopamidol, perform well in various diagnostic-imaging modali-ties. Comparatively, the introduced 2D GA-based CAs can exertsome specific or enhanced performances in these imagingmodalities such as tumor microenvironment-responsive MRimaging,120 significantly enhanced in vitro CT imaging perfor-mance100 and imaging-guided cancer therapy.100,120,134 However,these new 2D GA-based CAs have not been screened by systema-tic in vivo evaluations regarding their biosafety and performancecompared to the mostly explored CAs like Fe3O4 and Au NPs.Nevertheless, the preliminary results of 2D-GAs indicate theirpotential for clinical translations in diagnostic imaging.

5. Biosensors

In addition to the high performance of 2D-GAs for therapy anddiagnostic imaging of cancer, these 2D nanosystems can alsobe used as novel biosensing platforms for the detection ofbiomolecules and bio-effects. Compared to various NP-basedbiosensors, 2D-GAs have two apparent advantageous features inbiosensing applications. On the one hand, the large surfacearea of 2D planar structures allows the immobilization of largeamount of sensing molecules to concurrently cause the shortassay duration and low detection limit. On the other hand, theunique physical property of 2D nanosheets can exert someunusual performance such as the fluorescent quenching effectcaused by the photoinduced electron transfer (PET) from theexcited fluorophore to the conduction band of 2D nanosheets.

Recently, we demonstrated that single-layer MoS2 nanosheetscould be used as a sensing platform for detection of DNA basedon its strong fluorescence-quenching ability.166 As shown inFig. 12a, MoS2 nanosheet could absorb dye-labeled single-stranded DNA (ssDNA) via van der Waals force between nucleo-bases and the basal plane of MoS2, by which the fluorescence ofthe dye was quenched. Afterwards, the fluorescence-quenchingeffect can be significantly inhibited when the ssDNA was hybri-dized with its complementary target DNA to form double-stranded DNA (dsDNA). The availability of nucleobases to thebasal plane of MoS2 was substantially reduced due to therestriction of nucleobases between the densely negative-chargedhelical phosphate backbones, causing the weak interactionbetween dsDNA and MoS2 and subsequent retention of thefluorescence of the dye. This MoS2-based sensor shows a detec-tion limit of 500 pM to DNA (biomacromolecules) and 5 mm toadenosine (small molecules). This homogeneous process issimple and fast (within a few minutes), which shows greatpromise for molecular diagnostics.166 As another example, basedon the 2D g-C3N4 nanosheet, a DNA biosensor was designed byusing the affinity changes of g-C3N4 to DNA probes when theytargeted the analyte and the corresponding PET-based fluores-cence quenching effect.168 In addition, the atomistic and quan-tum simulations showed that the single-layer MoS2 is highly

Fig. 12 (a) The scheme of the fluorometric DNA assay based on 2D MoS2 nanosheets as the biosensor. Reprinted with permission from ref. 166.Copyright 2013 American Chemical Society. (b) The translocation of DNA through the single-layer MoS2 nanopore. Reprinted with permission fromref. 167. Copyright 2014 American Chemical Society.


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suitable for nanopore-based DNA sequencing with the signal-to-noise ratio (SNR) of more than 15 (Fig. 12b).167

Based on the fluorescence quenching effect of 2D WS2

nanosheets, Liu et al. established two biosensors, i.e., thenuclei acid hybridization model and protein–aptamer model,to detect the oligonucleotides and proteins.119 The binding ofbioprobes onto WS2 nanosheets is reversible, which indicatesthat the interactions between bioprobes and WS2 nanosheetscan be interrupted by the addition of other biomolecules. Sucha reversible process was reflected by the fluorescence quenchingand recovery based on a special energy-transfer process. Inaddition to being used as the fluorescent quencher, Jiang et al.found that WS2 nanosheets showed differential affinity towardsshort oligonucleotide fragment versus the ssDNA probe. Basedon this mechanism, they developed a WS2-based biosensor todetect microRNAs (miRNAs) with a detection limit of 300 fm.169

The intrinsic photoluminescence (PL) effect of 2D MoS2 canbe further used for the in situ sensing of biological systems, inwhich ion intercalation plays an important role.170 It has beendemonstrated that the PL effect of 2D MoS2 nanosheets is causedby the hybridization between Pz orbitals of S atoms and d orbitalsof Mo atoms, which can be manipulated by intercalating alkaline(Li+, Na+ and K+) and H+ ions. The quasi-2D MoS2 nanoflake wasrecently designed to monitor the ion transfer during enzymaticactivities and ion exchange in both viable and nonviable cells.170

The oxidation of glucose by glucose oxidase can generate H+ andelectrons, which can intercalate into the MoS2 nanoflake toquench its PL with the assistance of an external electric fieldbecause the intercalated H+ and electrons can transform thesemiconducting phase of MoS2 into the metallic HxMoS2 phase.The PL modulation of quasi-2D MoS2 by ion intercalation wasfurther used to investigate the ion exchange in yeast cells.Nonviable yeast cells could not quench the PL of quasi-2DMoS2 nanoflake because the ions (K+) could not intercalate intoMoS2 without the assistance of a driving force. Comparatively,

the viable yeast cells could generate an electric field across themembrane by the transfer of K+ ions to the exterior of cells, whichcould drive the intercalation of K+ ions into quasi-2D MoS2 toquench the fluorescence. The control experiment using poly-styrene particles (B5–10 mm) to substitute the yeast cells couldnot cause the PL quenching effect of MoS2 nanoflake, and theprocedure of cell death caused by introducing methanol couldgradually restore the PL of quasi-2D MoS2 nanoflake. Thus, thissensing ability of MoS2 could be used to reveal the cell viabilities.Importantly, this sensing procedure was expected to monitor theion-related biological and medical procedures.170

Compared to traditional one-dimensional carbon nanotubesand silicon nanowires, 2D nanosheets are more suitable for field-effect transistor (FET)-based biosensors because of their uniqueelectronic states and planar morphology. For example, there is nobandgap in pristine graphene while the single-layer MoS2 has adirect band gap of 1.8–1.9 eV. Sarkar et al. recently used MoS2 asthe channel material for fabricating a new FET-based biosensorfor the detection of pH and biomolecules.171 For the biosensingprocedure, the dielectric layer covering the MoS2 channel wasinitially modified with biotin to capture streptavidin. The chargedstreptavidin could induce a gating effect after the capture tomodulate the device current (Fig. 13a–d). For biosensing applica-tions, the device showed significant current decrease after addingstreptavidin in the 0.01� PBS solution while the addition of purebuffer could not cause the current change (Fig. 13e). This currentdecrease was due to the negative charge of the streptavidin in the0.01 � PBS solution. Comparatively, the addition of streptavidinsolution with the pH value lower than the isoelectric point (pI)could cause the current increase compared to that in pure PBSbuffer solution (Fig. 13f). This ultrasensitive and specific proteinbiosensing procedure could achieve the sensitivity of 196 evenat 100 fm concentration. This work gives strong evidence that2D MoS2 can be an excellent candidate for the next-generationlow-cost FET-based biosensors for diagnostics.

Fig. 13 (a) The scheme of the principle of the MoS2-based FET biosensor. (b) Optical image of the MoS2 flake on 270 nm SiO2 grown on Si substrate.(c) Optical image of the MoS2 FET biosensor device. (d) Photograph image and schematic illustration of the chip with the biosensor device andmacrofluidic channel. (e) The current change of a MoS2-based FET biosensor functionalized with biotin after the addition of streptavidin solution in purebuffer (0.01 � PBS). (f) The current change after the addition of streptavidin solution at a pH of 4.75, less than the pI of streptavidin. Reprinted withpermission from ref. 171. Copyright 2014 American Chemical Society.


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6. Biosafety/toxicity evaluations of2D-GAs

The biosafety and toxicity of 2D-GAs determine their furtherclinical translations. Similar to other organic/inorganic nano-systems, the biological effects such as the in vitro cellularuptake/location/toxicity and in vivo biodistribution/degradation/excretion of these 2D nanosheets are highly related to their crucialstructural/compositional parameters and physicochemical pro-perties such as the sheet size, morphology, dispersion, surfacestatus and hydrophilicity. Because of the use of various 2D-GAsin biomedicine, some crucial parameters of 2D-GAs, such assolubility, biodegradation and biocompatibility, are differentfrom one another. In addition, the biocompatibility/biosafetyevaluations of different types of 2D-GAs are still in progress.Therefore, it is still too early to claim the biosafety of 2D-GAs,though there are still some promising progresses indicating theirpotential in biomedicine.

It has been demonstrated that several members of TMDsexhibit low cytotoxicities towards living cells. For instance, thechitosan-modified MoS2 (MoS2–CS) sheets revealed low cytotoxi-cities against KB (human epithelial carcinoma cell line) andPanc-1 (pancreatic carcinoma, epithelial-like cell line) cell lines,even at the high concentration of 400 mg mL�1.141 After themodification of MoS2 with chitosan, the in vitro hemolytic effectof MoS2–CS against red blood cells (RBCs) was extremely loweven at the concentration of as high as 800 mg mL�1. PEGylatedMoS2 nanosheets also showed the low cytotoxicity against HeLacells at the high concentration of 0.16 mg mL�1.140 In addition,BSA-modified WS2 nanosheets exhibited low in vitro cytotoxici-ties against HeLa cancer cells by using a typical standard CellCounting Kit-8 (CCK-8) assay.114 Liu et al. found that PEGylatedWS2 nanosheets had no obvious in vitro toxicity using several celllines at the high concentration of up to 0.1 mg mL�1 by thetypical MTT assay,134 including murine breast cancer cells 4T1,human epithelial carcinoma cells HeLa and human embryokidney cells 293T. Further cytotoxicity investigation revealed thatthe release of lactate dehydrogenase (LDH) and reactive oxygenspecies (ROS) was at normal levels compared to the untreatedcells, further indicating the low cytotoxicities of PEGylated WS2

nanosheets. Very recently, exfoliated MoS2 and WS2 nanosheetswere found to be much less hazardous than GO or halogenatedgraphene by MTT and WST-8 assays against human lungcarcinoma epithelial cells A549.172

The in vivo potential cytotoxicity of PEGylated WS2 nanosheetsagainst Balb/c mice was systematically assessed by the typicalhematoxylin and eosin (H&E) assay, serum biochemistry assayand complete blood panel test (dose: 20 mg kg�1).134 It was foundthat no obvious abnormal behavior of mice was observed duringthe assay (45 days after PTT). H&E result showed high histo-compatibility and all the indexes of the blood test were inthe normal range. The biocompatibility of PEGylated MoS2

nanosheets were assessed by the same procedure, which showedthe low toxicities to Balb/c mice at the dose of 3.4 mg kg�1 andfeeding duration of 30 days.140 In addition, the surface statusand sheet size of 2D-GAs affected their in vivo biodistribution and

excretion. The kidney excretion was observed on the 8 nm-sizedPEGylated MnO nanoplates.164 This unique clearance behavior isof high significance for the biosafety of these nanosheets.143 Inaddition, the circulation time and enhanced permeability andretention (EPR)-mediated tumor targeting of 2D-GAs stronglydepend on the surface organic modifications, particle sizes,hydrophilicity and dispersity/stability, which are similar tomost explored nanosystems. The long blood-circulation timeand enhanced accumulation within tumor tissues can also beachieved as long as aforementioned compositional/structuralparameters are optimized. For instance, the PEGylated MoS2

nanosheets exhibited relatively long blood-circulation durationwith the blood level of 3.67% of injected dose per gram tissue(%ID/g) retained even after the post-injection for 24 h. Thetumor-accumulation amount at 24 h post-injection was deter-mined to be 6.62% ID/g.140

Compared to GO or rGO sheets, the research on the biosafetyof 2D-GA nanosheets is still at the very early stage. Only pre-liminary in vitro and in vivo assessments have been performed onthe cytotoxicities or acute toxicities to mice. Although these resultshave shown promising biosafety of these 2D nanomaterials underthe investigated doses, the evaluations on the biodistribution,tolerant threshold, degradation and clearance have not beensystematically conducted yet. Especially, some metal elements of2D-GAs (e.g., W, Mo, and Mn) are very rare in biological systems.Therefore, their retaining, excretion, potential toxicity and long-term influence to animals should also be revealed and deter-mined to guarantee their safe application clinically. In this regard,more detailed research results concerning the evaluations on thebiocompatibility and biosafety of these 2D-GAs are urgentlyrequired.

7. Conclusion and outlook

This review summarized some of the recent importantprogresses of inorganic 2D layered nanosheets for biomedicalapplications. Note that many of them only emerged over thepast few years. 2D-GAs with specific planar morphology andphysicochemical properties have been demonstrated to behighly effective in drug delivery, diagnostic imaging and bio-sensing. Compared to GO/rGO sheets, these 2D-GAs can bringmany more specific characteristics due to their abundantchemical compositions and diverse biological effects in bio-medicine. However, the investigation of these 2D-GAs in bio-medicine is still at their early stage, and several unresolvedcritical issues are to be addressed to further facilitate theadvances of this field (Fig. 14).

(i) From the materials point of view, one of the key challengesof 2D-GAs for biomedical applications is the lack of controllableand standard synthetic methodologies to obtain the nanosheetswith desired structural/compositional parameters such as sheetsize, dispersity, hydrophilicity and surface functionalities, whichare the critical factors to determine the in vivo biological effectsand therapeutic/diagnostic performances. Most of the reportednanosized 2D-GA nanosheets were fabricated by exfoliation from


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their bulk crystals. However, this method can only produce2D-GAs with wide sheet-size distribution. The bottom-up synth-esis is regarded as the alternative approach to solve this issue,while how to produce the 2D-GAs on a large scale is still a bigchallenge.

(ii) Detailed biological and biosafety assessments of these2D-GA nanosheets are urgently required to ensure their furtherclinical translations. Although the preliminary evaluation hasdemonstrated potentially low toxicities of several sub-familiesof 2D-GAs (e.g., MoS2, WS2, MnO2, and g-C3N4), their potentialrisks should be further determined and revealed from currentacute toxicity assessment to further chronic toxicity evaluation.The assessments should be further concentrated on the bio-distribution, biodegradation, excretion and potential toxicitiesto specific organs, including neurotoxicity, reproductive toxicityand the influences to embryonic development. In addition,animal models should be further updated to large animalssuch as pigs or primates. Only after the biosafety of these 2Dnanosheets is well proved, it will be possible to further carry outpotential clinical trials.

(iii) The application fields of these 2D-GA nanosheets aremuch less than the widely explored 2D GO/rGO, partially due tothe short research history of the 2D-GAs. In addition to thereported applications in drug delivery, diagnostic imaging andbiosensing, these 2D-GA nanosheets also show great potentialsin other biomedical aspects such as gene therapy, tissueengineering, and radiosensitization. In addition, the non-specific accumulation of these 2D nanosheets in the reticulo-endothelial system (RES) is still high due to the lack of targetingspecificities. Thus, the surface engineering of 2D-GAs withtargeting functions can endow these carriers with the capability

to accumulate at specific cells or sub-cellular organelles, andreduce the accumulation in RES organs. Based on this targetingstrategy, the high therapeutic/diagnostic performance isexpected to be enhanced and the side-effects to normal tissuescan be mitigated simultaneously.

(iv) The 2D-GA nanosheets are expected to be further inte-grated with many other biocompatible components, by whichmultiple diagnostic and therapeutic purposes can be realized.Integration of CAs with 2D layered nanosheets can bring withmultiple functions of concurrent diagnostic imaging and therapy(theranostics). In addition, integration of different therapeuticmodalities can realize the synergistically therapeutic outcome.However, it should be noted that such a multifunctionalizationprocess requires more strict and complex biosafety evaluations ofeach component and whole integrated nanosystems for furtherpotential clinical translations.

Compared to the mostly explored and nearly mature lipo-somes, micelles, Fe3O4 and Au NPs, the biomedical applicationsof 2D-GAs are now still at an early stage. We understand thatmuch more research effort is necessary to be conducted beforethese 2D GA-based materials come into the clinical stage.Encouragingly, these new material-based nanosystems in bio-medicine indeed provide new opportunities to promote thegeneration of new diagnostic-imaging and therapeutic modalitiessuch as enhanced/intelligent MR imaging, on-demand drugreleasing, and synergistic therapy, which are difficult to berealized by traditional liposomes, micelles and inorganic nano-crystals. The biomedical applications of 2D-GAs are now themuch focused research frontier in materials science, biomedicineand nanobiotechnology. Although there are still some unresolvedissues, the relatively high biosafety at appropriate doses and

Fig. 14 Summary of the development on 2D-GAs for biomedical applications, including the design/construction of 2D layered nanosheets based on thepractical clinical-use criteria, current status and future developments of these 2D-GAs in biomedicine.


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demonstrated performance in biomedicines shed light on newpathways towards efficient clinical translations. To realize thisgoal, much closer collaborations among the researchers andexperts from multidisciplinary fields should be further estab-lished to promote the clinical translations of these 2D-GAs. Giventhe very short research history and rapid development trajectoryof this field, we are confident that the biosafety and pharma-ceutical issues of the elaborately designed and synthesized 2D-GAnanosheets could be further addressed in the near future, whichmay eventually lead to clinical trials to benefit our human health.

In addition to the emerging applications in biomedicine, itshould be noted that these new 2D-GAs have shown importantroles in energy- and environment-related areas. For instance,they can be used for flexible supercapacitors30,173,174 or electrodematerials of lithium-ion batteries175–180 with enhanced electro-chemical performance. In light harvesting area, these 2D-GAscan be used as efficient and stable photocatalysts for solar fuelhydrogen production via a water splitting process.94,181–186 Inaddition, these 2D-GAs also exhibit high performance in gassensors,187 phototransistors188,189 and various device applica-tions.190–192 The new application opportunities of 2D-GAs inturn also promote the rapid development of new syntheticmethodologies of 2D-GAs with desirable structural and composi-tional parameters. On this ground, previous studies over thepast few years are expected to pave a solid base for using these2D-GAs to benefit our society not only in human healthcare butalso more broadly in other important areas such as energy andenvironment.

List of abbreviations

2D-GAs Two dimensional graphene analogues2D Two dimensionalTMDs Transition metal dichalcogenidesg-C3N4 Graphitic-phase carbon nitrideLDH Layered double hydroxidePTT Photothermal therapyPTA Photothermal agentPDT Photodynamic therapyMRI Magnetic resonance imagingCT Computed tomographyPAT Photoacoustic tomographyQDs Quantum dotsGO/rGO Graphene oxide/reduced graphene oxidePEG Polyethylene glycolPVP PolyvinylpyrrolidoneMB Methylene blueBSA Bovine serum albuminNPs NanoparticlesNIR Near-infraredCAs Contrast agentsDox DoxorubicinMDR Multidrug resistancePET Photoinduced electron transferFET Field-effect transistors

RBCs Red blood cellsROS Reactive oxygen speciesRES Reticuloendothelial systemCVD Chemical vapor depositionEPR Enhanced permeability and retention

Acknowledgements

L. Z. Wang acknowledges the financial support from AustralianResearch Council through its Discovery and Future Fellowshipschemes. Y. Chen acknowledges the financial support fromthe National Nature Science Foundation of China (Grant No.51302293), the Natural Science Foundation of Shanghai(13ZR1463500), the Shanghai Rising-Star Program (14QA1404100)and the Biomedical ECR Grant 2014. This work was also supportedby MOE under AcRF Tier 2 (ARC 26/13, No. MOE2013-T2-1-034),AcRF Tier 1 (RG 61/12, RGT18/13, and RG5/13), and Start-Up Grant(M4080865.070.706022) in Singapore. The NTU-HUJ-BGU Nano-materials for Energy and Water Management Programme underthe Campus for Research Excellence and Technological Enterprise(CREATE), which was supported by the National Research Founda-tion, Prime Minister’s Office, Singapore, is also acknowledged.

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