Biological interactions of carbon-based nanomaterials: From … · 2021. 1. 5. · We also discuss recent studies on nanomaterial ‘coronation’ and how this impacts on biodistribution

Nanomedicine: Nanotechnology, Biology, and Medicinexx (2015) xxx–xxx

nanomedjournal.com

Biological interactions of carbon-based nanomaterials: Fromcoronation to degradation

Kunal Bhattacharya, PhDa, Sourav P. Mukherjee, PhDa, Audrey Gallud, PhDa,Seth C. Burkert, MScb, Silvia Bistarelli, MScc, Stefano Bellucci, PhDc,

Massimo Bottini, PhDd,e, Alexander Star, PhDb, Bengt Fadeel, MD, PhDa,⁎aDivision of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

bDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA, USAcNational Institute of Nuclear Physics–INFN, Frascati, Province of Rome, Italy

dDepartment of Experimental Medicine and Surgery, University of Rome ‘Tor Vergata’, Rome, ItalyeSanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, United States

Received 19 August 2015; accepted 10 November 2015

Abstract

Carbon-based nanomaterials including carbon nanotubes, graphene oxide, fullerenes and nanodiamonds are potential candidates forvarious applications in medicine such as drug delivery and imaging. However, the successful translation of nanomaterials for biomedicalapplications is predicated on a detailed understanding of the biological interactions of these materials. Indeed, the potential impact of the so-called bio-corona of proteins, lipids, and other biomolecules on the fate of nanomaterials in the body should not be ignored. Enzymaticdegradation of carbon-based nanomaterials by immune-competent cells serves as a special case of bio-corona interactions with importantimplications for the medical use of such nanomaterials. In the present review, we highlight emerging biomedical applications of carbon-basednanomaterials. We also discuss recent studies on nanomaterial ‘coronation’ and how this impacts on biodistribution and targeting along withstudies on the enzymatic degradation of carbon-based nanomaterials, and the role of surface modification of nanomaterials for thesebiological interactions.© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Key words: Carbon nanotubes; Graphene oxide; Fullerenes; Nanodiamonds; Biodegradation; Bio-corona

Funding: This work was supported by grants from the EuropeanCommission (Flagship Project GRAPHENE, grant no. 604391; FP7-NANOSOLUTIONS, grant no. 309329; FP7-NANOREG, grant no.310584), the Swedish Research Council, and the Swedish Research Councilfor Environment, Agricultural Sciences and Spatial Planning (FORMAS) (toB.F.); National Institute of Environmental Health Sciences (NIEHS) grant no.R01ES019304 (to A.S.); Arthritis National Research Foundation (JohnVaughan Scholarship) (to M.B.); and SfP NATO grant no. SfP-984537, andItalian Ministry of Health grant no. PE-2011-02347026 (to S.B.).

Conflict of interest: The authors declare no conflicts of interest.Portions of the graphical abstract were reproduced with permission from

Nature Publishing Group and John Wiley & Sons.Acknowledgements.: We thank Dr. Kjell Hultenby, Karolinska

Institutet, for assistance with SEM.⁎Corresponding author at: Institute of Environmental Medicine,

Karolinska Institutet, Stockholm, Sweden.E-mail address: [email protected] (B. Fadeel).

http://dx.doi.org/10.1016/j.nano.2015.11.0111549-9634/© 2015 The Authors. Published by Elsevier Inc. This

Please cite this article as: Bhattacharya K., et al., Biological interactions of carNBM 2015;xx:1-19, http://dx.doi.org/10.1016/j.nano.2015.11.011

Engineered nanomaterials provide unique advantages andopportunities in several areas of medicine including therapeutics,diagnostics, imaging, and regenerative medicine.1,2 Carbon-basednanomaterials such as fullerenes, carbon nanotubes, carbon nano-horns, carbon nanodots, nanodiamonds, and graphene and itsderivatives have unique electronic, optical, thermal, andmechanicalproperties and have attracted considerable attention in recent yearsin nanomedicine.3-5 Hence, many studies have attempted to exploitthesematerials for drug delivery or imaging, or both. As pointed outin a recent editorial, the successful commercialization of nanome-dicines ultimately depends on demonstrating their superiority overexisting approaches and on documenting their safety.2 Indeed, adetailed understanding of the biological interactions of nanomater-ials, not least the interactions with cellular and other components ofthe immune system (Figure 1) is important both from an efficacy

is an open access article under the CC BY-NC-ND license

bon-based nanomaterials: From coronation to degradation. Nanomedicine:

http://dx.doi.org/10.1016/j.nano.2015.11.011




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Figure 1. Cellular and extracellular interactions of carbon nanotubes. Theupper panel shows an SEM image of isolated MWCNTs (single arrow) or abundle of MWCNTs (two arrows) entering human mesothelial cells.Reprinted from: Shi X, von dem Bussche A, Hurt RH, Kane AB, Gao H.Cell entry of one-dimensional nanomaterials occurs by tip recognition androtation. Nat Nanotechnol. 2011;6(11):714-9, with permission from NaturePublishing Group. The lower panel shows a cluster of short-cut SWCNTs(single arrow) entrapped in chromatin fibers (two arrows) of purifiedneutrophil extracellular traps [see Farrera et al6 for further details]. SEMcourtesy of K. Hultenby, Karolinska Institutet.

2 K. Bhattacharya et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2015) xxx–xxx

and safety point of view, as is the understanding of the ultimate fateof the nanomaterial– accumulation, degradation, and/or excretion–in the human body.7 To this end, particular attention should bedevoted to the role of adsorbed biomolecules which may confer anew biological ‘identity’ to nanomaterials,8 and is likely to play animportant role for cellular uptake and in vivo biodistributionof nanomaterials.9

Detailed accounts of the routes of synthesis and thephysicochemical properties of carbon-based nanomaterials arebeyond the scope of the present review, but a brief introduction isprovided here. Fullerenes are entirely composed of carbon andhave the form of spheres, ellipsoids or tubules. Spherical andcylindrical fullerenes are also referred to as buckyballs andbuckytubes (or carbon nanotubes), respectively. The firstrepresentative of the buckyball family, referred to as buckmin-sterfullerene, is composed of 60 carbon atoms (C60) and has theshape of a truncated icosahedron with 20 hexagons and 12pentagons and a diameter of approximately 1 nm, thusresembling a football (in the United States, a soccer ball);indeed, a picture of a football was included in the very firstpublication, and the authors even contemplated the alternativename, soccerene.10 Iijima is credited with the discovery ofcarbon nanotubes (CNTs)11 although some claim that these

structures (“graphitic carbon needles”) had been observeddecades earlier.12 CNTs are graphitic tubules, which can becapped with hemifullerenes at the ends, consisting of a singlegraphene sheet (single-walled carbon nanotubes, SWCNTs) orseveral concentric and nested sheets (multi-walled carbonnanotubes, MWCNTs). Both types of CNTs have nano-scaledimensions and display a very high aspect ratio, i.e., the ratiobetween the length and the diameter of the material. Hence,SWCNTs have a diameter of approximately 1 nmand lengths up toa few microns or more, whereas MWCNTs have diameters ofseveral tens of nanometers and lengths up to several tens ofmicronsor more. All of the aforementioned nanomaterials can be related toa parent material known as graphene consisting of a singleatomically thin sheet of hexagonally bound sp2 carbon atoms.13

For a comprehensive overview of the structural, electronic, andbiological properties and applications of graphene and other 2-Dmaterials, see Ferrari et al.14 Nanodiamonds represent yet anotherclass of nanoparticles in the carbon family, with highly versatilephysical and chemical properties.15 They are mainly composed ofcarbon sp3 structures in the core, with sp2 and disorder/defectcarbons on the surface, and display single-digit nm sizes.

In the present review, we will highlight emerging biomedicalapplications of various carbon-based nanomaterials. We will alsodiscuss bio-corona formation and the propensity for enzymaticdegradation, especially with regards to CNTs and grapheneoxide (GO), which are the most intensively investigatedcarbon-based nanomaterials to date in the field of nanomedicine,along with fullerenes and nanodiamonds. The impact of surfacemodifications, including grafting of polymers, on the biologicalinteractions of these materials is also highlighted.

Biocompatibility of carbon-based nanomaterials

Being small confers advantages in terms of negotiating biologicalbarriers, which may be desirable, but nanoscale size per se is notsufficient to qualify as a nanotechnology.16 Carbon-based nanoma-terials, however, possess intrinsic physicochemical properties thatcan potentially be exploited. For instance, CNTs display strongoptical absorption in the near infrared, Raman scattering as well asphoto-acoustic properties that widen the scope of in vivo applicationsas they can potentially have bio-imaging and tracing functionscoupled with drug delivery.4 Graphene is another material withmany promising areas of application as a result of its large surfacearea and possibility of easy functionalization, providing opportuni-ties for drug delivery.5 Moreover, its unique mechanical propertiessuggest tissue engineering and regenerativemedicine applications.17

Other carbon-based nanomaterials such as fullerenes and nanodia-monds have also received much attention in recent years, withemphasis mainly in the area of cancer medicine.4 In the presentreview, we will highlight some illustrative, pre-clinical examplesfrom recent literature.

However, safety first. The potential toxicity of carbon-basednanomaterials has been the subject of much concern in the pastdecade and much skepticism initially surrounded the notion ofusing, in particular, CNTs as drug delivery systems due to the factthat these fiber-like materials were presumed to be biopersistent,and, therefore, to possess asbestos-like pathogenicity.18-20

3K. Bhattacharya et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2015) xxx–xxx

However, more recent research has suggested strategies to improvethe biocompatibility of CNTs through surface modification of thematerials and has also demonstrated the susceptibility forenzymatic degradation of these nanomaterials (discussed below).Indeed, it is important to distinguish potentially harmful CNTs21

from more biocompatible ones. Moreover, there are importantlessons to be learned from these extensive toxicologicalinvestigations.22,23 Categorization or grouping of nanomaterialsaccording to their risk potential, taking into account indicators ofboth hazard and exposure, is needed to identify ‘nanomaterials ofconcern’.24 Overall, it is necessary to avoid generalizations aboutthe toxicity of ‘carbon nanotubes’ or, for that matter, of ‘graphene’,as these are not single nanomaterials, but classes of nanomaterialswith important differences in terms of their physicochemicalproperties (such as, aspect ratio or lateral dimensions, purity,surface functionalization, and so on) and, hence, in theirtoxicological profile. Thus, with careful evaluation of thebiological interactions of each nanomaterial, a more favorablescenario for their exploitation in medicine presents itself. Of keyimportance for any biocompatibility assessment of nanomaterialsis the evaluation of potential effects on the immune system.9

Indeed, the immune system has evolved to protect us frompathogens and other foreign intrusion. In brief, the immune systemcan be divided into the innate and the adaptive (or acquired)immune system. The innate immune system is composed ofinflammatory cells or ‘sensors’ and soluble ‘mediators’ (i.e.,complement factors, chemokines, and cytokines).20 It is this arm ofthe immune system that nanomaterials first encounter followingeither deliberate or accidental (occupational/non-occupational)exposures. The inflammatory cells encompass macrophages,professional phagocytic cells that differentiate from monocytesthat migrate from the circulation and extravagate into tissues. Themain functions of monocytes are phagocytosis, antigen presenta-tion and cytokine production. Functionalized CNTs have beenreported to activate immune-related pathways in monocytessuggesting that such carbon-based nanomaterials may functionas immunostimulatory agents.25 CNTs were also found to triggerso-called inflammasome activation in monocytes26 as well as inprimary human monocyte-derived macrophages27 leading to thesecretion of the pro-inflammatory cytokine, interleukin (IL)-1β[for a review, see Bhattacharya et al20]. In a related study, surfacefunctionalization of CNTs or carbon nano-onions (CNOs)attenuated the inflammatory properties of these nanomaterials,with a reduction in the recruitment of inflammatory neutrophils andmonocytes in vivo and reduced IL-1β production.28 Strategicallylocated macrophages act as sentinels against foreign materials andcan be divided into various subpopulations based upon theiranatomical location and functional phenotypes. The granulocytes,including neutrophils, basophils, and eosinophils, also form part ofthe innate immune system, along with mast cells, a tissue-residentgranulocytic cell that is closely related to basophils. Natural killer(NK) cells are a component of the innate immune system whichdoes not directly attack invading microbes. Instead, these cellsdestroy tumor cells or virus-infected cells. The interaction betweenimmune cells and tumors, and the role of immune cells as sentinelsin eliminating continuously arising transformed cells, are ofparticular importance for nanomedicine. Dendritic cells (DCs) areantigen-presenting cells that serve as a ‘bridge’ between the innate

and adaptive arms of the immune system. The adaptive immunesystem, in turn, is composed of B cells and T cells, and these cellsare responsible for immunological ‘memory’ which is ‘adaptive’because it occurs during the lifetime of an individual as anadaptation to encounters with a specific pathogen. Nanomaterialshave been shown to interact with cells of the innate immunesystem, while effects on the adaptive immune system occur, inmost but not all cases, via the innate immune system [see Farreraand Fadeel9 and Boraschi et al29 for a review]. To give one recentexample,GOwas shown to trigger a typical ‘foreign body’ reactionin mice upon subcutaneous implantation, with recruitment ofneutrophils, followed by monocytes; these cells secreted a varietyof soluble mediators resulting in the establishment of aninflammatory microenvironment.30 GO and CNTs have bothbeen reported to act directly on macrophages and DCs ex vivo andin animal models.31-33 As we shall discuss in the present review,the interactions between carbon-based nanomaterials and theimmune system can be reciprocal in the sense that immune-competent cells such as macrophages and neutrophils can ‘strikeback’ and digest nanomaterials.20

Biomedical applications of carbon-based nanomaterials

Carbon-based nanomaterials display excellent mechanical,thermal and optical properties making them potentially usefuland attractive in medicine, including for therapeutics and/ordiagnostics, as well as in regenerative medicine. In the followingsection, we shall discuss examples of each of these broad areasbeginning with therapeutics, which in turn may be divided into:carbon-based nanomaterials as drug or gene delivery vehicles, orcarbon-based nanomaterials as drugs per se.

CNTs have been studied intensively as drug carriers, withdoxorubicin being the most common model drug [see Chen et al4

for a recent review]. To this end, drugs may be loaded onto CNTsthrough noncovalent interactions, eg., π-π stacking as shown fordoxorubicin,4 although covalent binding has also been exploredfor hydrophilic drugs.34 In the latter study, the authors covalentlyattached not only the drug, cisplatin but also the targeting ligand,epidermal growth factor (EGF), and demonstrated that thesetargeted vectors were selectively taken up by head and necksquamous carcinoma cells overexpressing EGF receptors.34

Moreover, regression of tumor growth was rapid in mice treatedwith targeted SWCNT-cisplatin conjugates relative to thenon-targeted ones. CNTs may also serve as multi-functionaldevices for selective cancer cell destruction, by virtue of theirintrinsic physicochemical properties. For instance, Kam et al35

reported that SWCNTs can be deployed for targeted delivery ofoligonucleotides to cancer cells with near-infrared light-mediated killing of cancer cells due to the excessive localheating of the CNTs.

CNTs are known to interact with DNA and much interest hasbeen devoted to the potential use of CNTs for gene delivery ordelivery of small interfering RNA (siRNA). Some authors haveclaimed a passive, “needle-like” mechanism of cellular entry forCNTs,36 which could be exploited for gene delivery, if proven tobe specific for the intended target cells. In this context, theformation of a so-called bio-corona on the surface of the CNTsand its potential impact on cellular recognition and uptake needs


to be taken into account (discussed below). Al-Jamal et al37

provided evidence for efficient delivery of siRNA directly to theCNS through stereotactic administration of MWCNTs, resultingin neuroprotection in mice and rats. We recently demonstratedthat PEG-modified SWCNTs can be deployed as carriers forintra-articular delivery of antisense oligonucleotides to chon-drocytes in mice without affecting cartilage homeostasis oreliciting systemic side-effects.38 In another recent study, wedeveloped a novel strategy for delivery of microRNAs toendothelial cells to regulate angiogenesis, using polymerfunctionalized MWCNTs (submitted for publication). Wefound that endothelial cells displayed efficient uptake ofmiR-503 following administration of miR-503 bound to thefunctionalized CNTs, and a decrease of vessel formation wasobserved in a mouse model of angiogenesis. Moreover, thepolymer-coated CNTs displayed a reduced toxicity whencompared to the pristine CNTs.

Graphene is another promising material for drug delivery.Indeed, as pointed out by Novoselov et al,39 graphenederivatives can solubilize and bind drug molecules as a resultof their large surface area and delocalized π electrons, and thushave the potential to act as drug delivery vehicles if sufficientlyhigh drug loading and suitable in vivo drug distribution andrelease profiles can be achieved. In one of the earliest studies onthe potential biomedical uses of graphene, Yang et al40 showedthat intravenous administration of PEG-modified GO labeledwith a near-infrared fluorescence dye, but not carrying any drug,displayed significant passive tumor targeting in several mousexenograft models and relatively low retention in the reticuloen-dothelial system. The authors utilized the strong opticalabsorbance of the nanomaterial in the near-infrared region forin vivo photothermal therapy, achieving efficient tumor ablation.Moreover, a reduced GO-iron oxide nanoparticle complexfunctionalized with PEG was found to display excellentphysiological stability, strong near-infrared optical absorbance,and superparamagnetic properties.41 Using this novel theranosticprobe, in vivo tri-modal fluorescence, photoacoustic, andmagnetic resonance imaging was carried out, uncovering highpassive tumor targeting, and this was further used for photo-thermal ablation of tumors in mice.41 Furthermore, loading ofdoxorubicin onto the PEG-modified GO-iron oxide nanoparticlecomplex enabled magnetically targeted drug delivery.42 In thelatter study, magnetic resonance imaging of breast tumor-bearingmice was also demonstrated using GO–iron oxide NP–PEG ascontrast agent. In a recent study, doxorubicin was chemicallyconjugated to polymer (i.e., PEI-PEG) grafted GO via a matrixmetalloproteinase 2 (MMP2)-cleavable peptide linker.43 MMPsare a family of enzymes predominantly secreted by tumor cells.Under normal conditions the intrinsic fluorescence property ofdoxorubicin is quenched by GO; upon incubation with MMP2,the peptide is cleaved thereby permitting the unloading ofdoxorubicin for tumor cell killing and concurrent fluorescencerecovery of doxorubicin for tumor cell imaging,43 making this aversatile system, if not ‘theranostic’ in the conventional sense.Further studies are warranted to evaluate this approach usingrelevant in vivo tumor models, and to ascertain whether theadsorption of biomolecules leading to a bio-corona (discussedbelow) would obscure the peptide linker.

Fullerenes, especially C60, have received widespread atten-tion as drug and gene delivery vehicles.44 In one pertinentexample, gene delivery in vivo using water-soluble fullereneswas demonstrated.45 The in vivo biodistribution of the full-erene-DNA complexes and a lipid-based system (Lipofectin)showed similar patterns; however, levels of reporter geneexpression varied insofar as the fullerene-based system achievedup to 10-fold higher gene expression than Lipofectin in the liverand spleen, and no gene expression in the lung. The differencesin organ selectivity of the fullerene-based system could beexploited for diseases of the liver and spleen.45 Furthermore, asproof-of-principle, the authors demonstrated that the delivery ofan insulin gene using fullerenes increased plasma insulin levelsand reduced blood glucose concentrations in mice.

The metallofullerenol nanoparticles are fullerene derivativesconsisting of a metal atom inside a fullerene cage and arecurrently investigated for their unique mechanical, thermal andelectrochemical properties. In particular, gadolinium (Gd) basedmetallofullerenes are developed as innovative contrast agents,and may also act as anti-cancer agents.46 For example, themulti-hydroxylated metallofullerenol Gd@C82(OH)22 was re-cently shown to inhibit tumor metastasis through MMPinhibition rather than through direct killing of the cancercells,47 thus suggesting a new, nanomedicine-based approachin the management of tumor metastasis.48 In subsequent studies,based on computational and experimental approaches, theauthors proposed that Gd@C82(OH)22 suppress pancreaticcancer metastasis by inhibiting the interaction of histonedeacetylase 1 (HDAC1) and metastasis-associated protein 1(MTA1), thus acting as a novel HDAC inhibitor.49 Thesefullerene derivatives were also shown to possess intrinsicinhibitory activity against breast cancer cells blocking epithe-lial-to-mesenchymal transition with efficient elimination ofso-called breast cancer stem cells resulting in abrogation oftumor initiation and metastasis.50 Taken together, these studiesthus exemplify the use of nanoparticles as drugs per se.51

Chemoresistance is the main cause of treatment failure inadvanced, metastatic cancer. Drug efflux from tumor cells bydrug transporter proteins including multi-drug resistance protein1 (MDR1), also known as P-glycoprotein, is the most commonmechanism of chemoresistance.52 Doxorubicin is a standardtreatment for many cancers; however, its clinical use is limitedby its known dose-dependent toxicity (cardiotoxicity andmyelosuppression, i.e., decreased bone marrow activity), theemergence of so-called multi-drug resistance – which isexplained by drug efflux by transporter proteins – and its lowspecificity against cancer cells.53 Nano-based delivery systems,with or without targeting ligands, could potentially overcomethese limitations, by reducing the side-effects and increasing thetherapeutic effectiveness of the drug.54 Interestingly, novelapproaches to circumvent chemoresistance using nanodiamondswere recently reported. Chow et al55 showed that a complex ofnanodiamonds and doxorubicin (NDX) overcame drug effluxand significantly increased tumor growth inhibition in micebearing chemoresistant tumors. The authors found that nanodia-mond conjugation resulted in sustained drug release. To measuredrug retention in cells, the authors used cells overexpressing thedrug transporter MDR1, and found that treatment with NDX


resulted in a 10-fold increase in retained doxorubicin whencompared to the free drug.55 Moreover, NDX displayed lesstoxicity in mice (no myelosuppression, with no mortality at thehighest doses) when compared to standard treatment with freedoxorubicin.55 In a subsequent study, nanodiamonds were usedto deliver the related chemotherapeutic drug, epirubicin to cancercells. Epirubicin is favored over doxorubicin for its lowercardiotoxicity, but can also be effluxed by cancer cells via drugtransporters. Wang et al56 reported that nanodiamond-epirubicincomplexes displayed higher efficacy compared to unmodifiedstandard treatment in killing both normal cancer cells and cancerstem cells in vitro and in vivo, in a model of hepatic cancerenriched for chemoresistant cancer stem cells. The authors alsodocumented that the association of epirubicin to nanodiamondsprevented efflux of the drug by drug transporters.56 Notably, thiswas a function specific to nanodiamond-mediated drug deliveryas epirubicin delivery by liposomes failed to enhance drugretention. Together, these studies suggest novel approaches forovercoming chemoresistance using nanodiamonds. It will be ofinterest to learn whether nanodiamonds are susceptible todegradation, as shown for other carbon-based nanomaterials(below).

A second major area in nanomedicine is imaging anddiagnostics and carbon-based nanomaterials have receivedmuch attention also in this regard. Moreover, as already alludedto previously, therapeutic and diagnostic modalities can becombined in multi-functional theranostic devices.4 Here, wewill touch briefly on this topic [for a more comprehensivediscussion, refer to Chen et al4 and Yoo et al57].

CNTs have been studied intensively for multiple imagingmodalities including fluorescence imaging, photoacoustic andRaman imaging, and so on; some examples are provided here.De La Zerda et al58 demonstrated that SWCNTs conjugated withcyclic Arg-Gly-Asp (RGD) peptides can be used as a contrastagent for photoacoustic imaging of malignant glioma tumors inmice. Intravenous administration of these targeted nanotubes tomice bearing tumors showed eight times greater photoacousticsignal in the tumor than mice injected with non-targetednanotubes. Ghosh et al59 reported on the use of SWCNTs tovisualize deep, disseminated tumors in vivo which couldfacilitate surgical excision of model ovarian cancers withsubmillimeter precision. Delogu et al.60 provided evidence forthe use of MWCNTs as ultrasound contrast agents, in a largeanimal model (pig). The authors could demonstrate that theultrasound signal of functionalized MWCNTs was higher thanGO, pristine MWCNTs, and functionalized SWCNTs. Similarly,graphene and its derivatives are also investigated as optical ornon-optical imaging agents.57 For instance, as already men-tioned previously, novel, PEG-functionalized GO-iron oxidenanoparticle hybrid materials were recently developed for in vivotri-modal fluorescence, photoacoustic, and magnetic resonanceimaging.41 In another related example, Shi et al61 reported on theapplication of multi-functional sensors based on GO decoratedwith both iron oxide and gold nanoparticles and functionalizedwith PEG molecules. Additionally, graphene quantum dots, anemerging fluorescent material, were shown to act as photody-namic therapy agents, with a quantum yield that is higher than forany other known PDT agent.62

Fullerenes such as C60 have been functionalized using metalsfor use as contrast agents and radiotracers. Indeed, metalloful-lerenes have been explored as contrast agent for MRI for morethan a decade.46 Moreover, Shi et al63 recently developed ahybrid nanoplatform with multi-functional properties for com-bined cancer diagnosis, photodynamic therapy, radiofrequencythermal therapy, and magnetic targeting. Hence, the authorsproduced a C60-iron oxide nanoparticle composite functionalizedby PEG and decorated with folic acid, a widely used tumortargeting molecule, and were able to achieve synergistic,multi-modal ablation of tumors in sarcoma-bearing mice.63

More information on the biodistribution and long-term toxicity isneeded, but the approach aptly demonstrates the theranosticpotential of carbon-based nanomaterials. Nanodiamondspresenting nitrogen-vacancy centers have intrinsic fluorescenceproperties and nanodiamonds, as well as the metal hybridnanodiamonds, therefore present themselves as interesting toolsfor imaging and diagnostics [see Mochalin et al15 for a review].For instance, Fu et al64 reported on the use of fluorescentnanodiamonds as single-particle biomarkers for in vitro studies.

Biosensors are important tools in biomedical research and arebecoming an essential part of modern healthcare.65 By takingadvantage of their unique electrical and optical properties, CNTscan be integrated into highly sensitive sensors and probes.66 Forinstance, Iverson et al67 showed that single-stranded DNAoligonucleotide-functionalized SWCNTs can be used for theselective detection of local nitric oxide (NO) concentrationsin vivo in mice following intravenous injection. NO is animportant signaling molecule involved in many physiologicaland pathological processes. The authors also found that theSWCNTs can function as implantable inflammation sensors forNO detection, with no intrinsic immune reactivity or otheradverse responses. Due to the absence of photobleaching, theSWCNT-based sensors are highly stable (no negligible change ofactivity was noted after 400 days).67 In a recent study,biocompatible GO biosensors for detecting blood glucose levelsover a broad concentration range were developed by covalentlyattaching the amine groups of glucose oxidase to the carboxyl acidgroups of GO.68 Furthermore, Jiang et al69 reported a novelapproach for electrical sensing of NO using hemin-functionalizedgraphene. The graphene-hemin sensors could respond rapidly to NOin physiological environments with sub-nanomolar sensitivity.Additionally, in vitro studies showed that the sensors could beused for the detection of NO released from macrophages andendothelial cells.69

Finally, carbon-based nanomaterials are emerging as poten-tial candidates for the development of synthetic scaffolds intissue engineering [see Ku et al70 for a comprehensive review].CNTs offer several characteristics similar to those of theextracellular matrix, the environment in which cells physiolog-ically migrate and proliferate to form tissues and organs. Cellotet al71 provided theoretical and experimental evidence that CNTsmight improve neuronal performance by favoring electrical‘shortcuts’ between the soma or cell body of neurons and thedendrites. Bosi et al72 recently reported a biocompatible,synthetic polymer based-scaffold that allowed the developmentof 3-dimensional hippocampal cultures. Furthermore, the authorsendowed the scaffold with nano-topographies by incorporating


MWCNTs which enabled the nanotubes to interface and boostcultured neuronal circuits.72 In addition, nanodiamonds havebeen reported to act as a platform for neuronal growth73 whilehybrid structures of GO and silica nanoparticles promotedgrowth and alignment of human neural stem cells.74 Graphene isalso envisioned for artificial retinas, i.e., prosthetic devices thatinterface with the optical nerve; see the Science and Technologyroadmap of the Graphene Flagship Project.14 However, for suchapplications to be realized – indeed, for any biomedicalapplications of nanomaterials – a detailed understanding of thebiological interactions of the nanomaterial, including bio-coronaformation, is needed, and the propensity for degradation and/orclearance in vivo should also be evaluated.

Bio-corona formation on carbon-based nanomaterials

In biological environments, nanomaterials are rapidly coatedwith proteins, lipids and other biomolecules.75 This so-calledbio-corona formation confers a new biological ‘identity’ to thenanomaterial, and this is of key importance for the subsequentbiological (and toxicological) interactions of nanomaterials inliving systems.8,76 Moreover, it is important to consider the‘shifting identities’ of a nanomaterial as it translocates from onebiological compartment to another (for instance, from the lungsor the gastrointestinal tract to the systemic circulation) and fromthe extracellular environment to intracellular locations (cyto-plasm, lysosomes, etc).77 The bio-corona could also exhibitdynamic changes when passing through these different environ-ments, for instance as a result of enzymatic processing of thecorona constituents.75 For targeted nanomedicines, it isimportant to consider whether the acquired bio-corona could‘mask’ the ligands and thereby prevent targeting to the desiredlocation, for instance, to a tumor.78 On the other hand, it alsoremains possible that the bio-corona could display functionalepitopes that may engage specific cellular receptors79; indeed,nanomaterials could undergo ‘functionalization’ in vivo and animportant challenge is thus to decipher and to control thisphenomenon.80 In the following sections, we discuss bio-coronaformation in relation to the biological behavior of nanomaterials,and more specifically in relation to targeting of nano-carriers.

There are several experimental and theoretical studies onbio-corona formation on CNTs, and also some recent studies onGO. Dutta et al81 identified albumin as the major fetal bovine orhuman serum/plasma protein adsorbed onto SWCNTs and notedthat the bio-corona plays an important role in modulating cellularuptake of SWCNTs in murine RAW264.7 macrophage-likecells, presumably through interactions with scavenger receptors.Ge et al82 employed experimental and theoretical approaches tostudy the interaction of four major serum proteins – bovinefibrinogen (BFG), immunoglobulin, transferrin, and bovineserum albumin (BSA) – with SWCNTs and found that serumprotein-coated SWCNTs caused less cytotoxicity than uncoatedSWCNTs in the human leukemia cell line (THP-1) and humanumbilical vein endothelial cells (HUVECs), with BFG showingthe most pronounced effect. Notably, BFG was found torearrange themselves on the SWCNT surface in the mostcompact manner and the most layers (five layers as compared totwo or three layers for other proteins), which may potentially

explain why this protein was more effective at protecting cellsfrom the exposure of SWCNTs.82 Using an 80-membercombinatorial MWCNT library, Gao et al83 found that surfacechemistry modification reduced the immune perturbations ofMWCNTs both in vitro and in vivo. Furthermore, these authorsdemonstrated that the modified MWCNTs changed theirpreferred binding pattern from mannose receptor to scavengerreceptor, in the THP-1 macrophage model.83 While the role ofthe bio-corona was not investigated in the latter study, it is morethan likely that the surface modifications altered the binding ofserum proteins both in vivo and in cell culture which in turnmediated the ‘switch’ from mannose receptor to scavengerreceptor-mediated uptake. We recently noted that serum proteinsare accountable for the Toll-like receptor (TLR)-dependentsignaling of SWCNTs in primary monocyte-derived macro-phages, while GO did not display such effects (manuscript inpreparation). Taken together, macrophage recognition of CNTsseems to depend critically on the bio-corona and different CNTsurface properties may impart critical changes in the compositionof the bio-corona and hence affect the biological outcomes.

GO has an extremely high protein adsorption capacity. Hu etal84 noted that the cytotoxicity of GO toward human A549 lungcarcinoma cells was greatly mitigated in presence of 10% fetalbovine serum, the concentration usually employed in cell culturemedium. The authors noted that GO had a much higher capacityfor protein loading when compared to both SWCNTs andMWCNTs. Similarly, Chong et al85 also found that adsorption ofserum proteins onto GO drastically reduced their cytotoxicitytoward A549 lung carcinoma cells and found that GO exhibits adramatic enhancement of adsorption capacity compared toSWCNTs. In a subsequent study, coating of GO with BSAwas suggested to reduce cytotoxicity toward A549 cells byreducing the physical interaction of GO with the cellmembrane.86 It is noted, however, that A549 is a notoriouslyrobust carcinoma cell line not reflective of normal cellphysiology. It will therefore be of interest to perform similarstudies using professional phagocytic cells (macrophages) orother primary immune-competent cells. Using molecular dy-namics (MD) simulations, aromatic residues were found tocontribute significantly to the protein adsorption due to thestrong π-π stacking interactions between their aromatic rings andthe graphene sp2-carbons.87 In addition, basic residues likearginine played an equally or even stronger role during thisprocess. Furthermore, in another MD study, the dependence onsurface curvature was investigated for adsorption of BSA ontoCNTs of increasing radius versus a flat graphene sheet, and theresults confirmed that protein adsorption capacity is indeedenhanced on flatter surfaces.88

Most studies to date on the bio-corona have been conductedusing human plasma or bovine serum as a source ofbiomolecules reflective of the conditions in the blood or in cellculture, respectively.75 However, following the introduction ofnanomaterials into other compartments, such as the lung or thegastrointestinal tract, nanomaterials may encounter a differentenvironment leading to the formation of a distinct bio-corona. Inthe first study on the potential in vivo formation of a bio-coronain the lungs, Kapralov et al89 found that pharyngeal aspiration ofSWCNTs in mice resulted in adsorption of lung surfactant


proteins and surfactant lipids and, furthermore, that thisprotein-lipid bio-corona facilitated uptake of SWCNTs bymurine RAW264.7 macrophage-like cells. In a related in vitrostudy using amino- and carboxyl-modified MWCNTs, Gasseret al90 found that surfactant lipids (derived from Curosurf) bindunspecifically to the different functionalized MWCNTs, incontrast to plasma proteins which showed characteristic bindingpatterns. They also noted that the pattern of plasma proteinbinding was altered when MWCNTs had been previously coatedwith pulmonary surfactant. This could be interpreted to suggestthat nanomaterials retain a ‘memory’ of previous biologicalenvironments or compartments in vivo, for instance, upontranslocation across the lung–blood barrier.

As we have discussed, the adsorption of proteins and otherbiomolecules onto the surface of nanomaterials can influence the‘identity’ and biological behavior of the nanomaterials. Con-versely, the interactions between biomolecules and nanomater-ials can also lead to altered conformational and orientationalchanges of the biomolecules, potentially revealing crypticepitopes that could trigger immune responses via specific cellsurface receptors.79 Indeed, one may view the altered proteins onthe surface of nanomaterials as ‘nanomaterial-associated molec-ular patterns’ or NAMPs analogous to the pathogen-associatedmolecular patterns (PAMPs) displayed by microbes.9 Moreover,protein adsorption by nanomaterials, not least by GO, whichpresents a vast surface for protein binding, can lead to inhibitionof enzyme activity. Hence, recent studies have shown thatcarbon-based nanomaterials can inhibit the bacterial enzyme,VIM-2 belonging to the clinically relevant class of metallo-β-lactamases that provide resistance to a broad spectrum ofantibiotics including penicillin; the inhibition was noncompet-itive and was attributed to hydrophobic interactions with theenzyme.91 Moreover, adsorption of VIM-2 was further probedusing protein displacement assays and it could not displace or bedisplaced by BSA. We recently found that both SWCNTs andGO inhibit CYP3A4, a major drug-metabolizing enzyme andthat this was mitigated when the nanomaterials were pre-coatedwith BSA (submitted for publication). In addition, previousstudies have shown that GO is an inhibitor of α-chymotrypsin92

and β-galactosidase93 while, on the other hand, PEGylated GOcan apparently boost the activity of trypsin, but has no effect onchymotrypsin or proteinase K, which are also serine proteases.94

Shurin et al33 reported that GO can trigger alveolar macrophageproduction of chitinases, enzymes whose expression is associ-ated with asthma, in mice and theoretical and experimental datasuggested that GO could directly interact with and inhibitchitinase activity. Whether inhibition of chitinases also occurs ina complex biological environment, in the presence of lungsurfactant or other biomolecules, remains to be understood.

The complement system is a part of the innate immune systemthat helps or complements other humoral (antibodies) or cellular(phagocytes) components of the immune system to clearpathogens. Importantly, carbon-based nanomaterials, not leastCNTs, have been shown to bind complement factors and thisphenomenon thus represents a special case of bio-coronaformation which is of considerable relevance as complement-mediated toxicity is a major limiting factor for nanomedicineapplications following intravenous administration of the nano-

carrier [reviewed in Moghimi95]. There are three establishedpathways of complement activation: the so-called classical,lectin and alternative pathways. The majority of complementactivation studies with nanomaterials – including CNTs and GOwith or without polyethylene glycol (PEG) modification on thesurface – have focused mostly on the classical and alternativepathways.96-98 However, as recently pointed out by Moghimiet al,99 there is now evidence to suggest that many nanoparticlesmay trigger complement activation through the lectin pathway,which involves carbohydrate recognition, even though thesenanoparticles do not per se express surface-exposed sugars.Instead, according to Moghimi et al99, and see referencestherein], functionalized nanoparticles may ‘mimic’ pathogens byvirtue of the projected polymeric surface architecture thatresembles structural motifs of peptidoglycan constituents ofpathogens which then triggers the lectin pathway.

Nanomaterials intended for use as drug delivery vehicles arecommonly functionalized using long hydrophilic polymers suchas poly(acrylic acid), chitosan or PEG, as this increases thebiocompatibility of these systems and is thought to reducenon-specific protein adsorption and clearance by phagocyticcells of the reticuloendothelial system (RES), thereby promotingpassive targeting to the desired location, such as a tumor.100

However, as we shall discuss in more detail below, PEGfunctionalization does not completely prevent protein adsorp-tion. Moreover, to effectively counter the non-specific uptake byphagocytic cells, PEG molecules typically need to have amolecular weight in excess of 2 kDa, which adds considerably tothe overall hydrodynamic diameter of the nanoparticles. In recentyears, zwitterionic coatings have been explored as an alternativestrategy to endow nanoparticles with “stealth” properties [seePombo García et al101 for an excellent review]. Because suchcoatings can be constructed from low-molecular weightmaterials they provide an opportunity to develop ultra-small,excretable nanoparticles for biomedical applications.101 Choiet al102 demonstrated renal filtration and urinary excretion ofinorganic, metal-containing nanoparticles with zwitterionic orneutral organic coatings. Notably, zwitterionic coating using theamino acid cysteine prevented protein adsorption while yieldingthe highest solubility and the smallest hydrodynamic diameter.In comparison, although neutral, PEGylated nanoparticles didnot bind serum protein, it was not possible to synthesize suchparticles with a hydrodynamic diameter b10 nm; shorter PEGchains resulted in insoluble particles.102

Active targeting of nanoparticles is also frequently deployed.For instance, folic acid (FA) or transferrin, recognized by thefolate receptor and transferrin receptor, respectively, arecommonly used in an attempt to increase cellular uptake ofdrug-loaded carriers in cancer cells overexpressing thesereceptors.103 However, if it is true that all nanoparticles arerapidly coated with biomolecules in a living organism, then it isalso possible that the additional layer(s) of proteins (and otherbiomolecules) could obscure the targeting ligands that have beengrafted onto the surface of the nanoparticles. Indeed, in a recentin vitro study using transferrin-conjugated nanoparticles, Salvatiet al78 found that proteins in the cell culture medium can shieldtransferrin from binding to its cognate receptors on cells. Thus,although nanoparticles continued to enter the cells, the targeting


specificity of transferrin was lost. In contrast, we previouslyobserved that specific, i.e., FA-dependent uptake of FA-conjugatediron oxide nanoparticles by human ovarian cancer cells expressingthe corresponding receptor was observed only in the presence ofserum proteins, possibly due to a stabilizing effect of the serumproteins on the functionalized nanoparticles in vitro.104 However,while Fe3O4-SiO2-FA particles were specifically internalized,Fe3O4-PEG-FA nanoparticles did not undergo specific (targeted)uptake in the samemodel cell line; it is conceivable that the targetingligand (FA) on the PEGylated particles was embedded in amatrix ofpolymers and therefore not accessible for binding to its receptor.104

Indeed, Dai et al105 reported that backfilling the surface of a targetednanoparticle with PEG molecules reduces protein corona formationand noted that the length of the PEGmolecules must be less than thelength of the ligand linker; otherwise, PEG interferes with thebinding of the targeting ligand to its cellular receptor. More recently,it was demonstrated that the formation of a protein corona doesnot significantly influence the targeting ability of antibody-functionalized polymeric particles toward human colon cancercells.106 In another recent study. Hadjidemetriou et al107

reported on the formation of a bio-corona on clinically relevant,antibody-functionalized nanoparticles (liposomes) in mice. Theauthors found that both in vitro and in vivo formed proteincoronas significantly reduced cellular internalization of theantibody-conjugated liposomes, using human cervix or breastcancer cell lines (however, in vivo targeting was not evaluated);notably, the in vivo corona formation did not completely preventthe targeting capability.107 Thus, it appears that the bio-coronamayimpact on targetability of nanomedicines, but it is unlikely to be thesole critical factor determining their behavior.

Turning now to targeting of carbon-based nanomaterials, wepreviously reported on in vivo targeting of intratumoralregulatory T cells (Treg) using PEG-modified SWCNTs.108

We focused our attention on the glucocorticoid-inducedTNFR-related receptor (GITR), as it showed higher expressionon intratumoral versus peripheral (i.e., splenic) Treg compared toother reported Treg-specific markers. Our in vivo investigationsshowed that PEG-SWCNTs armed with GITR ligands targetedTreg residing in a melanoma xenograft more efficiently thenintratumoral non-Treg or splenic Treg.108 The latter result waslikely accomplished through a combination of passive tumortargeting (i.e., enhanced permeability and retention effect, EPR)due to enhanced tumor vascular permeability and active targetingof markers enriched in intratumoral Treg. This example ofintratumoral immune cell targeting thus points toward novel,nano-based immunotherapies against cancer. Further examplesof targeted SWCNTs are discussed in the following section.Hong et al109 demonstrated that GO can be specifically directedto the tumor neovasculature in vivo through targeting of CD105,a vascular marker for tumor angiogenesis (Figure 2). Notably,incorporation of an active targeting ligand (TRC105, amonoclonal antibody that binds to CD105) led to significantlyimproved tumor uptake of functionalized GO, which wasspecific for the neovasculature.109 The administration of ablocking dose of TRC105 before injection of the nano-graphenesignificantly reduced the tumor uptake which demonstratedCD105 specificity. Hence, although one might assume that themasking of targeting ligands would be of particular concern for

graphene-based materials, as the potential for protein adsorptionis considerable, this study suggests that relevant targeting can infact be achieved in vivo.

Finally, it is pertinent to note that the molecular compositionof biological fluids in patients suffering from cancer or otherdiseases is unlikely to resemble the normal situation. Indeed,recent studies on cellular uptake of GO have suggested thatattention should be focused on the ‘personalized bio-corona’resulting from differences in protein content in human plasmafrom various types of disease.110 This is a potentially importantchallenge for the nanomedicine community.

Biodistribution of carbon-based nanomaterials

Understanding the fate and behavior of carbon-basednanomaterials in vivo is imperative for the clinical translationof these materials. Pharmacokinetic (PK) profiling addresses theadsorption, distribution, metabolism, and excretion (ADME) of adrug or nanomaterial in vivo [see Moghimi et al111 for a review].Metabolism (or, degradation) of carbon-based nanomaterials isdiscussed in a subsequent section. Here, we focus on otheraspects of in vivo biodistribution of nanomaterials.

Drug molecules diffuse and distribute freely throughout thebody, causing unpredictable or undesirable effects in bystandertissues while also limiting the achievement of doses needed for atherapeutic response. One of the great promises of nanomedicineis the local or targeted delivery of drugs. Efficient targetingwould allow for a reduced systemic dosage meaning also areduced toxicity while resulting in relatively higher or moreefficient dosage at the desired target site.112 In an excellent andvery recent review, Ferrari and co-workers highlighted thebiological barriers that drug-loaded nanoparticles encounterupon intravenous administration.113 These barriers include, forinstance, opsonization and subsequent sequestration by the RES,as discussed at length in the present review, as well ashemorheological/blood vessel flow limitations, and they preventefficacious, site-specific delivery to tumors, as well as in otherclinical conditions. Tasciotti et al114 developed a multi-stagedelivery system designed specifically to circumvent severalbiological barriers after intravascular delivery. In this paradigm,stage-1 mesoporous particles were loaded with stage-2 nanopar-ticles, i.e., quantum dots (QDs) or SWCNTs, which in turn couldcarry active agents or higher-stage particles. The authorsreasoned that by loading the stage-2 nanoparticles inside thepores of the stage-1 particles, RES uptake would be prevented. Inthis manner, the mesoporous particles would transport andprotect a payload of nanoparticles and bioactive agentsthroughout their journey in the circulatory system.114 To escapecirculation, as in the case of drug delivery to a solid tumor, thesize of the nanoparticles is obviously critical, but it is alsoimportant to note that the EPR phenomenon may varydramatically with regard to the degree of tumor vascularity.115

Moreover, while the presence of a targeting ligand (see previoussection for a discussion on active targeting) does not seem tosignificantly affect extravasation of nanoparticles, inefficientextravasation could significantly affect targeted delivery.112 Thismeans that both passive and active targeting mechanisms arelikely to play a role.

Figure 2. Targeting of tumor vasculature with graphene oxide. In vivo PET/CT imaging of 64Cu-labeled GO conjugates in breast tumor-bearing mice. Left panelshows serial coronal PET images of tumor-bearing mice at different time points post-injection of 64Cu-NOTA-GO-TRC105, 64Cu-NOTA-GO, or64Cu-NOTA-GO-TRC105 after a pre-injected blocking dose of TRC105. Tumors are indicated by arrowheads. Right panel displays representative PET/CTimages of 64Cu-NOTA-GO-TRC105 in tumor-bearing mice. Reprinted from: Hong H, Yang K, Zhang Y, Engle JW, Feng L, Yang Y, Nayak TR, Goel S, BeanJ, Theuer CP, Barnhart TE, Liu Z, Cai W. In vivo targeting and imaging of tumor vasculature with radiolabeled, antibody-conjugated nanographene. ACS Nano.2012;6(3):2361-70, with permission from American Chemical Society.


In order to study the biodistribution of carbon-basednanomaterials, appropriately labeled nanomaterials are needed,or one may capitalize on their intrinsic physicochemicalproperties.116 In an early effort to monitor the fate of CNTs,Singh et al117 examined the PK behavior of water-soluble,SWCNTs functionalized with the chelating molecule DTPA andlabeled with 111In for imaging purposes. The authors noted thatthe CNTs were not retained in the liver or spleen uponintravenous administration in mice, and that the functionalizedCNTs were rapidly cleared from systemic blood circulationthrough the renal excretion route with a blood circulationhalf-life of 3.5 h. This ‘paradoxical’ glomerular filtration ofSWCNTs was also reported by others.118 Subsequent studies onthe retention of functionalized MWCNTs in the organs of miceshowed that the degree of chemical functionalization determinestissue distribution and excretion profile; hence, increasing thedegree of functionalization enhanced renal clearance, whilelower functionalization promoted RES accumulation (i.e., liverand spleen).119 Additionally, using similarly radiolabeledMWCNTs, the authors could show that the diameter of thefunctionalized MWCNTs also affects their organ distributionin vivo in mice.120 Using, 125I-labeled nanographene sheets (i.e.,GO) functionalized with PEG, Yang et al121 demonstrated thatthe nanomaterial mainly accumulated in the liver and spleen afterintravenous administration; substantial bone uptake was alsonoted at early time points, possibly owing to macrophage uptakein the bone marrow. However, the PEGylated GO was graduallycleared (and/or degraded), without appreciable toxicity up to3 months post-exposure.121 In a very recent study, Jasim et al122

studied the tissue distribution of radiolabeled and chemicallyfunctionalized GO and found that the injected materialaccumulated predominantly in the liver and spleen whileevidence for renal excretion was also provided. As discussedby the authors, the biological fate of graphene-based materials is

likely to depend both on lateral dimension and thickness (i.e.,layer number) as well as on the degree of functionalization,which may play an important role for subsequent biologicalinteractions in vivo including bio-corona formation.123

Cherukuri et al124 investigated the distribution of chemicallypristine, non-labeled SWCNTs upon intravenous administration.The authors made use of the intrinsic near-infrared fluorescence,a property of individualized or debundled SWCNTs, to measurethe blood elimination kinetics and to identify the target organs inrabbits exposed to the nanomaterials. First, as CNTs arehydrophobic and tend to form aggregates, the SWCNTs wereultrasonically dispersed in artificial surfactant, Pluronic F108.The results showed that the SWCNT concentration in the blooddecreased exponentially with a half-life of 1 h.124 Twenty-fourhours after administration, significant concentrations ofSWCNTs were found only in the liver. Notably, in separatein vitro experiments, the authors determined that the surfactantwas displaced within seconds by serum proteins suggesting thatthe PK results obtained are reflective of the fate of SWCNTs witha bio-corona of endogenous (serum) proteins rather than asynthetic surfactant.124 Nonetheless, the retention of the near-IRfluorescence implied that the SWCNTs remained disaggregatedin vivo. In another study using pristine, 13C-labeled SWCNTs,major accumulations were seen in liver, spleen, and lungfollowing intravenous injection.125 Thus, it is clear that thebiodistribution of pristine versus functionalized CNTs differsgreatly, with the former being predominantly trapped in the RESorgans, while the latter favor a renal excretion route.116

In a recent study, a novel approach was developed to monitorthe distribution of carbon-based nanomaterials at the organ andsub-organ level. Chen et al126 thus reported on label-free massspectrometry imaging to detect MWCNTs, single-layer GO, andcarbon nanodots (CDs) in mice based on their intrinsic carboncluster fingerprint signal. With this approach, it was observed


that MWCNTs and CDs were predominantly distributed in thekidneys, whereas all three nanomaterials were detected in the redpulp of the spleen, following intravenous administration.Evidence for clearance of the CNTs and CDs via the renalexcretion route was also provided, in line with previousstudies.117,127 The highest concentration of MWCNTs wasfound in the marginal zone of the spleen (the interface betweenthe non-lymphoid red pulp and the lymphoid white pulp), whereparticulate antigens from the circulation are trapped andpresented to the lymphocytes in the spleen126 (Figure 3). Thislevel of detail is difficult to achieve by other means. Overall, thisnew mass spectrometry method has the potential to be used as ageneral approach for the detection of carbon-based nanomater-ials in tissue samples. As pointed out,128 the method does notindicate whether the nanomaterials have been transformedin vivo. Nevertheless, other methods, such as Raman confocalimaging, could provide such information.129

In nanomedicine, it is common to modify the surface of thenanomaterial with polymers such as PEG in order to avoid rapidclearance by the immune system. This has been shown toincrease the circulation half-life of the nanomaterial. Liu et al130

reported on the biodistribution of radiolabeled SWCNTs in miceand determined the effect of PEG chain length on thebiodistribution and circulation of the SWCNTs. They notedthat effectively PEGylated SWCNTs exhibited relatively longblood circulation times and low uptake by RES organs.Moreover, efficient targeting of tumors in mice was achievedwith SWCNTs coated with PEG chains linked to an arginine-glycine-aspartic acid (RGD) peptide.130 Notably, injection of a

Figure 3. Sub-organ biodistribution of carbon nanotubes. Laser desorption/iontechnique that can map chemical compounds in biological samples. From left to riof MWCNTs; heat map showing the ion intensity distribution (m/z 72.0) of MWMWCNTs in the red pulp (red arrow), white pulp (white arrow) and marginal zonmass spectra of red and white pulp regions are depicted to the far right. ReprintedMass spectrometry imaging reveals the sub-organ distribution of carbon nanomPublishing Group.

blocking dose of RGD into mice bearing αvβ3-positive tumorssignificantly reduced the uptake of SWCNT-PEG-RGD in thetumor. However, while PEGylation has been shown to reduceprotein adsorption, this surface modification does not entirelyprevent bio-corona formation. The question, therefore, iswhether and to what extent the bio-corona influences thebiodistribution of PEGylated nanomaterials. In a recent study,we investigated the protein corona adsorbed onto SWCNTsmodified with 2 kDa PEG chains by using large-scale gel-basedproteomics.131 We identified more than 500 proteins in thebio-corona; a subset of these plasma proteins were selected andgrouped according to their physiological function. Coagulationproteins, immunoglobulins, apolipoproteins, and complementfactors were among the proteins bound by the PEGylatedSWCNTs.131 Interestingly, PEG conformation had a strongerinfluence on the protein corona repertoire than nanotube surfacecharge. Moreover, the bio-corona affected the biodistribution ofthe SWCNTs in mice. Hence, a change in PEG conformationfrom mushroom to mushroom-brush transition affected thecompetitive adsorption of the major constituents of the proteincorona and promoted shorter blood circulation time, faster renalexcretion, and higher relative spleen versus liver uptake ofPEG-SWCNTs.131 Our data thus suggest that the bio-corona,along with steric stabilization, may mediate the action of PEGconformation on the PK profile of PEG-modified SWCNTs.

As discussed above, the PEG chains have to be of highmolecular weight (N2 kDa) in order to avoid RES clearance.101

Zwitterions on the other hand provide a highly stable coating on thesurface of nanomaterials with little change in the hydrodynamic

ization (LDI) mass spectrometry imaging (MSI) is an emerging label-freeght: Optical image of a spleen tissue slice from mice following administrationCNTs in a spleen tissue slice; magnified view showing the distribution ofe (purple arrow) of the spleen. Scale bars, 2 mm. Finally, representative LDIfrom: Chen S, Xiong C, Liu H, Wan Q, Hou J, He Q, Badu-Tawiah A, Nie Z.aterials. Nat Nanotechnol. 2015;10(2):176-82, with permission from Nature


diameter. In order to both circumvent non-specific proteinadsorption and promote cellular uptake by tumor cells, Yuanet al132 synthesized surface charge switchable nanoparticles basedon zwitterionic polymers for drug delivery. The authors noted that,in physiological conditions the nanoparticles showed prolongedcirculation time as a result of the reduced protein absorptionafforded by the zwitterionic polymer. After accumulating in therelatively acidic tumor tissue, the zwitterionic polymer-basednanoparticle switched its surface to a positive charge, whichfacilitated tumor cell uptake and delivery of the anti-cancer drug,doxorubicin. Thus, zwitterionic coatings present an alternative toPEG and offer opportunities for the design of “smart” nanoparticlesfor biomedical applications.101

Finally, in an intriguing twist on tumor targeting, Smithet al133 recently reported that specific immune cell populations inthe blood may act as Trojan horses to deliver CNTs to tumors. Ingeneral, as pointed out by the authors, tumor targeting ofnanoparticles may transpire both via passive and activemechanisms, including extravasation from the blood streaminto the tumor (i.e., the EPR effect) and ligand-mediatedtargeting to tumor cells or to the tumor vasculature, examplesof which were provided in previous sections of the presentreview. Smith et al133 hypothesized that circulating cells in theblood take up nanoparticles and deposit them in the tumor, thusserving to complement the other mechanisms. Indeed, usingsevere combined immunodeficient (SCID) mice lacking func-tional B and T cells, the authors discovered that PEG-modifiedSWCNTs are selectively taken up by a single subset ofcirculating immune cells, the Ly-6Chi monocytes.133 Themechanism for this cellular uptake was not disclosed, but it isnoted that several known opsonins (i.e., phagocytosis-promotingfactors) are found in the bio-corona formed on PEGylatedSWCNTs.131 Notably, these monocytes are known to differen-tiate into so-called tumor-associated macrophages (TAMs) andare attracted to hypoxic regions of the tumor, which may be ofparticular relevance in cancer treatment.134 The authors foundthat the uptake of SWCNTs in circulating monocytes promotedthe delivery to tumors and, remarkably, that the conjugation of atargeting ligand (RGD) to the CNTs promoted the homing ofSWCNT-loaded monocytes to tumors when compared tonon-conjugated and control peptide-conjugated SWCNTs.133

These results suggest a novel mechanism for tumor targeting anddemonstrate that PEGylation does not necessarily preventimmune cell recognition. Several questions arise: What is themechanism of the selective (receptor-mediated) uptake of theSWCNTs? How does RGD functionalization of SWCNTspromote homing of these cells to tumors? Furthermore,following infiltration of this subset of monocytes into thetumor, are the SWCNTs (and their cargo) released? Finally, dothe PEG-SWCNTs undergo biodegradation in TAMs?

Biodegradation of carbon-based nanomaterials

As discussed in preceding sections, there has been awidespread concern that certain CNTs, in particular, may exhibitasbestos-like pathogenicity, in part due to the fiber-likemorphology, but also based on the assumption that CNTs arebiopersistent, like asbestos fibers. However, several groups have

reported in recent years that carbon-based nanomaterials aresusceptible to biodegradation.135 Importantly, these studies havehighlighted a key role for the innate immune system in theenzymatic ‘digestion’ of carbon-based nanomaterials.136 Theseobservations suggest that the potentially detrimental effects ofsuch materials may be mitigated thereby allowing the materialsto be more widely applied in nanomedicine. The fact that ourimmune system is capable of ‘sensing’ and destroyingcarbon-based nanomaterials through oxidative reactions maynot come as a surprise given that such nanomaterials have beenshown to occur abundantly in nature.137,138 Moreover, diamond,graphitic, fullerenic and amorphous carbon particles can form inthe flame of a candle.139 Thus, this suggests that mankind hasbeen exposed to carbon-based nanomaterials since the dawn oftime and it is conceivable that defense mechanisms have evolvedto protect us not only from microbes but also from other foreignparticles.

While chemical degradation of carbon-based nanomaterialswas demonstrated through either harsh chemical treatment withconcentrated mineral acids140 or destruction of graphitic latticesthrough high temperature treatment,141 neither of these processesare relevant once these nanomaterials find their way into a livingorganism. However, peroxidases which have strong redoxpotentials, are known to catalyze oxidation of foreign particlesand pathogens with hydrogen peroxide in biological systems.Allen et al142 initially demonstrated the degradation of SWCNTsusing a plant-derived enzyme, horseradish peroxidase (HRP).HRP contains a single protoporphyrin IX heme group which inits inactive form exists in its ferric (Fe3+) oxidation state andupon reaction with hydrogen peroxide forms a ferryl oxo iron(Fe4+ = O) known as Compound I.143 The high redox potentialof Compound I enables degradation of carboxylated SWCNTsdue to the close proximity of SWCNTs to the heme catalyticactive site.135 We and others have documented the degradationof single- and multi-walled CNTs and GO by HRP andH2O2.

142,144-147 Moreover, lignin peroxidase produced bywhite rot fungus also has the capacity to induce oxidativebiodegradation of fullerenes, SWCNTs, and graphenenanoribbons148-150 while recent studies have demonstratedbiodegradation of MWCNTs and GO by various bacteria.151,152

The catalytic heme active site is also characteristic ofmammalian peroxidases including neutrophil myeloperoxidase(MPO), eosinophil peroxidase (EPO), and lactoperoxidase(LPO), suggesting avenues for biological degradation ofcarbon-based nanomaterials. Indeed, MPO, EPO, and LPOhave all been shown to catalyze the degradation of carboxylatedSWCNTs in vitro in the presence of H2O2 and halide ionsthrough the production of reactive radical intermediates inaddition to hypohalous acids153-155 (Figure 4). Degradation ofoxidized SWCNTs has been demonstrated upon incubation withMPO and H2O2, in addition to incubation only with sodiumhypochlorite (NaOCl). However, only the combined effects ofMPO, H2O2, and NaCl (resulting in the production ofhypochlorous acid, HOCl) allowed for rapid degradation ofoxidized SWCNTs.153 In addition to electron microscopy-basedevidence (SEM and TEM), degradation has further been provenby tracking decreases in the intrinsic Raman peaks of SWCNTs,specifically the D-band (disorder in sp2 hybridized carbon

Figure 4. Enzymatic degradation of carbon nanotubes. Molecular modeling demonstrating possible SWCNT interaction sites on eosinophil peroxidase, EPO.Upper left: The two predicted interaction sites, site 1 and site 2 of oxidized SWCNTs modified at the edge. Upper right: Overlay of the possible interaction site 1of SWCNTs oxidized at the edge (colored in gray) and in the middle (colored in cyan). Lower left and right: The residues that are in close proximity (within 4 Å),stabilizing the binding sites (left) site 1 and (right) site 2. Positively charged residues (arginines) that are predicted to stabilize the oxidized groups on SWCNTsare colored in yellow. Reprinted from: Andón FT, Kapralov AA, Yanamala N, Feng W, Baygan A, Chambers BJ, Hultenby K, Ye F, Toprak MS, Brandner BD,Fornara A, Klein-Seetharaman J, Kotchey GP, Star A, Shvedova AA, Fadeel B, Kagan VE. Biodegradation of single-walled carbon nanotubes by eosinophilperoxidase. Small. 2013;9(16):2721-9, 2720, with permission from John Wiley and Sons.


~1350 cm−1) and the G-band (graphitic C-C bond stretching~1600 cm−1) with complete degradation occurring at 24 h ofincubation with MPO, H2O2, and NaCl.153 The peroxidase-catalyzed degradation of SWCNTs has been shown to proceedefficiently with oxidized SWCNTs where incorporated func-tionalities create defect sites for docking of the respectiveenzymes.143 The higher the degree of the incorporated defectsthe higher the rate of degradation, with pristine SWCNTsremaining unaffected by the degradation cycle.143 The degrada-tion of SWCNTs results in shortening of nanotube length,leading to the production of oxidized polyaromatic hydrocarbonsand, ultimately, CO2.

143 However, detailed determination ofdegradation intermediates has proven difficult as the systemcontains multiple complex molecular ions and fragmentsaccording to mass spectrometry (MS). In order to betterunderstand the degradation products an enzyme-free system ofGO degradation by the photo-Fenton reaction was investigatedin which the products were identified via multiple analyticaltechniques (FTIR, MS, and NMR).156 The degradation wasfound to proceed through an oxidation and decarboxylation

mechanism ultimately resulting in oxidized hydrocarbons,specifically aromatic rings functionalized with carboxylic acidgroups.156 These findings suggest that partially degraded GO orCNTs could trigger genotoxicity, as shown for extracts fromHRP-degraded SWCNTs.157 Further studies using mammalianperoxidases, in relevant in vitro and in vivo settings, are neededto address whether partial biodegradation of carbon-basednanomaterials elicits more or less genotoxicity when comparedto the undigested, as-delivered nanomaterials. Complete degra-dation, however, is not expected to do so.

In addition to test-tube experiments of SWCNT degradationusing recombinant peroxidases, we and others have shown thatprimary cells of the innate immune system are capable ofenzymatic degradation of SWCNTs.153,154,158 Notably, thisdegradation may take place both intracellularly and extracellu-larly. Kagan et al153 demonstrated that opsonization of SWCNTswith immunoglobulin (IgG) promotes neutrophil uptake ex vivowith subsequent degradation of the nanotubes. Moreover,evidence for MPO-dependent degradation of oxidized SWCNTsin vivo in the lungs of mice was also obtained.159 Importantly,


activated neutrophils and eosinophils are known to release theirgranule contents including MPO and EPO, respectively, whichenables extracellular destruction of microbes, and we cannotdiscount this pathway in the enzymatic degradation observed forSWCNTs.153,154 Moreover, neutrophils can produce so-calledneutrophil extracellular traps (NETs) consisting of nuclearchromatin fibers studded with granule proteins, and our studieshave shown that purified NETs can ‘capture’ and digestSWCNTs, testifying to the versatility of these cells.6 In additionto neutrophils and eosinophils, recent data also suggest thatmacrophages can ‘digest’ SWCNTs.160 Neutrophils are short-lived with a life-span of days. In contrast, tissue-residentmacrophages may persist for weeks in the context of chronicinflammation. However, in contrast to neutrophils, macrophagesdo not express significant amounts of MPO. Instead, theoxidative metabolism and destruction of foreign bodies includ-ing pathogens is driven by NADPH oxidase (producingsuperoxide) and the inducible isoform of nitric oxide synthase(iNOS) (producing nitric oxide), which conspire to produce ahighly potent oxidant, peroxynitrite (ONOO−).160 Using amodel of activated human THP-1 cells, Kagan et al160 recentlydemonstrated peroxynitrite-driven degradation of SWCNTs.Evidence for NADPH oxidase-dependent degradation ofSWCNTs in vivo was also provided.160 Moreover, Zhanget al161 reported that carbon nanohorns also undergo partialdegradation in macrophage-like cell lines (RAW264.7 andTHP-1) and degradation of MWCNTs has also been reportedrecently using differentiated THP-1 cells as a model.162

Importantly, in the studies cited above, degradation was shownto occur in a complex biological environment, i.e., in cell culturein the presence of fetal bovine serum, or in the lungs of mice,suggesting that bio-corona formation does not prevent degrada-tion of carbon-based nanomaterials. To probe this in furtherdetail, we recently investigated whether LPO-mediated degra-dation could occur in the presence of lung surfactant.155 LPO is asecreted enzyme that has been shown to be important for airwaydefense against infection. Indeed, efficient LPO-mediateddegradation of carboxylated SWCNTs pre-coated with porcinelung surfactant (Curosurf) was documented by Raman spectros-copy, and we also observed biodegradation of SWCNTs incell-free bronchoalveolar lavage fluid.155 Thus, the presence of aprotein-lipid corona89 does not appear to interfere withdegradation. One may postulate that several complementarypathways act together to ensure that foreign intruders – such asnanoparticles – are recognized and cleared from the lungs,including secreted enzymes as well as cell-based systems.Perhaps, as previously suggested, neutrophils are initiallyengaged while macrophages are called into action at a laterstage.160 Moreover, similar, macrophage-driven reactions maytake place in other compartments; for instance, recent studieshave suggested that MWCNTs stereotactically injected into themouse brain cortex are internalized by microglia, the residentmacrophages of the brain, and degraded.163

As mentioned before, PEGylation is commonly applied innanomedicine in order to reduce non-specific protein adsorptionand extend the half-life of nanomaterials in systemic circulation.However, it is important to ask whether such modifications makethe nanomaterials impervious to enzymatic degradation. In a

recent study, we noted that the presence of PEG chains on thesurface of SWCNTs may interfere with the degradation underin vitro conditions using recombinant MPO in a PEG chainmolecular weight dependent manner, suggesting that there couldbe some steric hindrance.164 However, when SWCNTs wereincubated with activated human neutrophils undergoing degran-ulation, effective degradation of SWCNTS was observedirrespective of whether they were PEG-modified or not, andwe provided evidence for a cooperative action of NE, aneutrophil protease, and MPO, suggesting that neutrophils releaseenzymes that can ‘strip’ the PEG chains of PEGylated SWCNTsallowing for efficient peroxidase-driven degradation.164 Further-more, other investigators have documented defunctionalization ofPEGylated SWCNTs in vivo following intravenous administrationin mice.165 Combined, these results imply that PEGylated CNTsmay undergo defunctionalization and degradation.

We previously developed so-called nitrogen-doped carbonnanotube cups.166 In a recent study, we found that these hollow,cup-shaped nano-containers can be effectively ‘corked’ withgold nanoparticles and we have shown that MPO can ‘open’ thecorked carbon nanotube cups through detachment of the goldnanoparticles, with subsequent enzymatic degradation of thegraphitic shells.167 Furthermore, the gold-corked carbon nano-tube cups were demonstrated to function as drug deliverycarriers, capable of delivery of the chemotherapeutic agent,paclitaxel to myeloid-derived suppressor cells (MDSC), withMPO-regulated release of the drug, resulting in the differenti-ation of MDSC into dendritic cells (DC), a property of MDSCthat has been reported to be lost in cancer.167 The findingsindicate the potential of the gold-corked carbon nanotube cups indrug delivery applications. MDSC are known to overexpressboth MPO and inducible nitric oxide synthase (iNOS),potentially providing a route for enzymatic degradation similarto the peroxidase-catalyzed and peroxynitrite-mediated degra-dation route of neutrophils and macrophages, respectively.160

Notably, not only the nano-carrier, but also the payload (drug)may undergo degradation. In a recent study, we were able todemonstrate that SWCNTs externally functionalized withdoxorubicin could be employed for drug delivery, and foundthat the nano-carriers improved the efficacy of the drug in anin vitro melanoma cell model due to the protection providedagainst oxidative degradation exerted byMDSC present in the cellculture.168 Thus, on the basis of these studies, one may concludethat it is crucial to understand and control the degradation ofcarbon-based nanomaterials, not only from the perspective of theirperceived toxicity, but also for the implementation of carbon-baseddrug delivery vehicles. Indeed, it is important to strike the rightbalance between degradation and resistance of the carrier and itspayload against oxidants generated by inflammatory cells in thetumor microenvironment.168

GO was also found to be degraded through the sameperoxidase cycle as CNTs; however, the degradation occursthrough a slightly different mechanism. Due to preferentialbinding of peroxidase enzymes such as HRP to the basal plane ofGO, as opposed to the edges of GO flakes, the graphitic lattice isdegraded from select points throughout the GO sheets resultingin the formation of holes169 (Figure 5). Moreover, analogous topristine, un-oxidized SWCNTs which are resistant to enzymatic

Figure 5. Enzymatic degradation of graphene oxide. Upper panels show atomic force microscopy (AFM) images with section analysis of GO and horseradishperoxidase (HRP) at day 0 (left) and at day 10 (right). GO with HRP has a sheet height of 5.37 nm and 9.81 nm. Holey GO has a sheet height of 1.10 nm, and theholes were authentic at a height of 0.01 nm. Lower panels display binding poses of HRP on (from left to right) GO, holey GO, and a small sheet of GO calculatedusing molecular docking studies. Reprinted from: Kotchey GP, Allen BL, Vedala H, Yanamala N, Kapralov AA, Tyurina YY, Klein-Seetharaman J, Kagan VE,Star A. The enzymatic oxidation of graphene oxide. ACS Nano. 2011;5(3):2098-108, with permission from American Chemical Society.


degradation, reduced GO with an absence of oxygen function-alities resisted degradation and remained intact with no evidenceof hole formation after 20 days of incubation with HRP andH2O2.

147 More recent studies have shown that the level ofoxidation of GO directly correlates with the efficiency ofMPO-mediated degradation in vitro.169 The oxygen functionalgroups provide better dispersibility of GO in aqueous media thusfacilitating the degradation of GO169; additionally, it can beargued that the incorporated oxygen functionalities may allowfor close proximity with the active site of MPO. Furthermore, werecently obtained evidence for degradation of GO by activatedneutrophils (manuscript in preparation). Degradation of carbox-ylated graphene has been reported in vivo in mice followingintravenous injection. Using Raman confocal imaging, Girish etal129 reported degradation of graphene from day 8 onwards,beginning from the edges and growing inwards. Spleen samplesshowed the most enhanced disorder leading to an almostcomplete amorphization of graphene over a period of 3 months.The authors provided evidence of phagocytosis of graphene byalveolar macrophages, Kupffer cells and spleen bound macro-phages, implying that the degradation of graphene was mainlyorchestrated by macrophages in the respective organs.129 Inaddition, in vitro studies using murine RAW264.7 macrophage-like cells showed development of structural disorder in theengulfed graphene when monitored up to 7 days, supporting therole of macrophages in their biodegradation.129 Together, theavailable data suggest that both neutrophil and macrophagemediated degradation of graphene and its derivatives is possible,as shown for CNTs.

Finally, Li et al170 reported that both PEG-coated andBSA-coated GO is fairly resistant to HRP-mediated biodegra-dation when compared to non-functionalized GO. In order toobtain functionalized GO that can still undergo enzymaticdegradation, the authors conjugated PEG to GO via a cleavabledisulfide bond, obtaining GO-SS-PEG with negligible toxicityand considerable degradability.170 This study thus points towarda safe-by-design approach that also takes degradability intoaccount. In fact, for drug delivery applications, it may beimportant to control the biodegradation of carbon-basednanomaterials both temporally and spatially (i.e., degradationat the right time and at the right place) and future efforts in thisarea should take into account the development of nanomedicinevectors that are degradable-on-demand.136 For some otheremerging biomedical applications, including in the field ofregenerative medicine, rapid degradation of the nanomaterial inquestion may not be desirable; for instance in the case of CNTsas scaffolds for neurons in spinal cord injury or graphene as anartificial retina (discussed above).

Concluding remarks

In synopsis, one may conclude that carbon-based nanomater-ials show tremendous promise as drug or gene delivery vehiclesand/or imaging agents, and as biosensors, in diverse areas ofmedicine, although there is certainly a preponderance to date ofstudies on cancer. In addition, there is emerging evidence forpotential applications of carbon-based nanomaterials in regen-erative medicine. However, as we have discussed at length in the


present review, studies on the biocompatibility, in vivobiodistribution, bio-corona formation, and biodegradation arenecessary in order to translate these promising nanomaterials intothe realm of clinical medicine. In fact, these various aspects ofnanomaterial behavior in a living system are intimatelyentwined: the bio-corona has been shown to dictate biodistribu-tion and may also impact on cellular recognition andbiodegradation, which is relevant for the safety or biocompat-ibility of the material. Moreover, a detailed understanding of howthese nanomaterials interact with the immune system is of criticalimportance. Indeed, coronation and degradation are directlyconnected to the role of the immune system in defending thebody from foreign intrusion, and it can be argued thatcarbon-based nanomaterials are ‘sensed’ as pathogens byimmune-competent cells.9 Careful engineering of nanomaterialsis needed to mitigate the potential toxicity of the material whileretaining its useful properties, thereby allowing for thenavigation of various compartments in the body and the timelyexecution of the intended function(s) at the desired location(s). Inother words, one should take care not to throw out the baby withthe bathwater. This is, in essence, the meaning ofsafe-by-design.171,172 With this in mind, one may anticipatemany exciting discoveries and novel applications of thecarbon-based nanomaterials in the years to come.

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