Available online at www.sciencedirect.com
ScienceDirect Nanotubes in biological applications Ruchir V Mundra1,2, Xia Wu1,2, Jeremy Sauer1,2, Jonathan S Dordick1,2,3,4,5 and Ravi S Kane1,2 Researchers over the last few years have recognized carbon nanotubes (CNTs) as promising materials for a number of biological applications. CNTs are increasingly being explored as potent drug carriers for cancer treatment, for biosensing, and as scaffolds for stem cell culture. Moreover, the integration of CNTs with proteins has led to the development of functional nanocomposites with antimicrobial properties. This review aims at understanding the critical role of CNTs in biological applications with a particular emphasis on more recent studies. Addresses 1 Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, CBIS 4105, 110 8th Street, Troy, NY 12180, USA 2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA 3 Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA 4 Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, USA 5 Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA Corresponding authors: Dordick, Jonathan S ([email protected]) and Kane, Ravi S ([email protected])
Current Opinion in Biotechnology 2014, 28:25–32 This review comes from a themed issue on Nanobiotechnology Edited by Jonathan S Dordick and Kelvin H Lee
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.10.012
Introduction Carbon nanotubes (CNTs), by virtue of their unparalleled electrical, mechanical, and optical properties, have emerged as ideal candidates for bioimaging, biosensing, and biomedical applications [1]. Moreover, the development of techniques for the facile functionalization of nanotubes (Table 1) has further expanded the range of potential applications [2]. For instance, nanotubes are not only being used to develop functional nanocomposites, but are also serving as potent vehicles for delivering a variety of drugs in vivo. This review focuses on the key biological applications of CNTs, with special emphasis on reports published within the last three years (Figure 1). www.sciencedirect.com
Nanotubes in drug delivery/cancer therapy On the basis of extensive research during the past decade, nanomaterials, particularly CNTs, have emerged as potent drug delivery vehicles [4]. CNTs are taken up passively into tumors without the assistance of antibodies or other large molecules, and this enhanced permeability and retention (EPR) effect has been exploited for the successful delivery of various anti-cancer drugs [4,5]. The ease of functionalization of CNTs provides opportunities for the attachment of multiple drugs [6]. P-glycoprotein is overexpressed in multi-drug resistant (MDR) mammalian cells and is responsible for the increased efflux of anti-cancer drugs out of the cells. To overcome the challenge posed by MDR cells, Li and colleagues [7] loaded single-walled CNTs with doxorubicin (DOX), a popular anticancer drug, and functionalized them with an antibody to P-glycoprotein. The resulting nanotubes specifically recognized MDR K562 leukemia cells, and exhibited effective loading and controlled release of DOX, thereby exhibiting 2.4-fold higher cytotoxicity towards these cells as compared to free DOX [7]. Liu and colleagues [8] used p–p stacking to load DOX onto single-walled nanotubes (SWNTs) functionalized with branched PEG and studied their biodistribution and pharmacokinetics. Their results clearly demonstrated an increase in the circulation half-life from 0.21 hours for free DOX to 2.22 hours for SWNT-DOX (Figure 2(a)). The branched PEG reduced the clearance by macrophages and repeated passage of these drug conjugates though tumor vessels caused increased tumor uptake (Figure 2(b)). Another advantage of the physical stacking of drugs onto nanotubes is that it does not compromise the drug chemistry. Lay and coworkers [9] extended this advantage to paclitaxel (PTX), another potent anticancer drug, but devoid of an extended p structure; hence, the formation of noncovalent CNT–drug conjugates may be more general than once believed. Despite these developments, a major limitation of CNTs as drug carriers in vivo has been the underlying concern about their toxicity. Karchemski et al. [10] addressed this concern by combining the efficient cell uptake of CNTs with the high drug loading capacity of liposomes in a synergistic fashion. One of the concerns about liposomal delivery is the rapid systemic clearance of the liposome via macrophages, which limits the therapeutic effectiveness of the loaded drug [10]. Current Opinion in Biotechnology 2014, 28:25–32
26 Nanobiotechnology
Table 1 Summary of reviewed functionalization chemistries Types of association Non-covalent attachment
Advantages
Disadvantages
Higher loading than covalent approaches Chemistry of attached molecule is unaltered
Weaker binding and higher leaching than covalent approaches Limited to molecules with affinity for nanotube surface
Molecules attached Paclitaxel
DNA
Doxorubicin
Covalent attachment (EDC-NHS chemistry)
Exhibits stronger/longer term binding
Works for molecules which do not have a high affinity for the nanotube surface
Loading is limited to the number of available functional sites on nanotube surface Alters structure of molecule attached which might prove detrimental for the desired application
M13 phage Lysostaphin AcT (perhydrolase) Protoporphyrin IX RNA aptamer
Potential applications
References
In vivo drug delivery for cancer therapeutics Detection of proteinprotein interactions
[9]
In vivo drug delivery for cancer therapeutics Tumor detection Antimicrobial nanocomposite films
[8]
[23] [45] [44] [50] [26]
Glycoprotein detection
Drug loaded liposomes
[21]
In vivo drug delivery for cancer therapeutics
[10]
Figure 1
Paint
Antimicrobial nanocomposite film
Scaffolds for stem cell culture
Biological applications of carbon nanotubes
Drug release and tumor targeting
SWNT – Analyte Binding
Blood vessel
Biosensors
Tumor Drug delivery vehicles Current Opinion in Biotechnology
Key biological applications of carbon nanotubes. Biological applications of nanotubes discussed in this review namely the use of nanotubes as biosensors (inspired by [26]), scaffolds for tissue engineering (to be reprinted with permission from [3]), vehicles for drug delivery in cancer therapeutics (inspired by [4]) and formation of antimicrobial surfaces. Current Opinion in Biotechnology 2014, 28:25–32
www.sciencedirect.com
Nanotubes in biological applications Mundra et al. 27
Figure 2
sensing, and imaging using engineered SWNTs [15,16]. SWNTs show photoluminescence (PL) from 650 to 1400 nm owing to their unique electronic structure, and NIR light in the range of 950–1400 nm has considerable penetration depth in biological tissues [17]. The intensity and wavelength of fluorescence is also extremely sensitive to the surface functionality and environment of SWNTs [18,19]. These properties make SWNTs ideal candidates for biosensing and bioimaging.
Dox uptake / % ID g–1
(a) 40 Free DOX 30
SWNT-DOX
20 10 0 1
0
3
2
4
5
6
7
tumor
muscle
t/h Dox uptake / % ID g–1
(b) 80
2 1.5 1 0.5 0
Free DOX SWNT-DOX
60 40 20 0
ar
he
t
ng
lu
er liv
ey
n
ee
l sp
dn
ki
st
or
e
h
ac
om
tin
in
s te
m
tu
e cl us m
Current Opinion in Biotechnology
Pharmacokinetics and biodistribution of free DOX and SWNT-DOX (data reprinted with permission from [8]). (a) SWNT-DOX showed prolonged blood circulation compared to free DOX. (b) SWNT-DOX had higher tumor specific uptake and reticulo-endothelial system (RES) uptake than free DOX.
Liposomes can be PEGylated to increase blood circulation times but the resulting steric hindrance may interfere with liposome entry into tumor cells. However, covalent loading of drug-loaded liposomes onto CNTs allowed large amount of drugs to be delivered in vivo without potential systemic effects, presumably because the high drug loading reduced the amount of CNTs required [10]. Expanding beyond anti-cancer drugs, Yang et al. [11] used SWNTs to deliver acetylcholine into the brain of mice to treat Alzheimer’s disease; the use of moderate doses of SWNT–acetylcholine conjugates resulted in selective targeting of lysosomes, thereby limiting the associated toxicity. While biological systems are nearly transparent to nearinfrared (NIR) light, the strong absorbance of CNTs in this spectral window can be exploited for designing cancer therapeutics [12,4]. Moon et al. [13] used the ability of the SWNTs to generate heat upon exposure to NIR light to induce cell death in vivo. Collectively, these studies illustrate the potential applications of CNTs in cancer therapeutics, both as delivery vehicles as well as heat sources.
Nanotubes as biosensors Following the discovery of the band-gap fluorescence of SWNTs in the near-infrared (NIR) range [14], several groups have described fluorescence-based detection, www.sciencedirect.com
Unfunctionalized SWNTs possess low fluorescence stability, intensity, and biocompatibility, thereby requiring chemical modification for sensitive bioimaging. M13 phage, by virtue of their threadlike structure, can interact with SWNTs in a multivalent mode and form stable complexes. Yi et al. [20] showed that SWNTs stabilized using genetically engineered M13 phage (M13-SWNT) could be used as in vivo fluorescence imaging probes of deep tissues when injected intravenously. In addition, M13-SWNT probes displaying antibodies specific to proteins overexpressed in prostate tumors accumulated in the tumors, facilitating fluorescence imaging of these tumors. This strategy has potential application in diagnosing hard-to-detect tumors. SWNTs can also be used to detect molecules of biological significance through modification with analyte-specific molecules. The modified SWNT complex is able to bind targets specifically and cause a conformational change of the analyte-specific molecules and/or the linkage between SWNTs and the analyte-specific molecules, resulting in a shift in the emission wavelength due to the altered local dielectric environment, or a change in fluorescence intensity resulting from the variation in charge transfer on the SWNT interface [15] (Figure 3). Zhang et al. [21] reported the first case of single molecular detection of cellular nitric oxide (NO), which is an important signaling molecule, using single-stranded d(AT)15 DNA nucleotide-wrapped SWNTs. With d(AT)15 having high selectivity toward NO, this DNA–SWNT complex could dynamically report NO adsorption or desorption. Similarly, Jin and colleagues illustrated SWNT-based single-molecule detection of H2O2 in human epidermal carcinoma cells stimulated by epidermal growth factor [22]. Ahn and coworkers [23] developed label-free microarrays for selective protein recognition and single protein detection. SWNTs were embedded in a chitosan matrix grafted with Ni2+–NTA, which could tether His-tagged capture proteins and act as a proximity PL quencher. When analyte proteins interacted with capture proteins, depending on whether the Ni2+ ions were closer to or farther away from the SWNTs, there was PL quenching or enhancing. This approach could be used for probing protein–protein interactions. Detection of blood glucose level and high-throughput profiling of glycans have also been reported [24,25]. Current Opinion in Biotechnology 2014, 28:25–32
28 Nanobiotechnology
Figure 3
Modified SWNT
Analyte
Binding
15000
Fluorescence (RFU)
Fluorescence (RFU)
15000 12000 9000 6000 3000 0 950
1000
1050
1100
1150
1200
1250
Wavelength (nm)
12000 9000 6000
OR 3000 0 950
1000
1050
1100
1150
1200
1250
Wavelength (nm) Current Opinion in Biotechnology
Schematic depiction of modified SWNTs used in fluorescent sensing of molecules. Upon binding of the analyte, there is either a shift in the emission wavelength or a change in fluorescence intensity. Figure inspired by [23].
In addition to fluorescence-based sensing, CNTs have been used for sensing based on their electrochemical properties, such as the ability to support charge transfer between heterogeneous phases and excellent double layer capacitance. These properties, combined with the large surface-to-volume ratio, endow CNTs with exceptional transducing ability for biosensor development. Zelada-Guillen et al. [26] described an aptasensor by coating SWNTs grafted with protein-specific RNA aptamers on an alumina electrode. This sensor was able to detect disease-related glycoproteins from crude blood samples in real-time in an ultrasensitive fashion. There are also demonstrations of cellular NO detection [27], epinephrine sensing [28], and in vivo dopamine monitoring [29] with functionalized CNTs. The application of CNTs in fluorescent and electrochemical sensing has proven to be promising in the past decade. With new probes and molecules being designed for functionalization of CNTs, modified CNTs will continue to find more use in life sciences, disease detection, and medical diagnosis.
Nanotubes as scaffolds for tissue engineering Due to the combined chemical, electrical, and mechanical properties of CNTs, they have been studied for a variety of tissue engineering applications. Recent work Current Opinion in Biotechnology 2014, 28:25–32
has shown that CNTs increase the number of synaptic junctions by 1.6-fold in cultured hippocampal neurons, resulting in an almost twofold increase in peak post-synaptic current and decreased transient desensitization [30]. Additionally, this increased connectivity has been shown to aid in spinal cord explant adhesion as well as an increase in fiber bundle growth and sensitivity to glutamate stimulation [31]. The favorable electrical properties of CNTs have been studied by growing myocardial tissues grown on CNT-gelatin methacrylate supports, resulting in a threefold increase in spontaneous beating and an 85% decrease in excitation threshold compared with cells grown on native gelatin methacrylate support [32]. Stem cells are keenly adept at sensing their physical surroundings and adjusting their cell fate according to external cues such as substrate stiffness, as shown in seminal work by Engler et al. [33]. CNTs can confer such stiffness to a substrate with the additional benefits of high surface area and varied levels of surface oxidation which allow for CNTs to effectively adsorb proteins. Li et al. showed that the nanotubes were able to bind serum proteins which increased hMSC attachment, proliferation, and ultimately differentiation into bone lineages as shown by increased COLIA1 and Cbfa1 mRNA levels, which regulate collagen production and osteogenic growth, respectively [34]. The electrical and mechanical properties of a silk matrix were www.sciencedirect.com
Nanotubes in biological applications Mundra et al. 29
(a)
(b)
Spontaneous Beats (min-1)
Figure 4
5.0mg/ml 3.0mg/ml 1.0mg/ml 0mg/ml (GM)
100 80 60
With CNT 40 Without CNT
20 0
10µm
3
4
5
6
7
8
9
Incubation Time (day) (c) NS
**
Axon length (XX unit)
50 40 30 20 10 0
PLO
Silk
Silk-CNT
PLO Silk Silk-CNT Current Opinion in Biotechnology
Cell attachment, electrical communication, and stem cell differentiation on CNT supports. (a) Increased cell adhesion on CNTs reinforced scaffold (to be reprinted with permission from ref [38]); (b) increased electrical communication between cardiomyoctes grown on CNTs increases spontaneous beating of cells (to be reprinted with permission from Ref. [32]); and (c) hESC grown on CNTs differentiate to neuronal lineages as shown by axon growth (to be reprinted with permission from Ref. [35]).
tuned by dispersing CNTs which resulted in directing hESC to differentiate toward neuronal lineages with efficiency similar to poly-L-ornithine, a substrate for neuronal differentiation [35] (Figure 4(c)). The strength of CNTs has been exploited by mixing them with traditionally fragile tissue scaffolds such as hydrogels. Pioneering work by Zanello et al. [36] showed that neutral charged CNTs can promote osteoblast adhesion and the production of bone crystals. Recent work has exploited the surface roughness and mechanical strength of CNTs by blending them with traditional scaffolds such as cellulose acetate and PLGA [36]. When CNTs were added to these traditional supports, a >30% increase in viable attached cells was observed 1 hour post plating and a 2.9-fold increase in cell attachment 24 hours after plating, respectively. In both studies, the increase in cellular attachment is attributed to the enhanced nano/ www.sciencedirect.com
micro roughness of the scaffold and the augmented ability to absorb proteins on the support [37,38] (Figure 4(a)). Additionally, the CNTs can transduce a phonon force wave upon the absorption of light [39]. When cells grown on CNTs were subjected to a pulse of a 527 nm laser, the added mechanical stress promoted superior bone formation, with levels of calcification 3.5-times that for control cells [39].
Nanotube-mediated decontamination The use of CNTs in antimicrobial nanocomposite films has evolved considerably during the past decade. This development stems from one of the earliest reports on protein–nanotube interactions by Karajanagi et al. [40], who analyzed the effect of the nanoscale environment on the structure and function of proteins. Asuri and coworkers [41,42] reported that SWNTs, owing to their Current Opinion in Biotechnology 2014, 28:25–32
30 Nanobiotechnology
surface curvature, can suppress unfavorable lateral protein-protein interactions and enhance the stability of adsorbed proteins under harsh denaturing conditions. Asuri et al. [42] also developed stable and active antifouling nanocomposite films by incorporating protease-nanotubes conjugates within paint/polymer matrix. The ease of functionalization and high surface area of the CNTs allowed for higher enzyme loadings, and the high aspect ratio of CNTs helped to retain the conjugates within the polymer matrix with minimal leaching [43,44]. Pangule and coworkers [45] developed nanocomposite, latex-based, paints and films that incorporated conjugates of CNTs with lysostaphin — an enzyme capable of degrading the cell wall of antibiotic resistant Staphylococcus aureus (MRSA). These enzyme–nanotube conjugates were highly effective in killing four different strains of MRSA (>99% killing within 2 hours), and were reusable and stable under dry storage conditions for at least six months. Such enzyme–nanotube based formulations could be potentially used in hospital settings to prevent the growth of pathogenic and antibiotic-resistant microorganisms on various common surfaces. Dinu and coworkers [46,47] incorporated conjugates of CNTs with the enzyme AcT into a latex based paint to form a sporicidal nanocomposite film. AcT, a perhydrolase, retained 40% of its activity on nanotubes and catalyzed the formation of peracetic acid, which is a potent decontaminant with broad effectiveness against spores, bacteria, fungi as well as viruses [44]. Similarly, laccase and haloperoxidases have been used to form potent polymernanotube based films with activity against gram positive as well as gram negative bacteria [43]. Beyond enzymes, Aslan and coworkers [48,49] reported that SWNTs when assembled layer-by-layer with polyelectrolytes, poly(Llysine) and poly(L-glutamic acid), possess antimicrobial activity inactivating 90% of Escherichia coli within 1 hour. With superior chemistries for conjugation available and recent advances in protein engineering making a diverse set of enzymes amenable to conjugation, the integration of nanotubes with biological proteins for decontamination is rapidly expanding. While carbon-based nanomaterials by themselves can induce the formation of reactive oxygen species (ROS), conjugation with a photoactive material, such as a porphyrin, can enhance this ROS generation. Banerjee and coworkers [50,51] synthesized MWNT-PPIX (Protoporphyrin IX) conjugates which upon irradiation with visible light inactivated bacterial spores [52], showed potent bactericidal activity against S. aureus and reduced the ability of influenza virus to infect mammalian cells [50]. These conjugates offered two critical advantages: they can be easily recovered by filtration; moreover, since ROS has a multi-targeted mechanism, it is much less likely that microbes would develop resistance against the conjugates [50,52]. Current Opinion in Biotechnology 2014, 28:25–32
Toxicity issues of nanotubes: challenges and future perspectives Given the tremendous potential of nanotubes in several biomedical applications, there have been extensive efforts, through both covalent and non-covalent functionalization, toward mitigating nanotube cytotoxicity and improving their biocompatibility. Surface functionalization has been used to introduce hydrophilic moieties and render CNTs more biocompatible [53]. Simmons and coworkers exploited a non-covalent functionalization scheme that allows carboxylic acid moieties to be attached to the CNT surface via p–p stacking interactions, thereby creating stable aqueous dispersions and limiting cytotoxicity [54]. Moreover the introduction of structural defects allows oxidative enzymes to degrade CNTs under environmentally relevant settings. Specifically, Allen et al. demonstrated biodegradation of SWNTs through natural enzymatic catalysis using horseradish peroxidase [55]. Amidst this prevalent optimism, it is critical to recognize that there are a host of factors affecting nanotube toxicity such as impurities in manufacturing, length, aspects ratios, dispersion, and type of chemical modification [56]. Manufacturing is critical in determining the extent of nanotube toxicity. CNT synthesis involves the use of metal catalyst like Fe, Co, Ni, Mo, etc. which if not removed during the purification step, can generate free radicals which in turn cause oxidative damage to cells and membranes [57]. Furthermore, the underlying mechanisms of nanotube toxicity are not yet completely understood, and the lack of consistent data on toxicity makes it difficult to predict an accurate timeline regarding future widespread use in humans. However, the advent of superior purification and functionalization methods coupled with studies aimed at a deeper understanding of their potential toxicity reinforces optimism for use of CNTs in biomedical applications.
Conclusion The studies reviewed here, collectively, provide a broad overview of the biological applications of carbon nanotubes. Owing to their special electrical, mechanical, optical, and superior physical properties, CNTs should continue to find various applications as biomaterials, and thereby play an immense role in the development of new technologies, from scaffolds for tissue regeneration and drug delivery to the development of functional surfaces. Despite the great promise of CNTs, their nonbiodegradable nature coupled with concerns over cytotoxicity has limited their clinical use. Newer techniques emerging for surface functionalization may help to mitigate these restrictions, thereby uncapping the innate potential of CNTs for the development of next generation biomaterials. www.sciencedirect.com
Nanotubes in biological applications Mundra et al. 31
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Jain KK: Advances in use of functionalized carbon nanotubes for drug design and discovery. Expert Opin Drug Discov 2012, 7:1029-1037.
2.
Vardharajula S, Ali SZ, Tiwari PM, Eroglu E, Vig K, Denis VA, Singh SR: Functionalized carbon nanotubes: biomedical applications. Int J Nanomedicine 2012, 7:5361-5374.
3.
Shin SR, Bae H, Cha JM, Mun JY, Chen YC, Tekin H, Shin H, Farshchi S, Docmeci M, Tang S et al.: Carbon nanotube reinforced hybrid microgels as scaffold materials for cell encapsulation. ACS Nano 2012, 6:362-372.
4.
Fabbro C, Ali-Boucetta H, Da Ros T, Kostarelos K, Bianco A, Prato M: Targeting carbon nanotubes against cancer. Chem Commun 2012, 48:3911-3926. The authors review the use of nanotubes in anti-cancer therapeutics: both as drug delivery vehicles as well as potent heat sources. CNTs may help overcome the lack of selectivity associated with existing anti-cancer therapies. 5.
Choi MR, Stanton-Maxey KJ, Stanley JK, Levin CS, Bardhan R, Akin D, Badve S, Sturgis J, Robinson JP, Bashir R: A cellular trojan horse for delivery of therapeutic nanoparticles into tumors. Nano Lett 2007, 7:3759-3765.
6.
Cheng J, Meziani MJ, Sun Y-P, Cheng SH: Poly (ethylene glycol)conjugated multi-walled carbon nanotubes as an efficient drug carrier for overcoming multidrug resistance. Toxicol Appl Pharmacol 2011, 250:184-193.
7.
Li R, Wu Ra, Zhao L, Wu M, Yang L, Zou H: P-glycoprotein antibody functionalized carbon nanotube overcomes the multidrug resistance of human leukemia cells. ACS Nano 2010, 4:1399-1408.
8.
Liu Z, Fan AC, Rakhra K, Sherlock S, Goodwin A, Chen X, Yang Q, Felsher DW, Dai H: Supramolecular stacking of doxorubicin on carbon nanotubes for in vivo cancer therapy. Angew Chem Int Ed 2009, 48:7668-7672. The authors used supramolecular p–p stacking to load Doxorubicin onto PEG-functionalized CNTs for drug delivery. Their approach resulted in superior biodistribution and pharmacokinetics when compared to traditional conjugation approaches.
9.
Lay CL, Liu HQ, Tan HR, Liu Y: Delivery of paclitaxel by physically loading onto poly (ethylene glycol)(PEG)-graftcarbon nanotubes for potent cancer therapeutics. Nanotechnology 2010, 21:065101.
10. Karchemski F, Zucker D, Barenholz Y, Regev O: Carbon nanotubes–liposomes conjugate as a platform for drug delivery into cells. J Control Release 2012, 160:339-345. The authors developed a novel drug delivery vehicle in which drug-loaded liposomes were covalently attached to CNTs, thereby synergistically combining the high drug loading capacity of liposomes with the efficient cell uptake of CNTs. 11. Yang Z, Zhang Y, Yang Y, Sun L, Han D, Li H, Wang C: Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomedicine 2010, 6: 427-441. 12. Shi Kam NW, Conell MO, Wisdom JA, Dai H: Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc Natl Acad Sci USA 2005, 102:11600-11605. 13. Moon HK, Lee SH, Choi HC: In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes. ACS Nano 2009, 3:3707-3713. 14. O’Connell MJ, Bachilo SM, Huffman CB, Moore VC, Strano MS, Haroz EH, Rialon KL, Boul PJ, Noon WH, Kittrell C et al.: Band gap fluorescence from individual single-walled carbon nanotubes. Science 2002, 297:593-596. www.sciencedirect.com
15. Boghossian AA, Zhang J, Barone PW, Reuel NF, Kim JH, Heller DA, Ahn JH, Hilmer AJ, Rwei A, Arkalgud JR: Near infrared fluorescent sensors based on single-walled carbon nanotubes for life sciences applications. ChemSusChem 2011, 4:848-863. 16. Reuel NF, Mu B, Zhang J, Hinckley A, Strano MS: Nanoengineered glycan sensors enabling native glycoprofiling for medicinal applications: towards profiling glycoproteins without labeling or liberation steps. Chem Soc Rev 2012, 41:5744-5779. 17. Smith AM, Mancini MC, Nie S: Bioimaging: second window for in vivo imaging. Nat Nanotechnol 2009, 4:710-711. 18. Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, Haddon RC: Solution properties of single-walled carbon nanotubes. Science 1998, 282:95-98. 19. Moore VC, Strano MS, Haroz EH, Hauge RH, Smalley RE, Schmidt J, Talmon Y: Individually suspended single-walled carbon nanotubes in various surfactants. Nano Lett 2003, 3:1379-1382. 20. Yi HJ, Ghosh D, Ham MH, Qi JF, Barone PW, Strano MS, Belcher AM: M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett 2012, 12:1176-1183. The authors describe the in vivo fluorescent detection of organs and tumors with M13 phage-stabilized single-walled carbon nanotubes. 21. Zhang JQ, Boghossian AA, Barone PW, Rwei A, Kim JH, Lin DH, Heller DA, Hilmer AJ, Nair N, Reuel NF et al.: Single molecule detection of nitric oxide enabled by d(AT)(15) DNA adsorbed to near infrared fluorescent single-walled carbon nanotubes. J Am Chem Soc 2011, 133:567-581. The authors demonstrate the single molecule detection of nitric oxide which is selectively recognized by d(AT)15, a single-stranded DNA oligonucleotide adsorbed onto near-infrared fluorescent single-walled carbon nanotubes. 22. Jin H, Heller DA, Kalbacova M, Kim J-H, Zhang J, Boghossian AA, Maheshri N, Strano MS: Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes. Nat Nano 2010, 5:302-309. 23. Ahn J-H, Kim J-H, Reuel NF, Barone PW, Boghossian AA, Zhang J, Yoon H, Chang AC, Hilmer AJ, Strano MS: Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis. Nano Lett 2011, 11:2743-2752. The authors report high-throughput, label-free single protein detection using Ni2+-NTA modified fluorescent single-walled carbon nanotubes and His-tagged capture proteins. 24. Yum K, Ahn J-H, McNicholas TP, Barone PW, Mu B, Kim J-H, Jain RM, Strano MS: Boronic acid library for selective, reversible near-infrared fluorescence quenching of surfactant suspended single-walled carbon nanotubes in response to glucose. ACS Nano 2011, 6:819-830. 25. Reuel NF, Ahn J-H, Kim J-H, Zhang J, Boghossian AA, Mahal LK, Strano MS: Transduction of glycan-lectin binding using nearinfrared fluorescent single-walled carbon nanotubes for glycan profiling. J Am Chem Soc 2011, 133:17923-17933. 26. Zelada-Guillen GA, Tweed-Kent A, Niemann M, Ulrich Goringer H, Riu J, Xavier Rius F: Ultrasensitive and real-time detection of proteins in blood using a potentiometric carbon-nanotube aptasensor. Biosens Bioelectron 2013, 41:366-371. 27. Santos RM, Rodrigues MS, Laranjinha Jo, Barbosa RM: Biomimetic sensor based on hemin/carbon nanotubes/ chitosan modified microelectrode for nitric oxide measurement in the brain. Biosens Bioelectron 2013, 44:152159. 28. Prasad BB, Prasad A, Tiwari MP, Madhuri R: Multiwalled carbon nanotubes bearing ‘terminal monomeric unit’ for the fabrication of epinephrine imprinted polymer-based electrochemical sensor. Biosens Bioelectron 2013, 45:114-122. 29. Lin L, Cai Y, Lin R, Yu L, Song C, Gao H, Li X: New integrated in vivo microdialysis-electrochemical device for determination Current Opinion in Biotechnology 2014, 28:25–32
32 Nanobiotechnology
of the neurotransmitter dopamine in rat striatum of freely moving rats. Mikrochim Acta 2011, 172:217-223. 30. Cellot G, Toma FM, Varley ZK, Laishram J, Villari A, Quintana M, Cipollone S, Prato M, Ballerini L: Carbon nanotube scaffolds tune synaptic strength in cultured neural circuits: novel frontiers in nanomaterial–tissue interactions. J Neurosci 2011, 31:12945-12953. 31. Fabbro A, Villari A, Laishram J, Scaini D, Toma FM, Turco A, Prato M, Ballerini L: Spinal cord explants use carbon nanotube interfaces to enhance neurite outgrowth and to fortify synaptic inputs. ACS Nano 2012, 6:2041-2055. The authors show that spinal cord explants grown on CNTs have more neuronal fibers than native support. 32. Shin SR, Jung SM, Zalabany M, Kim K, Zorlutuna P, Kim Sb, Nikkhah M, Khabiry M, Azize M, Kong J: Carbon-nanotubeembedded hydrogel sheets for engineering cardiac constructs and bioactuators. ACS Nano 2013, 7:2369-2380. The authors demonstrate that the increased electrical conductivity of a CNT support allows for increased excitability of cultured cardiomyocytes. 33. Engler AJ, Sen S, Sweeney HL, Discher DE: Matrix elasticity directs stem cell lineage specification. Cell 2006, 126:677-689. 34. Li X, Liu H, Niu X, Yu B, Fan Y, Feng Q, Cui F-z, Watari F: The use of carbon nanotubes to induce osteogenic differentiation of human adipose-derived MSCs in vitro and ectopic bone formation in vivo. Biomaterials 2012, 33:4818-4827. The authors report directing human adipose-derived mesenchymal stem cells to differentiate into bone lineages by growth on CNTs. 35. Chen C-S, Soni S, Le C, Biasca M, Farr E, Chen EYT, Chin W-C: Human stem cell neuronal differentiation on silk-carbon nanotube composite. Nanoscale Res Lett 2012, 7:1-7. 36. Zanello LP, Zhao B, Hu H, Haddon RC: Bone cell proliferation on carbon nanotubes. Nano Lett 2006, 6:562-567. 37. Cheng Q, Rutledge K, Jabbarzadeh E: Carbon nanotubes-poly(lactide-co-glycolide) composite scaffolds for bone tissue engineering applications. Ann Biomed Eng 2013:1-13. 38. Luo Y, Wang S, Shen M, Qi R, Fang Y, Guo R, Cai H, Cao X, Tomas H, Zhu M: Carbon nanotube-incorporated multilayered cellulose acetate nanofibers for tissue engineering applications. Carbohydr Polym 2013, 91:419-427. 39. Sitharaman B, Avti PK, Schaefer K, Talukdar Y, Longtin JP: A novel nanoparticle-enhanced photoacoustic stimulus for bone tissue engineering. Tissue Eng Part A 2011, 17:1851-1858. 40. Karajanagi SS, Vertegel AA, Kane RS, Dordick JS: Structure and function of enzymes adsorbed onto single-walled carbon nanotubes. Langmuir 2004, 20:11594-11599. 41. Asuri P, Karajanagi SS, Yang H, Yim T-J, Kane RS, Dordick JS: Increasing protein stability through control of the nanoscale environment. Langmuir 2006, 22:5833-5836. 42. Asuri P, Karajanagi SS, Kane RS, Dordick JS: Polymer– nanotube–enzyme composites as active antifouling films. Small 2007, 3:50-53. 43. Grover N, Borkar IV, Dinu CZ, Kane RS, Dordick JS: Laccase- and chloroperoxidase-nanotube paint composites with bactericidal and sporicidal activity. Enzyme Microb Technol 2012, 50:271-279. 44. Grover N, Douaisi MP, Borkar IV, Lee L, Dinu CZ, Kane RS, Dordick JS: Perhydrolase-nanotube paint composites with sporicidal and antiviral activity. Appl Microbiol Biotechnol 2013, 19:1-9.
Current Opinion in Biotechnology 2014, 28:25–32
The authors demonstrate that AcT (a perhydrolase) can be immobilized onto carbon nanotubes and the resulting conjugates can then be incorporated into latex-based paints to form highly effective biocatalytic coatings. 45. Pangule RC, Brooks SJ, Dinu CZ, Bale SS, Salmon SL, Zhu G, Metzger DW, Kane RS, Dordick JS: Antistaphylococcal nanocomposite films based on enzyme-nanotube conjugates. ACS Nano 2010, 4:3993-4000. 46. Dinu CZ, Borkar IV, Bale SS, Campbell AS, Kane RS, Dordick JS: Perhydrolase-nanotube-paint sporicidal composites stabilized by intramolecular crosslinking. J Mol Catal B Enzym 2012, 75:20-26. 47. Dinu CZ, Zhu G, Bale SS, Anand G, Reeder PJ, Sanford K, Whited G, Kane RS, Dordick JS: Enzyme-based nanoscale composites for use as active decontamination surfaces. Adv Funct Mater 2010, 20:392-398. 48. Aslan S, Deneufchatel M, Hashmi S, Li N, Pfefferle LD, Elimelech M, Pauthe E, Van Tassel PR: Carbon nanotube-based antimicrobial biomaterials formed via layer-by-layer assembly with polypeptides. J Colloid Interface Sci 2012, 388:268-273. 49. Aslan S, Maatta J, Haznedaroglu BZ, Goodman JPM, Pfefferle LD, Elimelech M, Pauthe E, Sammalkorpi M, Van Tassel PR: Carbon nanotube bundling: influence on layer-by-layer assembly and antimicrobial activity. Soft Matter 2013, 9:2136-2144. The authors demonstrate the layer-by-layer assembly (LbL) of carbon nanotubes with charged polymers and the antimicrobial action of the resultant films. 50. Banerjee I, Douaisi MP, Mondal D, Kane RS: Light-activated nanotube–porphyrin conjugates as effective antiviral agents. Nanotechnology 2012, 23:105101. The authors report the formation of protoporphyrin (PPIX)–nanotube conjugates, which under visible light significantly reduce the ability of Influenza A virus to infect mammalian cells. 51. Banerjee I, Mondal D, Martin J, Kane RS: Photoactivated antimicrobial activity of carbon nanotube–porphyrin conjugates. Langmuir 2010, 26:17369-17374. 52. Banerjee I, Mehta KK, Dordick JS, Kane RS: Light-activated porphyrin-based formulations to inactivate bacterial spores. J Appl Microbiol 2012, 113:1461-1467. 53. Bianco A, Kostarelos K, Prato M: Making carbon nanotubes biocompatible and biodegradable. Chem Commun 2011, 47:10182-10188. 54. Simmons TJ, Justin B, Hashim DP, Linhardt RJ, Ajayan PA: Noncovalent functionalization as an alternative to oxidative acid treatment of single wall carbon nanotubes with applications for polymer composites. ACS Nano 2009, 3(4): 865-870. 55. Allen B, Kichambare PD, Pingping G, Vlasova II, Kapralov AA, Nagarjun K, Kagan VE, Star A: Biodegradation of single-walled carbon nanotubes through enzymatic catalysis. Nano Lett 2008, 8(11):3899-3903. 56. Cui HF, Vashist SK, Rubeaan KA, Luong J, Sheu FS: Interfacing carbon nanotubes with living mammalian cells and cytotoxicity issues. Chem Res Toxicol 2010, 23: 1131-1147. 57. Jain S, Singh SR, Pillai S: Toxicity issues related to biomedical applications of carbon nanotubes. J Nanomed Nanotechnol 2012, 3:5.
www.sciencedirect.com
ScienceDirect Nanotubes in biological applications Ruchir V Mundra1,2, Xia Wu1,2, Jeremy Sauer1,2, Jonathan S Dordick1,2,3,4,5 and Ravi S Kane1,2 Researchers over the last few years have recognized carbon nanotubes (CNTs) as promising materials for a number of biological applications. CNTs are increasingly being explored as potent drug carriers for cancer treatment, for biosensing, and as scaffolds for stem cell culture. Moreover, the integration of CNTs with proteins has led to the development of functional nanocomposites with antimicrobial properties. This review aims at understanding the critical role of CNTs in biological applications with a particular emphasis on more recent studies. Addresses 1 Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, CBIS 4105, 110 8th Street, Troy, NY 12180, USA 2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA 3 Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA 4 Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, USA 5 Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA Corresponding authors: Dordick, Jonathan S ([email protected]) and Kane, Ravi S ([email protected])
Current Opinion in Biotechnology 2014, 28:25–32 This review comes from a themed issue on Nanobiotechnology Edited by Jonathan S Dordick and Kelvin H Lee
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.10.012
Introduction Carbon nanotubes (CNTs), by virtue of their unparalleled electrical, mechanical, and optical properties, have emerged as ideal candidates for bioimaging, biosensing, and biomedical applications [1]. Moreover, the development of techniques for the facile functionalization of nanotubes (Table 1) has further expanded the range of potential applications [2]. For instance, nanotubes are not only being used to develop functional nanocomposites, but are also serving as potent vehicles for delivering a variety of drugs in vivo. This review focuses on the key biological applications of CNTs, with special emphasis on reports published within the last three years (Figure 1). www.sciencedirect.com
Nanotubes in drug delivery/cancer therapy On the basis of extensive research during the past decade, nanomaterials, particularly CNTs, have emerged as potent drug delivery vehicles [4]. CNTs are taken up passively into tumors without the assistance of antibodies or other large molecules, and this enhanced permeability and retention (EPR) effect has been exploited for the successful delivery of various anti-cancer drugs [4,5]. The ease of functionalization of CNTs provides opportunities for the attachment of multiple drugs [6]. P-glycoprotein is overexpressed in multi-drug resistant (MDR) mammalian cells and is responsible for the increased efflux of anti-cancer drugs out of the cells. To overcome the challenge posed by MDR cells, Li and colleagues [7] loaded single-walled CNTs with doxorubicin (DOX), a popular anticancer drug, and functionalized them with an antibody to P-glycoprotein. The resulting nanotubes specifically recognized MDR K562 leukemia cells, and exhibited effective loading and controlled release of DOX, thereby exhibiting 2.4-fold higher cytotoxicity towards these cells as compared to free DOX [7]. Liu and colleagues [8] used p–p stacking to load DOX onto single-walled nanotubes (SWNTs) functionalized with branched PEG and studied their biodistribution and pharmacokinetics. Their results clearly demonstrated an increase in the circulation half-life from 0.21 hours for free DOX to 2.22 hours for SWNT-DOX (Figure 2(a)). The branched PEG reduced the clearance by macrophages and repeated passage of these drug conjugates though tumor vessels caused increased tumor uptake (Figure 2(b)). Another advantage of the physical stacking of drugs onto nanotubes is that it does not compromise the drug chemistry. Lay and coworkers [9] extended this advantage to paclitaxel (PTX), another potent anticancer drug, but devoid of an extended p structure; hence, the formation of noncovalent CNT–drug conjugates may be more general than once believed. Despite these developments, a major limitation of CNTs as drug carriers in vivo has been the underlying concern about their toxicity. Karchemski et al. [10] addressed this concern by combining the efficient cell uptake of CNTs with the high drug loading capacity of liposomes in a synergistic fashion. One of the concerns about liposomal delivery is the rapid systemic clearance of the liposome via macrophages, which limits the therapeutic effectiveness of the loaded drug [10]. Current Opinion in Biotechnology 2014, 28:25–32
26 Nanobiotechnology
Table 1 Summary of reviewed functionalization chemistries Types of association Non-covalent attachment
Advantages
Disadvantages
Higher loading than covalent approaches Chemistry of attached molecule is unaltered
Weaker binding and higher leaching than covalent approaches Limited to molecules with affinity for nanotube surface
Molecules attached Paclitaxel
DNA
Doxorubicin
Covalent attachment (EDC-NHS chemistry)
Exhibits stronger/longer term binding
Works for molecules which do not have a high affinity for the nanotube surface
Loading is limited to the number of available functional sites on nanotube surface Alters structure of molecule attached which might prove detrimental for the desired application
M13 phage Lysostaphin AcT (perhydrolase) Protoporphyrin IX RNA aptamer
Potential applications
References
In vivo drug delivery for cancer therapeutics Detection of proteinprotein interactions
[9]
In vivo drug delivery for cancer therapeutics Tumor detection Antimicrobial nanocomposite films
[8]
[23] [45] [44] [50] [26]
Glycoprotein detection
Drug loaded liposomes
[21]
In vivo drug delivery for cancer therapeutics
[10]
Figure 1
Paint
Antimicrobial nanocomposite film
Scaffolds for stem cell culture
Biological applications of carbon nanotubes
Drug release and tumor targeting
SWNT – Analyte Binding
Blood vessel
Biosensors
Tumor Drug delivery vehicles Current Opinion in Biotechnology
Key biological applications of carbon nanotubes. Biological applications of nanotubes discussed in this review namely the use of nanotubes as biosensors (inspired by [26]), scaffolds for tissue engineering (to be reprinted with permission from [3]), vehicles for drug delivery in cancer therapeutics (inspired by [4]) and formation of antimicrobial surfaces. Current Opinion in Biotechnology 2014, 28:25–32
www.sciencedirect.com
Nanotubes in biological applications Mundra et al. 27
Figure 2
sensing, and imaging using engineered SWNTs [15,16]. SWNTs show photoluminescence (PL) from 650 to 1400 nm owing to their unique electronic structure, and NIR light in the range of 950–1400 nm has considerable penetration depth in biological tissues [17]. The intensity and wavelength of fluorescence is also extremely sensitive to the surface functionality and environment of SWNTs [18,19]. These properties make SWNTs ideal candidates for biosensing and bioimaging.
Dox uptake / % ID g–1
(a) 40 Free DOX 30
SWNT-DOX
20 10 0 1
0
3
2
4
5
6
7
tumor
muscle
t/h Dox uptake / % ID g–1
(b) 80
2 1.5 1 0.5 0
Free DOX SWNT-DOX
60 40 20 0
ar
he
t
ng
lu
er liv
ey
n
ee
l sp
dn
ki
st
or
e
h
ac
om
tin
in
s te
m
tu
e cl us m
Current Opinion in Biotechnology
Pharmacokinetics and biodistribution of free DOX and SWNT-DOX (data reprinted with permission from [8]). (a) SWNT-DOX showed prolonged blood circulation compared to free DOX. (b) SWNT-DOX had higher tumor specific uptake and reticulo-endothelial system (RES) uptake than free DOX.
Liposomes can be PEGylated to increase blood circulation times but the resulting steric hindrance may interfere with liposome entry into tumor cells. However, covalent loading of drug-loaded liposomes onto CNTs allowed large amount of drugs to be delivered in vivo without potential systemic effects, presumably because the high drug loading reduced the amount of CNTs required [10]. Expanding beyond anti-cancer drugs, Yang et al. [11] used SWNTs to deliver acetylcholine into the brain of mice to treat Alzheimer’s disease; the use of moderate doses of SWNT–acetylcholine conjugates resulted in selective targeting of lysosomes, thereby limiting the associated toxicity. While biological systems are nearly transparent to nearinfrared (NIR) light, the strong absorbance of CNTs in this spectral window can be exploited for designing cancer therapeutics [12,4]. Moon et al. [13] used the ability of the SWNTs to generate heat upon exposure to NIR light to induce cell death in vivo. Collectively, these studies illustrate the potential applications of CNTs in cancer therapeutics, both as delivery vehicles as well as heat sources.
Nanotubes as biosensors Following the discovery of the band-gap fluorescence of SWNTs in the near-infrared (NIR) range [14], several groups have described fluorescence-based detection, www.sciencedirect.com
Unfunctionalized SWNTs possess low fluorescence stability, intensity, and biocompatibility, thereby requiring chemical modification for sensitive bioimaging. M13 phage, by virtue of their threadlike structure, can interact with SWNTs in a multivalent mode and form stable complexes. Yi et al. [20] showed that SWNTs stabilized using genetically engineered M13 phage (M13-SWNT) could be used as in vivo fluorescence imaging probes of deep tissues when injected intravenously. In addition, M13-SWNT probes displaying antibodies specific to proteins overexpressed in prostate tumors accumulated in the tumors, facilitating fluorescence imaging of these tumors. This strategy has potential application in diagnosing hard-to-detect tumors. SWNTs can also be used to detect molecules of biological significance through modification with analyte-specific molecules. The modified SWNT complex is able to bind targets specifically and cause a conformational change of the analyte-specific molecules and/or the linkage between SWNTs and the analyte-specific molecules, resulting in a shift in the emission wavelength due to the altered local dielectric environment, or a change in fluorescence intensity resulting from the variation in charge transfer on the SWNT interface [15] (Figure 3). Zhang et al. [21] reported the first case of single molecular detection of cellular nitric oxide (NO), which is an important signaling molecule, using single-stranded d(AT)15 DNA nucleotide-wrapped SWNTs. With d(AT)15 having high selectivity toward NO, this DNA–SWNT complex could dynamically report NO adsorption or desorption. Similarly, Jin and colleagues illustrated SWNT-based single-molecule detection of H2O2 in human epidermal carcinoma cells stimulated by epidermal growth factor [22]. Ahn and coworkers [23] developed label-free microarrays for selective protein recognition and single protein detection. SWNTs were embedded in a chitosan matrix grafted with Ni2+–NTA, which could tether His-tagged capture proteins and act as a proximity PL quencher. When analyte proteins interacted with capture proteins, depending on whether the Ni2+ ions were closer to or farther away from the SWNTs, there was PL quenching or enhancing. This approach could be used for probing protein–protein interactions. Detection of blood glucose level and high-throughput profiling of glycans have also been reported [24,25]. Current Opinion in Biotechnology 2014, 28:25–32
28 Nanobiotechnology
Figure 3
Modified SWNT
Analyte
Binding
15000
Fluorescence (RFU)
Fluorescence (RFU)
15000 12000 9000 6000 3000 0 950
1000
1050
1100
1150
1200
1250
Wavelength (nm)
12000 9000 6000
OR 3000 0 950
1000
1050
1100
1150
1200
1250
Wavelength (nm) Current Opinion in Biotechnology
Schematic depiction of modified SWNTs used in fluorescent sensing of molecules. Upon binding of the analyte, there is either a shift in the emission wavelength or a change in fluorescence intensity. Figure inspired by [23].
In addition to fluorescence-based sensing, CNTs have been used for sensing based on their electrochemical properties, such as the ability to support charge transfer between heterogeneous phases and excellent double layer capacitance. These properties, combined with the large surface-to-volume ratio, endow CNTs with exceptional transducing ability for biosensor development. Zelada-Guillen et al. [26] described an aptasensor by coating SWNTs grafted with protein-specific RNA aptamers on an alumina electrode. This sensor was able to detect disease-related glycoproteins from crude blood samples in real-time in an ultrasensitive fashion. There are also demonstrations of cellular NO detection [27], epinephrine sensing [28], and in vivo dopamine monitoring [29] with functionalized CNTs. The application of CNTs in fluorescent and electrochemical sensing has proven to be promising in the past decade. With new probes and molecules being designed for functionalization of CNTs, modified CNTs will continue to find more use in life sciences, disease detection, and medical diagnosis.
Nanotubes as scaffolds for tissue engineering Due to the combined chemical, electrical, and mechanical properties of CNTs, they have been studied for a variety of tissue engineering applications. Recent work Current Opinion in Biotechnology 2014, 28:25–32
has shown that CNTs increase the number of synaptic junctions by 1.6-fold in cultured hippocampal neurons, resulting in an almost twofold increase in peak post-synaptic current and decreased transient desensitization [30]. Additionally, this increased connectivity has been shown to aid in spinal cord explant adhesion as well as an increase in fiber bundle growth and sensitivity to glutamate stimulation [31]. The favorable electrical properties of CNTs have been studied by growing myocardial tissues grown on CNT-gelatin methacrylate supports, resulting in a threefold increase in spontaneous beating and an 85% decrease in excitation threshold compared with cells grown on native gelatin methacrylate support [32]. Stem cells are keenly adept at sensing their physical surroundings and adjusting their cell fate according to external cues such as substrate stiffness, as shown in seminal work by Engler et al. [33]. CNTs can confer such stiffness to a substrate with the additional benefits of high surface area and varied levels of surface oxidation which allow for CNTs to effectively adsorb proteins. Li et al. showed that the nanotubes were able to bind serum proteins which increased hMSC attachment, proliferation, and ultimately differentiation into bone lineages as shown by increased COLIA1 and Cbfa1 mRNA levels, which regulate collagen production and osteogenic growth, respectively [34]. The electrical and mechanical properties of a silk matrix were www.sciencedirect.com
Nanotubes in biological applications Mundra et al. 29
(a)
(b)
Spontaneous Beats (min-1)
Figure 4
5.0mg/ml 3.0mg/ml 1.0mg/ml 0mg/ml (GM)
100 80 60
With CNT 40 Without CNT
20 0
10µm
3
4
5
6
7
8
9
Incubation Time (day) (c) NS
**
Axon length (XX unit)
50 40 30 20 10 0
PLO
Silk
Silk-CNT
PLO Silk Silk-CNT Current Opinion in Biotechnology
Cell attachment, electrical communication, and stem cell differentiation on CNT supports. (a) Increased cell adhesion on CNTs reinforced scaffold (to be reprinted with permission from ref [38]); (b) increased electrical communication between cardiomyoctes grown on CNTs increases spontaneous beating of cells (to be reprinted with permission from Ref. [32]); and (c) hESC grown on CNTs differentiate to neuronal lineages as shown by axon growth (to be reprinted with permission from Ref. [35]).
tuned by dispersing CNTs which resulted in directing hESC to differentiate toward neuronal lineages with efficiency similar to poly-L-ornithine, a substrate for neuronal differentiation [35] (Figure 4(c)). The strength of CNTs has been exploited by mixing them with traditionally fragile tissue scaffolds such as hydrogels. Pioneering work by Zanello et al. [36] showed that neutral charged CNTs can promote osteoblast adhesion and the production of bone crystals. Recent work has exploited the surface roughness and mechanical strength of CNTs by blending them with traditional scaffolds such as cellulose acetate and PLGA [36]. When CNTs were added to these traditional supports, a >30% increase in viable attached cells was observed 1 hour post plating and a 2.9-fold increase in cell attachment 24 hours after plating, respectively. In both studies, the increase in cellular attachment is attributed to the enhanced nano/ www.sciencedirect.com
micro roughness of the scaffold and the augmented ability to absorb proteins on the support [37,38] (Figure 4(a)). Additionally, the CNTs can transduce a phonon force wave upon the absorption of light [39]. When cells grown on CNTs were subjected to a pulse of a 527 nm laser, the added mechanical stress promoted superior bone formation, with levels of calcification 3.5-times that for control cells [39].
Nanotube-mediated decontamination The use of CNTs in antimicrobial nanocomposite films has evolved considerably during the past decade. This development stems from one of the earliest reports on protein–nanotube interactions by Karajanagi et al. [40], who analyzed the effect of the nanoscale environment on the structure and function of proteins. Asuri and coworkers [41,42] reported that SWNTs, owing to their Current Opinion in Biotechnology 2014, 28:25–32
30 Nanobiotechnology
surface curvature, can suppress unfavorable lateral protein-protein interactions and enhance the stability of adsorbed proteins under harsh denaturing conditions. Asuri et al. [42] also developed stable and active antifouling nanocomposite films by incorporating protease-nanotubes conjugates within paint/polymer matrix. The ease of functionalization and high surface area of the CNTs allowed for higher enzyme loadings, and the high aspect ratio of CNTs helped to retain the conjugates within the polymer matrix with minimal leaching [43,44]. Pangule and coworkers [45] developed nanocomposite, latex-based, paints and films that incorporated conjugates of CNTs with lysostaphin — an enzyme capable of degrading the cell wall of antibiotic resistant Staphylococcus aureus (MRSA). These enzyme–nanotube conjugates were highly effective in killing four different strains of MRSA (>99% killing within 2 hours), and were reusable and stable under dry storage conditions for at least six months. Such enzyme–nanotube based formulations could be potentially used in hospital settings to prevent the growth of pathogenic and antibiotic-resistant microorganisms on various common surfaces. Dinu and coworkers [46,47] incorporated conjugates of CNTs with the enzyme AcT into a latex based paint to form a sporicidal nanocomposite film. AcT, a perhydrolase, retained 40% of its activity on nanotubes and catalyzed the formation of peracetic acid, which is a potent decontaminant with broad effectiveness against spores, bacteria, fungi as well as viruses [44]. Similarly, laccase and haloperoxidases have been used to form potent polymernanotube based films with activity against gram positive as well as gram negative bacteria [43]. Beyond enzymes, Aslan and coworkers [48,49] reported that SWNTs when assembled layer-by-layer with polyelectrolytes, poly(Llysine) and poly(L-glutamic acid), possess antimicrobial activity inactivating 90% of Escherichia coli within 1 hour. With superior chemistries for conjugation available and recent advances in protein engineering making a diverse set of enzymes amenable to conjugation, the integration of nanotubes with biological proteins for decontamination is rapidly expanding. While carbon-based nanomaterials by themselves can induce the formation of reactive oxygen species (ROS), conjugation with a photoactive material, such as a porphyrin, can enhance this ROS generation. Banerjee and coworkers [50,51] synthesized MWNT-PPIX (Protoporphyrin IX) conjugates which upon irradiation with visible light inactivated bacterial spores [52], showed potent bactericidal activity against S. aureus and reduced the ability of influenza virus to infect mammalian cells [50]. These conjugates offered two critical advantages: they can be easily recovered by filtration; moreover, since ROS has a multi-targeted mechanism, it is much less likely that microbes would develop resistance against the conjugates [50,52]. Current Opinion in Biotechnology 2014, 28:25–32
Toxicity issues of nanotubes: challenges and future perspectives Given the tremendous potential of nanotubes in several biomedical applications, there have been extensive efforts, through both covalent and non-covalent functionalization, toward mitigating nanotube cytotoxicity and improving their biocompatibility. Surface functionalization has been used to introduce hydrophilic moieties and render CNTs more biocompatible [53]. Simmons and coworkers exploited a non-covalent functionalization scheme that allows carboxylic acid moieties to be attached to the CNT surface via p–p stacking interactions, thereby creating stable aqueous dispersions and limiting cytotoxicity [54]. Moreover the introduction of structural defects allows oxidative enzymes to degrade CNTs under environmentally relevant settings. Specifically, Allen et al. demonstrated biodegradation of SWNTs through natural enzymatic catalysis using horseradish peroxidase [55]. Amidst this prevalent optimism, it is critical to recognize that there are a host of factors affecting nanotube toxicity such as impurities in manufacturing, length, aspects ratios, dispersion, and type of chemical modification [56]. Manufacturing is critical in determining the extent of nanotube toxicity. CNT synthesis involves the use of metal catalyst like Fe, Co, Ni, Mo, etc. which if not removed during the purification step, can generate free radicals which in turn cause oxidative damage to cells and membranes [57]. Furthermore, the underlying mechanisms of nanotube toxicity are not yet completely understood, and the lack of consistent data on toxicity makes it difficult to predict an accurate timeline regarding future widespread use in humans. However, the advent of superior purification and functionalization methods coupled with studies aimed at a deeper understanding of their potential toxicity reinforces optimism for use of CNTs in biomedical applications.
Conclusion The studies reviewed here, collectively, provide a broad overview of the biological applications of carbon nanotubes. Owing to their special electrical, mechanical, optical, and superior physical properties, CNTs should continue to find various applications as biomaterials, and thereby play an immense role in the development of new technologies, from scaffolds for tissue regeneration and drug delivery to the development of functional surfaces. Despite the great promise of CNTs, their nonbiodegradable nature coupled with concerns over cytotoxicity has limited their clinical use. Newer techniques emerging for surface functionalization may help to mitigate these restrictions, thereby uncapping the innate potential of CNTs for the development of next generation biomaterials. www.sciencedirect.com
Nanotubes in biological applications Mundra et al. 31
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Jain KK: Advances in use of functionalized carbon nanotubes for drug design and discovery. Expert Opin Drug Discov 2012, 7:1029-1037.
2.
Vardharajula S, Ali SZ, Tiwari PM, Eroglu E, Vig K, Denis VA, Singh SR: Functionalized carbon nanotubes: biomedical applications. Int J Nanomedicine 2012, 7:5361-5374.
3.
Shin SR, Bae H, Cha JM, Mun JY, Chen YC, Tekin H, Shin H, Farshchi S, Docmeci M, Tang S et al.: Carbon nanotube reinforced hybrid microgels as scaffold materials for cell encapsulation. ACS Nano 2012, 6:362-372.
4.
Fabbro C, Ali-Boucetta H, Da Ros T, Kostarelos K, Bianco A, Prato M: Targeting carbon nanotubes against cancer. Chem Commun 2012, 48:3911-3926. The authors review the use of nanotubes in anti-cancer therapeutics: both as drug delivery vehicles as well as potent heat sources. CNTs may help overcome the lack of selectivity associated with existing anti-cancer therapies. 5.
Choi MR, Stanton-Maxey KJ, Stanley JK, Levin CS, Bardhan R, Akin D, Badve S, Sturgis J, Robinson JP, Bashir R: A cellular trojan horse for delivery of therapeutic nanoparticles into tumors. Nano Lett 2007, 7:3759-3765.
6.
Cheng J, Meziani MJ, Sun Y-P, Cheng SH: Poly (ethylene glycol)conjugated multi-walled carbon nanotubes as an efficient drug carrier for overcoming multidrug resistance. Toxicol Appl Pharmacol 2011, 250:184-193.
7.
Li R, Wu Ra, Zhao L, Wu M, Yang L, Zou H: P-glycoprotein antibody functionalized carbon nanotube overcomes the multidrug resistance of human leukemia cells. ACS Nano 2010, 4:1399-1408.
8.
Liu Z, Fan AC, Rakhra K, Sherlock S, Goodwin A, Chen X, Yang Q, Felsher DW, Dai H: Supramolecular stacking of doxorubicin on carbon nanotubes for in vivo cancer therapy. Angew Chem Int Ed 2009, 48:7668-7672. The authors used supramolecular p–p stacking to load Doxorubicin onto PEG-functionalized CNTs for drug delivery. Their approach resulted in superior biodistribution and pharmacokinetics when compared to traditional conjugation approaches.
9.
Lay CL, Liu HQ, Tan HR, Liu Y: Delivery of paclitaxel by physically loading onto poly (ethylene glycol)(PEG)-graftcarbon nanotubes for potent cancer therapeutics. Nanotechnology 2010, 21:065101.
10. Karchemski F, Zucker D, Barenholz Y, Regev O: Carbon nanotubes–liposomes conjugate as a platform for drug delivery into cells. J Control Release 2012, 160:339-345. The authors developed a novel drug delivery vehicle in which drug-loaded liposomes were covalently attached to CNTs, thereby synergistically combining the high drug loading capacity of liposomes with the efficient cell uptake of CNTs. 11. Yang Z, Zhang Y, Yang Y, Sun L, Han D, Li H, Wang C: Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomedicine 2010, 6: 427-441. 12. Shi Kam NW, Conell MO, Wisdom JA, Dai H: Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc Natl Acad Sci USA 2005, 102:11600-11605. 13. Moon HK, Lee SH, Choi HC: In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes. ACS Nano 2009, 3:3707-3713. 14. O’Connell MJ, Bachilo SM, Huffman CB, Moore VC, Strano MS, Haroz EH, Rialon KL, Boul PJ, Noon WH, Kittrell C et al.: Band gap fluorescence from individual single-walled carbon nanotubes. Science 2002, 297:593-596. www.sciencedirect.com
15. Boghossian AA, Zhang J, Barone PW, Reuel NF, Kim JH, Heller DA, Ahn JH, Hilmer AJ, Rwei A, Arkalgud JR: Near infrared fluorescent sensors based on single-walled carbon nanotubes for life sciences applications. ChemSusChem 2011, 4:848-863. 16. Reuel NF, Mu B, Zhang J, Hinckley A, Strano MS: Nanoengineered glycan sensors enabling native glycoprofiling for medicinal applications: towards profiling glycoproteins without labeling or liberation steps. Chem Soc Rev 2012, 41:5744-5779. 17. Smith AM, Mancini MC, Nie S: Bioimaging: second window for in vivo imaging. Nat Nanotechnol 2009, 4:710-711. 18. Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, Haddon RC: Solution properties of single-walled carbon nanotubes. Science 1998, 282:95-98. 19. Moore VC, Strano MS, Haroz EH, Hauge RH, Smalley RE, Schmidt J, Talmon Y: Individually suspended single-walled carbon nanotubes in various surfactants. Nano Lett 2003, 3:1379-1382. 20. Yi HJ, Ghosh D, Ham MH, Qi JF, Barone PW, Strano MS, Belcher AM: M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett 2012, 12:1176-1183. The authors describe the in vivo fluorescent detection of organs and tumors with M13 phage-stabilized single-walled carbon nanotubes. 21. Zhang JQ, Boghossian AA, Barone PW, Rwei A, Kim JH, Lin DH, Heller DA, Hilmer AJ, Nair N, Reuel NF et al.: Single molecule detection of nitric oxide enabled by d(AT)(15) DNA adsorbed to near infrared fluorescent single-walled carbon nanotubes. J Am Chem Soc 2011, 133:567-581. The authors demonstrate the single molecule detection of nitric oxide which is selectively recognized by d(AT)15, a single-stranded DNA oligonucleotide adsorbed onto near-infrared fluorescent single-walled carbon nanotubes. 22. Jin H, Heller DA, Kalbacova M, Kim J-H, Zhang J, Boghossian AA, Maheshri N, Strano MS: Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes. Nat Nano 2010, 5:302-309. 23. Ahn J-H, Kim J-H, Reuel NF, Barone PW, Boghossian AA, Zhang J, Yoon H, Chang AC, Hilmer AJ, Strano MS: Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis. Nano Lett 2011, 11:2743-2752. The authors report high-throughput, label-free single protein detection using Ni2+-NTA modified fluorescent single-walled carbon nanotubes and His-tagged capture proteins. 24. Yum K, Ahn J-H, McNicholas TP, Barone PW, Mu B, Kim J-H, Jain RM, Strano MS: Boronic acid library for selective, reversible near-infrared fluorescence quenching of surfactant suspended single-walled carbon nanotubes in response to glucose. ACS Nano 2011, 6:819-830. 25. Reuel NF, Ahn J-H, Kim J-H, Zhang J, Boghossian AA, Mahal LK, Strano MS: Transduction of glycan-lectin binding using nearinfrared fluorescent single-walled carbon nanotubes for glycan profiling. J Am Chem Soc 2011, 133:17923-17933. 26. Zelada-Guillen GA, Tweed-Kent A, Niemann M, Ulrich Goringer H, Riu J, Xavier Rius F: Ultrasensitive and real-time detection of proteins in blood using a potentiometric carbon-nanotube aptasensor. Biosens Bioelectron 2013, 41:366-371. 27. Santos RM, Rodrigues MS, Laranjinha Jo, Barbosa RM: Biomimetic sensor based on hemin/carbon nanotubes/ chitosan modified microelectrode for nitric oxide measurement in the brain. Biosens Bioelectron 2013, 44:152159. 28. Prasad BB, Prasad A, Tiwari MP, Madhuri R: Multiwalled carbon nanotubes bearing ‘terminal monomeric unit’ for the fabrication of epinephrine imprinted polymer-based electrochemical sensor. Biosens Bioelectron 2013, 45:114-122. 29. Lin L, Cai Y, Lin R, Yu L, Song C, Gao H, Li X: New integrated in vivo microdialysis-electrochemical device for determination Current Opinion in Biotechnology 2014, 28:25–32
32 Nanobiotechnology
of the neurotransmitter dopamine in rat striatum of freely moving rats. Mikrochim Acta 2011, 172:217-223. 30. Cellot G, Toma FM, Varley ZK, Laishram J, Villari A, Quintana M, Cipollone S, Prato M, Ballerini L: Carbon nanotube scaffolds tune synaptic strength in cultured neural circuits: novel frontiers in nanomaterial–tissue interactions. J Neurosci 2011, 31:12945-12953. 31. Fabbro A, Villari A, Laishram J, Scaini D, Toma FM, Turco A, Prato M, Ballerini L: Spinal cord explants use carbon nanotube interfaces to enhance neurite outgrowth and to fortify synaptic inputs. ACS Nano 2012, 6:2041-2055. The authors show that spinal cord explants grown on CNTs have more neuronal fibers than native support. 32. Shin SR, Jung SM, Zalabany M, Kim K, Zorlutuna P, Kim Sb, Nikkhah M, Khabiry M, Azize M, Kong J: Carbon-nanotubeembedded hydrogel sheets for engineering cardiac constructs and bioactuators. ACS Nano 2013, 7:2369-2380. The authors demonstrate that the increased electrical conductivity of a CNT support allows for increased excitability of cultured cardiomyocytes. 33. Engler AJ, Sen S, Sweeney HL, Discher DE: Matrix elasticity directs stem cell lineage specification. Cell 2006, 126:677-689. 34. Li X, Liu H, Niu X, Yu B, Fan Y, Feng Q, Cui F-z, Watari F: The use of carbon nanotubes to induce osteogenic differentiation of human adipose-derived MSCs in vitro and ectopic bone formation in vivo. Biomaterials 2012, 33:4818-4827. The authors report directing human adipose-derived mesenchymal stem cells to differentiate into bone lineages by growth on CNTs. 35. Chen C-S, Soni S, Le C, Biasca M, Farr E, Chen EYT, Chin W-C: Human stem cell neuronal differentiation on silk-carbon nanotube composite. Nanoscale Res Lett 2012, 7:1-7. 36. Zanello LP, Zhao B, Hu H, Haddon RC: Bone cell proliferation on carbon nanotubes. Nano Lett 2006, 6:562-567. 37. Cheng Q, Rutledge K, Jabbarzadeh E: Carbon nanotubes-poly(lactide-co-glycolide) composite scaffolds for bone tissue engineering applications. Ann Biomed Eng 2013:1-13. 38. Luo Y, Wang S, Shen M, Qi R, Fang Y, Guo R, Cai H, Cao X, Tomas H, Zhu M: Carbon nanotube-incorporated multilayered cellulose acetate nanofibers for tissue engineering applications. Carbohydr Polym 2013, 91:419-427. 39. Sitharaman B, Avti PK, Schaefer K, Talukdar Y, Longtin JP: A novel nanoparticle-enhanced photoacoustic stimulus for bone tissue engineering. Tissue Eng Part A 2011, 17:1851-1858. 40. Karajanagi SS, Vertegel AA, Kane RS, Dordick JS: Structure and function of enzymes adsorbed onto single-walled carbon nanotubes. Langmuir 2004, 20:11594-11599. 41. Asuri P, Karajanagi SS, Yang H, Yim T-J, Kane RS, Dordick JS: Increasing protein stability through control of the nanoscale environment. Langmuir 2006, 22:5833-5836. 42. Asuri P, Karajanagi SS, Kane RS, Dordick JS: Polymer– nanotube–enzyme composites as active antifouling films. Small 2007, 3:50-53. 43. Grover N, Borkar IV, Dinu CZ, Kane RS, Dordick JS: Laccase- and chloroperoxidase-nanotube paint composites with bactericidal and sporicidal activity. Enzyme Microb Technol 2012, 50:271-279. 44. Grover N, Douaisi MP, Borkar IV, Lee L, Dinu CZ, Kane RS, Dordick JS: Perhydrolase-nanotube paint composites with sporicidal and antiviral activity. Appl Microbiol Biotechnol 2013, 19:1-9.
Current Opinion in Biotechnology 2014, 28:25–32
The authors demonstrate that AcT (a perhydrolase) can be immobilized onto carbon nanotubes and the resulting conjugates can then be incorporated into latex-based paints to form highly effective biocatalytic coatings. 45. Pangule RC, Brooks SJ, Dinu CZ, Bale SS, Salmon SL, Zhu G, Metzger DW, Kane RS, Dordick JS: Antistaphylococcal nanocomposite films based on enzyme-nanotube conjugates. ACS Nano 2010, 4:3993-4000. 46. Dinu CZ, Borkar IV, Bale SS, Campbell AS, Kane RS, Dordick JS: Perhydrolase-nanotube-paint sporicidal composites stabilized by intramolecular crosslinking. J Mol Catal B Enzym 2012, 75:20-26. 47. Dinu CZ, Zhu G, Bale SS, Anand G, Reeder PJ, Sanford K, Whited G, Kane RS, Dordick JS: Enzyme-based nanoscale composites for use as active decontamination surfaces. Adv Funct Mater 2010, 20:392-398. 48. Aslan S, Deneufchatel M, Hashmi S, Li N, Pfefferle LD, Elimelech M, Pauthe E, Van Tassel PR: Carbon nanotube-based antimicrobial biomaterials formed via layer-by-layer assembly with polypeptides. J Colloid Interface Sci 2012, 388:268-273. 49. Aslan S, Maatta J, Haznedaroglu BZ, Goodman JPM, Pfefferle LD, Elimelech M, Pauthe E, Sammalkorpi M, Van Tassel PR: Carbon nanotube bundling: influence on layer-by-layer assembly and antimicrobial activity. Soft Matter 2013, 9:2136-2144. The authors demonstrate the layer-by-layer assembly (LbL) of carbon nanotubes with charged polymers and the antimicrobial action of the resultant films. 50. Banerjee I, Douaisi MP, Mondal D, Kane RS: Light-activated nanotube–porphyrin conjugates as effective antiviral agents. Nanotechnology 2012, 23:105101. The authors report the formation of protoporphyrin (PPIX)–nanotube conjugates, which under visible light significantly reduce the ability of Influenza A virus to infect mammalian cells. 51. Banerjee I, Mondal D, Martin J, Kane RS: Photoactivated antimicrobial activity of carbon nanotube–porphyrin conjugates. Langmuir 2010, 26:17369-17374. 52. Banerjee I, Mehta KK, Dordick JS, Kane RS: Light-activated porphyrin-based formulations to inactivate bacterial spores. J Appl Microbiol 2012, 113:1461-1467. 53. Bianco A, Kostarelos K, Prato M: Making carbon nanotubes biocompatible and biodegradable. Chem Commun 2011, 47:10182-10188. 54. Simmons TJ, Justin B, Hashim DP, Linhardt RJ, Ajayan PA: Noncovalent functionalization as an alternative to oxidative acid treatment of single wall carbon nanotubes with applications for polymer composites. ACS Nano 2009, 3(4): 865-870. 55. Allen B, Kichambare PD, Pingping G, Vlasova II, Kapralov AA, Nagarjun K, Kagan VE, Star A: Biodegradation of single-walled carbon nanotubes through enzymatic catalysis. Nano Lett 2008, 8(11):3899-3903. 56. Cui HF, Vashist SK, Rubeaan KA, Luong J, Sheu FS: Interfacing carbon nanotubes with living mammalian cells and cytotoxicity issues. Chem Res Toxicol 2010, 23: 1131-1147. 57. Jain S, Singh SR, Pillai S: Toxicity issues related to biomedical applications of carbon nanotubes. J Nanomed Nanotechnol 2012, 3:5.
www.sciencedirect.com
