Carbon nanotubes in hyperthermia therapy

Advanced Drug Delivery Reviews 65 (2013) 2045–2060

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Advanced Drug Delivery Reviews

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Carbon nanotubes in hyperthermia therapy☆

Ravi Singh a, Suzy V. Torti b,⁎a Department of Cancer Biology, Wake Forest School of Medicine, Winston Salem, NC 27157, USAb Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA

☆ This review is part of the Advanced Drug Delivery Renanotubes in medicine and biology — Therapy and dia⁎ Corresponding author. Tel.: +1 860 679 6503.

E-mail address: [email protected] (S.V. Torti).

0169-409X/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.addr.2013.08.001

a b s t r a c t
a r t i c l e i n f o
Article history:Accepted 1 August 2013Available online 8 August 2013

Keywords:Photothermal therapyCarbon nanotubesSingle-walled nanotubesMultiwalled nanotubesCancer therapy

Thermal tumor ablation therapies are being developed with a variety of nanomaterials, including single- andmultiwalled carbon nanotubes. Carbon nanotubes (CNTs) have attracted interest due to their potential for simul-taneous imaging and therapy. In this review, we highlight in vivo applications of carbon nanotube-mediatedthermal therapy (CNMTT) and examine the rationale for use of this treatment in recurrent tumors or those resis-tant to conventional cancer therapies. Additionally, we discuss strategies to localize and enhance the cancerselectivity of this treatment and briefly examine issues relating the toxicity and long term fate of CNTs.

© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20452. CNT-mediated thermal therapy: rationale and current status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2046

2.1. In vivo anticancer efficacy of CNT-mediated thermal therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20462.2. Selective delivery of CNTs to tumors for CNT-mediated thermal therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20472.3. Efficacy of CNMTT for treatment of resistant or recurrent cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20492.4. Enhancement of conventional cancer therapy by CNMTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20492.5. Beyond cancer therapy: novel applications of photothermal properties of CNTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2051

3. Clinical translation of CNMTT: accomplishments and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20513.1. Intratumoral dissemination of CNTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20513.2. Monitoring intratumoral distribution of CNTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20513.3. Optimizing heat localization and treatment efficacy of CNMTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20533.4. CNT toxicity and long-term fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2053

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2056Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2056References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2056

1. Introduction

Carbon nanotubes (CNTs) consist of one or more seamless, cylin-drical graphitic sheets of sp2 carbon atoms bonded together in anedgeless, hexagonal network [1]. Their combination of electrical,thermal and spectroscopic properties has evoked great interest; bio-medical researchers are using CNTs to develop new technologies for

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the detection, monitoring and therapy of diseases including cancer[2–6]. CNTs can be internalized easily by cells [7–9] and act as deliv-ery vehicles for drugs, nucleic acids, and imaging agents [7,10–23].Their unique optical, thermal and cancer selective properties affordthe possibility to engineer multiple diagnostic and therapeutic func-tions into a single particle [2,24]. This makes CNTs extremely suitablefor further biomedical development [25].

Minimally invasive, rapidly administered, and highly selectivenanotechnology-based thermal tumor ablation therapies are beingdeveloped with a variety of nanomaterials (reviewed in [26]).These include single walled carbon nanotubes (SWCNTs) [27],multiwalled carbon nanotubes (MWCNTs) [28], graphene [29], iron

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oxide nanoparticles [30], gold nanorods [31] and gold nanoshells [32,33].Heat-based cancer therapy requires elevation of malignant tissues tosupraphysiologic temperatures [34–39]. Exposure to high temperaturefor a sufficient amount of time causes physical damage such as proteindenaturation and membrane lysis and can increase oxidative stress[34,35,40,41]. These effects may be cytotoxic on their own, causingcoagulative necrosis or apoptosis [37,39]. Additionally, hyperthermictreatments that increase temperatures more modestly may enhance theanti-cancer efficacy of ionizing radiation or systemic chemotherapy regi-mens [40,42,43].

Dose limiting toxicities resulting from diffuse heating of non-tumortissues and the relative invasiveness of thermal ablative instrumen-tation have limited wider clinical use of thermal ablation for cancertherapy [44]. However, with recent refinements in technology, therole of such therapies should increase in the near future [26,45,46],and nanotechnology is playing a key role in these advances. Humanclinical trials are ongoing for a gold nanoshell based photothermalcancer therapy (Id: NCT00848042 on ClinicalTrials.gov) and for ironoxide nanoparticle-based magnetic thermotherapy [47,48]. Althoughthe results of these studies are promising, there remains significantroom to improve upon both the generation and localization of heat forthermal therapy.

In this arena, CNTs have emerged as promising, next generationagents for thermotherapy of cancer. Currently, significant efforts arebeing applied to develop CNT-based, clinical treatments. In this review,we will specifically high-light in vivo applications of CNT-mediatedthermal therapy (CNMTT) and examine the rationale for use of thistreatment in recurrent tumors or those resistant to conventional cancertherapies. Additionally, we will identify strategies to localize andenhance the cancer selectivity of this treatment, and briefly examineissues relating the toxicity and long term fate of CNTs.

2. CNT-mediated thermal therapy: rationale and current status

Carbon nanotubes offer an exceptional combination of attributes forthe development of the next generation of photothermal agents; chiefamong these is their ability to efficiently convert near infrared radiation(NIR) into heat [49]. Compared to other wavelengths of light, the trans-mission of NIR through the body is poorly attenuated by biologicalsystems [50,51]. Penetration of light through tissue is fundamental tophotothermal applications of nanomaterials for the treatment of non-superficial cancerous lesions in vivo [28]. Following exposure to NIR,CNTs enter an excited state and release vibrational energy that istransformed into heat, which can induce cell death [27,52].

CNTs possess an extremely broad electromagnetic absorbance spec-trum, covering the full spectra of both the NIR I and II windows [53],which correspond to the “optical transmission window” of biologicaltissues [50,51], and the radio frequency and microwave bands as well[54]. Further tuning of the photophysical properties of CNTs can beachieved by tailoring the wall number, diameter, and length of thenanotubes according to the “nano-attenna” effect [52]. Critically, NIRabsorption and energy transduction efficiency remains high across awide frequency range [53], which allows for flexibility in the choiceof both material characteristics (diameters from b2 nm to N30 nm;lengths from b100 nm to N 1 μm) and excitation wavelengths. Thisprovides great versatility to tailor size, shape, and surface properties tooptimize the tissue distribution of CNTs without a significant loss inthermal conversion efficiency. The broad electromagnetic absorbancespectrum of CNTs also offers a significant advantage over plasmonicallyheated nanomaterials (such as gold nanoshells and nanorods) forwhichthe excitation spectra are highly dependent upon the size and shape ofthe particles [2]. While direct comparisons are difficult to make, someestimates indicate that CNTs can achieve thermal destruction of tumorsat 10-fold-lower doses and 3-fold-lower power than is needed for goldnanorods [53]. However, in contrast to gold nanoparticles, which can besynthesized with great uniformity and have already been tested in

human clinical trials, production of uniform, well-characterized CNTsremains a significant hurdle for clinical translation.

CNTs exhibit both advantages and limitations in thermal therapywhen compared to iron oxide nanoparticles. The magnetic field usedto excite iron oxide nanoparticles as a means of generating heatoffers superior depth of energy penetration as compared to NIR,but the slower rate of heating induced by this technique leads to sig-nificant thermal diffusion away from the targeted area (discussed inSection 2.3), potentially increasing collateral damage to neighboringhealthy tissue [55]. A second drawback of this technique is that italso requires the removal of all metallic materials within the mag-netic field covering the treatment area including dental fillings,crowns and implants. On the other hand, iron oxide nanoparticles,like gold nanoparticles, share a significant advantage over CNTsbecause uniform preparations of iron-based nanoparticles can besynthesized and have already been tested in human clinical trials.

Inmost cases, the clinicalmodel for the use of CNTs as heat transduc-tion agents is based upon laser-induced thermotherapy (LITT) [33,39], aphotothermal ablation technique in which an NIR laser is used to heat atarget tissue, such as a tumor, above the thermal ablation temperaturethreshold of approximately 55 °C [33,56]. A major limitation of LITThas been an inability to consistently achieve thermoablative tempera-tures throughout the target lesion and to confine treatment exclusivelyto the tumor [57,58]. Therefore, to be of clinical benefit, CNTs mustgreatly improve the deposition of heat following NIR exposure withoutcausing any significant toxicity on their own.

2.1. In vivo anticancer efficacy of CNT-mediated thermal therapy

The efficacy of CNMTT for the treatment of locoregional tumorsin vivo has been demonstrated using syngeneic models of cancer inmice [53,59], rabbits [54], and in human xenografts grown in mice(reviewed in [49]). In most cases, CNTs are injected systemically ordirectly into the tumor, which is then exposed to an external NIR(typically a YAG or diode laser) or microwave source. As highlightedin Table 1, CNMTT has proven effective for the treatment of a wide vari-ety of cancer types both in vitro and in vivo. In this review, wewill focusour discussion upon recent developments in CNMTT treatment ofhuman cancer xenografts in animal models.

Initial in vivo studies of CNMTT focused on the use of SWCNTs, andthe results were mixed. Moon et al. demonstrated that followingintratumoral injection of SWCNTs into mice bearing flank xenograftsof human mouth carcinoma cells and NIR irradiation (3 W/cm2;3 min), the tumorswere completely destroyed [60]. Normal tissue adja-cent to the treated area was spared; however, the irradiation procedureitself resulted in significant burning of the heated area even in theabsence of SWCNTs. Significantly, no indication of tumor recurrence orapparent side effects of treatmentwere observed during severalmonthsof follow-up [60]. Huang et al. achievedmoremodest results following asimilar treatment [59]. Usingmice bearing syngeneic murine squamouscell tumors, the researchers intratumorally injected SWCNTs and irradi-ated the tumor with a low power (200 mW/cm2) NIR laser for 10 min.They observed that treatment resulted in a maximum tumor tempera-ture of approximately 55 °C, indicating that the thermal ablationthreshold was reached. However, while a reduction in tumor growthand a modest survival advantage were documented, this treatmentfailed to achieve a durable cancer remission. In contrast to Moon et al.[60], necrotic normal tissue was present adjacent to the treatmentsite, indicating significant heat transfer away from the targeted siteand into the surrounding non-tumor region. As discussed inmore detailin Section 2.3, recent work by Xie et al. [61] modeling the effects ofenergy deposition rate on the efficacy of CNMTT suggest that the slowerrate of energy deposition used byHuang et al. [59] as compared toMoonet al. [60] may have resulted in greater heat diffusion leading to lessertreatment efficacy and more collateral damage to surrounding tissue.

Table 1Preclinical assessment of CNMTT in cancer models.

Cancer type Experimental model Material (effective dose) Laser (wavelength; radiantexposure)

Reference

Brain (human) In vitro; adherent primary glioblastoma cells SWCNT (2.5 μg/well of 24 well plate) 808 nm; 600 J/cm2 [83]In vitro; non-adherent U251 glioblastoma cells SWCNT (10 μg/ml) 808 nm; 600 J/cm2 [184]In vivo murine flank tumor; primary glioblastoma cells MWCNT (10 μg — ex vivo) 808 nm; 600 J/cm2 [83]

Breast (human) In vitro; transformed human mammary epithelial cells MWCNT (50 μg/ml) 1064 nm; 90 J/cm2 [84]In vitro; adherent BT-474 cells SWCNT (5–10 μg/ml) 808 nm; 5130 J/cm2 [75]In vitro; adherent SK-BR-3 cells SWCNT (4 μg/ml) 808 nm; 900 J/cm2 [76]In vitro; non-adherent BT474 cells SWCNT (100 μg/ml) 800 nm;12 J/cm2 [185]In vitro; non-adherent MCF-7 cells SWCNT (not reported) 808 nm; 144 J/cm2 [78]In vitro; non-adherent MDA-MB-231 cells gold coated SWCNTs

(estimated 0.5–1 × 104 SWCNTs per cell)850 nm; 0.5 J/cm2 [91]

In vivo murine flank tumor; MDA-MB-231 cells MWCNT (100 μg IT) 1064 nm; 90 J/cm2 [22]In vivo murine flank tumor; transformedhuman mammary epithelial cells

MWCNT (100 μg IT) 1064 nm; 90 J/cm2 [84]

Breast (murine) In vitro; adherent EMT6 cells SWCNT (3.5 μg/ml) 980 nm; 120 J/cm2 [82]In vitro; adherent EMT6 cells SWCNT (50 μg/ml) 980 nm; 60–150 J/cm2 [106]In vivo murine flank tumor; 4T1 cells SWCNT (70 μg IV) 808 nm; 180 J/cm2 [53]In vivo murine flank tumor; 4T1 cells SWCNT (100 μg IV) 808 nm; 300 J/cm2 [69]In vivo murine flank tumor; EMT6 cells SWCNT (20–25 μg IT) 980 nm; 300 J/cm2 [73]In vivo murine flank tumor; EMT6 cells SWCNT (1 mg IT) 980 nm; 450 J/cm2 [82]

Cervical (human) In vitro; adherent Hela cells MWCNT (10 μg/ml) 1064 nm; 128 J/cm2 [144]In vitro; non-adherent HeLa cells SWCNT (25 μg/ml) 808 nm; 168 J/cm2 [27]

Erlich ascites(murine)

In vitro; non-adherent Erlich ascitic carcinoma cells MWCNT (100 μg/ml) 780–1400 nm; 315 J/cm2 [186]

Kidney(human)

In vitro; adherent CRL 1932 cells Nitrogen-doped MWCNT(approx. 83 μg/ml)

1064 nm; 720 J/cm2 [52]

Kidney (murine) In vitro; non-adherent RENCA cells MWCNT (100 μg/ml) 1064 nm; 135 J/cm2 [28]In vitro; non-adherent RENCA cells MWCNT (100 μg/ml) 1064 nm; 4590 J/cm2 [98]In vivo murine flank tumor; RENCA cells MWCNT (100 μg IT) 1064 nm; 90 J/cm2 [28]

Liver (human) In vitro; adherent HepG2 or CRL 4020 cells MWCNT (1–50 μg/ml) 808 nm; 7680 J/cm2 [49]in vitro; adherent SK-BR-3 cells SWCNT (20 μg/ml) 1064 nm; 4 J/cm2 [74]

Lymph (human) In vitro; non-adherent Daudi Burkitt's lymphoma cells SWCNT (90 μg/ml) 808 nm; 2100 J/cm2 [94]Mouth (human) In vivo murine flank tumor; KB epidermoid mouth

carcinoma cellsSWCNT (12 μg IT) 808 nm; 684 J/cm2 [60]

neuroendocrine (human) In vitro; adherent stNB-V1 neuroblastoma cells MWCNT (5–10 μg/ml) 808 nm; 3000 J/cm2 [77]Prostate (human) In vitro; non-adherent PC-3 cells MWCNT (100 μg/ml) 1064 nm; 4590 J/cm2 [98]

In vivo murine flank tumor; PC-3 cells MWCNT (50 μg IT) 1064 nm; 175 J/cm2 [63]Skin (murine) In vivo murine flank tumor; SCCVII squamous

carcinoma cellsSWCNT (60–100 μg IT) 785 nm; 120 J/cm2 [59]

IT = intratumoral injection; IV = intravenous injection.

2047R. Singh, S.V. Torti / Advanced Drug Delivery Reviews 65 (2013) 2045–2060

Following synthesis, SWCNTs are amixture ofmetallic and semicon-ducting SWCNTs. Differences in the density of state at the Fermi level ofmetallic SWCNTs (which have a finite value ≠ 0) and semiconductingSWCNTs (which have a value of zero) imply that metallic SWNTsmore efficiently absorb and convert NIR into heat, and this was recentlyconfirmed by Murakami et al. [62]. Consistent with the concept thatCNTs with a more metallic nature possess enhanced NIR thermalconversion efficiency, both Burke et al. [28] and Ghosh et al. [63] report-ed that MWCNTs, which are inherently more metallic than unpurifiedSWCNTs, were 20–100 fold more efficient than bulk SWCNTs atconversion of NIR into heat. As shown in Fig. 1, in vivo studies demon-strated durable remission and long-term survival (N6 months post-treatment) after intratumoral injection of MWCNTs into tumors im-planted in the flanks of nude mice combined with exposure to NIR(3 W/cm2; 30 s) [28]. Significantly, this result was achieved usingone-sixth of the energy Moon et al. [60] required to achieve a similarresult using SWCNTs. Because of this, laser treatment in the absenceof MWCNTs resulted in only minimal superficial burning [28]. In thesame study, electron microscopy studies revealed that at least a portionof MWCNTs remained intact at the injection site, but no toxicity wasdetected [28]. Ghosh et al. observed similar results for in vivo treatmentof tumors using MWCNTs and NIR [63]. Moreover, they confirmedthe importance of CNT dispersion on the efficacy of such therapy. Byself-assembly, they encased MWCNTs in DNA, which disentangled thetubes. Compared to dispersions of MWCNTs prepared in the absence

of DNA, well dispersed DNA encased MWCNTs were more efficient atconverting NIR into heat, which led to more effective tumor treatmentin mice [63].

As previously noted, CNTs can be heated by externally deliveredmi-crowave radiation. Therefore, CNTs may be capable of non-invasivelytreating tumors in any part of the body, a capability currently not sharedby NIR laser-based treatments. In the first study to investigate cancertreatment using an external (non-contacting) microwave source toheat intratumoral SWCNTs, treatment of VX2 hepatocellular carcinomaxenografts in rabbits resulted in complete thermal necrosis of thetumor. No toxicity was seen but there was a 2–5 mm zone of thermalinjury to the surrounding liver [54]. To date, studies on microwave orradiofrequency heating of CNTs for cancer therapy are extremely limit-ed and more research will be needed to determine if this promisingstrategy is clinically viable.

2.2. Selective delivery of CNTs to tumors for CNT-mediated thermal therapy

Fueled by the promise demonstrated by these early studies,researchers are now evaluating strategies to selectively deliver CNTsto tumor sites following systemic administration. One such strategy,known as the enhanced permeation and retention effect (EPR), pro-poses that increases in tumor accumulation can be achieved bynanoparticles that remain in circulation for long periods due to thenanoparticles' ability to extravasate through the leaky vasculature

Laser only

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*p<0.0001**p=0.0004

***p=0.0395C

Fig. 1. Treatment of tumor bearingmice by CNMTT reduces tumor growth and increases long term survival. Nu/numicewere implanted s.c.with RENCA tumors and divided into groups of10. Micewere either left untreated, treatedwithMWCNT alone, treatedwith laser alone, or treatedwith the combination ofMWCNT and laser (3 W/cm2; 30 s). (A) Photographs at day 21post-treatment of representativemice from groups treatedwith laser only, untreated controls, ormice treatedwith 100 μg ofMWCNT plus laser. (B)Mice treatedwith the combination ofMWCNTs and laserwere injectedwith a range ofMWCNT doses. Tumor sizesweremeasured every 2 days. Means and standard errors are shown. Control groups (untreated, treatedwithMWCNTs alone, or treatedwith laser alone)were statistically identical. There is a dose-dependent attenuation in tumor growth after 30 s of NIR laser treatment ofMWCNT-loaded tumors(P b 0.0001). (C) MWCNT-based photothermal therapy increases long-term survival of tumor-bearingmice. Survival of mice treated as described in (C)was assessed for 10 months aftertreatment. Kaplan–Meier curves demonstrate a significant increase in survival inmice treated with all doses of MWCNTs plus laser (P b 0.0001 vs all controls). Survival curves for controlgroups were statistically identical (P N 0.775).Adapted and reproduced with permission from Burke et al. PNAS 106 (2009) 12897–12902.


surrounding the tumor and enter the tumor site [64]. Like manynanoparticles, the blood circulation half-life and tumor accumulationof CNTs can be increased in mice by coating nanoparticles with stericstabilizers such as polyethylene glycol (PEG),which inhibit nanoparticleblood clearance bymacrophages and other components of themononu-clear phagocyte system [10,65–68].

Recently, a significant step toward the development of tumor selec-tive, systemically delivered CNTs for photothermal ablationwas achievedby Robinson et al. [53]. The researchers coated, short (140 nm) SWCNTswith a PEG-funtionalized phospholipid by self-assembly and theninjected the CNTs into mice bearing 4T1 murine breast tumors at adose of 3.6 mg/kg via the tail vein. Three days later, the inherent NIRphotoluminescence of the SWCNTswas quantified using an optical imag-ing system to confirm the accumulation of SWCNTs at the tumor site.Following exposure to NIR (0.6 W/cm2; 5 min), complete tumor ablationwas observed onmice previously administered SWCNTs. Thesemice sur-vived without recurrence for the duration of the six month study and notoxicity was seen [53].

In another study, Liu et al. systematically evaluated the relationshipbetween the length and density of PEGylation on the SWCNT surfaceand the biodistribution, tumor accumulation and photothermal ablationefficacy of PEG-functionalized SWCNTs injected intravenously intomice[69]. They observed long blood circulation half-lives (16–21 h) forheavily PEGylated SWCNTs and high tumor uptake. However, thiscame at the cost of extensive accumulation of SWCNTs in the mouseskin dermis. They found that by controlling the degree of PEGylationto achieve a blood circulation half-life of 12–13 h, theywere able to bal-ance high tumor uptake of the SWCNTs with low skin retention. Twodays after intravenous injection of the SWCNTs into tumor bearingmice, the researchers exposed the tumors to NIR, achieving a surfacetemperature of over 50 °C. In contrast, the temperature rise in tumorstreated with NIR in the absence of SWCNT injection was only 1–2 °C.

Significant reduction in tumor growth was observed in the SWCNTsand NIR treated mice, and three of the seven mice treated exhibitedcomplete regression for the two week study [69]. This study suggeststhat fine tuning the surface properties of CNTs is critical to enhancingtheir passive tumor targeting capabilities.

Notably, the intravenously injected nanotubes used by Robinson[53] or Liu [69] were not modified to display a specific targeting ligand,nor is it known if they were they internalized by the cancer cells them-selves. Attachment of ligandswith an enhanced binding affinity for can-cer specific biologicalmoieties is one strategywhichmay allow formoreselective therapy. This can be accomplished by conjugation of peptides,proteins or antibodies to the surface of CNTs, and has been shown to in-crease the specificity of CNT tumor targeting following intravenousinjection in mice [27,68,70,71]. Targeting CNTs to cancer cells in vitrousing agents including folic acid [72–74], the breast cancer associatedreceptor Her2 [75,76], and anti-GD2, a monoclonal antibody targetinga carbohydrate antigen overexpressed in neuroblastomas [77] hasbeen shown to increase the specificity and efficacy of CNMTT as com-pared to non-targeted tubes. Additionally, CNTs can effectively displaymore than one targeting ligand on their surface which is a strategythat can be used to expand both the tropism and specificity of cancer-targeted CNTs. For example, SWCNTs conjugated to antibodies targetingHer2 and insulin-like growth factor 1 receptor (IGF1R) attached toSWCNTs [78] were shown to be effective at targeting CNMTT to cellsexpressing one or both of the targeted receptors.

The benefits of actively targeting nanoparticles to tumors in vivoremain the subject of much debate [79], and more research is neededto determine if active targetingwill offer an additional benefit for deliv-ery of CNTs. The underlyingmechanisms bywhich CNTs are taken up bycells are still under investigation with both energy-dependent internal-ization mechanisms (e.g. phagocytosis; clatherin or caveolae mediatedendocytosis) and passive diffusion through the plasma membrane


(e.g. “nanoneedle” effects) apparently playing a role [9]. As noted above,comparative studies between actively targeted and non-targeted CNTsgenerally indicate that targeted CNTs are taken up to a greater degreethan untargeted tubes. Give the many non-specific mechanisms throughwhich CNTs can bind to and enter cells, precisely how targeting increasesthe binding and/or uptake of actively targeted CNTs remains to be deter-mined. It is conceivable that the PEG coating commonly used tofunctionalize the CNT surface may reduce the interaction of coatedCNTs with cells; introduction of a binding moiety to the PEGylated CNTwould then greatly increase the targeting specificity. To date, direct com-parisons of the binding and uptake of PEGylated and uncoated CNTs havenot been conducted. Comparative studies of nanoparticle binding kinet-ics are complicated because the physical properties that influence cellularinteractions of nanoparticles also affect their solution dynamics [80].Nanoparticles diffuse, settle and agglomerate in cell culture media as afunction of systemic and particle properties (e.g. media density andviscosity, particle size, shape and density). Therefore the delivered doseof nanoparticles in cell culture is a function not only of cell binding anduptake pathways, but of the rate of transport in solution as well. Todate,most studies using targeted CNTs fail to account for changes in solu-tion dynamics induced by structural alterations in the CNTs caused by theaddition of targeting ligands, and further researchwill be needed to thor-oughly understand how such alterations influence binding specificity.Nevertheless, there is strong evidence that internalization of CNTs priorto exposure to NIR can improve the treatment efficacy of CNMTT [75].For example, Zhou et al. demonstrated that SWCNTs coated with aphospholipid-PEG conjugate selectively accumulate intracellularly atthe mitochondrial membrane [81]. Their proximity to mitochodriaallowed for selective destruction of these organelles following NIR expo-sure, inducing mitochondrial depolarization, cytochrome c release, andcaspase 3 activation. Treatment of murine breast cancer tumors in vivowith these modified SWCNTs reduced tumor growth and induced com-plete tumor regression in some mice [82].

2.3. Efficacy of CNMTT for treatment of resistant or recurrent cancers

Thermal ablation therapies based upon CNTs are being tested for thetreatment of cancers that are highly resistant to current therapies, in-cluding stem-cell like cancer sub-populations [83,84]. In many typesof tumors, cancer stem cells (CSCs) have been putatively identified asself-renewing, therapy-resistant populations [85].

In glioblastomas and other brain tumors, the CD133 receptor ap-pears to be a CSCmarker associatedwithmalignancy, tumor recurrence,and poor survival [86–88]. CD133+ subpopulations in glioblastoma areenriched following radiotherapy, are radio and chemotherapy resistant,and may be responsible for tumor recurrence following treatment[86,89,90]. Treatment strategies based on targeting this subpopulationmay prevent the development of resistance to therapy. To test this hy-pothesis, Wang et al. conjugated a monoclonal antibody directedagainst CD133 to MWCNTs [83]. They observed specific internalizationof these targeted MWCNTs in primary clinical isolates of glioblastomathat expressed CD133, but not in cells which did not. To determine thein vivo efficacy of this treatment, CD133 expressing glioblastoma cellswere pre-treated with targeted MWCNTs before inoculation into mice.The cells took up the MWCNTs, and xenograft growth was abolishedafter NIR exposure. This key study demonstrated the potential forCNTs to treat glioblastomas and other currently untreatable cancers.

Another recent report describes a novel application of the CNMTTtechnique to target invasive CSCs in systemic blood circulation [91].In this study, Galanzha et al. used the photoacoustic (PA) andphotothermal (PT) properties of CNTs for the detection and eliminationof circulating CSCs, which are thought to be the primary drivers of met-astatic tumor spread [91]. The development of technology to purgethese cells from the vasculature of cancer patients could reduce the in-cidence of metastatic disease. To accomplish this, the researchersconstructed NIR-absorbing, gold plated SWCNTs and conjugated them

to anti-human CD44 antibodies. These particles selectively labeledcirculating human breast CSCs (which overexpress CD44 [92]) in theblood stream. Rare CD44+ circulating cancer stem cells bindingnanoparticles were identified in the vasculature of nude mice whichbore human breast cancer xenografts by detection of photoacousticwaves generated by excitation of the nanoparticle-labeled cancer cellsusing a low powered laser [91]. Furthermore, these cells could be ablat-ed following more extended irradiance with NIR [91].

Importantly, CNTs may be superior to other heat deliverymodalitiesin ablating cancer stem cells. Burke and co-authors compared theresponse of breast cancer stem cells (BCSCs) to both conventionalhyperthermia and CNMTT to determine the relative therapeutic efficacyof each approach for the treatment of these cancer cells [84]. Key resultsof this study are shown in Fig. 2. Notably, BCSCs exhibited high basalexpression levels of heat shock protein 90 (HSP 90) which contributedto their ability to tolerate conventional hyperthermia treatments(modeled by water bath heating) that were lethal to non-stem breastcancer cells [84]. BCSCs were found to be resistant to hyperthermiaacross a range of temperatures, and heat treatments did not reducethe long-term proliferative capacity of these cells. A significant enrich-ment of BCSCs was detected in the surviving fraction of a mixedpopulation of stem and non-stem breast cancer cells treated with con-ventional hyperthermia. In contrast, the researchers were able to over-come the resistance to hyperthermia observed in BCSCs through the useof CNMTT. Furthermore, BCSCs that survived CNMTT did not retainlong-term proliferative capabilities. The researchers generated precisetemperature increases in mixed or isolated populations of BCSCs andnon-stem breast cancer cells by exposing the cells toMWCNTs followedby NIR irradiation, which resulted in cell death that was proportional tolaser exposure time. In vivo treatment by CNMTT induced completeregression of BCSC-driven tumors in mice for the duration of the30 day study. In contrast, control groups exhibited N80% mortality atidentical time points. Based on these findings, CNMTT may represent arapid, minimally invasive approach for the simultaneous eliminationof both the bulk breast tumor and the BCSC components of tumorsand represents a significant therapeutic advance for the treatment ofrefractory, stem cell-driven cancers [84].

Flow cytometric characterization of the cells following treatment in-dicated that CNMTT, but not conventional hyperthermia, led to rapidmembrane permeabilization and necrotic death in treated cancer cells[84]. The high surface temperature of NIR-stimulated MWCNTs [93]may irreversibly permeabilize cell membranes [94,95], leading to therapid cytolysis observed in CNMTT-treated cells. Therapies that prefer-entially cause necrotic death may be therapeutically advantageous bybypassing resistance mechanisms to apoptotic cell death because theydo not provide selective pressure toward the emergence of treatmentresistant cancer cell clones [96,97]. The significance of this for CNMTTis exemplified by the fact that elevated HSP90 in BCSCs conferred atleast partial protection to classical hyperthermic cell death, but did notprotect BCSCs fromnanotube-mediated hyperthermic death [84]. Fisheret al. also indicated that CNMTT leads to a necrotic rather than apoptoticcell death mechanism [98]. Nevertheless, necrosis has not been univer-sally observed following treatment with NIR and MWCNTs [99], and todate, extensive research has not been conducted to determine factorsthat may influence the mechanism(s) of cell death induced by CNMTT.

2.4. Enhancement of conventional cancer therapy by CNMTT

Hyperthermia synergistically enhances tumor cell cytotoxicitywhencombinedwith chemotherapy or radiotherapy, in part by increasing thepermeability of tumor vasculature, which can enhance the delivery ofdrugs into tumors [41]. Thus, in addition to ablation of cancer cells,the thermal effects generated by CNMTT may enhance the efficacy ofother therapies. Such strategies may enable the development of thera-peutic agents with increased cancer selectivity, reduce the dose

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Fig. 2. Breast cancer stem cells are resistant to conventional hyperthermic cell death but sensitive to CNMTT. (A) Relative viability of cancer cells 24 h after water bath heat treatment as amodel of conventional hyperthermia. Stem cell-like breast cancer cells (BCSCs) or bulk breast cancer (non-stem) cells were heated in awater bath at 47 °C for 0–60 min. MTT absorbancevalues were normalized to the untreated condition (“0” minutes heat shock). The results clearly show that BSCC subpopulations are more resistant to heat than breast cancer cells as awhole. (B) Sub-lethal hyperthermia enriches for the BCSC phenotype in bulk breast cancer cells. Changes in the CD44high/CD24low stem cell fraction of surviving bulk breast cancercells 24 h after water bath heat shock at 43 °C, 45 °C or 47 °C were determined by flow cytometry. Shown are mean percent changes in the CD44high/CD24low cell fraction normalizedto the Untreated condition (which is set as 1.0, i.e. “0 percent change”). Dashed lines indicate the 95% C.I. for the Untreated condition. All heat treatments led to significant increases inthe stem cell fraction (P b 0.0001) relative to Untreated. (C) In contrast, BCSC and non-stem breast cancer cells are equally sensitive to CNMTT. Cancer cells were heat treated to specifictemperatures by the combination of MWCNTs and NIR laser irradiation and the relative viability of BCSC and non-stem breast cancer cells was determined byMTT 24 h later and normal-ized to the “Untreated” conditions. “CNT Only” describes samples that were mixed withMWCNTs but were not laser treated. “CNT + Laser” describes samples that were heat shocked tothe indicated final temperatures by the combination of 50 mg/ml MWCNTs and 3 W laser radiation. In contrast to the water bath heating results, no significant difference between thesensitivity of stem-like and non-stem breast cancer cells was observed, and (D) no enrichment of the stem cell phenotype (quantified as in (B)) in viable cells 24 h after CNMTT wasdetected.Adapted and reproduced with permission from Burke et al. Biomaterials 33 (2012) 2961–2970.


necessary for efficacy, decrease the toxicity of such treatments, and thusincrease their therapeutic index.

For example, NIR irradiation of MWCNTs to sublethally heated cancercells increased the uptake of co-delivered chemotherapeutic drugs andenhanced cancer cell death both in vitro and in vivo in a murine ascitestumor model [100]. Excitation of CNTs by exposure to an appropriatelytuned radio frequency field has been shown to permeabilize cell mem-branes to allow cell uptake of normally impermeable drugs or geneexpression vectors both in in vitro and in vivo in brain tissue [101]. Sim-ilarly, treatment of cancer cells or tumor bearing mice using SWNTschemically conjugated with platinum-based chemotherapeutics [102]or loaded with doxorubicin by hydrophobic interactions [103] and com-bined with NIR mediated photothermal heating were significantly more

effective than either therapy alone. Dual mode carbon nanomaterialshave been developed to allow excitation by a single wavelength of lightto initiate both photothermal and photodynamic events, allowing forsimultaneous generation of therapeutic heat and reactive oxygen species[62,104].

Conjugation of antigens to the surface of CNTsmay enhance immuneresponses by increasing the uptake of such antigens by dendritic cellsand macrophages [105]. Recently, the combination of an immunologi-callymodified nanotube and CNMTTwas shown to generate a sustainedanti-tumor immune response in mice following treatment [106].Glycated chitosan (GC), an immunoadjuvant, was used as a surfactantto disperse SWCNTs. BALB/cmice bearing syngeneic EMT6 flank tumorswere injected intratumorally with GC coated SWCNTs (SWCNT-GC)


then exposed to a 980 nm laser (0.75 W/cm2 for 10 min). All micetreated with the combination of laser and SWNT-GC survived for atleast 100 days following treatment. In contrast, only 43.75% of micetreated with laser and SWNTs without GC, 25% of mice treated withlaser and GC without SWCNTs, and 12.5% of mice in the laser onlygroup survived. Importantly, mice that were successfully treated bythe combination of laser irradiation and SWNT-GC did not developtumors following rechallenge with EMT6 cells100 days after the initialtumor inoculation. This study shows the combination of laser irradiationand immunologicallymodifiednanotubes can induce systemic antitumorresponse through a local intervention, with few adverse side effects[106].

2.5. Beyond cancer therapy: novel applications of photothermal propertiesof CNTs

Although most studies have focused on anti-tumor applications ofCNMTT, additional applications have also been proposed. Kosuge et al.recently investigated the use of SWNT-mediated photothermal ablationof inflammatory macrophages in a mouse model of atherosclerosis[107]. Following the creation ofmacrophage-rich atherosclerotic lesionsin the carotid arteries, mice were injected with fluorescently taggedSWNTs, which accumulated in themacrophages. Excision of the arteriesand ex-vivo illumination with NIR induced heating and apoptosis selec-tively in SWNT-containing macrophages, providing a proof of principledemonstration that the photothermal properties of CNTs may be usefulin treatment of vascular inflammation. Another group used laser excita-tion of carbon nanohorns to induce expression of genes driven by a heatshock protein-responsive promoter in livingmice [108]. The use of NIR-activated SWNTS for ‘remote control’ of gene expression in livinganimals may represent both a powerful experimental tool and a newtherapeutic venue. Similarly, a recent report indicates that CNTs alsomay be useful for the development of implantable bioelectronic devicesoperated by laser irradiation from outside the body [109]. In this study,a unique CNT-based photothermal-electrical (PTE) converter is de-scribed. This device is able to convert the thermal energy generated byNIR irradiated with NIR light and convert it to electrical power. Devicespowered by this system could be implanted beneath the skin and acti-vated by laser irradiation, providing a new opportunity for biomechan-ical development or possibly for a remotely activated drug deliverysystem.

3. Clinical translation of CNMTT: accomplishments and challenges

As described above, proof of principle studies in animals haveestablished that CNMTT can address several limitations of contem-porary clinical thermal ablation methodologies: (1) the procedure isminimally-invasive, potentially expanding the type and location oftumors that can be treated; (2) because each nanoparticle generatesheat in response to stimulation electromagnetic radiation, a uniformtemperature distribution can be generated throughout the tumormass; and (3) the heated region is defined by both the location of theCNTs and the position of the irradiation source, confining treatment tothe intended lesion and diminishing off-target toxicities. However, asdescribed in the following sections, significant hurdles still must beaddressed before CMNTT will be a clinically viable modality.

3.1. Intratumoral dissemination of CNTs

Delivery of CNTs and other engineered nanoparticles across large(N1 cm) tumors represents a major, under-investigated need for bio-medical use of nanomaterials [110–116]. As highlighted in Fig. 3, multi-ple inter-related properties influence particle distribution; these aredependent not only on the characteristics of the particular type ofCNT, but also upon the environment in which the particle is to beused and the strategy bywhich itwill be delivered. Asmore information

on the use of CNTs in animal models becomes available, it is clear thatlike other nanoparticles, even minor structural or chemical changes tothe particle surface significantly alter the biodistribution of CNTs [117].

The in vivo intratumoral distribution of CNTs largely is related to thephysicochemical properties of the tubes, which include length, diame-ter, degree of agglomeration, surface chemistry, and flexibility [25].The interactions of these properties with the pathophysiology and anat-omy of the target site will dictate the biological interactions of the CNTs[118]. Furthermore, it is important to realize that small molecules andproteins frequently adsorb onto the surface of nanoparticles, creatingan interface at which all further interactions occur. The environmentobviously influences the composition of this interface, and creates anadditional factor that is rarely taken into account when evaluating theefficacy and distribution of CNT preparations.

Current delivery strategies, which are limited to either intravenousinjection or direct intratumoral injection do not address the issue oflimited macromolecular diffusion of CNTs once they enter the tumormicroenvironment. Recent work suggests that this may be an issue ofconsiderable complexity [119]. Using intravital microscopy, the authorsdemonstrated that SWNTs and spherical particles with similar surfacecoating, area, and charge nevertheless exhibit different abilities toextravasate into tumors. Rather remarkably, the relative penetrance ofthese particleswas dependent on tumor type: spherical particles prefer-entially extravasated into LS174T colon tumors, whereas SWNTs accu-mulated preferentially into U87MG gliobastoma tumors; neitherparticle extravasated into SKOV-3 ovarian adenocarcinoma tumors.The mechanistic basis for this difference remains to be determined,but these results suggest that biological characteristics of the tumor it-self will be an important determinant in the optimization of therapeuticnanoparticles.

Among the options for enhancing intratumoral dissemination, adense PEG coating is known to improve penetration of largenanoparticles in tissue [120], but the role of PEG coating on CNT diffu-sion in tumors has not yet been assessed. Furthermore, pressure-driven infusion techniques such as convection enhanced delivery(CED),which rely on a pressure gradient rather than diffusion to distrib-ute macromolecules, may be of benefit. CED of nanomaterials is an areaof current research [121] but has not yet been tested for the deliveryof CNTs. Positive indications for use of CED for CNTs include: (1) CNTscan be synthesized with diameters (b30 nm) far smaller than the esti-mated pore size in the extracellular matrix (ECM) of many tumors[112,122,123]; (2) CNTs tend to align parallel to the direction of fluidflow [124]; and (3) their diffusion can be enhanced by selectivelytuning their surfaces to the physical properties of porous media[119,125–128] like the ECM.

More research is needed to identify specific properties or strategiesthat will enhance intratumoral transport of CNTs both during andafter infusion. It will be particularly important to these studies thatCNTs for use in biomedical applications have well-defined preparationchemistries combined with rigorous physicochemical characterization,and, as discussed below, that their in vivo performance can be trackedby non-invasive imaging techniques.

3.2. Monitoring intratumoral distribution of CNTs

For the clinical application of CNMTT for cancer treatment, CNTsmust be specifically engineered to be compatible with an imagingmodality to spatially define the margins of the target lesion, assess thedistribution of injected CNTs within the tumor, and to allow for place-ment of the electromagnetic radiation source. The versatility of CNTsas a platform technology has seen the development of CNT-basedimaging agents for use as magnetic resonance imaging (MRI) contrastenhancers[22,129–132], positron and single photon emission correla-tion spectroscopy (PET/SPECT) [19,70,133,134], fluorescence imaging[71,129,135] and photoaccoustic and Raman imaging [136]. Carbonnanotubes offer several advantages for targeted molecular imaging

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Nanotube parameters• Length• Diameter/wall number• Functionalization/coating• Concentration• Aggregation

Infusion parameters• Hydration• pH• Flow rate• Infusion volume• Viscosity• Osmolarity• Co-infusate

Fig. 3. Effective delivery of CNTs to the tumor is a central challenge for using CNTs to treat cancer. In spite of the dynamic growth of innovative nanoplatforms, the path from bench tobedside is still challenging. Barriers within the tumor microenvironment, including vascular heterogeneity, extracellar matrix pore size, high interstitial pressure, and immune cell infil-tration, limit the uniform penetration of nanotherapeutics, lead to inefficient delivery and reduce the potential efficacy ofmany treatments. Currently, there is an insufficient understand-ing of how CNTs interact with the tumor microenvironment. As shown here, there is a patchwork of inter-related properties that are dependent not just on the characteristics of thenanoparticle, but also upon the environment in which the particle is to be used and the strategy by which it will be delivered. These properties influence the pharmacologic behaviorof the CNTs in vitro and in vivo. Failure to appropriately characterize CNTs has led to a bottleneck in their clinical translation due to inadequately designed studies at the pre-clinicallevel that could bridge cell-culture-to-rodent-to-human studies. Identification of CNT physicochemical and surface characteristics under specific infusion parameters and considerationof how the pathophysiology and anatomy of the target site influence the pharmacologic properties of CNTs will be necessary for the development of pharmaceutical grade material forclinical use.


techniques, including their ability to deliver large numbers of imagingagents for each targeted molecular recognition, which can improvethe sensitivity of imaging, and their ability to simultaneously displayseveral different types of agents to perform multimodality imaging.

A unique feature of SWCNTs is that they possess an intrinsic fluores-cence in the secondnear-infraredwindow (NIR II, 1.1–1.4 μm)upon ex-citation by laser emission in the traditional near-infrared region (NIR I,0.75–0.9 μm) [137]. Compared to the NIR I window, the longer wave-length emission in NIR II offers great promise for the development ofSWCNT imaging agents due to minimal autofluorescence and tissuescattering, which allows for deeper anatomical penetration and greaterspatial resolution than is currently available for traditional NIR imagingagents [137]. Studies by Welsher et al. indicate the SWCNT-basedfluorophores can be imaged deep inside mice following excitationwith a modest laser power [135]. Similarly, injection of SWCNTs intothe blood allows for quantifications of blood velocity and mapping ofsmall vessels with an accuracy that is far beyond the capabilities ofultrasonography [137]. Initial studies show that intravenously injectedSWNTs could be used as photoluminescent agents for in vivo tumorimaging in the 1.0–1.4 μm emission region and as NIR absorbers andheaters at 808 nm for photothermal cancer therapy [53]. Additionalclinical applications of these tubes potentially could include contrastenhancement for delineation of tumors/metastasis, or for mappingintratumoral vasculature.

Several additional studies have examined dual modality CNTs forboth imaging and photothermal applications. In one study, Ding et al.

constructed MWCNTs containing increasing amounts of ferrocene, aniron-based catalyst used in the production of CNTs [22]. These iron-containing MWCNTs were studied for their potential use as dualmodality agents for both MR contrast enhancement and photothermalenergy transduction. The researchers demonstrated that iron-containingMWCNTswere effectiveMRcontrast agents. In vivoMR imagingprovideda clear indication of intratumoral distribution of the CNTs, allowing forimage guided intervention. Following multiple rounds of NIR exposure,the contrast enhancing and heating properties of the MWCNTs did notchange, even after reaching thermal ablative temperatures [22]. Wheninjected into tumor bearing mice, the iron-containing MWCNTs couldbe detected at the injection site before and during heating, and until theend of the study one week later [22]. An additional advantage conferredby this imaging strategy was the ability to monitor temperature in realtime usingMR thermometry (see Section 2.3). Conceptually, this demon-strates that multiple or fractionated laser treatments could be targeted tothe tumorwithout the need for additional injections, and the distributionof the MWCNTs could be monitored over time.

In addition to MR imaging, photoacoustic molecular imaging usingCNTs has shown some early promise [138]. Photoaccoustic imagingrelies upon detection of the shock wave that is generated followingheating of the CNTs by an external NIR source. Recent studies by Zhouand colleagues indicate that CNTs targeted to αvβ3 integrin could bedesigned to possess both high photoaccoustic contrast and efficienttargeting of αvβ3 integrin positive U87 human glioblastoma tumors inmice, suggesting that photoacoustic molecular imaging with targeted


SWNTs has the potential for use as a tumor diagnostic method [138].In another study, Zarov and colleagues developed golden carbonnanotubes (GNTs) consisting of SWCNTs encased in gold layers [139].The gold coating not only enhanced the photothermal transduction effi-ciency of the tubes, it introduced a high plasmon resonance at 850–900 nm, indicating the GNTs could be used for both non-invasivephotoaccoustic (PA) imaging and photothermal cancer therapy [140].Moreover, cancer diagnosis and therapy applications using these tubescould be performed with the same laser in real time using a uniquein vivo flow cytometry based imaging system [139]. Using anti-CD44labeled GNTs, the researchers were able to detect single CD44+ circu-lating breast cancer stem cells in the blood stream of tumor bearingmice following NIR irradiation. They postulate that these cells could beeliminated by increasing the laser power [139].

From these studies, it is clear that CNTs can be developed for bothcancer imaging and effective photothermal therapy and are compatiblewith current imaging technologies. Future developments of such agentswould have a dramatic effect on non-invasive cancer diagnosis, targetedtherapies, selection of patient specific treatments, and monitoring ofcancer progression or recurrence.

3.3. Optimizing heat localization and treatment efficacy of CNMTT

Heat delivery for photothermal applications is dependent upon thetotal laser energy incident upon the target and the efficiency of the tar-get at converting that energy into heat. Temperature elevation byCNMTT is limited by the maximum laser output, penetration of thelight, the CNT concentration in the tumor target, and heat dissipationaway from the tumor target. Heat dissipation away from the tumor tar-get is a complex process that is dependent upon environmental factorsincluding proximity to heat absorbers (i.e. blood vessels and neighbor-ing normal tissue), solvent, and the substrate (tissue type) into whichthe CNTs are dispersed. This process is dynamic, making it essential todevelop strategies to reproducibly control the spatial and temporaldistribution of heat used to ablate tumors [141].

To reduce off-target damage due to thermal diffusion, the total ener-gy deposited into the tissue should be minimized such that only theamount of heat needed for treatment is delivered to the targeted area.To accomplish this, it is necessary to monitor in real-time spatio-temporal changes in temperature resulting from CNMTT using infraredcameras [59,141] ormagnetic resonance imaging (MRI)-basedmethods[28,61]. Infrared cameras are useful for optimizing both CNT concentra-tion and NIR irradiation parameters in model tissue and in tumor bear-ing mice, but cannot accurately map temperature deep within tissue.However, a more clinically relevant method of non-invasive tempera-ture mapping is a MRI-based thermometry method known as protonresonance frequency (PRF) MR temperature mapping (reviewed in[142]). This technique allows for superposition of both temperatureinformation and anatomical images at any depth, and is currentlyused clinically to monitor the efficacy of thermal ablation techniques.By relating the treatment temperature to actual thermal tissue damage,PRFMR temperature mapping can be useful in predicting the treatmentoutcome [61].

Extensive work by Torti and colleagues has demonstrated the com-patibility of PRF MR temperature mapping with CNMTT [22,28,61]. Forexample, Burke et al. used PRF MR temperature maps to measure thetemperature increase in kidney tumor xenografts following CNMTT[28]. Use of MRI contrast enhancing CNTs allows for monitoring ofnanomaterial distribution [22] in the tumor and can aid in placementof the NIR source [143]. Ding et al. showed that iron-containingMWCNTs act as dual mode MRI T2 contrast agents and photothermalmediators [22]. However, a potential limitation of iron-containingMWCNTs is their propensity to attenuate MR signals, which can inter-fere with temperature mapping by PRF MR thermometry [22].

Though dependent upon tumor type and location, generation ofsufficiently high temperatures to ablate large (N3 cm) tumors while

minimizing damage to healthy tissue surrounding the tumor due toheat delocalization is the major limitation to all current approaches tothermal ablation therapy [144]. Experimental use of nanosecond pulsedlasers has been successfully applied to localize heat on the cancer celllevel in the presence or absence of CNTs [144–147]. However, use ofcontinuous irradiation for tenths of seconds or longer (rather thannanosecond pulses) to heat a large number of nanoparticles dispersedthroughout a tumor produces an overall temperature rise several ordersof magnitude larger than the localized (nanoscale) temperature risenear each particle [141,148]. Recently, Xie et al. used a combination ofexperimental data in tissue phantoms and diffusive heat modeling todetermine the thermal distribution CNT-bearing tumors during andfollowingNIR irradiation and used clinically relevant damage predictingalgorithms to quantify the likely efficacy of CNMTT following treatmentwith different laser pulse strengths and durations (summarized inFig. 4). Fitting of the data to diffusive heat models indicates that itmay be possible to achieve thermally ablative temperatures withinsharply defined regions (b1 mmmargins) in as little as 2 s using clini-cally available continuous NIR lasers in combination with modest CNTconcentrations [61]. In this short time scale, heat deposition is morerapid than thermal diffusion and convective cooling, whichwill simplifytreatment planning and reduce heat diffusion to surrounding normaltissue as compared to current clinical thermal ablation techniqueswhich require tissue to be heated for up to several minutes to achieveablative temperatures.

More extended NIR treatment exposure may be necessary fortreating bulky tumors because the generated heat can effectively spreadover centimeter size regions [61,141]. Moreover, modeling and experi-mental data in breast cancer tissue phantoms have shown that the pres-ence of CNTs can extend the effective treatment volume for a fixed NIRenergy input [149,150]. Burke et al. made similar observations in vivousing the induction of heat shock proteins (HSPs) 27, 70, and 90 as en-dogenous cellular markers of thermal stress for indirect measurementof heat generation in full-depth tissue sections taken from tumors ofmice after NIR exposure [28]. In tumors treatedwith NIR plusMWCNTs,HSPs were seen at deeper tissue levels than in mice treated only withNIR, confirming that CNTs combined with NIR can be used to extendthe effective volume of thermal therapy.

There remain several areas, summarized in Fig. 5, for further study.The temperature distribution following CNMTT is determined by therate of energy input and efficiency of NIR conversion to heat, which isinfluenced by concentration [28], aggregation [63], length [52] andwall number of the CNTs [63]. Therefore, careful matching of both CNTtype and distribution with NIR irradiation parameters can more effec-tively treat targeted cell populations. It is not clear how intratumoraldistribution of CNTs will affect the efficacy of CNMTT. A homogeneousdistribution of CNTs throughout the tumor mass may allow generationof a more predictable and uniform temperature in the targeted area fol-lowing laser irradiation. However, the effective tissue penetration depthof NIRmay be far smaller than the tumor volume to be treated, and thusa moremodest distribution of CNTsmay be needed. Additionally, a bet-ter understanding of the contribution of heat dissipation caused by thepresence of blood and body fluids will be required to determine boththe optimal concentration of CNTs and NIR irradiation parametersrequired for clinical applications. Finally, a greater understanding ofhow different cancer cell populations respond to CNMTT – includingheat resistant stem-like cells and tumor cells that have received priortreatment – may greatly assist in the development of new therapeuticstrategies for non-resectable tumors that are resistant to current thera-peutic modalities.

3.4. CNT toxicity and long-term fate

Progress towards the clinical use of CNTs has been limited in partdue to the lack of a comprehensive understanding of the basic determi-nants of CNT toxicity and clearance from the human body. Fear of

Fig. 4.MRthermography combinedwith damagemodeling algorithms can be used to optimize thedelivery of therapeutic heat to tumor targets. (A) Schematic of experimental design. Theentire set-upwas placedwithin themagnet of a 7 TMRI. A YAG laser tuned to 1064 nm served as the laser source and the phantomwas exposed to a total fixed radiant energy of approx-imately 100 J/cm2 using either a high power, short duration pulse (10 W/cm2; 9.6 s; referred to as the “short pulse”) or a low power, longer duration pulse (3 W/cm2; 32 s; referred to asthe “long pulse”). (B) Time evolution of the average temperature measured by MR thermometry achieved in the MWCNT inclusion: Temperature increased approximately linearly withtime in response to both long and short pulse irradiationwith a rate of average temperature increase of 1.89 and 0.38 °C s−1 and for the short and long pulse heating, respectively. Notably,the ratio of these heating rates is about 5 and is significantly greater than the ratio of heating powers (3.3), suggesting more heat diffuses away from the heated region during long pulseheating. (C) Temperaturemaps calculated by PRFMR thermometry of a coronal slice across theMWCNT inclusion and surrounding nanotube-free phantom generated at the time the laserwas turnedoff. A temperature contour forwhich the peak temperature reached43 °C (green curve) is shown. The images clearly show that heat ismore localized following the short pulsetreatment. Thermal damage contours calculated for 100 or 200 cumulative equivalentminutes at 43 °C (100CEM43 (pink curve) and the 240CEM43 thresholds (blue curve)) and contourscalculated using the Arrhenius damage integral are shown. Thermal necrosis is predicted to begin when the Arrhenius integral equals 1 (light blue curve), which is roughly equivalent to100CEM43. Ninety-ninepercent of cells are predicted to diewhen theArrhenius integral equals 4.6 (dark blue curve),which is roughly equivalent to 240CEM43.Note that 99% cell death ascalculated by the Arrhenius damage integral was not achieved using long laser pulse heating. Thus, for a fixed radiant exposure, short pulse heating of CNTs leads to a higher maximumtemperature, a more rapid rate of temperature increase, more localized heating, and greater therapeutic efficacy.Adapted and reproduced with permission from Xie et al. Phys. Med. Biol. 57 (2012) 5765–5775.


exposure to CNTs has been exacerbated because CNTs can act as ‘nano-needles’, easily piercing cell membranes [8,9], traversing to the nucleus[23] and potentially inducing genotoxicity [151]. One preliminary studyon the toxicity of CNTs indicated that when they are injected into theabdominal cavity or lungs of mice, they can induce inflammatoryresponses and granuloma formation similar to those associated withasbestos exposure [152,153]; amore recent study further demonstratedmesothelial cell injury and formation of mesotheliomas following peri-toneal injection of pristine CNTs [154]. In contrast, a two year study onthe potential carcinogenic nature of CNTs injected into the peritonealcavity of rats found no evidence of CNTs causing cancer [155]. In lightof these conflicting results, the future outlook for biomedical applicationof CNTs continues to be met with skepticism. Fears over CNT exposureincreased due to the perception that they were not degraded in thebody, though now it is known that some types of CNTs can be degradedin biological systems by oxidative enzymes [156,157]. The persistenceof these concerns indicates that for biomedical use of CNTs to progress,the toxicity, and therefore the potential risk–benefit balance for thesematerials, must be resolved.

Use of CNTs in cancer therapy requires bypassing the body's naturaldefenses, generating concern that toxicities not previously observed

might be induced. However, many strategies have evolved to reducethe toxic effects of CNTs by altering lengths, diameters, or chemicallymodifying their surfaces [158]. Biomedical use of CNTs requires modifi-cation of the highly hydrophobic surface of the tubes to render themhydrophilic [159], but not all chemical treatments alleviate the toxicityrisks associated with CNTs. It is essential to identify which characteris-tics CNTs should possess to be “safe-for-use”. It is becoming clear thatmodifications that render carbon nanotubes short (b1000 nm), flexibleand permit stable suspension in biological fluids are necessary to ame-liorate the risks identified for exposure to pristine (unmodified) CNTs[160].

For example, work byDonaldson and colleagues established that theinflammation and fibrosis induced by CNTs introduced into the pleuralcavity of mice could be reduced by shortening the length of CNTs[161]. Shorter tubes (b1000 nm)were not retained in the pleural cavityand eventually were excreted. In another related study, Ali-Boucettaet al. described two different chemical modifications to CNT surfacesand asked if either could reduce the inflammatory toxicity of the CNTsfollowing intraperitoneal injection of the tubes [160]. They found thatCNTs functionalized using the 1,3-dipolar cycloaddition of azomethineylidesfirst described by Prato and colleagues [23] to introduce pyrrolidine

Irradiation parameters• Wavelength (NIR; RF; Microwave)• Power • Pulse duration• Pulse rate/frequency• Location (internal/external)

Nanotube thermal conversion efficiency• Length• Diameter/wall number• Metallic/semiconducting• Functionalization• Aggregation/distribution• Concentration

Tissue properties• Electromagnetic absorbance• Blood flow (convective cooling)• Thermal diffusion coefficient• Water content

Tumor properties• Cancer type• Cancer stem cells• Tumor location• Prior treatment• Tumor size/shape

Thermal mapping• Imaging technique• Compatibility with CNTs• Compatibility with

irradiation source.

Fig. 5. Summary of important parameterswhichmust be assessed for successful clinical translation of CNMTT. Tomaximize treatment efficacy andminimize collateral damage due to heatspread, laser irradiation parameters (irradiance, duration, duty cycle) must be carefully matched to the NIR absorptive characteristics and distribution of the CNTs as well as the thermalproperties of the targeted and surrounding tissue. These parameters are highly interrelated and must be balanced with the underlying biology and biophysics of the tumor target. Anunderstanding of both the NIR absorptive and thermal dispersive qualities of the tumor target is needed. Specific issues related to the sensitivity of the tumor and surrounding tissueto thermal ablation must be identified. Treatment should be compatible with a non-invasive thermal mapping modality (such as proton resonance frequency shift magnetic resonance(MR) thermography) to monitor temperature localization.


rings bearing an ammonium-terminated tri(ethylene glycol) chain to theside wall of CNTs do not induce inflammation. In contrast, CNTs function-alized using the method of Billups et al. [162] to introduce an octyl chainon the side walls induce an inflammatory response comparable to thatobserved for long, pristine CNTs [163]. Functionalization of CNTs usingthe 1,3-dipolar cycloaddition method shortened and debundled thetubes following aqueous dispersion while CNTs functionalized by theBillups method remained aggregated in aqueous suspension [160]. Theauthors suggest that not all chemical treatments will alleviate the toxicityrisks associated with CNTs; only chemical reactions to CNT surfacesthat lead to CNT shortening and aid in disentangling/debundling ofCNTs following aqueous dispersion will reduce the risks [160].

In addition to length and dispersibility in aqueous solvents, a thirdcritical factor which determines the potential of CNTs to cause cell inju-ry appears to be CNT diameter. In a recent study, pristine MWCNTsof differing thickness were suspended in saline solution containingalbumin, and their uptake by mesothelial cells was compared to thatof asbestos fibers [154]. Mesothelial cells were shown to take upMWCNTs and asbestos by two distinct mechanisms: MWCNTs directlypierced mesothelial plasma and nuclear membranes, whereas asbestosfiberswere internalized by endocytosis. Consistentwith the observationthat the force needed to penetrate cell membranes is proportional toCNT diameter [164], thickMWCNTs (150 nmdiameter) were less likelyto pierce mesothelial cells than thin MWCNTs (50 nm diameter) [154].In contrast, asbestos fibers, even those thicker than 150 nm, were easilyinternalized by the cells because they do not necessarily entermesothelial cells in the same way as CNTs. This work suggests that not

only should CNT toxicology be evaluated differently than asbestos toxi-cology, but also that control of the diameter of MWCNTs could reducethe potential for mesothelial injury and other hazards caused by CNTs.

The potential risks due to inflammatory responses caused by chronicexposure to CNTs in different tissues are compounded by the possibilityof acute risks associated with blood-borne delivery of CNTs [165–168].Despite the fact that no long-term, or chronic toxicity has beenexhibited by mice injected with CNTs that have been chemically func-tionalized to improve aqueous dispersion [6,67,133,169–171], concernshave persisted regarding the potential thrombogenic nature of CNTs. Inthis regard, several groups have shown that CNTs have a high propensi-ty to activate elements of the coagulation cascade in vitro [166,167,172].However, in an extensive study comparing the role played by chemicalfunctionalization of CNTs on blood toxicity in vitro to indicators ofthrombosis following intravenous injection into mice (platelet counts;D-dimer levels; murine Von Willebrand factor; histology), Burke et al.found that in vitro analysis of CNT induced coagulation was a poor pre-dictor of actual in vivo outcomes [173]. Specifically, the researchersfound that pristine MWCNTs appeared to be less thrombogenic thanchemically functionalized MWCNT (acid oxidized; acid oxidized thenamidated) in vitro as indicated by activation of the coagulation cascade,coagulation time, and platelet activation, which is consistent with otherrecent reports [174]. However, following intravenous injection in mice,pristine MWCNTs were found to rapidly deplete platelets, and induceelevated D-dimer and Von Willebrand factor levels, indicating severethrombosis, disseminated intravascular coagulation, and endothelialcell injury [173]. At a dose of 250 μg, pristine MWCNTs were acutely

image of Fig.�5


lethal, inducing blockage of the pulmonary vasculature. In contrast,the sole measurable effect of an equivalent dose of the two types offunctionalized MWCNTs was a transient depletion of platelets [173].Although more detailed studies must be undertaken before the toxicityprofile of CNTs is fully understood, these results support the hypothesisthat CNTs can be designed to be suitable for systemic delivery.

Importantly, chemical functionalization of CNTs is also known togreatly influence their blood clearance profile. Following early debatesregarding whether the primary clearance route for intravenouslyadministered CNTs was via renal or biliary pathways [133,175,176], itis now known that the type and degree of chemical functionalization,degree of agglomeration, and length of the tubes determine the tissuedistribution and excretion of CNTs [177–179]. Renal clearance of CNTsin particular has caused much debate as many believe that 5 nm is themaximum diameter that permits for clearance of nanoparticles via theurine [180]. However, several groups have shown that intact CNTs aslong as 500 nm with diameters of up to 25–30 nm can exit the bloodstream and be excreted in urine by orienting themselves along theirsmaller axis to pass through the fenestrated endotheliumof the glomer-ular capillary wall, across the glomerular basement membrane, andthrough the filtration slits [133,134,179,181] despite these openingsbeing approximately 43 nm in diameter [182]. A more recent study byKostarelos et al. convincingly confirms these previous works, showing:1) individualization of CNTs resulting from a high degree of chemicalfunctionalization to introduce hydrophilic groups to the CNT surfaceaids in rapid renal clearance and low tissue retention; 2) shorter CNTsexhibit greater renal clearance than longer CNTs; and 3) in general,increasing hydrophilic chemically functionalization is associated withrenal clearance while decreasing functionalization favors clearance bythe mononuclear phagocyte system [177].

Combined, the studies described above indicate that it should bepossible to engineer CNTs suitable for future use in humans. New strat-egies to synthesize andmodify CNTs in a highly controlledmanner con-tinue to evolve and show tremendous potential for designing CNTswithoptimal in vivo characteristics. As more research evaluating the rela-tionship between CNT characteristics and in vivo kinetics, excretion,and toxicity emerges, the clinical use of CNTs is becoming more andmore feasible.

4. Conclusions

The translation of carbon nanotubes from an interesting nanomaterialto an effective cancer therapeutic agent is an actively evolving field [25].As discussed in Section 1 of this review, CNTs offer enormous possibilitiesfor the development of the next generation of cancer therapeutic anddiagnostic agents. Their desirable features include the following: 1) theyefficiently convert incident NIR into heat for use in cancer thermal abla-tion or imaging; 2) they can be used for additional therapeutic applica-tions including chemotherapeutic drug or gene delivery; 3) their highsurface area to volume ratio per particle allows for incorporation of ahigh density of imaging agents which can improve the sensitivity ofimaging; 4) they can simultaneously deliver several different types ofagents to perform multimodality imaging and cancer therapy; 5) theyeasily cross cellmembranes and can be engineered to be exhibit a positivebiocompatibility profile.

As research in this field has advanced, a number of issues in the useof CNTs for CNMTT, identified in Section 2 in this review, remain to beaddressed. Size of the tumor is one consideration. Many cancers donot present until tumors are significantly larger than one or severalcentimeters in diameter,which is larger thanmost tumors treated in an-imal models to date. Delivery of both CNTs and therapeutic heat acrossthese larger tumors represents a major challenge in the pre-clinicaldevelopment of CNMTT. Most applications to date have relied on NIR,which has a finite range of tissue penetration. Although biologicaltissues are relatively transparent to NIR, treatment of deep-seatedtumors may require either the use of RF or longer wavelength

electromagnetic radiation; such strategies are being actively pursued[54,183]. Alternatively, the development of companion technology,such as fiber optic probes that can deliver NIR to the target site, mayenable the utilization of NIR for thermal activation of CNTs in evendeep-seated tumors. Additionally, strategies must be developed tomonitor both the distribution of the CNTs and the deposition, localiza-tion, and efficacy of heat treatment.

Conversely, it is also desirable to treat small micrometastases. Cur-rently, CNMTT relies on conventional clinical imaging to detect andlocalize tumors prior to treatment, and tumors that are too small to bevisualized by such methodology will remain untreated. Use of targetednanoparticles conjugated to imaging moieties may overcome this limi-tation by allowing the detection and treatment of such small tumors.Targeted nanotubes may also permit dose reduction, since each nano-tube would have a higher probability of associating with its tumortarget. Of particular interest would be the development of CNT-basedPET agents, which would allow for extremely high detection sensitivityand depth of penetration. Because the risk of nanoparticle toxicity willgo downwith decreased exposure, the use of PET as an imagingmodal-ity may further push the envelope on clinical development of CNT-based therapeutic and diagnostic agents. A number of potential agentshave been tested in mice [19,70,133,134], but their utility in cancerdiagnosis has yet to be evaluated.

Most importantly, the long term fate and toxicity of CNTs must bedetermined. It has become clear that specifics matter: that each changein particle design creates an entirely new particle with novel toxicolog-ical, pharmacological and therapeutic properties. This necessitates thatboth the biomedical and toxicological investigations of these materialstake place simultaneously. The debate over toxicity of CNTs continuesdespite research showing that appropriate design can amelioratemany unwanted effects of CNTs; thus, overcoming the perceived andreal potential toxicities of nanotubes may represent the largest hurdletowards their clinical application. Based upon the current evidence, aseries of guidelines for future development of CNT-based therapeuticshas been proposed by Kostarelos and colleagues [183]. Briefly sum-marized, these recommendations are: (1) use aqueous preparations ofsmall, hydrophilic, well dispersed CNTs that can easily be internalizedby target cells and that macrophages can efficiently take-up and clearafter the therapeutic effect is achieved; (2) use colloidal dispersions ofCNTs that are stable under physiologic pH and salinity to minimize ag-gregation in vivo; and (3) use easily excreted or chemically-modifiedCNTs that are readily degraded in vivo.

The promise of CNTs in photothermal cancer therapy still re-mains; realization of this potential will depend on production ofwell-characterized, biocompatible, homogeneous materials andwell-designed efficacy and toxicology studies.

Acknowledgments

This work was supported in part by grants RO1CA12842 (SVT)from the National Cancer Institute/National Institutes of Health,W81XWH-10-1-0434 (SVT) from the Department of Defense, and byR00CA154006 (RS) from the National Cancer Institute/National Insti-tutes of Health. The content is solely the responsibility of the authorsand does not necessarily represent the official views of the NationalCancer Institute, the National Institutes of Health, or the Departmentof Defense.

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