the application of carbon nanotubes for targetted drug delivery system for cancer therapies

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N A N O R E V I E W Open Access

The application of carbon nanotubes in targetdrug delivery systems for cancer therapiesWuxu Zhang1, Zhenzhong Zhang2* and Yingge Zhang1*

Abstract

Among all cancer treatment options, chemotherapy continues to play a major role in killing free cancer cells and

removing undetectable tumor micro-focuses. Although chemotherapies are successful in some cases, systemic

toxicity may develop at the same time due to lack of selectivity of the drugs for cancer tissues and cells, which

often leads to the failure of chemotherapies. Obviously, the therapeutic effects will be revolutionarily improved if

human can deliver the anticancer drugs with high selectivity to cancer cells or cancer tissues. This selective delivery

of the drugs has been called target treatment. To realize target treatment, the first step of the strategies is to buildup effective target drug delivery systems. Generally speaking, such a system is often made up of the carriers and

drugs, of which the carriers play the roles of target delivery. An ideal carrier for target drug delivery systems should

have three pre-requisites for their functions: (1) they themselves have target effects; (2) they have sufficiently strong

adsorptive effects for anticancer drugs to ensure they can transport the drugs to the effect-relevant sites; and (3)

they can release the drugs from them in the effect-relevant sites, and only in this way can the treatment effects

develop. The transporting capabilities of carbon nanotubes combined with appropriate surface modifications and

their unique physicochemical properties show great promise to meet the three pre-requisites. Here, we review the

progress in the study on the application of carbon nanotubes as target carriers in drug delivery systems for cancer

therapies.

Keywords:carbon nanotubes, cancer therapies, drug delivery systems, target chemotherapy

Introduction

Cancers are a kind of the diseases that are hardest tocure, and most cancer patients definitely die even whentreated with highly developed modern medicinal techni-ques. Surgery can remove cancer focuses but cannot dothe same for the micro-focuses and neither can extin-guish the free cancer cells that are often the origin ofrelapse. Chemotherapy with anticancer drugs is themain auxiliary treatment but often fails because of theirtoxic and side effects that are not endurable for thepatients. Over the past few decades, the field of cancer

biology has progressed at a phenomenal rate. However,despite astounding advances in fundamental cancer biol-ogy, these results have not been translated into

comparable advances in clinics. Inadequacies in the abil-ity to administer therapeutic agents with high selectivityand minimum side effects largely account for the discre-pancies encompassing cancer therapies. Hence, consid-erable efforts are being directed to such a drug deliverysystem that selectively target the cancerous tissue withminimal damage to normal tissue outside of the cancerfocuses. However, most of this research is still in thepreclinical stage and the successful clinical implementa-tion is still in a remote dream. The development of sucha system is not dependent only on the identification of

special biomarkers for neoplastic diseases but also onthe constructing of a system for the biomarker-targeteddelivery of therapeutic agents that avoid going into nor-mal tissues, which remains a major challenge [1]. Withthe development of nanotechnology, few nanomaterial-based products have shown promise in the treatment ofcancers and many have been approved for clinicalresearch, such as nanoparticles, liposomes, and polymer-drug conjugates. The requirements for new drug

* Correspondence: [email protected];[email protected] of Pharmacology and Toxicology and Key Laboratory of

Nanopharmacology and Nanotoxicology, Beijing Academy of Medical

Science, Zhengzhou, Henan, Peoples Republic of China2Nanotechnology Research Center for Drugs, Zhengzhou University,

Zhengzhou, Henan, Peoples Republic of China

Full list of author information is available at the end of the article

Zhang et al. Nanoscale Research Letters 2011, 6 :555

http://www.nanoscalereslett.com/content/6/1/555

2011 Zhang et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.
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delivery systems to improve the pharmacological profileswhile decreasing the toxicological effects of the delivereddrugs have also envisaged carbon nanotubes (CNTs) asone of the potential cargos for the cancer therapy.CNTs belong to the fullerene family of carbon allotropeswith cylindrical shape. The unique physicochemicalproperties [2,3] of CNTs with easy surface modificationhave led to a surge in the number of publications in thisinteresting field. Apart from their uses in the cellularimaging with diagnostic effects in nanomedicine [4,5],CNTs are promising drug carriers in the target drugdelivery systems for cancer therapies. Unlike other nao-carriers, such as liposomes/micelles that emerged in the1960s and nanoparticles/dendrimers that emerged in1980s, it has emerged no more than 20 years for carbonnanotubes to be envisaged as target drug carriers. Inthis chapter, the works that have been carried out with

CNTs in the f ield of cancer therapy are briefly introduced.

Physicochemical properties of CNTs

Carbon nanotubes are a huge cylindrical large moleculesconsisting of a hexagonal arrangement of sp2 hybridizedcarbon atoms (C-C distance is about 1.4 ). The wall ofCNTs is single or multiple layers of graphene sheets, ofwhich those formed by rolling up of single sheet arecalled single-walled carbon nanotubes (SWCNTs) andthose formed by rolling up of more than one sheets arecalled multi-walled CNTs (MWCNTs). Both SWCNTsand MWCNTs are capped at both ends of the tubes ina hemispherical arrangement of carbon networks calledfullerenes warped up by the graphene sheet (Figure1A).The interlayer separation of the graphene layers ofMWCNTs is approximately 0.34 nm in average, eachone forming an individual tube, with all the tubes hav-ing a larger outer diameter (2.5 to 100 nm) thanSWCNTs (0.6 to 2.4 nm). SWCNTs have a betterdefined wall, whereas MWCNTs are more likely to havestructural defects, resulting in a less stable nanostruc-ture, yet they continue to be featured in many publica-tions due to ease of processing. As for their use as drugcarriers, there remain no conclusive advantages of

SWCNTs relative to MWCNTs; the defined smaller dia-meter may be suitable for their quality control while thedefects and less stable structure make their modificationeasier. CNTs vary significantly in length and diameterdepending on the chosen synthetic procedure. SWCNTsand MWCNTs have strong tendency to bundle togetherin ropes as a consequence of attractive van der Waalsforces. Bundles contain many nanotubes and can beconsiderably longer and wider than the original onesfrom which they are formed. This phenomenon couldbe of important toxicological significance [6,7]. CNTsexist in different forms depending upon the orientation

of hexagons in the graphene sheet and possess a veryhigh aspect ratio and large surface areas. The availablesurface area is dependent upon the length, diameter,and degree of bundling. Theoretically, discrete SWCNTshave special surface areas of approximately 1300 m2/g,whereas MWCNTs generally have special surface areasof a few hundred square meters per gram. The bundlingof SWCNTs dramatically decreases the special surfacearea of most samples of SWCNT to approximately 300m2/g or less, although this is still a very high value [8,9].The markedly CNTs have various lengths from severalhundreds of nanometers to several micrometers and canbe shortened chemically or physically for their suitabilityfor drug carriers (Figure1B) [10] by making their twoends open with useful wall defects for intratube drugloading and chemical functionalization (Figure1B).

Functionalization of CNTsAs drug carriers, the solubility of CNTs in aqueous sol-

vent is a prerequisite for gastrointest inal absorption,blood transportation, secretion, and biocompatibility andso on; hence, CNT composites involved in therapeuticdelivery system must meet this basic requirement. Simi-larly, it is important that such CNT dispersions shouldbe uniform and stable in a sufficient degree, so as toobtain accurate concentration data. In this regard, thesolubilization of pristine CNTs in aqueous solvents isone of the key obstacles in the way for them to be devel-oped as practical drug carriers owing to the rather hydro-phobic character of the graphene side walls, coupled withthe strong -interactions between the individual tubes.These properties cause aggregation of CNTs into bun-dles. For the successful dispersion of CNTs, the mediumshould be capable of both wetting the hydrophobic tubesurfaces and modifying the tube surfaces to decreasetubes bundle formation. To obtain desirable dispersion,Foldvari et al. have proposed four basic approaches [11]:(1) surfactant-assisted dispersion, (2) solvent dispersion,(3) functionalization of side walls, and (4) biomoleculardispersion. Among the above described approaches, func-tionalization has been the most effective approach. Inaddition, functionalization has been shown capable of

decreasing cytotoxicity, improving biocompatibility, andgiving opportunity to appendage molecules of drugs, pro-teins, or genes for the construction of delivery systems[12]. Up to now, there have been a lot of literatures onthe functionalization of CNTs with various molecules(Figure2A). The functionalization can be divided intotwo main subcategories: non-covalent functionalizationand covalent functionalization (Figure2B).

Non-covalent functionalization

Many small, as well as large, polymeric anticanceragents can be adsorbed non-covalently onto the surface



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Figure 1The formation of SWCNT and its physical and chemical treatment for use as drug carriers . (A) The schematic illustration of the

structure formation of SWCNTs with the two ends closed. (B) The schematic illustration of the strategy for the preparation of the CNT-based

drug delivery systems.

Figure 2The modification of CNTs. Schematic illustration of modification of CNTs with various molecules. 1, Dhar et al. [70]; 2, Jia et al. [13]; 3,

Georgakilas et al. 2002 [16]; 4, Peng et al. 1998; 5, Liu et al. [91]; 6, Gu et al. 2008; 7, Son et al. 2008; 8, Klingeler et al. 2009.



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of pristine CNTs. Forces that govern such adsorptionare the hydrophobic and - stacking interactionsbetween the chains of the adsorbed molecules and thesurface of CNTs. Since many anticancer drugs arehydrophobic in nature or have hydrophobic moieties,the hydrophobic forces are the main driving forces forthe loading of such drugs into or onto CNTs. The pre-sence of charge on the nanotube surface due to chemi-cal treatment can enable the adsorption of the chargedmolecules through ionic interactions [13,14]. Aromaticmolecules or the molecules with aromatic groups can beembarked on the debunching and solubilization ofCNTs using nucleic acids and amphiphilic peptidesbased on the - stacking interactions between theCNT surface and aromatic bases/amino acids in thestructural backbone of these functional biomolecules.Noncovalent functionalization of CNT is particularly

attractive because it offers the possibility of attachingchemical handles without affecting the electronic net-work of the tubes.

Oxide surfaces modified with pyrene through -stacking interactions have been employed for the pat-terned assembly of single-walled carbon nanomaterials[15]. The carbon graphitic structure can be recognizedby pyrene functional groups with distinct molecularproperties. The interactions between bifunctional mole-cules (with amino and silane groups) and the hydroxylgroups on an oxide substrate can generate an amine-covered surface. This was followed by a coupling stepwhere molecules with pyrene groups were allowed toreact with amines. The patterned assembly of a singlelayer of SWCNT could be achieved through -stack-ing with the area covered with pyrenyl groups. Alkyl-modified iron oxide nanoparticles have been attachedonto CNT by using pyrenecarboxylic acid derivative aschemical cross-linker [16]. The resulting material hadan increased solubility in organic media due to the che-mical functions of the inorganic nanoparticles.

Surfactants were initially involved as dispersing agents[17] in the purification protocols of raw carbon material.Then, surfactants were used to stabilize dispersions ofCNT for spectroscopic characterization [18], optical lim-

iting property studies, and compatibility enhancement ofcomposite materials.

Functionalized nanotube surface can be achieved sim-ply by exposing CNTs to vapors containing functionali-zation species that non-covalently bonds to thenanotube surface while providing chemically functionalgroups at the nanotube surface [19]. A stable functiona-lized nanotube surface can be obtained by exposing it to

vapor stabilization species that reacts with the functio-nalization layer to form a stabilization layer against des-orption from the nanotube surface while depositingchemically functional groups at the nanotube surface.

The stabilized nanotube surface can be exposed furtherto at least another material layer precursor species thatcan deposit as a new layer of materials.

A patent [20] is pertinent to dispersions of CNTs in ahost polymer or copolymer with delocalized electronorbitals, so that a dispersion interaction occurs betweenthe host polymer or copolymer and the CNTs dispersedin that matrix. Such a dispersion interaction has advan-tageous results if the monomers of the host polymer/copolymer include an aromatic moiety, e.g., phenylrings or their derivatives. It is claimed that dispersionforce can be further enhanced if the aromatic moiety isnaphthalenyl and anthracenyl. A new non-wrappingapproach to functionalizing CNTs has been introducedby Chen et al. [21]. By this approach, the functionaliza-tion can be realized in organic and inorganic solvents.With a functionally conjugated polymer that includes

functional groups, CNT surfaces can be functionalizedin a non-wrapping or non-packaging fashion. Throughfurther functionalization, various other desirable func-tional groups can be added to this conjugated polymer.This approach provided the possibility of further tailor-ing, even after functionalization. A process registered byStoddart et al. [22] involves CNTs treated with poly{(5-alkoxy-m-phenylenevinylene)-co-[(2,5-dioctyloxy-p-phe-nylene) vinyl-ene]} (PAmPV) polymers and their deriva-tives for noncovalent functionalization of the nanotubeswhich increases solubility and enhances other propertiesof interest. Pseudorotaxanes are grafted along the wallsof the nanotubes in a periodic fashion by wrapping ofSWCNTs with these functionalized PAmPV polymers.Many biomolecules can interact with CNTs withoutproducing of covalent conjugates. Proteins are animportant class of substrates that possess high affinitywith the graphitic network. Nanotube walls can adsorbproteins strongly on their external sides, and the pro-ducts can be visualized clearly by microscopy techni-ques. Metallothionein proteins were adsorbed onto thesurface of multi-walled CNT, as evidenced by high-reso-lution transmission electron microscopy (TEM) [23].DNA strands have been reported by several groups tointeract strongly with CNT to form stable hybrids effec-

tively dispersed in aqueous solutions [24,25]. Kim et al.[26] reported the solubilization of nanotubes with amy-lose by using dimethyl sulfoxide/water mixtures. Thepolysaccharide adopts an interrupted loose helix struc-ture in these media. The studies of the same group onthe dispersion capability of pullulan and carboxymethylamylase demonstrated that these substances could alsosolubilize CNTs but to a lesser extent than amylose.There are also some literatures that reported severalother examples of helical wrapping of linear orbranched polysaccharides around the surface of CNT[27].



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Covalent functionalization

Covalent functionalization gives the more secure con-junction of functional molecules. CNTs can be oxidased,giving CNTs hydrophilic groups as OH, COOH, and soon. Strong acid solution treatment can create defects inthe side walls of CNTs, and the carboxylic acid groupsare generated at the defect point, predominantly on theopen ends. Excessive surface defects possibly change theelectronic properties and cut longer CNTs into shortones. as drug carriers may need CNTs with differentelectronic properties and different lengths. In the pre-paration of some drug delivery systems, CNTs are delib-erately cut into short pieces. The functional groups onthe oxidized CNTs can further react with SOCl and car-bodiimide to yield functional materials with great pro-pensity for reacting with other compounds [28,29].

Covalent functionalization of SWCNTs using addition

chemistry is believed to be very promising for CNTmodification and derivatization. However, it is difficultto achieve complete control over the chemo- and regionselectivity of such additions and require very special spe-cies such as arynes, carbenes, or halogens, and the reac-tions often occur only in extreme conditions for theformation of covalent bonds. Furthermore, the charac-terizatiuon of functionalized SWCNTs and the determi-nation of the precise location and mode of addition arealso very difficult [30]. The covalent chemistry of CNTsis not particularly rich with respect to variety chemicalreactions to date. As regard to functionalization beha-

vior of SWCNTs and MWCNTs, it has been reportedthat functionalization percentage of MWCNTs is lowerthan that of SWCNTs with the similar process [ 31],which is assumingly attributed to the larger outer dia-meter and sheathed nature of MWCNTs that rendermany of their sidewalls inaccessible; nonetheless, a com-parative study on functionalizing single-walled andmulti-walled carbon nanotubes is scarce hitherto inopen literature.

In comparison with non-covalent functionalization,there are new substances to develop and therefore mostpatents regarding functionalization of CNTs registeredto date are based on covalent chemistry. Though cova-

lent procedures are not highly diverse yet, the end pro-ducts vary exceedingly in terms of characteristicsdepending upon the incorporated species.

Methotrexate functionalization can be realizedthrough 1,3-cycloaddition reaction [32]. Azomethine

ylides consisting of a carbanion adjacent to an immo-nium ion are organic 1,3-dipoles, which give pyrrolidineintermediates upon cycloaddition to dipolarophiles.Through decarboxylation of immonium salts obtainedfrom the condensation ofa-amino acids with aldehydesor ketones, azomethine ylides can be easily produced.These compounds can make CNTs fused with

pyrrolidine rings with varied substituents depending onthe structure of used a-amino acids and aldehydes.

Using acyl peroxides can generate carbon-centeredfree radicals for functionalization of CNTs [33]. Thepromising method allows the chemical attachment of a

variety of functional groups to the wall or end-cap ofCNTs through covalent carbon bonds without destroy-ing the wall or end-cap structure of CNTs [34], unlikein the case of treating with strong acid. Carbon-centeredradicals generated from acyl peroxides can have terminalgroups that render the modificated sites capable offurther reaction with other compounds. For example,organic groups with terminal carboxylic acid functional-ity can further react with acyl chloride and an amine toform an amide or with a diamine to form an amide withterminal amine. The reactive functional groups attachedto CNTs not only render solvent dispersibility improved

but also offer reaction sites for monomers to incorpo-rate in polymeric structures. Free radicals for functiona-lization can also be produced by organic sulfoxides. Thekey feature of this free radical method is its simplicitycoupled with a reasonable choice of radical generatingcompounds [35].

A method for producing polymer/CNTs compositesinvented by Ford et al. [36] allows covalent attachmentsof polymers to CNTs. The resultant composites dispersein liquid media to form stable colloidal dispersions with-out separating for prolonged periods ranging from hoursto months. The polymer functionalized CNTs are alsocapable of being dispersed into the parent polymer. Themethod has been effectively and conveniently used inthe functionalization, solubilization, and purification ofCNTs, although the stabilization of these dispersions isgreatly dependent upon given colloidal systems.

A three-step method has been proposed by Barrera etal. [37], in which functionalized CNTs are used to pre-pare polymer composite in first place and then theseCNTs are defuntionalized therein returning them to ori-ginal chemistry. The first step is dispersing functiona-lized CNTs in a solvent to form a dispersion; the secondis incorporating the dispersion of functionalized CNTsinto a polymer host matrix to form a functionalized

CNTs-polymer composite; and the third is modifyingthe functionalized CNTs-polymer composite with radia-tion, wherein the modifying comprises defunctionaliza-tion of the functionalized CNTs via radiation selectedfrom the group consisting of protons, neutrons, alphaparticles, heavy ions, cosmic radiation, etc. The featureof this method is that the functionalization is carriedout only as assist dispersion, and CNTs are returned toits original characteristics after incorporating in polymermatrix.

A method to create new polymer/composite materialshas been devised by Tour et al. [38] by blending



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derivatized carbon nanotubes into polymer matrices.Modification with suitable chemical groups using diazo-nium chemistry made CNTs chemically compatible witha polymer matrix, which allows the properties of CNTsto transfer to that of the product composite material asa whole. This method is simple and convenient. Thereaction can be achieved by physical blending of deriva-tized CNTs with the polymeric material, no matter atambient or elevated temperature. This method can beused in the fixation of functional groups to CNTsfurther covalently bonding to the matrix of host poly-mers or directly between two tubes themselves. Further-more, CNTs can be derivatized with a functional groupthat is an active part of a polymerization process, result-ing in a composite material in which CNTs are chemi-cally involved as generator of polymer growth. Thisprocedure ensures an excellent interaction between the

matrix and CNTs since CNTs aid polymerization andgrowth of polymer chains that render them more com-patible with the host polymer, although it does notaddress the question of CNT dispersion. Stanislaus et al.[39] functionalized the sidewalls of a plurality of CNTswith oxygen moieties. This procedure exposed CNT dis-persion to an ozone/oxygen mixture to form a pluralityof ozonized CNTs. The plurality of ozonized CNTsreacted with a cleaving agent to form a plurality of side-wall-functionalized CNTs.

As mentioned above, functionalization of CNTs canbe achieved in acidic media [40]. Bundled CNTs can beseparated as individual CNTs by dispersing them in anacidic medium, which exposes the sidewalls of CNTs,facilitating the functionalization. Once CNTs are dis-persed in unbundled state, the functionalizing reactionoccurs. This method is of great promising because it iseasily scalable, providing for sidewall-functionalizedCNTs in large, industrial quantities. In acidic medium,CNTs can be shortened, which causes loss of someproperties of CNTs, but this shortening are sometimesneeded for special purposes such as in the case of CNTsare used as oral drug carriers [10].

For studies on the use of CNTs in neurology at thenanometer scale, Mark et al. constructed an implant sys-

tem [41], composed of CNTs and neurons growing fromthere. CNTs are functionalized with neuronal growthpromoting agents selected from a group chemicals con-sisting of 4-hydroxynonenal, acetylcholine, dopamine,GABA (g-aminobutyric acid), glutamate, serotonin,somatostatin, nitrins, semaphorins, roundabout, calcium,etc. Functionalized CNTs in this system are employedfor promoting the growth of neurons, which are clini-cally significant because it is possible to be used foreffectively promoting nerve regeneration, bringingopportunity for stroke patients to recover from theirparalyzed states.

CNTs have been demonstrated to be rather inert dueto the seamless arrangement of hexagon rings withoutany dangling bonds in the sidewalls. The fullerene-liketips in the ends of the tubes are more reactive than thesidewalls. Various chemical reagents can react with thetips to attach chemical groups on them. However, itremains a challenge to realize the asymmetric functiona-lization of CNTs with each of their two endtips attachedby different chemical reagents. The method of asym-metric end-functionalization has been tried by Dai andLee [42] who employ physicochemical process to pro-duce asymmetric end-functionalization of CNTs.

A method for functionalizing CNTs with organosilanespecies has been devised by Enrique et al. [43]. Hydro-xyl-functionalized CNTs are prepared by reacting fluori-nated CNTs with moieties comprising terminal hydroxylgroups and then to obtain organosilane-functionalized

polymer-interacting CNTs by reacting the hydroxyl-functionalized CNTs with organofunctionalized silanol(hydrolyzed organoalkoxysilanes) bearing polymer-interactingfunctional moieties. Such CNTs can interactchemically with a polymer host material. This methodhas two benefits. The first is that the functionalizedCNTs can provide strong attachment to both fiber(other CNTs) and matrix (polymer) via chemical bonds.With polymer compatible organofunctional silane, func-tionalized CNTs can be directly included into polymermatrices. The second is a high level of CNT unropingand the formation of relatively soluble materials in com-mon organic solvents, offering opportunity for homoge-neous dispersion in polymer matrices. Valery et al. [44],also invented a method regarding the functionalizationof SWCNT sidewall through C-N bond substitutionreactions with fluorinated SWCNTs (fluoronanotubes).Ford et al. patented a very convenient and simplemethod of solubilizing CNTs that involves mixing andheating of CNTs and urea to initiate a polymerizationreaction of the isocyanic acid and/or cyanate ion to

yield modified CNTs [45].As a summary, there have been a lot of literatures and

patents regarding the functionalization of CNTs. Ofthese techniques, most have not been used, but they are

identical with those used in drug delivery systems.These functionalization methods provided candidatetechniques, and there are great possibilities for them tobe used in the construction of drug delivery systems innot too long a time. The functionalization of CNTsused in the construction of drug delivery systems will bediscussed in later sections.

In vivo behavior of functionalized CNTs

For all pharmaceuticals, precise determination of phar-macological parameters, such as the absorption, trans-portation, target delivery effects, blood circulation time,



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clearance half-life, organ biodistribution, and accumula-tion, are essential prerequisites for them to be developedinto practically usable drugs [46]. For their drug carrieruse, CNTs must be absorbed from the administrationsite into the body. There are quite a few ways for theadministration of drugs, such as oral, vein injection,muscle injection, subcutaneous injection, and localinjection and so on. The absorbed CNTs must be trans-ported from the administration sites to the effects-related sites, such as cancer focuses, infection focuses,ischemia focuses, and so on. For the excretion, CNTsmust be transported from everywhere in the body to theexcretion organs such as kidney, liver, and so on. All ofthese questions must be made clear for the biosafety ofCNTs used as drug carriers. Unfortunately, the dataabout these questions are still insufficient, althoughremarkable progress has been achieved.

Administration, absorption, and transportation

As drug carriers, the administration, absorption, andtransportation of CNTs must be considered for obtain-ing desired treatment effects. The studied ways of CNTadministration include oral and injections such as sub-cutaneous injection, abdominal injection, and intrave-nous injection. There are different ways for theabsorption and transportation when CNTs are adminis-tered in different ways. The absorbed CNTs are trans-ported from the administration sites to the effects-relevant sites by blood or lymphatic circulation.

After administration, absorption is the first key stepfor drug carriers to complete their drug-delivering mis-sion. However, there have been very few literatures onthe absorption of CNTs from their administration sites.Yukako et al. studied the absorption of erythropoietin(EPO) loaded in CNTs from rat small intestine and theeffect of fiber length on it. Erythropoietin-loaded carbonnanotubes (CNTs) with surfactant as an absorptionenhancer were prepared for the oral delivery of EPOusing two types of CNTs, long and short fiber lengthCNTs. The results of ELISA measurements revealedthat serum EPO level reached to Cmax, 69.0 3.9 mIU/ml, at 3.5 0.1 h, and the area under the curve (AUC)

was 175.7 13.8 mIU h/ml in free EPO group, whichwas approximately half of that obtained with that loadedinto short fiber length CNTs, of which Cmax was 143.1 15.2 mIU/ml and AUC was 256.3 9.7 mIU h/ml[47]. When amphoteric surfactant, lipomin LA, sodiumb-alkylaminopropionic acid, was used to accelerate thedisaggregation of long fiber length CNTs, Cmaxwas 36.0 4.9 and AUC was 96.9 11.9, showing less bioavail-ability of EPO. These results suggest that CNTs them-selves are capable of being absorbed and that the shortfiber length CNTs deliver more both EPO and absorp-tion enhancer to the absorptive cells of the rat small

intestine and the aggregation of CNTs is not the criticalfactor for the oral delivery of EPO. Our recent worksfurther demonstrated that the physically shortenedCNTs orally administered can be absorbed through thecolumnar cells of intestinal mucous membrane, whichwas confirmed by transmission electron microscope(Figure3) [10]. In the experiment, high-speed shearing-shortened SWCNTs were used. The absorb ability ofintestinal tract for CNTs is of great significance becausethis makes it possible to develop oral drug delivery sys-tems based on CNTs.

When subcutaneously and abdomenally administered,a part of CNTs exist persistently in the local tissueswhile some of them may be absorbed through lymphaticcanal. Because the fenestra in the endothelial cells ofblood capillaries are 30 nm to approximately 50 nmwhile that in the endothelial cells of lymphatic capil-

laries are larger than 100 nm in diameter, the lymphabsorption of bundled CNTs seemed to be easier thanblood absorption. The lymphatically absorbed CNTsmigrate along the lymph canal and are accumulated inthe lymph node, which is in fact a lymphatic targeteffects. This is clinically important because lymphaticmetastasis occurs extensively in cancers, resulting in fre-quent tumor recurrence, even after extended lymphnode dissection. If anticancer drugs are loaded intoCNTs, they will be delivered into lymph system, wherethe drugs will be released to kill metastatic cancer cells.Ji et al. successfully delivered gemcitabine to lymphnodes with high efficiency by using lymphatic targeteddrug delivery system based on magnetic MWCNTsunder the magnetic field guidance [48,49]. The resultsuggests that the anticancer drug delivery system basedon CNTs is advantageous over the current ways to deli-

ver chemotherapeutic agents to lymph nodes. In anotherapproach [50], water-soluble MWCNTs were subcuta-neously injected into the left rear foot pad of rat; thebiopsy found that the accumulation of MWCNTs in leftpopliteal lymph nodes was more obvious than in otherregions, and micropathology revealed large MWCNTcollections in the popliteal lymph nodes. At the sametime, the biopsy experiments found no presence of

MWCNTs in the major internal organs such as liver,kidney, and lung, which suggests the properties ofMWCNT lymphatic targets.

When administered through veins, CNTs can directlyget into blood circulation and distribute in many inter-nal organs, such as liver, spleen, heart and kidney(unpublished date). Some studies demonstrated that theblood clearance of intravenously injected CNTs largelydepends upon the surface modification. Singh et al.found that, following intravenous administration, 111In-labeled water-soluble SWCNTs functionalized withdiethylenetriaminepenta acetic acid (average diameter, 1



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rapid, although the blood half-lives have not beenreported [52].

Polyethylene glycol(PEG)ylation is believed to be oneof the most important strategies to prolong the circula-tion time of CNTs in blood because the surface cover-age with PEG lowers the immunogenicity of the carriersand prevents their nonspecific phagocytosis by the reti-culoendothelial system (RES); thus, their half-life inblood circulation is prolonged. In fact, it has been foundthat PEGylated CNTs can persistently exist within liverand spleen macrophages for 4 months with excellentcompatibility [53]. In a recent investigation, it wasobserved that fluorescein isothiocyanate (FITC)-labeledPEGylated SWCNTs can penetrate the nuclear mem-brane and get into nucleus in an energy-independentway [54]. The presence of FITC-PEG-SWCNTs innucleus did not produce any significant ultrastructural

change in the nuclear organization and had no signifi-cant effects on the growth kinetics and cell cycle distri-bution for up to 5 days. Surprisingly, upon removal ofthe FITC-PEG-SWCNTs from the culture medium, theinternalized FITC-PEG-SWCNTs rapidly moved out ofthe nucleus and were released from the cells, suggestingthat the internalization of CNTs into and excretion ofCNTs from the cells are a bidirectional reversible pro-cess. These results illustrated well the successful exploi-tation of SWCNTs as ideal nanovectors for biomedicaland pharmaceutical applications and, they will drive theconcern about the excretion problems out of peoplesheart.

Distribution

Distribution indicates the sites or places the absorbedCNTs can arrive and exist there, which are of greatimportance in clinical pharmacology and toxicology ofCNTs as drug carriers.

There have been experiments to investigate in vivoand ex vivo biodistributions, as well as tumor targetingability of radiolabeled SWCNTs (diameter, approxi-mately 1 to 5 nm; length, approximately 100 to 300 nm)noncovalently functionalized with phospholipids(PL)-PEG in mice using positron emission tomography and

Raman spectroscopy, respectively. It was interesting tonote that the PEG chain lengths determine the biodistri-bution and circulation of CNTs. PEG-5400-modifiedSWCNTs have a circulation time ( t1/2 = 2 h) muchlonger than that of PEG-2000-modified counterpart (t1/2= 0.5 h), which may be attributed to the lower uptake ofthe former by the RES as compared with that of thelater. By further functionalization of these PEGylatedSWCNTs with arginine-glycine-aspartic acid (RGD)peptide, the accumulation in integrin-positive U87MGtumors was significantly improved from approximately3% to 4% to approximately 10% to 15% of the total

injected dose (ID)/g, owing to the specific RGD-integrinavb3 recognition. Raman signatures of SWCNTs werefurther used to directly probe the presence of CNTs inmice tissues and confirmed the radiolabel-based results[55]. In another experiment to evaluate the influences ofPEG chain lengths on cellular uptake of PEGylatedSWCNTs, it has been found that adsorbing shorterchain PEG (PL-PEG-2000) to SWCNTs was incapable ofprotecting CNTs from macrophagocytosis both in vitroand in vivo, while adsorbing longer chain PEG (PL-PEG-5000) effectively reduced their nonspecific uptakeof CNTs in vivo [56]. Functionalization of SWCNTswith PEG grafted branched polymers, namely poly(mal-eicanhydride-alt-1-octadecene)-PEG methyl ethers(PMHC18-mPEG) and poly (g-glutamic acid)-pyrine(30%)-PEG methylethers (70%) (gPGA-Py-mPEG), theblood circulation time was remarkably prolonged (half-

life of 22.1 h for gPGA-Py-mPEG and 18.9 h forMHC18-mPEG) after intravenous injection into mice[57]. Further research reveals that the tumor accumula-tion of PEG-SWCNTs was 8% ID/g and 9% ID/g of theintravenously administered doses in EMF6 model (breastcancer in BABL/c mice) and the Lewis model (lung can-cer in C57BL mice), respectively. SWCNTs covalentlymodified with PEG showed longer half-life in blood cir-culation in comparison with those noncovalently modi-fied with PEG of similar chain lengths. SWCNTscovalently conjugated with branched chains of 7-kDaPEG effectively increased the half-life of SWCNTs up to1 day, which is the longest among all of the testedPEGs. And this length chain PEG-modified SWCNTshad near-complete clearance from the main organs inapproximately 2 months. There seemed to be a lengthlimits in the relations between PEG chain lengths andtheir effects to increase the blood circulation time.Further increase in molecular weight from 7 to 12 kDahad no influence on the blood circulation time and RESuptake [58].

There are few literatures on the in vivo biodistributionproperties of radionuclide-filled CNTs, although theyhave been extensively used as drug delivery systems orradiotracers. A very recent study revealed that surface

functionalization of 125

I-filled SWCNTs offers versatilitytowards modulation of biodistribution of these radio-emitting crystals, in a manner determined by the systemthat delivers them, which gave great promises for thedevelopment of organ-based therapeutics [59]. Nanoen-capsulation of iodide within SWCNTs facilitated its bio-distribution in tissues, and SWCNTs was completelyredirected from tissue with intrinsic affinity (thyroid) tolungs. In this experiment, Na125I-filled glyco-SWCNTswere intravenously administered into mice and trackedin vivo by single photon emission computed tomogra-phy. Tissue-specific accumulation (lung in this case),



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coupled with high in vivo stability, prevented excretionor leakage of radionuclide to other high-affinity organs(thyroid/stomach), allowing ultrasensitive imaging anddelivery of unprecedented radiodose density [60].

Metabolism and excretion

The nonbiodegradability in the body and non-eliminat-ability from the body interrogate on the possibility oftheir successful use in clinical practice, which has beenalways concerned about.

Functionalized SWCNTs seem to be metabolizable inanimal body. For example, SWCNTs with carboxylatedsurfaces have demonstrated their unique ability toundergo 90-day degradation in a phagolysosomal simu-lant, resulting in shortening of length and accumulationof ultrafine solid carbonaceous debris. Unmodified, ozo-nolyzed, aryl-sulfonated SWCNTs exhibit no degrada-

tion under similar conditions. The observed metabolismphenomenon may be accredited to the unique chemistryof acid carboxylation, which, in addition to introducingthe reactive, modifiable COOH groups on CNT surfaces,also induces a collateral damage to the tubular graphe-nic backbone in the form of neighboring active sitesthat provide points of attack for further oxidative degra-dation [59]. Some experiments demonstrated that CNTspersisted inside cells for up to 5 months after adminis-tration; short (< 300 nm) and well-dispersed SWCNTseffectively managed to escape the RES and finally wereexcreted through the kidneys and bile ducts [61].

A very recent investigation reveals that the biodegra-dation of SWCNTs can be catalyzed by hypochloriteand reactive radical intermediates of the human neutro-phil enzyme myeloperoxidase in neutrophils. The phe-nomenon of CNT metabolism can also be seen inmacrophages to a lesser degree. Molecular modelingfurther reveals that the interaction between basic aminoacid residues on the enzyme backbone and carboxylacid groups of CNTs is favorable to orient the nano-tubes close to the catalytic site. Notably, when aspiratedinto the lungs of mice, the biodegradation of the nano-tubes does not engender an inflammatory response.These findings imply that the biodegradation of CNTs

may be a key determinant of the degree and severity ofthe inflammatory responses in individuals exposed tothem. However, further studies are still required inorder to draw an appropriate conclusion [62].

CNT-based drug delivery

While attachment of drugs to suitable carriers signifi-cantly improves their bioavailability, owing to theirincreased residence time in blood circulation andenhanced solubility, the therapeutic efficacy of the drugcan be improved by the site-selective accumulation inthe pathological zone of interest that sometimes were

called therapeutic-effects-related sites. The unique cap-ability of CNTs to penetrate cell membranes paves theroad for using them as carriers to deliver therapeuticagents into the cytoplasm and, in many cases, into thenucleus. The intrinsic spectroscopic properties of CNTs,such as Raman and photoluminescence, afford addi-tional advantages for tracking and real-time monitoringof drug delivery efficacy in vivo.

Intracellular drug delivery

To study the cellular drug delivery, in vitro experimentshave unique advantages, which are convenient to carryout; experiment conditions are easy to control and cangive reliable results, although they cannot completelyrepresent in vivo case.Small molecules

Most of the anticancer agents are small molecules and

can be loaded into or onto CNTs either by physicaladsorption through p-p stacking interactions betweenpseudoaromatic double bonds of the graphene sheet andthe drug molecules, or covalent immobilization of theinterest drug molecules onto the reactive functionalgroups present on the sidewalls of CNTs.

Recently, Borowiak-Palen et al. reported that cisplatin,a small molecule, can be loaded into SWCNTs with adiameter of 1.3 to 1.6 nm [63]. The cisplatin incorpo-rated into the tubes was proved with Raman spectro-scopy, infrared spectroscopy, and high-resolutiontransmission electron microscopy (TEM). Drug-releasestudy using dialysis membrane method revealed that cis-platin was continually released for almost a week, withmaximum release during 72 h and up to 1 week. Theencapsulation was 21 g of drug per 100 g of SWCNTsas revealed by thermogravimetric analysis. Cytotoxicitystudies carried out on DU145 and PC3 human prostatecancer cell lines using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) cell proliferationassay showed that the cell viability decreased with anincrease in the concentration of the CNT-based nano-

vector , whereas blank CNTs showed no signif icanteffects. Computational methods revealed the feasibilityof interactions between CNTs and drug molecules [64].

For cisplatin acceptance or incorporation, CNTs musthave a radius of at least 4.785 (0.4785 nm). In fact,most of the experimentally used CNTs have diametersgreater than 4.785 . So, it is inferred that cisplatin islikely to be encapsulated inside the nanotubes [65].

Doxorubicin can be loaded on CNT to form supramo-lecular complexes based on p-p stacking interactions bysimply mixing the drug with an aqueous dispersion ofCNTs stabilized by Pluronic F127 (nonionic surfactant).The doxorubicin loading on MWCNTs was observed bymeasuring the emission spectrum of doxorubicin viafluorescence spectrophotometry. With the increase in



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the concentration of MWCNTs from 5 to 20 g/ml, thefluorescence intensity of doxorubicin dramaticallydecreased with the final concentration in the suspensionremaining constant (10 g/ml), a part of which is attrib-uted to the quenching of fluorescence. It was found thata mass ratio of 1:2 is optimum for maximum interac-tion/quenching ratio. TEM structural characterizationrevealed that CNTs present as well-individualized, dis-persed nanotubes, confirming the polymer moleculesability to disperse the CNTs effectively. On MCF-7human breast cancer cell line, it was revealed that thedoxorubicin-MWCNT complex shows enhanced cyto-toxity in comparison with both doxorubicin alone anddoxorubicin-Pluronic complexes. The enhanced cyto-toxicity obtained with the doxorubicin-MWCNT com-plex indicates that MWCNTs can effectively enhancethe delivery of doxorubicin and hence improve the cel-

lular uptake of the drug [66], although in vivo studiesare essential in order to further validate the efficiency ofthe reported system. Some other groups have also devel-oped doxorubicin-loaded nanotubes but with more com-plex system structures. The system was composed ofoxidized SWCNTs trifunctionalized with doxorubicin, amonoclonal antibody (mAb), and a fluorescent markerand therefore can be used for targeting, imaging, andtherapeutic effects simultaneously. Confocal microscopyobservation revealed that the complex was efficientlytaken up by cancer cells and the doxorubicin wasreleased subsequently and then translocated to thenucleus, while SWCNTs remain in the cytoplasm [67].Of course, such a complex system requires rigorouslyinvestigating in order to check the integrity of theSWCNT hybrids during the course through biologicalmilieu for its in vivo use. Zhang et al. have successfullyprepared biocompatible and water-dispersible multifunc-tional drug delivery system with doxorubicin-loadedpolysaccharide functionalized SWCNTs, which presentsstimuli-responsive drug-release characteristics in addi-tion to simultaneous targeting. The chitosan/alginatepolymer chains have been wrapped around the CNTssimply by sonication and stirring of a chitosan/alginatesolution containing CNTs. At acidic pH, hydrophilicity

of doxorubicin is enhanced, which facilitates its detach-ment from the CNT surface. Compared with normal tis-sues, physiological pH condition of tumor environmentand intracellular lysosomes is more acidic, and therefore,this system seemed to be able to intelligently releasedrugs in tumor tissues. Through tethering the freeamino groups of chitosan with folic acid (FA), the tar-geting effects may be further improved. Such nanocar-rier-based drug delivery systems for doxorubicin couldbe more selective and effective than the free drug andhave the promise to result in reduced toxicity and sideeffects in patients, along with a smaller drug dose

needed for chemotherapy [68]. Thus, such CNT-basedsupramolecular systems with structural uniqueness ofthe doxorubicin are capable of self-targeting due to theaforementioned mechanisms. Furthermore, this can bebolstered through ligand-based targeting by incorpora-tion of special ligands, similar to RGD peptide, whichtargets integrin receptors, making it a multitargetedmodality complementing each other for selective actionat cancerous tissue [69].

Antioxidants have been considered to play a signifi-cant role in cancer therapy owing to their ability tocombat oxidative stress. However, their poor solubilitymitigates the reaping of the benefits from these com-pounds. This drives us to bring them under the canopyof nanocarriers in order to use them as practical phar-maceuticals. Covalently PEGylated ultrashort SWCNTscan be linked to the antioxidant, amino butylated

hydroxy toluene, through ionic interactions by simplestirring of the mixture. Residual carboxylic acid groupson the oxidized CNT allow the ionic interactions withthe amine group of the butylated hydroxy toluene. Theformulation was evaluated using an oxygen radicalabsorbance capacity assay, in which a fluorescent probesloss of fluorescent intensity is monitored in the presenceof oxygen radicals. Oxygen-radical scavengers can keepthe fluorescence of the probes. The fluorescence inten-sity remains unaffected until the radical scavenger isconsumed when oxygen-radical scavengers are added tothe system. The assay readout can be compared to theradical scavenging ability of a known radical scavenger,Trolox, a vitamin E derivative. The radical scavengingability of the composite was found to be as high as1,240 times that of Trolox. However, when the butylatedhydroxy toluene functionalization was carried outthrough covalent addition to the sidewall, the antioxi-dant activity of the system was found to be decreased[14], suggesting that not all functionalizations are bene-ficial for antioxidant activity.

Poor blood circulation times of platinum anticancerdrugs result in insufficient uptake by tumor tissues andintracellular DNA binding due to their unusually lowsize, making them suitable candidates for a nanoparti-

cle-based drug delivery system to improve their pharma-cological performance. For this purpose, a longboatdelivery system has been prepared for the platinumwarhead. In this system, a platinum complex [Pt (NH3)2Cl2(O2CCH2CH2CO2H) (O2CCH2CH2CONH-PEG-FA)derivatized with PEG and folate (FA) was attached tothe surface of SWCNT functionalized with aminogroups (SWNT-PL-PEG-NH2) through multiple amidelinkages. Such a unique surface design facilitates activetargeting of the prodrug to the tumor cell, where cispla-tin is released upon intracellular reduction of Pt(IV) toPt(II) after endocytosis. Internalization studies revealed



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observe the target effects in a model of disseminatedhuman lymphoma and in cells by flow cytometry andcell-based immunoreactivity assays versus appropriatecontrol cells. Chakravarty et al. in a pioneering study,used biotinylated polar lipids (1,2-distearoyl sn-glycero-3-phosphoethanolamine-N-[biotinyl (PEG)2000] [DSPE-PEG(2000)-biotin]) to prepare stable, biocompatible,noncytotoxic CNT dispersions. Then, CNTs were func-tionalized by one of two different neutralite avidin-deri-

vatized mAbs against either human CD22 or CD25. Theperipheral blood mononuclear cells activated by CD22+CD25- and CD22-CD25+ cells can be bound only bythe CNTs bearing the corresponding mAbs, respectively.And only the cells bound by the corresponding nanosys-tems were ablated after exposure to NIR light [79]. Kamet al. reported an approach similar to that of Chakra-

varty et al., in which targeting effects were achieved by

using PL-PEG-FA functionalized SWCNTs [80].Tumor lysate proteins (TLP) are composed of varioustumor proteins that develop in a large number oftumors and patients, irrespective of the genetic originsof tumors. However, defined tumor markers, such astarget antigens, are still in a lack, rendering the antibodyresponses difficult to assess, and since there is a lack ofthe nature of the immunogens, the efficacy of therapyinvolving the lysate proteins was generally assessed bytumor rejection, tumor growth retardation, or prolongedsurvival of the immunized mice rather than by antibodyproduction. The anticancer immune response of TLPagainst multiple tumors [81] has been improved byCNTs. The efficacy of the RGD peptide for targetingtumors with overexpressed avb3integrin receptor is wellestablished. An application example has exploited asimilar strategy for active targeting of RGD-functiona-lized, PEGylated CNTs to integrin-positive tumors inmice. Based on similar lines, avb3 mAbs were used totarget the PL-PEG-modified SWCNTs. As targetingligands, avb3 mAbs have their advantages in terms oftheir high specificity towards antigen avb3 and greaterstability in vivo relative to RGD peptide. In spite of cellculture-based studies in U87MG (human glioblastomacancer cells) and MCF-7 (human breast cancer cells)

revealing good targeting efficiency, the behavior of sucha macromolecular structure needs to be traced insidethe complex biological system to confirm its value incancer therapy [82].RNA, DNA, or genes

The application of CNTs as gene carriers in gene deliv-ery has been considered quite promising. Gene therapyinvolves not only the gene-based treatment for cancersbut also that for the infectious diseases by introducinggenetic materials. It is generally believed that the tumorformation is the results of the gene alterations and genetherapy aims to correct them. For all of the gene-based

therapeutic strategies, efficient and safe gene deliverysystems have become imperative to develop, especiallythe gene vectors because it is relatively easy to obtaincorresponding genes. There have been two subcategoriesof gene vectors including many viral and nonviral vec-tors. Viral vectors have been modified to eliminate theirtoxicity and maintain their high gene transfer capability.However, their limited capacity for transgenic materialsand safety, particularly immunogenicity, has compelledresearchers to increasingly shift attention upon nonviral

vectors as an alternative. Nonviral vectors are mainlybased on cationic polymers. It is just recent thing thatCNTs emerge as DNA carriers owing to their uniquephysical, chemical, and biological properties [83].

Charged hybrid DNA/SWCNT complexes can beobtained by sonicating the suspensions composed of sin-gle-stranded DNA and CNTs. The aromatic nucleobases

are believed to bind to the graphene side walls through- stacking effects. DNA molecules can be confinedand oriented by CNTs that acted as scaffolds, whichwere wrapped around by DNA macromolecules. More-over, there are different interaction energies with nano-tubes for different nucleobases. The sugar andphosphate groups remain at the periphery relative to thebases, playing the roles of enhancing the dispersibility ofCNTs. The spontaneous wrapping of DNA aroundnanotubes has been also confirmed by atomic-forcemicroscopy and spectroscopic studies. Such a systemcan definitely prove useful in gene delivery. Severalexperimental studies in this area [84] were discussedhere.

A material capable of binding negatively chargedsiRNA through electrostatic interactions can be obtainedby functionalizing SWCNTs with hexamethylenediamine(HMDA) and poly(diallyldimethyl ammonium chloride)(PDDA). PDDA was bound by noncovalent interactions,whereas HMDA was covalently linked to the oxidizedCNTs. The experiments on isolated rat heart cellsrevealed that the effect of ERK1/ER K2 genes loadedonto HMDA-PDDA-SWCNTs enhanced efficiency forERK1and ERK2by a factor of nearly 80%. Furthermore,the biocompatibility has also been improved. No signifi-

cant cytotoxicity was caused by PDDA-HMDA-SWCNTcomplexes at concentrations of 10 mg/l [85]. Pantarottoet al. used CNTs functionalized with ammonium as vec-tors for transfecting plasmid (b-galactosidase encodedgene, b-gal) into HeLa and CHO cell lines. The transfec-tion efficiency was calculated based on the experimentaldata to be proportional to the CNT/DNA charge ratio,with the highest at 6:1. In comparison with DNA alone,transfection efficacy of CNT-DNA conjugates was tentimes higher [86]. Through layer-by-layer electrostaticself-assembly of polycationic agents such as polyethyle-nimine (PEI), PDDA, polyamidoamine dendrimers, and



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chitosan on nanotube side walls, cationic polyelectrolytefunctionalization of CNTs can be obtained, which areconsidered to be effective vehicles for gene delivery [87].For example, green fluorescence protein (EGFP) reporterprotein expression has been enhanced with functiona-lized CNTs complexed with nanochitosan as the genecarriers for the delivery of encoding DNA. In compari-son with blank chitosan, CNT-chitosan nanocomplexeshave exhibited significantly higher transfection efficiency[88]. The feasibility of bifunctional, MWCNT-quantumdot-based hybrids as gene transfer vector systems havealso been explored by recent investigations. Mercaptoa-cetic acid-capped CdTe quantum dots, as fluorescentprobes, were linked to antisense oligodeoxynucleotidesto suppress telomerase expression. Their activity is unu-sually high in 90% of cancer cells compared with normalcells. Then, these complexes further complexed with

functionalized MWCNTs through ionic interactions.Based on confocal and flow-cytometric studies, efficientintracellular transporting, strong cell nucleus localiza-tion, and high delivery efficiency of antisense oligodeox-

ynucleotides were exhibited by this system incomparison with free antisense oligodeoxynucleotides inHeLa cells. When the EGFPgene transfected using PEI-MWCNT as gene vectors, EGFP was overexpressed,further confirming the role played by the nanostruc-tures. The nanosystems and the enhanced protonsponge effects of PEI coating on the surface of MWCNTs prevent DNA from enzyme degradation [13]in cytoplasm and increase the ratio of the gene to beexpressed afterwards. However, efficacy of such deliverysystems was critically dependent on the chemistry ofsurface functionalization. Kam et al. have demonstratedthat effective transporting, enzymatic cleaving, andreleasing of DNA from SWCNT transporters and subse-quent nuclear translocation of DNA oligonucleotides inmammalian cells can also be achieved by incorporatingcleavable disulfide bonds on the surface of PL-PEGfunctionalized SWCNTs. Such functionalization showednot only that the delivery of siRNA was highly efficientbut also that the RNAi functionality achieved in thisway was more potent than the conventional transfection

agents that have been widely used [89].

In vivo studies on drug delivery

As drug carriers, they will be finally used in living ani-mals and human. Although the results of the in vitroexperiments have provided a lot of useful informationabout the application of CNTs as drug carriers, only thein vivo experiments can give corroboration for the use-fulness of CNTs in practical gene delivery for cancertherapies. However, there have been only limited in vivostudies reported on the application of CNTs as themolecular transporter in drug delivery.

Drug delivery targeted to lymphatic system

Many cancers metastasize through lymphatic canal. Thedrug delivery systems targeted to the lymphatic systemcan block the metastasis of cancers effectively. Usingradical polymerization, polyacrylic acid (PAA) can beappended onto CNTs, making them highly hydrophilic.Through coprecipitation, Fe3O4-based magnetic nano-particles can be adsorbed on the PAA-CNT surface.Through the interaction with COOH groups of graftedPAA, the nanoparticles can be stabilized from clustering.By stirring the solution containing PAA-CNT, Fe3O4-based magnetic nanoparticles, and gemcitabine for 24 h,gemcitabine was loaded into the nanosystem with aloading efficiency of 62%. It was found that CNTs wereseen only in the local lymphatic nodes and were absentin the major organs, such as liver, kidney, heart, spleen,and lungs, after 3 h of subcutaneous injection. Without

the help of such a nanostructures [90], gemcitabine can-not preferentially distribute in the lymphatic system.Drug delivery targeted to tumor

To deliver anticancer drugs into cancer focus is the pre-requisite for the drugs to develop their effects. However,some drugs cannot arrive or enter cancer tissuesbecause of their short residence time in blood circula-tion. For example, the efficacy of paclitaxel (PTX), awidely used chemotherapeutic agent in cancer therapy,is often limited by its poor solubility in aqueous mediumand nonspecific cytotoxicity, thereby preventing it fromefficiently reaching the cancer focuses. Furthermore, thesolubilizer cremophor in current formulation (Taxol)has exhibited allergenic activity, prompting the searchfor alternative delivery systems. For this purpose, Liu etal. successfully conjugated PTX to branched PEG chainson SWCNTs via a cleavable ester bond to obtain awater-soluble SWCNT-PTX complex. In a murine 4T1breast cancer model, the SWCNT-PTX complex showedan efficacy higher than that of the clinically used Taxolin suppressing tumor growth. The blood circulationtime almost was sixfold higher than Taxol, which wasattributed to the PEG fictionalization. PTX uptake oftumor for SWCNT-based PTX delivery system is likelythrough an enhanced permeability and retention effect

(EPR effects). Hepatic macrophage showed considerableuptake [91], but histopathological and biochemical stu-dies found no obvious morphological changes, althoughthe function of the liver requires examining for anypotential side effects of SWCNT-PTX.

Wu et al. successfully constructed a novel MWCNT-based drug delivery system by tethering anticancer agenthydroxycamptothecin (HCPT) onto the surface ofMWCNTs. By carboxyl enrichment via optimized oxidi-zation treatment, CNTs were surface-functionalized,which was followed by amidation with a hydrophilic dia-minotriethylene glycol, and subsequent conjugation of



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succinylated HCPT to hydroxyl derivatized MWCNTswas achieved via a cleavable ester linkage. In compari-son with the clinical, presently used HCPT formulation,the MWCNT-HCPT complexes demonstrated superiorantitumor activity and low toxicity both in vitro and invivo. The prepared complexes had longer blood circula-tion time and higher tumor-specific drug accumulationas in vivosingle photon emission computed tomographyimaging and ex vivo g-scintillation counting analysis dis-closed. These properties synergistically boost the antitu-mor efficacy of the conjugate [92]. Through in vivoobservation of the killing effects on cancer cells, Bhirdeet al. demonstrated superior efficacy of drug-SWCNTbioconjugates over nontargeted bioconjugates [93]. Inorder to specifically target squamous cancer, anticanceragent cisplatin and epidermal growth factor (EGF) wereattached to SWCNTs. SWCNT-cisplatin without EGF

was used as a nontargeted control. It was revealed thatSWCNT-quantum dot-EGF bioconjugates internalizedrapidly into the cancer cells as confirmed by imagingstudies with head and neck squamous carcinoma cells(HNSCC) overexpressing EGF receptors (EGFR) usingquantum dot (linked covalently) luminescence and con-focal microscopy. Control cells showed limited uptakeand cellular uptake can be blocked by siRNA knock-down of EGFR, revealing the importance of EGF in thesystem. Imaging with three color, two-photon intravital

video showed that injected SWCNT-quantum dot-EGFwas selectively taken up by HNSCC tumors causingrapid regression, but in those control animals, SWCNT-quantum dot was cleared from the tumor region in lessthan 20 min. HNSCC cells were also killed selectively bySWCNT-cisplatin-EGF, while control systems caused noeffect on the proliferation of these cells [93].

Dendritic cells are of importance for the inductionand regulation of immune responses. To carry siRNAinto the antigen-presenting dendritic cells in vivo, catio-nic 1,6-diaminohexane functionalized CNTs were pre-pared. Splenic CD11c+ dendritic cells, CD11b+ cells andalso Gr-1+CD11b+ cells comprising dendritic cells,macrophages, and other myeloid cells showed activetake up for the complexes. The complexes silenced sup-

pressor of cytokine signaling 1 (SOCS1) expression andretarded the growth of B16 tumor in mice [94]. It iswell known that telomerase reverse transcriptase plays acritical role in tumor development and growth throughthe maintenance of telomere structure. The same groupof authors coupled siRNA to SWCNTs via a CONH-(CH2)6-NH3

+ Cl - spacer, demonstrating that siRNAdelivered via SWCNT complexes silences the expressionof telomerase reverse transcriptase and inhibits the pro-liferation and growth of tumor cells both in vitroand inmouse models. By 48 h, all of the cells, such as LLC,TC-1, and 1H8 cells, treated with telomerase reverse

transcriptase SWCNT-siRNA showed an almost com-plete inhibition of proliferation in murine tumor models[95]. Unmodified siRNA with pristine SWCNTs havealso been complexed noncovalently to deliver iRNA tocancer cells in a more recent study. The complex wasprepared by simple sonication of pristine SWCNTs in asolution of siRNA that plays the dual role of cargo anddispersant for CNTs. It was envisioned that there wasstrong specific inhibition of cellular HIF-1 activity whensiRNA complexes targeted to hypoxia-inducible factor 1a (HIF-1a) were added in serum-containing culturemedia. The ability to response biologically to SWCNT-siRNA complexes have been observed in a wide varietyof cancer cell types. Moreover, the activity of tumorHIF-1a was significantly inhibited by intratumoraladministration of SWCNT-HIF-1a siRNA complexes inmice bearing MiaPaCa-2/HRE tumors, suggesting that

such SWCNT-siRNA complexes promise considerablyas therapeutic agents [96].Drug delivery targeted to central nervous system

To deliver drugs to central nervous system is still a ser-ious challenge in anticancer drug delivery system for thetreatment of the tumors in the central nervous systembecause of the blood-brain barrier. Recently, our groupdevised a simple drug delivery system of acetylcholinefor treatment of Alzheimer disease. Acetylcholine is nat-ural neurotransmitter of the cholinergic nervous systemand related with high-level nervous activities, such aslearning, memory, and thinking. Because of the synthesisimpairment, acetylcholine is decreased in the neurons inthe Alzheimer disease brain, leading to the incapabilityof learning, memory, and thinking. Providing acetylcho-line for the neurons would be able to prove the intellec-tual activities of the patients with Alzheimer disease.But there have been no way to deliver acetylcholine intobrain because the acetylcholine is a compound withstrong polarities, making it difficult to pass through theblood-brain barrier. In our newly prepared drug deliverysystem, the adsorption of acetylcholine on SWCNT wasconfirmed by Raman spectrum, although it wasunknown whether acetylcholine was adsorbed on thesurfaces or in the tubes of SWCNT. This system suc-

cessfully delivered acetylcholine into the neurons in thebrain through the axoplasma transformation of neurites(Figure4) and significantly improved the learning andmemory capabilities of the model animals with Alzhei-mer diseases [10]. This delivery system provided thefirst instance for nanocarriers to deliver drugs into neu-rons in the central nervous system, opening the door forthe drug delivery system for the treatment of the tumorsin central nervous system.

As regards the in vivoanticancer drug delivery system,it should be mentioned that the results of CNT-baseddrug del iver y has b een com pared w ith som e



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commercially available formulations. The results demon-strated several advantages of CNTs over other nanoma-terial-based drug delivery systems. For example, Liu etal. compared the efficacy and the toxicity of doxorubi-cin-loaded CNTs with that of doxorubicin-loaded nano-liposomes [97]. However, a decisive conclusion has not

been reached about whether CNTs are the best carriersin anticancer drug delivery systems because there aremany other anticancer drug delivery systems based onother nanomaterials, such as cationic polymers [98],fibrinogens [99], and PVP/PVAL nanoparticles [100],that have also shown great promises as drug carriers forcancer treatments. More comparison works are requiredto make sure of the position of CNTs as drug carriersin anticancer therapies. In addition, the reproducibilityof pharmacokinetic data is severely challenged by thefacts that the volume, diameter, and length of CNTs arenot well controlled in the studies, nor is there any

reliable data on their size distribution. The commerciali-zation of drug delivery systems based on CNTs will beimpeded by these problems. To the best of our knowl-edge, no such system has gone into clinical trial andmany difficulties are yet to be overcome before success-ful application of CNT-based drug delivery systems in

practical therapy comes into fruition.

The biosafety of SWCNT used as drug carriers

Although there are exciting prospects for the applicationof CNTs as drug carriers in medicine, one of the keyobstacles in the way for the use of SWCNT as drug car-riers is their biosafety. This problem has been quite con-troversial. There are many studies that believe SWCNTis safe, while many literatures have reported the damageeffects on cells in vitro and on tissues in vivo. Fortu-nately, excitement encompassing the attractive physico-chemical properties of CNTs has been tempered by

2 m500 nm2 m

200 nm 500 nm

BA C

ED

Figure 4SWCNTs enter the neurons in brain through axoplasmic transportation [10]. (A) SWCNTs in the lysosomes of a neuron (arrows);

(B) the magnification of the two lysosomes containing SWCNTs; (C) there are no SWCNTs in glial cells; ( D) a bundle of SWCNTs parallel to the

neurite in the section along the longitudinal axis of the neurite; (E) the SWCNTs are dot-like in the section vertical to the longitudinal axis of the

neurite.



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issues concerning their potential health hazards, despitesome toxic effects of CNTs have been observed, whichgive rise to concern about the biosafety of CNTs. Theobserved toxicity has been largely attributed to thestructural analogy between CNTs and asbestos fibers inthe high aspect ratio, large surface area, induction ofinflammation [101-103], fibrosis [104-106], and malig-nant mesothelioma [107,108] on inhalation, and, moreimportantly, their biopersistence [109-111]. A recent invitro study has revealed that there are close relationsbetween the biodurability of CNTs and the chemistry ofCNT surface functionalization. However, it was revealedby recent investigations that the toxicity of SWCNTs invivo is resulted from the aggregation rather than thelarge aspect ratio of individual CNTs [112-114]. Theexperiment demonstrated that granuloma-like structureswith mild fibrosis observed in mice treated with aggre-

gated SWCNTs were completely absent in mice treatedwith nanoscale dispersed SWCNTs, even after 30 daysof intratracheal administration. Histopathological sec-tions from the lung of the mice treated with nanoscaledispersed SWCNTs revealed uptake of the SWCNTs bymacrophages and demonstrated the gradual clearanceover time, corroborating the biosafety and biocompat-ibility of CNTs in the form of nanoscale dispersions inin vivo applications. Further attempt has been tried toevaluate the toxicity of SWCNTs in Swiss mice as afunction of dose, length, and surface chemistry. Someresearchers have observed that oral administration ofCNTs, even at very high doses (1,000 mg/kg body-weight), induces neither death, growth, nor any beha-

vioral dilemma. When intraperitoneally administered,SWCNTs were found to coalesce inside the body, form-ing fiber-like structures. It was further observed thatsmaller aggregates almost induced no granuloma.Rafeeqi and Kaul explored the interactions betweenmulti-walled carbon nanotubes and cell culture mediumby spectroscopy, and the results supported biocompat-ibility of these nanotubes [115]. From these literatures,both positive and negative biological effects of SWCNTscan be seen, which has been attributed to the methodsused in the experiments. For example, Mittal et al.

found that clear interference of CNTs with conventionalin vitro cytotoxicity assays (MTT, NRU, and LDH) wasfound, which was confirmed by cellular system, butmorphological changes, and flow cytometry showed thecharacteristics of cytotoxicity [116] . Brown et al .reported that the cytotoxicity of CNTs is related to theirlengths [117].

More recently, we carefully and extensively investi-gated the biosafety problem of SWCNTs [10]. Theresults demonstrated that SWCNT are considerably safewith a safe range of 12 as revealed by the ratio of ED99/LD99, where the ED99 is the doses effective for half of

the model animals with Alzheimer disease. It was alsodemonstrated that the pharmacological and toxicologicaleffects of SWCNT are mediated separately by lysosomesand mitochondria. I n v i tr o, SWCNT induced theincrease of reactive oxygen species (ROS) in lysosomeswhile had no influences on the ROS level in mitochon-dria. In vivo, SWCNT exclusively distributed in lyso-somes when the doses was under 300 mg/kg, but therewas no ultrastructural damage as revealed by transmis-sion electron microscope. SWCNT began to enter mito-chondria when the doses got over 400 mg/kg. OnceSWCNT entered, the pathological ultrastructuralchanges developed in mitochondria. The mitochondriadilated, the crestae decreased or disappeared, and eventhe whole mitochondria became a vacuole. Once themitochondria damaged developed, the lysosomes alsoappeared as damaged. The membrane of lysosomes

destroyed, the contents of lysosomes decreased orexcreted, and even the whole lysosomes became a hugevacuole. All of these results indicated that mitochondriaare the original organelles for SWCNT damage and thelysosomal damage is secondary to mitochondrialdamage, namely, mitochondria are the toxicological tar-get organelles and lysosomes are the pharmacologicaltarget organelles of SWCNT. Further experimentsdemonstrated that the damage of SWCNT on mito-chondria was resulted by its influences on the mito-chondrial membrane potentials (MMP). The decrease inMMP means there is leakage of free electrons, whichresults in the increase of the ROS, further leading to theultrastructural damage of the cells including lysosomes.The mechanism of SWCNT damage can be summarizedin Figure5.

The progress in several aspects of the safety studies onSWCNT is of great importance for its practical use asdrug carriers for cancer treatment. Firstly, the modifica-tion or functionalization can significantly decrease thetoxicity of CNTs, making it possible to select safe CNTderivatives as drug carriers. Secondly, it has been foundthat CNTs can be metabolized in liver and eliminatedthrough kidneys and hap-bile systems, making less con-cern about the persistence residence of them in bodies.

Thirdly, it has been illustrated that the pharmacologicaland the toxicological effects of CNTs are mediated bydifferent target organelles and the distribution of CNTsin organelles can be regulated by some chemicals, mak-ing it possible to use the advantage and decrease oravoid the disadvantage of them. Fourthly, the dose dif-ferences exist between the pharmacological and toxico-logical effects of CNTs, which means that thetoxicological effects may be avoid by controlling thedoses. Through these progresses, it can be predictedthat the safety problems will be solved in not too long atime.



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The future of CNTs used as drug carriers for cancer

treatments

Summarizing the above described progress in the studies onthe application of CNTs as drug carriers, it may be seen thatthe chemistry on the modification of CNTs now has consid-erably grown up. The previous studies have provided us var-ious chemical methods to solve some fundamental problems

in the use of CNTs as drug carriers such as their water solu-bility and target properties. Many reported results, includingthose obtained from in vitroand in vivoexperiments, havedemonstrated that CNTs can increase the treatment effectswhile decrease the side and toxic effects of the drugs loadedon them, indicating a considerably bright future for them tobe used as drug carriers. However, there is a long way to go

ATPADPPi

e-

e-

e-

e-

e-

O

O

O

O

e-

O

e-

O

O

e-

O

ROS

e-

e-

e-

e-O

e-

O

e-

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mitochondria electron transmission chains

Figure 5The schematic illustration of the mechanisms of SWCNT to induce cell damage. Based on the studies of Yang et al. [10]. SWCNTs

interact with the mitochondria electron transmission chains (ETC) (by binding to ETC?) after they enter into mitochondria (1); The interaction of

SWCNTs with ETC blocks the transmission of electrons, which results in the increase of the leaking of free electrons from ETC (2); The leaked free

electrons form free radicals H2O2 or reactive oxygen species (ROS) (3); The free radicals or ROS attack the membrane system of mitochondria

through peroxidation (4); Then the free radicals or ROS diffuse through the damaged mitochondrial membrane to lysosomes to destroy the

membrane of the lysosomes (5); The injured lysosomes release digestive enzymes, leading to the damage or death of the whole cells. On the

other hand, the blocking of ETC makes mitochondria incapable of producing ATP (7), which results in the depletion of energy for the living

activities of the cells, also leading to the damage or death of the whole cells (8).



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for CNTs to get into practical use. Particularly, the pharma-cological and toxicological profiles must be made completelyclear and the advantages and the disadvantages of CNTsmust be carefully weighed before they are used as drug car-

riers in human body.On the bases of matured chemical modification, the

remaining key to the successful practical use of CNTs asdrug carriers is to make clear of the mechanisms fortheir pharmacological and toxicological effects. Theunderstanding of the pharmacological mechanismsmakes it possible to take the advantage of CNTs to theoutmost and to avoid or limit the disadvantages to pos-sibly low degree (Figure6). The weighing of the advan-tage and disadvantage in the treatment of a specialdisease is also very important because CNT-based drugdelivery system also has its indication and contraindica-

tion just like any other drugs.After all, the studies on the application of CNTs asdrug carriers for the treatment of cancers have achievedimportant progress. Some key obstacles in the way topractical use have been overcome. Although there is stilla long way to go for the practical use, it may be pre-dicted that, on one day in the future, CNTs will becomean important class of drug carriers for cancer treatment.

Acknowledgements

This work is supported by National Basic Research Program of China No

2010CB933904 and Major New Drug Creations No 2011ZX09102-001-15

Author details1Institute of Pharmacology and Toxicology and Key Laboratory of

Nanopharmacology and Nanotoxicology, Beijing Academy of Medical

Science, Zhengzhou, Henan, Peoples Republic of China 2Nanotechnology

Research Center for Drugs, Zhengzhou University, Zhengzhou, Henan,

Peoples Republic of China

Authors contributions

ZWX drafted the manuscript. ZZZ and ZYG go over and corrected themanuscript. All authors read and approved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 27 July 2011 Accepted: 13 October 2011

Published: 13 October 2011

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to outmost

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the application of carbon nanotubes for targetted drug delivery system for cancer therapies