Carbon Nanotubes for Delivery of Small Molecule Drugs

Carbon nanotubes for delivery of small molecule drugs

Bin Sheng Wong a,, Sia Lee Yoong b, Anna Jagusiak c, Tomasz Panczyk d, Han Kiat Ho a,Wee Han Ang e, Giorgia Pastorin a,b,a Department of Pharmacy, National University of Singapore, S4 Science Drive 4, Singapore 117543, Singaporeb NUS Graduate School for Integrative Sciences & Engineering (NGS), National University of Singapore, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456, Singaporec Chair of Medical Biochemistry, Jagiellonian University Medical College, ul. Kopernika 7, 31034 Cracow, Polandd Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30239 Cracow, Polande Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore

either the main carrier or adjunct material for the delivery of various non-anticancer drugs.small molecule drugs is expounded, with special attention paid to the current prog-

. . .arbon n. . .

2.1.3. Anthracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1992

Advanced Drug Delivery Reviews 65 (2013) 19642015

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j ourna l homepage: www.e lsev ie r .com/ locate /addrAbbreviations: AAS, Atomic absorption spectroscopy; AMB, Amphotericin B; BBB, Blood brain barrier; BCEC, Brain capillary endothelial cells; BSA, Bovine serum albumin; CDDP,Cisplatin; Ce6, Chlorin e6; CEA, Carcinoembryonic antigen; CHI, Chitosan; CNF, Carbon nanober; CNTs, Carbon nanotubes; CP, Carboplatin; CPT, Camptothecin; CT, Catechin; DAU,Daunorubicin; dC, 2,2-Diuoro-2-deoxycytidine; DDS, Drug delivery system; DEX, Dexamethasone; DMAAM, N-dimethylacrylamide; DNA, Deoxyribonucleic acid; DOX, Doxorubicin;DSPE-mPEG 2000, 1,2-Distearoyl-phosphatidylethanolamine-methoxy-polyethylene glycol conjugate-2000; DTX, Docetaxel; DWCNTs, Double-walled CNTs; EAT, Ehlrich ascitestumor; EC, Ethyl cellulose; EDBE, 2,2-(Ethylene dioxy) bis(ethylene amine); EDX, Energy dispersive X-ray analysis; EGF, Epidermal growth factor; EGFR, EGF receptors; EPC,Endothelial progenital cell; EPI, Epirubicin; EPR, Enhanced permeability and retention; ER, ES receptor; ES, Estradiol; FA, Folic acid; FITC, Fluorescein isothiocyanate; FR, FA receptor;FTIR, Fourier transform infrared spectroscopy; GelCT, Gelatincatechin; GEM, Gemcitabine; GNP, Gold NP; HA, Hyaluronic acid; HCPT, 10-Hydroxycamptothecin; HET-CAM, Hen's eggtest-chorioallantoic membrane; HMM, Hexamethylmelamine; HMME, Hematoporphyrin monomethyl ether; HR, Hyaluronan receptor; HUVEC, Human umbilical vein endothelialcells; ICP-OES, Inductively coupled plasma optical emission spectroscopy; LcL, Luciola cruciate luciferase; LRP, Lipoprotein receptor-related protein; mACs, Magnetic activated carbonparticles; MAPK, Mitogen-activated protein kinase; MDR, Multidrug resistance; MTX, Methotrexate; MWCNTs, Multi-walled CNTs; NIPAM, N-isopropylacrylamide; NIR, Near infrared;NP, Nanoparticles; NSAID, Non-steroidal anti-inammatory drugs; ODT-f-GNP, 1-Octadecanethiol functionalized GNP; P-gp, P-glycoprotein; PAA, Poly(acrylic acid); PAMAM,

Poly(amidoamine); PBS, Phosphate buffered saline; PCA,therapy; PEG, Polyethylene glycol; PEG PSS, Poly (ethylePoly(lactide); PSS, Poly(sodium 4-styrene sulfonate); Pt,system; RF, Radiofrequency; Rh, Rhodamine; ROS, ReactivSmall interference ribonucleic acids; SWCNTs, Single-waCNTs; UVvis, Ultravioletvisible; XPS, X-ray photoelectro This review is part of the Advanced Drug Delivery Revi Corresponding author. Tel.: +65 6516 1876; fax: +6 Correspondence to: G. Pastorin, Department of Pharma

E-mail addresses: [email protected] (B.S. Wong), ph

0169-409X/$ see front matter 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.addr.2013.08.005. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1991. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1991

s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1991

2.1.1. Topoisomerase I inhibitors2.1.2. Topoisomerase II inhibitorContents

1. Introduction . . . . . . . . . .2. Delivery of anticancer drugs with c

2.1. Topoisomerase inhibitors .on inevitable complications that hamper successful disease intervention with CNTs. 2013 Elsevier B.V. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1965anotubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1991Anticancer drugsNon-anticancer drugs

ress of in vitro and in vivo research involving CNT-basedDDSs, before nally concludingwith some considerationDrug deliverySmall molecule drugs In this review, the delivery ofKeywords:Carbon nanotubes

displaying superior efcacy, eery system (DDS) had been ea b s t r a c ta r t i c l e i n f o

Article history:Accepted 5 August 2013Available online 14 August 2013

In the realm of drug delivery, carbon nanotubes (CNTs) have gained tremendous attention as promisingnanocarriers, owing to their distinct characteristics, such as high surface area, enhanced cellular uptake andthe possibility to be easily conjugated with many therapeutics, including both small molecules and biologics,

nhanced specicity and diminished side effects. While most CNT-based drug deliv-ngineered to combat cancers, there are also emerging reports that employ CNTs asPolycitric acid; PDM, Polyamholyte poly [2-(dimethylamino) ethyl methacrylate]-co-(methacrylic acid); PDT, Photodynamicne glycol-b-propylene sulde); PEI, Polyethylenimine; PEO, Poly-ethylene oxide; PK, Pharmacokinetic; PL, Phospholipid; PLA,Platinum; PTT, Photothermal therapy; PTX, Paclitaxel; PVA, Poly(vinyl alcohol); QD, Quantum Dot; RES, Reticuloendotheliale oxygen species; SCID, Severe combined immunodecient; SD, Sprague Dawley; SEM, Scanning electron microscopy; siRNA,lled CNTs; TEM, Transmission electron microscopy; TPGS, Tocopheryl PEG succinate; Trf, Transferrins; US-CNTs, Ultra-shortn spectroscopy.ews theme issue on Carbon nanotubes in medicine and biology Therapy and diagnostics.5 6779 1554.cy, National University of Singapore, S4 Science Drive 4, Singapore 117543, Singapore. Tel.: +65 6516 1876; fax: +65 6779 [email protected] (G. Pastorin).

ights reserved.

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novel and functional microelectronics, energy storage devices, lledcomposites, nanoprobes, sensors and templates [11].

system (DDS), targetingmolecules, such as folic acid (FA) [45], antibodies[46] and even magnetic NP [47] can be further incorporated onto the

1965B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015In terms of biomedical applications, CNTs have also demonstratedimmense potentials, particularly in the areas of tissue engineering, ther-mal ablation and drug delivery [12,13]. As scaffolding materials, CNTsare able to support the growth of bone cells [14,15], neurons [16,17]and cardiomyocytes [18], and even direct or promote the differentiationof stem cells into specic lineages, such as from human mesenchymalstemcells into bone cells [1921]. The ability for CNTs, especially SWCNTs,to absorb and convert electromagnetic radiation, specically near infrared(NIR), into heat or sound energy has been exploited for successfulphotothermal therapy (PTT) or photoacoustic therapy against cancercells [2225]. Regarding their application in the delivery of therapeuticagents, CNTs have also been popularly employed as carriers for controlledand targeteddrug delivery to improve thepharmacological activity of bio-activemolecules and simultaneously diminish their undesirable systemicside effects. Indeed, various therapeutic agents, ranging from small mole-cules such as chemotherapeutic drugs [2632], antimicrobials [33,34] andanti-inammatory agents [35], to more complex biologics like peptide-based vaccines [36,37], antibodies [38] and small interference ribonucleicacids (siRNA) [39], have been successfully delivered with CNTs using amultitude of strategies, demonstrating superior efcacy and reducedtoxicity.

In fact, CNTs possess many intriguing features that make them attrac-tive drug delivery carriers. Firstly, nanocarriers, including nanoparticles(NP), liposomes, and CNTs, experience the enhanced permeability and re-

drug-loaded CNTs (covalently or non-covalently) to confer eitheractive targeting capabilities via receptor-mediated endocytosis orlocal nanocarrier accumulation induced by external magnetic eld.In addition, imaging tags like radioactive nuclides [48] and uorescenceprobes [46] can also be conjugated with CNTs to observe their intracellu-lar trafcking and biodistribution in vitro and in vivo easily and non-invasively. Coupled with the NIR absorption capability of CNTs, multi-modal DDSs can also be created by combining NIR-induced PTT or drugrelease with conventional drug molecules or biologics [49,50].

Despite the above-mentioned advantages of CNTs for the purpose ofdrug delivery, such as high aspect ratio, functionalizable surface, fastcellular uptake, etc., the issues of toxicity surrounding the biomedicalapplications of CNTs still remain controversial to this date, with studiesdemonstrating conicting results regarding their safety proles [51,52].As a result, despite various successful attempts of delivering drugs withCNTs in vitro and, to a lower degree, in vivo, CNT-based DDSs are stillconsidered far from being accepted for use in actual clinical settings.Having said that, some preliminary understandings regarding the toxicityof CNTs have been unveiled. In general, CNTs with non-functionalizedhydrophobic surfaces and high degree of residual heavy metal contami-nation tend to be more cytotoxic [53,54]. The problem of heavy metalcontamination can be easily rectied by purication [48], while the issuesof poor aqueous dispersibility and high aggregation tendency of pristineCNTs can be resolved by appropriate surface functionalization [55].2.2. Platinum-based drugs . . . . . . . . . . . . . . . . . . .2.3. Antimetabolites . . . . . . . . . . . . . . . . . . . . . .

2.3.1. Antifolates . . . . . . . . . . . . . . . . . . . .2.3.2. Purine/pyrimidine antagonists . . . . . . . . . . .

2.4. Antimicrotubules . . . . . . . . . . . . . . . . . . . . .2.5. Other anticancer drugs . . . . . . . . . . . . . . . . . .

3. Delivery of non-anticancer drugs with carbon nanotubes . . . . . .3.1. Antimicrobials . . . . . . . . . . . . . . . . . . . . . .3.2. Anti-inammatories . . . . . . . . . . . . . . . . . . . .3.3. Antihypertensives . . . . . . . . . . . . . . . . . . . . .3.4. Antioxidants . . . . . . . . . . . . . . . . . . . . . . .3.5. Other non-anticancer drugs . . . . . . . . . . . . . . . .

4. Progress of in vivo research on CNT-based drug delivery systems . . .4.1. Anticancer drugs . . . . . . . . . . . . . . . . . . . . .

4.1.1. Topoisomerase inhibitors . . . . . . . . . . . . .4.1.2. Platinum-based drugs . . . . . . . . . . . . . . .4.1.3. Antimetabolites . . . . . . . . . . . . . . . . .4.1.4. Antimicrotubules . . . . . . . . . . . . . . . . .4.1.5. Other anticancer drugs . . . . . . . . . . . . . .

4.2. Non-anticancer drugs . . . . . . . . . . . . . . . . . . .5. Concerns regarding CNT-based drug delivery systems . . . . . . . .6. Conclusion & future perspectives . . . . . . . . . . . . . . . . .Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Following the discovery of their presence in the insoluble soot of arc-burned graphite rods in 1991 by Japanese physicist Sumio Iijima, carbonnanotubes (CNTs) had since gained tremendous attention as a versatilenanomaterial with abundant applications [1]. First of all, with excep-tionally high tensile strength and elastic modulus, CNTs represent oneof the strongest and stiffest materials to be discovered [2,3]. CNTs arealso excellent thermal [4,5] and electrical conductors [6,7], with addi-tional abilities to absorb optical intensity [8], photoluminesce [9] andgenerate strong Raman signals [10] that enable their facile and non-destructive characterization. Equippedwith all these tunable distinctivefeatures, CNTs have been investigated and applied successfully to createtention (EPR) effect, i.e. they exhibit higher accumulation in tumor tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1996

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as compared to normal tissues due to poorly formed blood and lymphaticvessels that supply rapidly proliferating tumors [40]. The EPR effectenables CNTs to transport chemotherapeutic agents preferentially totumor sites [41]. Secondly, the needle-like shape of CNTs facilitates trans-membrane penetration and intracellular accumulation of drugs viathe nanoneedle mechanism that is independent of additional CNTfunctionalization and cell types [42]. Aside from direct translocationthrough cellular membranes, CNTs have also been shown to enter cellsvia energy-dependent endocytic pathways [43]. Thirdly, as a platformfor drug attachment, CNTs, owing to their high aspect ratios and surfaceareas, display extraordinary ability for drug loading onto the surface orwithin the interior core of CNTs via both covalent and non-covalent inter-actions [44]. To further augment the efcacy of CNT-based drug deliveryFunctionalization of CNTs can be achieved by either non-covalently

Table 1Summary of the CNT-based DDS described in this review.

Drug CNT system Drug loading Release control Targeting mechanism Biological studies Ref

In vitro In vivo

Anticancer drugsTopoisomerase I inhibitorsHCPT MWCNTs covalently functionalized with

diaminotriethylene glycol spacersCovalent conjugation viaester linkage

lEsterases Uptake & cytotoxicity inMKN-28

Biodistribution, efcacy &toxicity inhepatic H22 tumorbearing ICR mice

[48]

CPT Oxidized MWCNTs functionalized with PVA Physical adsorption Cytotoxicity in MDA-MB-231 & A-5RT3

[64]

CPT Oxidized MWCNTs coated with Pluronic P123 Physical adsorption Uptake & cytotoxicity inHeLa

[65]

Irinotecan MWCNTs with open tips Physical encapsulation Acidic pH [66]

Topoisomerase II inhibitorsEtoposide Carboxyl SWCNTs functionalized with CHI & EGF Physical adsorption Acidic pH due to CHI

disruptionEGF against EGFR Uptake & cytotoxicity in

A549 [67]

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AnthracyclinesDOX SWCNTs functionalized with PEG with cyclic RGD Physical adsorption Acidic pH

CNT diameterCyclic RGD against integrinv3

Uptake & cytotoxicity inU87MG & MCF-7

[26]

DOX MWCNTs dispersed with Pluronic F127 Physical adsorption Cytotoxicity in MCF-7 [27]

DOX SWCNTs functionalized with branched PEG Physical adsorption PK, biodistribution, efcacy &toxicity in SCID mice withRaji lymphoma xenografts

[57]

DOX Oxidized SWCNTs functionalized with anti-CEAantibody & uorescein using BSA as multifunctionallinker

Physical adsorption Antibody against CEA Uptake in WiDr [46]

(continued on next page)

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Table 1 (continued)


In vitro In vivo

DOX SWCNTs functionalized with polysaccharide coating(CHI &/or sodium alginate) & FA

Physical adsorption Acidic pHCHI & sodium alginate ratio

FA against FR Uptake & cytotoxicity inHeLa

[45]

DOX SWCNTs covalently linked to P-gp antibody labeledwith FITC

Physical adsorption NIR Antibody against P-gp Uptake & cytotoxicity inK562 sensitive & resistantcell lines

[50]

DOX SWCNTs functionalized with FA-conjugated CHI Physical adsorption Acidic pH disruption of CHI FA against FR [86]

DOX MWCNTs dispersed with PEG-PSS labeled with FITC Physical adsorption Uptake in HeLa.Cytotoxicity in MDA-MB-435

[87]

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DOX MWCNTs functionalized withFA-hexamethylenediamine conjugate & iron NP.

Physical adsorption NIR FA against FRIron oxide NP for magnetictargeting

Uptake & cytotoxicity inHeLa

[85]

DOX Oxidized MWCNTs Physical adsorption Acidic pHSerum proteinShorter loading time

[75]

DOX SWCNTs functionalized with branched PEG2500-NH2 & FA

Physical adsorption Acidic pHSerum protein

FA against FR Uptake & cytotoxicity inHeLa

[71]

DOX PAA grafted MWCNTs functionalized withFA & iron NP

Physical adsorption Acidic pHIron oxide NP for magnetictargeting

FA against FR Uptake in U87, cytotoxicityin U87 & 3 T3

[76]

DOX EDBE-conjugated MWCNTs covalently functionalizedwith HA

Physical adsorption Acidic pH HA against HR Uptake & cytotoxicity inA549

Biodistribution in EATbearing miceEfcacy in chemically-induced breast cancerbearing SD ratsToxicity in mice & rats

[73]

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Table 1 (continued)


In vitro In vivo

DOX Oxidized MWCNTs functionalized withmulti-branched GNP& PEG methyl ether thiol

Physical adsorption Acidic pH Uptake in A549 [77]

DOX Iron NP-lled PSS modied CNTs conjugated withpoly(allylamine)-functionalized SiO2-coated CdTeQDs linked to transferrin

Physical adsorption Acidic pH Transferrin againstIron NP for magnetictargeting

Uptake in HeLa & HEK 293Cytotoxicity in HeLa

[47]

DOX CHI-coated SWCNTs covalently functionalized withFITC

Physical adsorption Acidic pH Uptake in EPC [78]

DOX CHI-coated SWNCTs chemically attached with FA Physical adsorption Acidic pH FA against FR Cytotoxicity in SMMC-7721 Efcacy & toxicity in nudeBALC/c mice inoculatedsubcutaneously with SMMC-7721

[79]

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DOX PEGylated oxidized MWCNTs modied withangiopep 2

Physical adsorption Acidic pH Angiopep 2 peptide againstLRP receptors

Uptake & cytotoxicity in C6& BCED

Biodistribution, efcacy &toxicity in glioma bearingBALB/cmice injectedwith C6into right striatum

[80]

DOX CNTs coated with zipper comprising PEI & PVA viahydrogen bonding

Physical adsorption Heat Uptake in breastadenocarcinomaCytotoxicity in lungbroblast, breastadenocarcinoma, HeLa,adult & neonatal HDF

[88]

DOX Amine-MWCNTs conjugated covalently with DEXmesylate

Physical adsorption Acidic pH DEX mesylate for nucleartargeting

Uptake & cytotoxicity inA549

[82]

DOX SWCNTs non-covalently functionalized with FA-terminated methoxy-PEG

Physical adsorption Acidic pH FA against FR Uptake & cytotoxicity inHeLa & 3 T3

[81]

DOX MWCNTs linked with EDBE conjugated with FA, HAor -estradiol-17-hemisuccinate

Physical adsorption Acidic pH FA against FRHA against HRES against ER

Uptake & cytotoxicity inA549, HeLa & MCF-7

[84]


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Table 1 (continued)

Drug CNT system Drug loading Release control Targeting mechanism Biolo al studies Ref

In vi In vivo

DOX MWCNTs covalently functionalized with amine-terminated PAMAM dendrimers modied with FITC& FA

Physical adsorption Acidic pH FA against FR Upta & cytotoxicity inhigh ow FR expressing KB

[74]

DOX Poloxamer 188 modied SWCNTs functionalizedwith AS1411 aptamer with NIR-inducedhyperthermia

Physical adsorption Acidic pH AS1411 aptamer againstnucleolin

Upta & cytotoxicity in EC109

[83]

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ke

DOX SWCNTs labeled with recombinant thermostable LcL Physical adsorption Biodistribution in FVB mice [93]

DOX SWNCTs functionalized with Cremophor EL Physical adsorption Acidic pH PK, biodistribution, efcacy &toxicity in S180 sarcomabearing ICR mice

[72]

DOX PEGylated SWCNTs with non-covalently attachedpyrene

Chemical conjugation ontopyrene with carbamatelinker

Enzymatic cleavage ofcarbamate

Uptake & cytotoxicity inB16-F10

Efcacy & toxicity in C57/BL/6 mice with subcutaneousimplantation of B16-F10

[99]

DOX PEGylated SWCNTs with hydroxinobenzoic acidlinker

Physical adsorption &chemical conjugation viahydrazone bonds

Acidic pH Uptake & cytotoxicity inHepG2 & HeLa

[100]

DOX Isolated SWCNTs dispersed in NIPAM & DMAAMhybrid gel

Physical adsorption Acidic pHNIR

[101]


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Table 1 (continued)


In vitro In vivo

EPI MWCNTs with or without carboxylic groups &SWCNTs

Physical adsorption Acidic pH [102]

DAU SWCNTs functionalized with PL-PEG Physical adsorption [26]

DAU SWCNTs functionalized with sgc8c aptamer Physical adsorption Acidic pH Sgc8c aptamer againsttyrosine kinase-7

Uptake & cytotoxicity inMolt-4 & U266

[103]

Pirarubicin SWCNTs functionalized with PL-branched PEG Covalent conjugation viaester bond

Cytotoxicity in BIU-87 &C2C-12

Efcacy & toxicity inchemically-induced SD ratbladder cancer model

[104]

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Mitoxantrone SWCNTs functionalized with branched PEG-NH2 Physical adsorption Acidic pH Cytotoxicity in HeLa [71]

Platinum-based drugsc,c,t-[Pt(NH3)2Cl2(OEt)(O2CCH2CH2CO2H)]

SWCNTs non-covalently functionalized with PL-PEG-NH2

Chemical conjugation Cellular reductiveenvironment

Cytotoxicity & intracellularPt content in Ntera-2

[29]

c,c,t-[Pt(NH3)2Cl2(O2CCH2CH2-CO2H)2]

SWCNTs non-covalently functionalized with PL-PEG-NH2. FAwas attached to the remaining axial ligand onPt (IV) prodrug

Chemical conjugation Cellular reductiveenvironment

FA against FR Uptake & cytotoxicity in JAR,KB & NTera-2

[115]

CDDP Oxidized SWCNTs functionalized with EGF Chemical conjugation viaester bond

EGF against EGFR Uptake in HN13 with EGFR& EGFR-knockdown control.Cytotoxicity in HN13 with &without EGFR, NIH-3T3 &SAA

Biodistribution & efcacy inHN12 xenograft mice

[116,117]


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Table 1 (continued)


In vitro In vivo

CDDP Oxidized SWCNTs covalently functionalized with EGF& PEG5000

Chemical conjugation viaester bond

EGF against EGFR Cytotoxicity in HN12 Biodistribution, efcacy &toxicity in HN12 xenograftmice

[199]

CP Open-ended oxidized MWCNTs Physical encapsulation Cytotoxicity in EJ28.Cytotoxicity in PC-3, DU145,EJ28, A498.

[122,125]

CDDP SWCNTs Physical encapsulation Cytotoxicity in PC-3 &DU145

[124]

CDDP Pristine MWCNTs with 1-octadecanethiol-coatedGNP caps

Physical encapsulation GNP cap Cytotoxicity in MCF-7 [126]

CDDP US-CNTs wrapped with Pluronic F108 Physical encapsulation Pluronic coatRF eld

Cytotoxicity & intracellularPt content in MCF-7 &MDA-MB-231Cytotoxicity in Hep3B &HepG2

[128,131]

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Pt (IV) prodrug MWCNTs Physical encapsulation Reductive environment (e.g.ascorbic acid)

Intracellular Pt content inA2780

[28]

Oxaliplatin Oxidized MWCNTs covalently functionalized withPEG600

Physical encapsulation PEG coating Cytotoxicity in HT29 [132]

AntifolatesMTX MWCNTs with 1,3-dipolar cycloaddition of

azomethine ylides with orthogonally protectedamino functions, tagged with FITC

Covalent conjugation Uptake & cytotoxicity inhuman Jurkat Tlymphocytes

[30]

MTX Oxidized MWCNTs with 2 different cleavable linkers,tetrapeptide Gly-Leu-Phe-Gly or 6-hydroxyhexanoicester

Covalent conjugation Proteases or esterases Uptake & cytotoxicity inMCF-7

[136]


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Table 1 (continued)


In vitro In vivo

MTX SWCNTs covalently functionalized with Oligo-HA-NH2

Covalent conjugation [137]

MTX MWCNTs non-covalently functionalized with DSPE-mPEG 2000

Physical adsorption Acidic pH [134]

MTX MWCNTs linked with EDBE conjugated with FA, HAor -estradiol-17-hemisuccinate

Physical adsorption Neutral pH FA against FRHA against HRES against ER


[84]

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Purine/pyrimidine antagonistsGEM PAA-grafted MWCNTs deposited with iron magnetic

NPPhysical adsorption Iron NP for magnetic

targetingUptake & cytotoxicity inBxPC-3 & SW1990

Biodistribution & toxicity inSD rats.Efcacy & toxicity inmetastatic nude BALB/c nu/nu mice subcutaneouslyinoculated with BxPc-3

[31,140]

GEM MWCNTs covalently conjugated with FA Physical adsorption Acidic pH FA against FR Cytotoxicity in MCF-7 Biodistribution & PK inalbino rats

[141]

dC SWCNTs covalently functionalized with PEI Physical adsorption Endoscopic ultrasound [142]

AntimicrotubulesSB-T-1214 SWCNTs covalently functionalized with biotin &

uoresceinChemical conjugation viacleavable disulde bond

Intracellular thiols Biotin against surface biotinreceptors

Uptake & cytotoxicity inL1210FR, L1210 & W138

[155]


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Drug CNT system Drug loading Release control Targeting mechanism Biolo al studies Ref

In vit In vivo

PTX SWCNTs adsorbed with branched PEG phospholipids Chemical conjugation viaester bond

Esterases Cytot icity in 4T1 PK, biodistribution, efcacy &toxicity in BALB/c micesubcutaneously injectedwith 4T1

[32]

PTX MWCNTs functionalized with hyperbranched PCA Chemical conjugation viaester bond

EsterasesAcidic pH

Cytot icity in A549 &SKOV

[156]

PTX PEGylated SWCNTs & MWCNTs Physical adsorption pH depending of nature ofCNTs

Cytot icity in MCF-7 &HeLa

[147]

PTX Supramolecular complex of SWCNTs & PDM Physical adsorption Uptak & cytotoxicity inCaco-

[148]

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ox

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PTX Hydroxy-functionalized MWCNTs covalently coatedwith PLA-PEG

Physical adsorption Uptake & cytotoxicity in U87& HUVEC.Inammatory proteinexpression in rat epithelialcells.

Biodistribution, toxicity &inammatory responses inBALB/c mice

[157]

PTX & C6-ceramide CNTs (no mention on the specic type) non-covalently functionalized with PL-PEG-NH2

Physical encapsulation Inductive heating withexternal alternating currentor magnetic eld pulse

Synergism study betweenPTX & C6-ceramide in L3.6,PANC-1 & MIA PaCa-2Uptake & cytotoxicity inL3.6.

[158]

PTX MWCNTs linked with EDBE conjugated with FA, HAor -estradiol-17-hemisuccinate

Physical adsorption Neutral pH FA against FRHA against HRES against ER


[84]


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In vitro In vivo

DTX SWCNTs non-covalently functionalized with PVP K30& DSPE-PEG-Maleimide linked with NGR peptide,combined with NIR-induced PTT

Physical adsorption NGR peptide against CD13 Uptake & cytotoxicity in PC-3

PK & biodistribution inhealthy mice.Efcacy & toxicity in murineS180 BALB/c mice

[49]

Other anticancer drugsTamoxifen Oxidized SWCNTs with octa(ethyleneglycol) linker Chemical conjugation via

ester bond [159]

Thalidomide PEGylated oxidized SWCNTs functionalized withcyclic RGD & Rh

Chemical conjugation Cyclic RGD against integrinv3

Uptake in U87MG & MCF-7. Biodistribution in wild typezebrash embryos.Targeting ability intransgenic zebrashembryos with greenuorescent protein-producing endothelial cells.Angiogenesis assay intransgenic zebrash embryoxenografted with HT1080

[165]

CT Hybrid of non-covalent inclusion of MWCNTs tocovalent complex of gelatin & CT

Physical adsorption Cytotoxicity in HeLa [166]

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HMM SWCNTs or DWCNTs with open ends sealed with C60 Physical encapsulation C60 caps [121]

Ce6 Oxidized SWCNTs wrapped with CHI Physical adsorption Uptake & cytotoxicity inHeLa

[169]

Ce6 Oxidized SWCNTs non-covalently wrapped withthrombin aptamers

Chemical conjugation ontoaptamers

Thrombin Cytotoxicity in Ramos [170]

5-Aminolevulinicacid

PAMAMmodied MWCNTs Physical adsorption Uptake & cytotoxicity inMGC-803

[171]

Bodipy-basedPDT sensitizer

SWCNTs Physical adsorption [172]


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In vitro In vivo

HMME Amine-functionalized SWCNTs covalently linked toHA

Physical adsorption HA against HR Uptake & cytotoxicity inB16-F10

Efcacy & toxicity in C57mice subcutaneouslyinjected with B16-F10

[173]

Non-anticancer drugsAntimicrobialsAMB Ammonium-functionalized MWCNTs & SWCNTs Chemical conjugation Uptake & cytotoxicity in

Human Jurkat Lymphoma TcellsAntifungal activity inCandida & Cryptococcusfungi

[34]

AMB Oxidized MWCNTs & PEGylated SWCNTs Chemical conjugation Antifungal activity against acollection of fungi

[33]

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AMB MWCNTs functionalized with ethylene diamine Chemical conjugation Antileishmanial activity inintramacrophageamstigotes

Antileishmanial efcacy inSyrian Golden Hamsterinfected with L. donovaniToxicity in healthy BALB/cmiceAntileshmanial efcacy inSyrian en Hamster (Oral ad-ministration)

[174,201]

AMB Mannosylated MWCNTs Physical adsorption Mannose to targetmacrophages

Uptake in J774 Biodistribution & toxicity inalbino rats

[175]

Dapsone Oxidized MWCNTs Chemical conjugation Uptake & cytotoxicity in ratperitoneal macrophages

[177]

Pazuoxacinmesilate

MWCNTs functionalized with ethylene diamine Physical adsorption Acidic pH [178]

Gentamicin Bullfrong collagen hydrogel doped with 1% w/woxidized CNTs (Type of CNTs not specied)

Physical adsorption Presence of CNTs [179]


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In vitro In vivo

Chloroquine DWCNTs coated with PEI & plasmid encodingluciferase

Physical adsorption Acidic pH Transfection ability &cytotoxicity in HeLa

[180]

Anti-inammatoriesDEX phosphate Oxidized MWCNTs sealed with a lm of polypyrrole

via electropolymerizationPhysical encapsulation Polypyrrole lm Electrical

stimulation Anti-inammatory activity

demonstrated inlipopolysaccharide-activated microglial cell

[35]

DEX phosphate CHI/SWCNTs hybrid lm Physical encapsulation Electrical stimulationPresence of CNTs

[181]

Diclofenac sodium Spherical gelatin/MWCNTs hybrid microgel Physical encapsulation Electrical stimulationPresence of CNTs

[182]

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Diclofenac sodium Carboxymethyl guar gum/oxidized MWCNTs hybridhydrogel

Physical encapsulation Amount of CNTs [183]

Diclofenac sodium MWCNTs Physical adsorption [188]

Ketoprofen Electrospun bers comprising PEO & pentaerythritoltriacrylate interspersed with MWCNTs

Physical encapsulation Electrical stimulationPresence of CNTs

Biocompatibility in L929 [184]

Indomethacin Osmotic pump tablet system coated with celluloseacetate membrane containing MWCNTs

Present in core tablet Presence of CNTs [185]


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In vitro In vivo

AntihypertensivesDiltiazemhydrochloride

Composite membrane of PVA & oxidized MWCNTs Physical encapsulation Presence of CNTs [186]

Metoprolol tartrate EC microsphere impregnated with MWCNTs Physical encapsulation Presence of CNTs [187]

CandesartancilexetilDiltiazemhydrochloride

MWCNTs Physical adsorption [188]

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Carvedilol Pristine & oxidized MWCNTs Physical adsorption [189]

AntioxidantsTPGS SWCNTs & MWCNTs Physical adsorption [192]

QuercetinRutin

SWCNTs (pristine, hydroxylated & carboxylated) Physical adsorption [193]


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In vitro In vivo

Gallic acid Pristine MWCNTs Covalent conjugation Biocompatibility with HET-CAM

[194]

Other non-anticancer drugsAcetylcholine SWCNTs Physical adsorption Lysosomal & mitochondrial

damageEfcacy & toxicity in kainicacid-induced Alzheimer'sKunming mice

[195]

Theophylline Hybrid microspheres of alginate & CNTs (types notspecied) dispersed with triblock copolymer ofPEO137-b-PPO44-b-PEO137

Physical encapsulation Presence of CNTs Cytotoxicity in L929 [196]

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1991B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015coating CNTs with amphiphilic macromolecules like lipid, polymers andsurfactants, or covalently modifying the backbone of CNTs with hydro-philic functional groups [56]. Besides improving the water dispersibilityand reducing the cytotoxicity of CNTs, surface functionalization also pro-vides extra attachment sites for additional chemical or supramolecularloading of drugs, for targeting strategies or for imaging purposes [46]. Inaddition, properly functionalized CNTs, specically those with polyethyl-ene glycol (PEG), are also able to achieve prolonged circulation half-lifeand improved bioavailability by escaping opsonization-induced reticulo-endothelial system (RES) clearance [57]. The physical dimensions ofCNTs, such as length and diameter, also have some bearings on the toxic-ity of CNTs, where longer and thinner structures tend to inict greater cy-totoxicity [58,59]. With all these modications, CNTs with improvedbiocompatibility and solubility have been successfully created. The abilityfor someof these functionalizedCNTs to be cleared by renal excretion alsoaddresses some of the pharmacokinetic (PK) safety concerns related tothe elimination of CNTs following administration [6063].

In this review, the use of CNTs as carriers or adjuncts for the deliveryof various small molecule drugs, including both anticancer and non-anticancer drugs, is examined, with specic focus on their loadingmethod, release characteristics, targeting ability (if any) and resultanttherapeutic efcacy and toxicity (please refer to Table 1 for a summaryof all the CNT-based DDSs described in this review). Even though bio-logics like peptides and nucleic acids have also been delivered withCNTs, they were not encompassed in this review. As in vivo works arehighly imperative for estimating the clinical feasibility of CNTs as viableDDSs, a section of this review is dedicated to the current progress ofin vivo research involving CNT-based DDSs. Last but not the least, cer-tain limitations and considerations regarding the use of CNTs for drugdelivery are also discussed briey.

2. Delivery of anticancer drugs with carbon nanotubes

While chemotherapy has long been employed to manage cancers,either alone or in combination with other treatment modalities likesurgery and radiation, it is often associated with undesirable systemictoxicity due to non-specicity, narrow therapeutic window and devel-opment of drug resistance. Therefore, novel ways of selectively deliver-ing anticancer drugs to tumors with improved therapeutic efcacy andreduced adverse effects are highly desired. In this section of the review,the use of CNTs for the delivery of anticancer drugs of various pharma-cological classes is examined.

2.1. Topoisomerase inhibitors

Topoisomerases are a group of enzymes that relieve the torsionalstrain of supercoiled double helical deoxyribonucleic acid (DNA) bymaking either single or double stranded nicks at the DNA phosphatebackbone and allowing the DNA to be unwind, before eventuallyresealing the cleaved DNA. As failure to relieve these tensions couldlead to the arrest of DNA replication and subsequently apoptosis,some chemotherapeutic agents have leveraged on this property toslow down cancer cell growth by inhibiting the activity of eukaryotictopoisomerases. These agents are collectively known as topoisomeraseinhibitors.

2.1.1. Topoisomerase I inhibitorsTopoisomerase I catalyzes a transient break of 1 strand of duplex

DNA and allows the unbroken complementary strand to unwindthrough the enzyme-linked strand. After successful DNA relaxation,topoisomerase I also relegates the broken DNA. Examples of clinical-ly used topoisomerase I inhibitors include irinotecan, topotecan andcamptothecin (CPT).

In an attempt to raise its water solubility and antitumor effect, a con-gener of CPT, namely 10-hydroxycamptothecin (HCPT), was covalently

conjugated to MWCNTs via a cleavable ester bond [48]. With a HCPTloading of 16%w/w, the conjugate remained stable in the absence of es-terases in buffer solution, and released HCPT readily in fetal bovineserum after hydrolysis of ester linkages by esterases present in theserum.While the uptake study of the conjugate with additional uores-cein isothiocyanate (FITC) tag in human gastric carcinomaMKN-28 cellsrevealed successful internalization of the CNT conjugate, no comparisonwas made to the uptake of free HCPT. Thus, it is not possible to conclu-sively assert if CNTs enhanced the cellular uptake of HCPT. Nonetheless,in vitro cytotoxicity of the HCPTCNT conjugate was observed to behigher than that of lyophilized clinical HCPT injection at equivalentHCPT concentration, with the non-HCPT loaded CNT carrier inictingonly negligible killing in MKN-28 cells.

Employing the technique of non-covalent supramolecular attach-ment, a nanocarrier comprising poly(vinyl alcohol) (PVA)-functional-ized MWCNTs loaded with CPT via interactions was reported bySahoo et al. [64]. The loading of CPT was estimated to be around 0.1 gper g of PVA-MWCNT by ultravioletvisible (UVvis) spectra. The re-lease of CPT, however, was observed to be rather slow, achieving onlyaround 20% cumulative release by 72 h in buffer of pH 7.4 at 37 C.While the slow release prole is indicative of a strong association be-tween CPT and CNTs, strategies to improve or even trigger the releaseof CPT, if devised successfully, would be extremely useful for controlleddrug release purposes. In spite of this, the construct was found to be ap-proximately 15 fold more potent than free CPT against MDA-MB-231human breast cancer cells by MTT assay. Similar chemo-enhancing ef-fect was also observed in metastatic skin tumor cell line A-5RT3.

In another study, CPT was supramolecularly loaded onto MWCNTscoatedwith tri-block copolymer Pluronic P123 via stacking interac-tions [65]. The formation of the supramolecular complex was veriedand quantied by UVvis spectra and photoluminescence. Approxi-mately 8 1016 of CPT molecules were estimated to be present onevery mg of the coated MWCNTs. With enhanced water solubility com-pared to free CPT, the complex could be internalized into the cytoplasmand onto the cell membrane of human cervix adenocarcinoma HeLacells, as indicated by the uorescence signal of CPT. However, it is notpossible to ascertain if the uorescence signals observed were due tofree CPT that had been released from the CNTs or CPT molecules thatwere still attached on the CNTs, especially since an in vitro releasestudy of the construct was not conducted. Without a comparison withfree CPT, it also could not be assessed if CNTs were able to enhancethe cellular uptake of CPT. Nevertheless, the construct demonstratedsignicant improvement in cell killing ability over free CPT in MTTassay against HeLa cells.

Irinotecan, a more water soluble semisynthetic analog of CPT, wasencapsulated into the cavity of puried MWCNTs with opened tips,achieving a loading of around 32% as determined by thermogravimetricanalysis [66]. The release of irinotecan was slightly improved in mildlyacidic condition (pH 6.0 versus 7.0), possibly due to increased stabilityand hydrophilicity of irinotecan in acidic medium. Intriguingly, furtherdecrease of pH to 5.0 appeared to have no additional inuence on therate of drug release. The anticancer activity of this constructwas howev-er not investigated.

2.1.2. Topoisomerase II inhibitorsUnlike topoisomerase I inhibitors, which only nick at a single strand,

topoisomerase II inhibitors cleave both strands of DNA, which then en-ables the passage of another unbroken DNA duplex through the brokenpoints, before nally resealing the strands. Inhibitors of topoisomeraseII, such as etoposide and teniposide, prevent the rejoining of the nickedstrands, resulting in double strain breaks and consequently cell death.

A targeted DDS comprising carboxyl SWCNTs functionalized withchitosan (CHI) and epidermal growth factor (EGF) physically loadedwith etoposide was fabricated by Chen et al. [67]. In this system, CHI, acationic polysaccharide, was non-covalently attached onto the surfaceof carboxyl SWCNTs to improve the water dispersibility of CNTs, and

to serve as a linker for subsequent covalent conjugation of EGF against

1992 B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015EGF receptors (EGFR)-overexpressing cancer cells. The loading capacityof etoposide was around 25 to 27% w/w via stacking and electro-static interaction. Release of etoposide from the systemwas acceleratedunder lowpH conditions of 5.5, presumably due to increased amine pro-tonation and enhanced solubility of CHI in acidic condition. The abilityfor this system to release more drugs at low pH is particularly advanta-geous for cancer therapy, as the intracellular environment of canceroustissues tends to be more acidic [68]. When tested on human alveolarcarcinoma epithelial cell line A549, the delivery system was foundto be more potent than free etoposide. Surface attachment of EGFfurther contributed to the cytotoxicity of etoposide by facilitating EGF-mediated energy-dependent endocytosis. Interestingly, the same con-struct that was taggedwith FITCwas shown to accumulate in the nucleusof A549 cells after 3 h of incubation. However, therewas no clearmentionin the study if FITC was covalently or non-covalently linked to the CNTconstruct. Even if FITC was covalently attached to CHI, CHI was itselfnon-covalently attached onto the surface of CNTs. Therefore, it is not pos-sible to conrm that all the molecules previously incorporated onto theCNT carrier were preserved throughout the experiment as a single entity.The uorescent signal observed in the nucleus is thus unable to unequiv-ocally support the nuclear localization of the complex, as the signal couldarise from dissociated FITC and not from the entire uorescently taggedCNT complex.

2.1.3. AnthracyclinesAnthracyclines, such as doxorubicin (DOX), daunorubicin (DAU) and

epirubicin (EPI), represent a unique class of anticancer drugs that canalso inhibit topoisomerase II. However, anthracyclines differ from theother topoisomerase II inhibitors by exhibiting multiple mechanisms ofaction. With a at and aromatic tetracyclic ring structure, anthracyclinesare able to intercalate between DNA base pairs and inhibit the synthesisof DNA. In addition, the hydroquinone moiety of anthracyclines can alsobe metabolized and generate iron-mediated free oxygen radicals thatdamage DNA and cell membranes. In spite of their high clinical effective-ness against many cancers, the use of anthracyclines is unfortunatelyplagued with dose limiting myelosuppression, alopecia, acute nauseaand vomiting, vesicant effects and, most notably, cardiotoxicity. More ef-fective and saferways of delivering anthracyclines are hence of signicantresearch interest. Already, liposomal formulation of DOX, for instanceDoxil and Myocet, have been invented and employed clinically withdiminished incidence of cardiotoxicity [69,70].

By exploiting the ability for the at aromatic tetracyclic structure ofDOX to establish strong and hydrophobic interactionswith the alsoaromatic surfaces of CNTs, Liu et al. have created a novel DDS compris-ing PEG-functionalized SWCNTs supramolecularly attached to DOXwith an ultrahigh loading capacity of around 400% [26]. Shortlyafter the report by Liu et al., another similar strategy of DOX deliv-ery, this time with MWCNTs dispersed with 1% Pluronic F127, wasreported by Ali-Boucetta et al., validating the results of Liu et al.and suggesting that non-covalent attachment of DOX via inter-actions are applicable to both SWCNTs and MWCNTs [27]. Remarkably,theMWCNTDOX complexwas observed to enhance the cytotoxicity ofDOXonhumanbreast cancer cellsMCF-7 signicantly.More important-ly, the DOX-free carrier of MWCNTs dispersed with Pluronic alone didnot depress cell viability, implying that the cytotoxicity effect observedwas attributable entirely to improved DOX efcacy rather than any in-herent toxicity of CNTs.

A thorough understanding on the various factors that govern thenon-covalent adsorption and desorption behaviors of DOX on CNTs isuseful in helping researchers to devise strategies to control the loadingand release of DOX from CNTs. These factors include loading and releasepH, loading DOX concentration, time allocated for adsorption, diameterof CNTs, coating/functionalization on CNTs, temperature, presence ofcompeting proteins and external radiation.

With an amine group present in its structure, the physicochemical

properties of DOX are highly sensitive to changes in environmentalpH. Typically, DOX remains unionized and hydrophobic in neutral andbasic pH. In acidic condition, the amine on DOX can be protonated, rais-ing its hydrophilicity and solubility. This change in hydrophobicity is anessential feature that controls the loading and release of DOX fromCNTs. Several studies have consistently veried higher degree of DOXloading in basic conditions, as DOX can maintain its unionized stateand associate stronger with CNTs via and hydrophobic interactions[47,7174]. Conversely, it was shown that DOX could be released morereadily in acidic environment after protonation [26,45,47,7284]. Thisdifferential rate of drug release is useful in targeted delivery of DOX tocancer cells, as tumormicroenvironments tend to bemore acidic. In ad-dition, as the internal pH environment of lysosomes is acidic (pH 5.5),release of DOX from CNTs can also be triggered automatically afterreceptor-mediated endocytosis and internalization of the CNTDOXcomplex into lysosomal compartments, liberating free DOX to enter nu-cleus and exert its cytotoxic effect. In fact, the importance of acidic pHfor ensuring adequate release of DOX was demonstrated by the loss ofanticancer activity against A549 with MWCNTDOX complex co-incubated with ammonium chloride, as a result of lysosomal accumula-tion of ammonium ions [73]. While the supramolecular loading of DOXon CNTs is promoted by high pH, care however must be taken not toincubate CNTs with DOX in an environment that is too basic, as DOXcan start to destabilize above pH 6 and becomes totally inactivated atpH 9 in daylight at 25 C [71].

Loading of DOX is affected by the concentration of DOX in the loadingsolutions. As there exist a nite surface area on CNTs to which DOX canbind, there is thus a saturated adsorption capacity for each system. Inthe loading of DOX onto SWCNTs functionalized with P-glycoprotein(P-gp) antibody, it was observed that the adsorption capacity of DOXinitially increasedwith increasing DOX concentration in the loading solu-tion, but eventually reaching a plateau with a maximum loading capacityfollowing continual rise of DOX concentration in the loading solution.Similar adsorption isotherm prole was also observed in other studies[74,75,85]. Interestingly, in the loading of DOX onto FA conjugated mag-neticMWCNTs, a linear increase inDOX loading content and a nearly con-stant DOX loading efciency of above 96% were observed when the ratioof DOX to MWCNTs was increased from 0.2 to 2.0 [76]. This discrepancymight be attributed to the fact that saturated adsorption capacity hasnot been reached.

The time allocated for DOX adsorption/incubation can signicantlyalter the level of DOX loading as well as its rate of release from oxidizedMWCNTs [75]. As the adsorption kinetic of DOX on CNTs is slow,10 days were required for complete saturation and equilibrium to beattained. Nevertheless, 2 h of incubation could easily accomplish a load-ing that is considered adequate for chemotherapy. Comparing the ad-sorption behaviors of DOX with 2 h and 10 days of incubation, mistylayer coatings and sludge-like substances were observed on the surfaceof CNT samples under transmission electron microscopy (TEM) respec-tively, indicating stronger and more favorable adsorption of DOX onCNTs with prolonged incubation time. Desorption of DOX, on theother hand, was more favored with shorter incubation time underboth neutral and acidic conditions. While the de-sorption percentagefor 10 days-incubated sample was low, the total amount of DOX re-leased was actually higher than that of 2 h-incubated sample.

The diameter of CNTs is also able to inuence thebinding and releaseof DOX from CNTs. Specically, DOXwas able to bindmore strongly butbe released slower from MWCNTs of larger diameter, as larger tubespossess bigger and atter graphitic sidewalls that can facilitatemore ef-cient interactions between DOX and CNTs [26].

The coatings on functionalized CNTs can also be manipulated tomodify the loading and release efciency of DOX. CHI, being a naturalcationic hydrophilic polymer, has been used to coat CNTs. CHI is stableat pH 7.4 but degrades readily in acidic pH. In one study, oxidizedSWCNTs supramolecularly attached with DOX were coated with FA-decorated CHI, and it was shown that the CHI-FA conjugate coating

lowered the rate of DOX release compared to the uncoated samples,

1993B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015by lengthening diffusion path length and forming additional hydrogenbonds between FA and DOX [86]. Degradation of CHI coating in acidicpH, as indicated by scanning electronmicroscopy (SEM), was suggestedto be another factor contributing to the enhanced release of DOX inacidic condition in addition to DOX protonation.

Sodium alginate and CHI, either alone or in combination, were coat-ed non-covalently onto oxidized SWCNTs, and the effects of differentpolysaccharides coating on the loading of DOX were studied [45]. AsCHI is cationic and alginate is anionic, they confer the coated SWCNTswith different zeta potential. Alginate-coated SWCNTs resulted in thehighest DOX loading, as the negative charges on alginate facilitated as-sociation with cationic DOX. Conversely, low level of DOX loading wasobserved for CHI-coated CNTs due to mutual repulsion. The release ofDOX followed an inverse relationship to the loading study, in whichconstructs with higher drug loading efciency released DOX slower.From these results, it is revealed that, in addition to stacking, elec-trostatic interaction also plays an important role in the adsorption ofDOX on CNTs. It is possible to achieve desirable DOX loading and releaseprole by modulating the CHI-to-alginate ratio used for coating CNTs.

The amount of adsorbed coating molecules also has some bearingson the amount of DOX that can be loaded on the surface of CNTs. Poly(ethylene glycol-b-propylene sulde) (PEG PPS), a biocompatible am-phiphilic diblock copolymer, was used to disperse MWCNTs, and theamount of DOX loaded was observed to be inversely proportional tothe concentration of PEG PPS used, suggesting that DOX loadingwas de-pendent on the surface area left free from PEG PPS adsorption [87]. Sim-ilar trend of increasing coating density leading to decreasing DOXbinding was also observed for DOX being loaded on oxidized SWCNTsfunctionalized with branched PEG 2500-NH2 [71]. Notably, while PEGcoatings generally reduced the extent of DOX binding to SWCNTs by10% as compared to uncoated SWCNTs, different PEGs of varyingmolec-ularweights, irrespective if theywere covalently or non-covalently con-jugated to the SWCNTs, appeared to have no signicant impact on theloading of DOX. Similar negligible inuence is also observed in anotherstudy [80]. Yet in the report by Niu et al. that compared the loading andrelease of DOX from PEGylated SWCNTs functionalized with or withoutFA, while similar drug release proles were observed for both con-structs, the loading efciency of the construct with FA was slightlyhigher than thatwithout. This was possibly due to the carboxylic groupsof FA conferring negative surface potential to the SWCNTs and enrichingthe electrostatic attraction between DOX and FA-PEG-SWCNT [81]. Thiscomplicated inuence of surface coating on drug loading is further evi-dent by the contradictory observation made by Wen et al., where theloading, encapsulation efciency and release proles of DOX on multi-functional dendrimer-modied MWCNTs were similar with and with-out FA functionalization [74]. With all these conicting results, it istherefore important to always evaluate the extent of DOX loading andrelease individually for every CNT construct created with different coat-ing and/or targeting molecules.

It is also possible to manipulate the release of DOX from CNTswith temperature. Capitalizing on the unique property for 2 polymers,polyethylenimine (PEI) and PVA, to complex at low temperature via hy-drogen bonding and de-complex at high temperature, a thermosensitiveDOX DDS based on polymer-gated CNTs was created [88]. OxidizedCNTs were rst covalently conjugated with PEI, and then coated withPVA via hydrogen bonding complexation, forming zippers. At 40 C,the zippers opened due to weakening of hydrogen bonding betweenPEI and PVA, allowing DOX to penetrate through the sparse polymericnetwork and attach on the surface of CNTs. After cooling back toroom temperature (25 C), hydrogen bonding between PEI and PVAreestablished, forming back the zippers and limiting the movement ofDOX. When tested on lung broblasts, breast adenocarcinoma and HeLacells, while free DOX demonstrated non-discriminatory killing at temper-ature ranging from 35 C to 40 C, the zippers DOXCNT construct wasrelatively non-toxic below 37 C but reaches comparable cell inhibition

level as free DOX at 40 C. The ability for such delivery system todiscriminately release DOX at elevated temperaturemaybe advantageousfor cancer treatment, as tumors tend to display higher temperature thannormal healthy tissues [89].

One of themajor differences between in vitro drug release studies inphosphate buffered saline (PBS) and the actual release of drugs fromnanocarriers in the biological system is the presence of proteins in bio-logical uids. Many proteins possess aromatic rings and hydrophobiccavities that can interact with the hydrophobic surface of CNTs [90].The phenomenon of proteins binding to nanoparticles (includingCNTs), known as the corona effect, has already been demonstratedby a few studies [91,92]. Inevitable protein adsorption on CNTs follow-ing in vivo administration may therefore alter the release of DOX bycompetitively displacing DOX from the surface of CNTs. In fact, the de-sorption of DOX from oxidized MWCNTs was accelerated by 2 times inrelease medium of neutral pH comprising of bovine serum albumin(BSA) or immunoglobulin G versus blank PBS [75]. Similar promotionof DOX release in cell culture medium was also demonstrated forPEGylated SWCNTs loadedwithDOX [71]. A fewstudies, however, dem-onstrated negligible difference between the release proles of DOXfrom MWCNTs covalently functionalized with hyaluronic acid (HA)and SWCNTs non-covalently functionalized with FA-terminated PEGin buffer and serum, suggesting possibly the effects of protein bindingon DOX release may be inuenced by the different functional groups/coating present on the surface of CNTs [73,81].

As CNTs, especially SWCNTs, are able to absorb energy in the NIR re-gion, NIR can hence also be used as a trigger to stimulate and enhancethe release of DOX, by favoring the desorption process of DOX fromSWCNTs, which is an endothermic process [50]. In another study, NIRwas used to control and induce the release of DOX loaded onto FA andiron di-functionalized MWCNTs in PBS, while still maintaining asustained release prole [85].

In addition to recognizing the different factors capable of altering theloading and release of DOX, it is also imperative to understand the uptakeprocess and intracellular distribution of CNTDOX complexes in cancercells. Typically, in order to visualize the intracellular distribution ofCNTs, uorescence dyes, such as uorescein [46,50,71,74,76,78,80,83],Alexa-uor-647 [73,84], rhodamine (Rh) [84] or even CdTe quantumdots (QD) [47], are used to conjugate the CNTs. In a study that investigat-ed the subcellular trafcking of CHI-coated SWCNTs conjugatedwith FITCand supramolecularly loaded with DOX in endothelial progenital cells(EPC), a time-dependent release of DOXwas observed. DOXappeared ini-tially as red uorescence of darker intensity due to quenching from CNTinteraction near the FITC-labeled green-colored CNTs in lysosomes, andsubsequently detach from the SWCNTs inside the acidic environment oflysosomes to yield free DOX with brighter red uorescence that movesinto nucleus, leaving the CNT carrier in the perinuclear region with-out signicant exocytosis after 3 h [78]. This release and localizationpattern of DOX ensuing CNTDOX complex uptake is consistentwith many other studies conducted with FITC uorescent label inother cell types [47,71,74,76].

Non-uorescentmolecules have also been used to label CNTs loadedwithDOX for intracellular uptake study and other imagingpurposes. Forinstance, star-shaped andmulti-branched gold NP (GNP) capable of en-gaging with surface enhanced Raman scattering have been successfullyadsorbed onto PEGylated MWCNTs with supramolecularly loaded DOXto visualize the uptake of this CNT complex in A549 cells [77]. A novelmethod for in vivo imaging of SWCNTswas recently reported by chem-ically linking recombinant thermostable Luciola cruciate luciferase (LcL)on SWCNTs carrying DOX [93]. Unlike the use of uorophores or QDs,which need external excitation source and are unable to image non-supercial tissues, LcL engages in bioluminescence that requires no ex-citation source and is able to penetrate deep within biological tissueswith high spatial resolution.

To further enhance the therapeutic efcacy and safety prole ofDOX, a plethora of strategies has been employed to enable CNT-

based DDSs to specically target selective cancer cells. Different

1994 B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015small targeting molecules have been successfully conjugated ontoCNTs, and FA is one molecule that has been popularly employedagainst tumor cells with overexpressed FA receptors (FR). It wasdemonstrated in many similar studies that supramolecularly loadedCNTDOX complexes with additional FA functionalization were ableto be taken up more efciently by cancer cells that overexpress FR(e.g. HeLa) via FR-mediated endocytosis, and were more cytotoxicthan non-targeting CNTDOX constructs without FA [45,71,81]. Whilesome studies have demonstrated the superiority of FA-conjugated CNTDOX complexes in inhibiting the growth of FR-expressing cancer cellsversus free DOX, no comparisons were made with non-targeting con-structs without FA, leaving some concerns on the true targeting abilityand specicity of these FA-functionalized CNT-based DDSs [76,79].In an interesting study, carboxyl MWCNTs that have been covalentlyattached to amine-terminated generation 5 poly(amidoamine)(PAMAM) dendrimers modied with FITC and FA, and subsequentlyloaded with DOX, were able to be selectively internalized by humanepithelial carcinoma KB cells with high level of FRs and caused morecytotoxicity than in KB cells with low level of FRs [74]. The sameconstructs but without FA attachment, on the other hand, demon-strated indiscriminately low uptake and cytotoxicity in both KBcell types. Alas, the potency of DOX delivered by the multifunctionalMWCNT complex was at most equivalent to free DOX against KBcells, with free DOX demonstrating signicant higher level of cellular up-take than the construct. Similar result of FA-conjugated CNTDOX systembeing more effective than non-FA-conjugated construct (but less potentthan free DOX) was also seen in another study, and it was presumablydue to slow rate of DOX release [71].

HA is a naturally occurring glycosaminoglycan and overexpressionof activated hyaluronan receptors (HR), such as CD44 and receptor forhyaluronan-mediated motility, has been detected on tumor cells to en-hance cell adhesion [94]. Against A549 cells, which are known tooverexpress HR, DOX-loaded oxidized MWCNTs covalently tetheredto HA via 2,2-(ethylene dioxy) bis(ethylene amine) (EDBE) werearound 2.4 times more cytotoxic and induced apoptosis more ef-ciently than free DOX at equivalent DOX concentration [73]. Fur-thermore, HR-mediated endocytosis facilitated the internalizationof the HA-functionalized construct and subsequent translocationinto lysosomes.

As themajormolecular targets of DOX, namely topoisomerase II andDNA, are located in nucleus, it is thus postulated that higher cancer cellkill can be achieved by delivering DOX specically to nucleus. Steroidscould be employed to accomplish nuclear targeting, as the complexformed between steroid and its receptors after binding in cytoplasmwould be translocated to nucleus, dilating nuclear pores up to 60 nmduring the process [95]. CNTs functionalized with steroid can thusexploit this nuclear translocation mechanism and deliver their cargospecically into nucleus. Estradiol (ES) is one such nuclear targetingmolecule that has been explored. -Estradiol-17-hemisuccinatewas co-valently conjugated onto oxidized MWCNTs with a EDBE linker andsubsequently loaded with DOX [84]. The uptake and intracellular distri-bution of this ES-conjugated construct was determined in A549, HeLaand MCF-7 cells, together with MWCNTs functionalized with othertargeting molecules such as HA and FA. As expected for steroids, ES-CNTs were localized mainly in nuclear and perinuclear region, unlikewith HA-CNTs and FA-CNTs where no nuclear co-localization wasobserved. As a nuclear targeting device, ES-CNTs were more efcientin enhancing the cytotoxicity of DOX in A549 and MCF-7 lineages. Thechemo-enhancing effect of ES-CNTs was also found to be dependenton cell types, as the improvement in cytotoxicity was more apparentin ER positive A549 and MCF-7 but not in ER negative HeLa cells.

Besides ES, glucocorticoid like dexamethasone (DEX) mesylate hasalso been covalently linked to amine-modied MWCNTs to create anuclear targeting device [82]. While the authors claimed that theDOX-loaded CNT construct with DEX mesylate was more cytotoxic,

and was more internalized by A549 cells as compared to free DOX dueto ligand-receptor specic targeting, these claims were not fully sub-stantiated by the doseresponse curves that were almost overlappingand the lack of non-DEX mesylate conjugated CNT control. To betterevaluate the nuclear targeting ability of such system, techniques suchasuorescent confocalmicroscopy to look at the degree of nuclear accu-mulation of DOX and/or uorescently tagged CNTs with samples withand without the additional DEX mesylate nuclear targeting moietycould also be performed.

Other than small chemical molecules, biological molecules, such aspeptides, antibodies and even DNA, have also been engaged to equipDOX-loaded CNT-based DDSs the ability to attack specic cancer cells.By conjugating cyclic RGD (arginineglycineaspartic acid) peptide, thatcan recognize integrinv3 unregulated inmany tumors, on the terminalgroup of PEG-functionalized SWCNTs loaded with DOX, higher degree ofdrug uptake and cell killing were observed in integrin v3 positiveU87MG human glioblastoma cancer cells compared to non-targetedconstruct without cyclic RGD [26]. While the IC50 values obtained for cy-clic RGDPEG-SWCNTDOX were still higher than that of free DOX, thetargeted-construct was found to be more selective for tumors thatoverexpress integrin v3, as it was relatively less cytotoxic to integrinv3 negative MCF-7.

A triple functionalized SWCNT comprising DOX, a uorescent mark-er (uoresceine) and a monoclonal antibody capable of recognizingcarcinoembryonic antigen (CEA, which is a glycoprotein expressedonly in cancer cells, especially adenocarcinoma such as colon cancers),was fabricated [46]. While DOX was non-covalently attached, bothuoresceine and CEA antibody were linked to the SWCNTs covalentlyvia BSA as a hydrophilic multifunctional linker. The complex could beinternalized by CEA expressing WiDr colon cancer cells. While similarconstruct without CEA antibody resulted in lower complex uptake,which alluded to the role of CEA antibody in facilitating cell penetration,free DOX seemed to have however equal or even superior cellular up-take to the CEA antibody-tethered construct, as qualitatively assessedby confocalmicroscopy.Moreover, the specicity of the targeting abilityand the efcacy of this DDS were not assessed in this study, leavingdoubts on whether such system is really more specic than or superiorto free DOX.

P-gp is a transmembrane efux pump that can be found on cancercells to promote multidrug resistance (MDR). Targeting DOX to cancercells with oxidized SWCNTs covalently bound to antibody against P-gpuorescently labeled with FITC showed enhanced cellular uptake by23 fold and cytotoxicity against MDR P-gp overexpressing K562human leukemia cells compared to free DOX and non-targeted con-struct without P-gp antibody at equivalent DOX concentration [50].The targeting role of P-gp antibodywas further validated by the inabilityfor human serum albumin-functionalized SWCNTs to be taken up ef-ciently by resistant K562. Intracellular delivery of DOX was boosted,due to the difculty for P-gp to pump out the entire CNTDOX complex.Moreover, steric hindrance presented by the interaction between P-gpand its antibody attached on the SWCNTs also prevented efcient efuxof DOX. Further investigation on the construct's efcacy on non-P-gpexpressing cancer or healthy cell lines, though, could be conducted toconrm that the cytotoxicity of the construct was indeed selective.

Transferrins (Trf) are a group of glycoproteins involved in the trans-port of iron. Overexpression of Trf has been observed in many cancercell types due to heightened iron demand for heme synthesis and rapidcell division [96]. CdTe QD-conjugated and iron NP-lled poly (sodium4-styrene sulfonate) (PSS)-modied CNTs coated with Trf and DOXwere developed as a 3-in-1 system with biologically targeting, magnetictargeting and optical imaging properties [47]. Due to its targeting ability,Trf was able to enhance the uptake of Trf-functionalized CNT constructin Trf positive HeLa but not in Trf negative HEK 293 human kidney cells.Compared to free DOX and non-Trf-conjugated construct at equivalentDOX concentration, Trf-functionalized construct was the most cytotoxictoHeLa cells, corroboratingwith the greater degree of internalization pre-

viously demonstrated.

1995B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015With the aim to achieve efcient targeting of DOX to brain tumorsacross blood brain barriers (BBB), angiopep-2, a peptide capable ofbinding to lipoprotein receptor-related protein (LRP) receptors thatare overexpressed on both BBB and glioma [97], was covalently at-tached to phospholipid (PL)PEG-MWCNTs supramolecularly loadedwith DOX [80]. Higher uptake of the angiopep-2 tethered constructwas observed in lysosomes of both brain capillary endothelial cells(BCEC) and C6 glioma cells compared to non-targeted construct with-out angiopep-2 functionalization. In terms of efcacy, the constructwas also more cytotoxic compared to free DOX and the non-targetedconstruct. Intriguingly, the non-targeted CNT construct was found tobe almost a fold less active than free DOX, though the authors did notprovide any explanation. Also, to further substantiate the targetingability of angiopep-2, it would have been useful to repeat the uptakeand cytotoxicity experiments on cancer and normal cell lines that donot overexpress LRP.

Aptamers are single stranded DNA or RNA nanomaterials with spe-cic 3-dimensional structures that can selectively bind to other smallmolecules or even an entire cell. Being a 26mer guanine rich oligonucle-otide aptamer, AS1411 is capable of interacting with nucleolin, anoverexpressed multifunctional protein that contributes to rapid tumorproliferation [98]. Poloxamer 188-dispersed DOXSWCNTs complexnon-covalently functionalized with AS1411 aptamer could recognizenucleolin receptors found on the surface of EC-109 human esophagealcancer cells with high afnity, thereby elevating its cellular uptake andits growth inhibitory ability in a time- and dose-dependent manner[83]. The efcacy, however, was only compared against free DOX. Inorder to validate the targeting ability of AS1411, in vitro growth inhibi-tion studies with non-targeted construct without AS1411 attachmentand on other cell lines that do not overexpress nucleolin could beconducted. Interestingly, the therapeutic efcacy of this DDS could befurther improvedwithNIR. NIR irradiation at 808 nmwas able to increasethe cytotoxicity of the conjugate in a time- and dose-dependent manner.

Incorporation of ironNP to CNTs can confermagnetic property to theCNTs, enabling one to utilize external magnetic eld to guide the mag-netic CNTs to specic cells or tissues. With this aim, a dual targeted ox-idized MWCNTs-based nanocarrier di-functionalized with FA and ironNP was created for DOX delivery [85]. This system was amendable totargeting using external magnetic eld, by enriching its local concentra-tion in the tumor extracellular environment. To assess the effect ofmagnetic targeting, the FA-DOXmagneticMWCNTs constructwas incu-batedwithHeLa cells for 8 h in the presence or absence of externalmag-netic eld, followed by replacement of culture medium to simulatein vivo drug clearance. The cytotoxicity of the magnetic construct wasenhanced by 23 fold with external magnetic eld, and this was esti-mated to be around 6 fold higher than that of free DOX. Another similarconstruct comprising poly(acrylic acid) (PAA)-grafted MWCNTs func-tionalized with FA and iron oxide magnetic NP also achieved greaterkilling of U87 human glioblastoma with external magnetic eld [76].In this study, the effect of magnetic targeting was observed by creating2 separate non-overlapping circular U87 growth zones in each well of a6 well plate. After treatment with either free DOX or the magnetic CNTconstruct, a magnet was placed below only 1 growth zone and the 2separate zones were observed microscopically for cell growth.While no difference in cell death was observed in the 2 growthzones treated with free DOX, no cells were found in the magneticallytargeted zone treated with the magnetic complex. Conversely, cellsin the non-magnetically targeted zone continued to grow beyondthe original circular boundaries, revealing the ability for externalmagnetic eld to concentrate the magnetic nanocarrier within a xedlocation. Similarly, the uptake and cytotoxicity of the 3-in-1 system ofQDs-conjugated magnetic CNTs loaded with Trf and DOX created byChen et al. in HeLa cells were also further improved by enriching theconcentration of the CNT construct in a conned area using externalmagnetic eld [47]. Interestingly, in this magnetic CNT DDS, iron NPs

were specically encapsulated within the interior of CNTs so as toprotect the NPs from agglomeration, enhance their chemical stability,free up the external surface of CNTs for improved DOX binding and last-ly minimize magnetic-induced uorescence quenching of the QDs con-jugated on the CNT exterior.

While the CNTDOX DDSs discussed so far have all been developedbased on the supramolecular interaction between DOX and CNTs,there are also studies that adopt other methods of loading DOX ontoCNTs. In one study, DOX was rst covalently linked to a pyrene via anenzymatically cleavable carbamate bond, and the pyreneDOX complexwas subsequently irreversibly attached onto PEG-SWCNTs via stronghydrophobic and interactions [99]. In vitro release demonstratedefcient liberation of DOX in cancer cell lysate, as a result of enzymaticcleavage by carboxylesterase, but inefcient hydrolysis in pH 7.4 buffer.The absence of pyrene in the dissolution medium suggested that anyDOX measured was not due to pyreneDOX complex desorption butcarbamate hydrolysis. Even though the construct was able to be internal-ized into lysosomes and induce time- and concentration-dependent celldeath in vitro against B16-F10melanoma cells, it was less potent as com-pared to free DOX especially at lower drug concentration. Furthermore,while the authors claimed that DOX was attached to SWCNTs via pyrenelinkers, they have neglected the possibility of direct interactionbetween DOX and SWCNTs. Having said that, being a universal linker,the use of pyrene actually offers the possibility to connect CNTs withother drugs, targeting or tracking molecules that are otherwise unableto associate with CNTs non-covalently.

Taking into consideration the natural tendency for DOX to associatewith the surface of CNTs, Gu et al. attempted to further enhance theloading, release and activity of DOX by covalently attaching DOX via ahydrazine linker onto PEGylated SWCNTs [100]. Loading of DOX wasimproved from 160 to 220% compared to PEGylated CNTs that wereonly supramolecularly attached with DOX, as DOX was now able to as-sociate with CNTs via 2 different interactions (i.e. covalent and non-covalent). The release of DOX could be further accelerated at low pHas hydrazine bonds are cleavable under acidic condition. As a result ofenhanced drug loading and release efciency, the construct was ableto be better uptaken and it exerted greater cytotoxicity in both humanhepatocellular carcinoma HepG2 and HeLa cells at equivalent DOX dos-age compared to supramolecular-only DOXPEGylated CNT complex.However, no comparison was made to free DOX.

Insteadof serving as a drug carrier, CNTs can also beused as an adjuncttomodulate the loading and release of DOX from another parent DDS. Forinstance, SWCNTs were incorporated as isolated bers in a hybrid gelsystem of N-isopropylacrylamide (NIPAM) and N-dimethylacrylamide(DMAAM), acting as amolecular reservoir to store DOX in basic conditionand release the drug in acidic environment [101]. This composite gelSWCNT system could also respond to NIR irradiation and released the en-capsulated DOX cargo by inducing rapid volume phase transition of thegel between shrinkage and swelling.

Aside from DOX, other members of the anthracyclines drug classhave also exhibited promising potentials to be amendable for CNT deliv-ery. Since all anthracyclines share a similar planar aromatic tetracycliccore structure, it is of no surprise that the other members of this drugclass can also be supramolecularly attached onto CNTs via interac-tions like DOX.

With the aim to investigate the adsorption behavior of EPI hydro-chloride on MWCNTs, Chen et al. had identied several key factorsthat inuence the loading of EPI [102]. Adsorption of EPI was favoredby high pH, low temperature, large overall surface area and smallerCNT diameter. Functionalizing MWCNTs with carboxylic acids also im-proved EPI loading, by increasing free surface area through reducingCNT aggregation and forming additional hydrogen bonds between EPIand CNTs. Specically regarding the inuence of diameter, where theauthors observed more rapid EPI adsorption and higher drug loadingfor CNTs with lower diameter, this observation is inconsistent withthe one made by Liu et al., who demonstrated instead stronger binding

and lower rate of DOX release from SWCNTs with higher diameter due

1996 B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015to atter graphitic side walls that promote stronger stacking [26].More investigations are therefore needed to resolve this discrepancyto fully understand the impact of CNT diameter on the loading and re-lease of anthracyclines.

In their rst attempt of demonstrating DOX attachment on CNTs, Liuet al. had also applied the same supramolecular binding strategy toother aromatic molecules like DAU, albeit with different degree of load-ing than DOX [26]. In another paper, DAU was non-covalently loadedonto SWNCTs functionalized with sgc8c aptamer, a three-dimensionalsingle stranded DNA structure capable of targeting leukemia biomarkerprotein tyrosine kinase-7, with high loading efciency of 157%w/w anda similar pH dependent release prole as DOX [103]. At equivalent DAUconcentration, this DDSwas found tohave higher uptake and greater se-lectivity towards Molt-4 (target cell line, acute lymphoblastic leukemiaT cells) than U266 (non-target cell line, B lymphocyte humanmyeloma)compared to free DAU. However, the construct was only as effective asfree DAU against Molt-4. The study also lacks a non-targeted controlwithout aptamer functionalization. Nonetheless, the targeting role ofaptamer was alluded by the inability for the construct to induce signi-cant cell death upon co-incubation with an antisense of sgc8c aptamer,indicating also the potential for concomitant administration of the anti-sense sequence to be an effective antidote for this DDS.

Pirarubicin was covalently attached to PL-branched PEG functional-ized SWCNTs via a cleavable ester bond. The complex demonstrated su-perior efcacy in vitro against human bladder cancer cells BIU-87 to freepirarubicin [104]. However, no in vitro release study was performed.Moreover, the authorsmade nomention about the extent of pirarubicinloading and the possibility of additional unintentional supramolecularattachment of pirarubicin on the CNT surface. There was also no indica-tion if the doses of pirarubicin used in the in vitro study were standard-ized to the concentration of free pirarubicin.

While technically not an anthracycline, mitoxantrone is ananthracenedione that bears similar structure and mechanisms of ac-tion as the other anthracyclines. Loading of mitoxantrone has beenattempted on PEGylated SWCNTs and its loading and release pat-terns were almost identical to that of DOX, namely, the loading ofmitoxantrone was promoted at higher pH while its release was fa-vored at lower pH [71]. In terms of cytotoxicity, the mitoxantrone-loaded SWCNTs construct was found to be around one fold less po-tent than that of free mitoxantrone in HeLa cells, presumably due toslow rate of drug release.

2.2. Platinum-based drugs

Platinum (Pt) based compounds constitute an effective class of anti-cancer agents for a wide array of malignancies [105], by chelating DNAand forming intrastrand adducts that affect key cellular processes,like transcription and replication, and ultimately triggering apoptosis[106,107]. While highly effective, the use of Pt based drugs is unfortu-nately limited by severe dose limiting nephrotoxicity, neurotoxicityand myelosuppression, arising from pre-mature aquation and non-specic target interactions [108,109]. As a result, sub-lethal doses of Ptcompounds are often used clinically, which consequently promote thedevelopment of resistance [110]. Particularly for active Pt (II) com-plexes, pre-matured loss of activity is also related to poor circulationand limited tumor delivery, in addition to the presence of inactivationmechanisms in living biological systems [107]. Hence, in order to cir-cumvent pre-matured inactivation of Pt (II) drugs, designing of moreinert Pt (IV) prodrugs or combining Pt (II) drugs with drug carriershave been investigated extensively [111114].

An earlier attempt inmerging Pt based anticancer agentwith the useof CNTs as drug carrier has been reported jointly by Lippard andDai's re-search groups, whereby a SWCNT tethered Pt (IV) prodrug conjugatewas constructed and demonstrated to effectively deliver a lethal doseof cisplatin (CDDP) upon selective intracellular reduction [29]. cis,cis,

trans-[Pt(NH3)2Cl2(OEt)(O2CCH2CH2CO2H)], a Pt (IV) complex, wassynthesized and covalently linked through one of its axial COOH ligandsto the surface of SWCNTs which have been non-covalently functional-ized with amine-ended PL-PEG chain (SWCNT-PL-PEG-NH2). ThisSWCNT-based longboat carried an average of 65 Pt (IV) centers pernanotube as determined by atomic absorption spectroscopy (AAS). Asevidenced by a positive shift in the reduction potential of the Pt (IV)complex under pH 6.0, it is speculated by the authors that theSWCNTPt (IV) entered cells by endocytosis and the lower pH environ-ment of endosomes served to facilitate Pt release by reductively cleav-ing the axial ligands by which the Pt (IV) complex was covalentlylinked to the SWCNTs. Remarkably, in testicular carcinoma cellsNTera-2, the conjugate showed more than 25-fold enhancement in cy-totoxicity as compared to the relatively inactive Pt (IV) prodrug, whilea 2.5-fold improvement in efcacy was observed with respect to CDDPas assessed by MTT assay. Pt concentration of the cytoplasmic and nu-clear fractions of cells treated with the SWCNTPt (IV) conjugate were68 and 2 times higher than those of cells incubated with free Pt (IV)prodrug and CDDP alone, respectively.

A further development of the above-mentioned SWCNTPt (IV) con-jugate was proposed by the same research group by further conjugatingthe remaining axial ligand of the Pt (IV) complex with a targeting mole-cule, FA, forming Pt (IV)-FA. The synthesized complexes were then teth-ered in the same way as reported previously to SWCNTPL-PEGNH2[115]. The authors demonstrated the selectivity of SWCNTPt (IV)-FAby performing efcacy studies in a panel of cell lines encompassing FRoverexpressing human choriocarcinoma JAR and human nasopharyngealcarcinoma cells KB, and non-overexpressing control NTera-2. Both FAcontaining constructs, SWCNTPt (IV)-FA and Pt (IV)-FA, exhibited supe-rior cytotoxicity to CDDP. Indeed, their respective IC50 values obtainedfrom MTT assay were reported to be 0.01 M, 0.086 M, and 0.15 M inKB cells, indicating that FA was able to enhance the cytotoxicity of thePt (IV) complex in FR positive cells, and that such enhancement couldbe further potentiated by 8-fold with the use of SWCNTs as drug carrier.Moreover, when KB cells were incubated with 1 M of SWCNTPt (IV)-FA, their nuclear fraction showed a considerable amount of Pt. Conversely,in Pt (IV)-FA treated cells, Pt content could not be detected, showing thatSWCNTs were able to improve Pt nuclear uptake. Additional proof of FRselectivity is provided with the nding that the IC50 of SWCNTPt (IV)-FA and CDDP did not differ much (0.048 M versus 0.044 M) in FR neg-ative NTera-2 cells. Nevertheless, while the IC50 of SWCNTPt (IV)-FA inJAR cells was reported to be as low as 0.019 M, comparisons forFR selectivity were not substantiated as there was a lack of cytotox-icity data from Pt (IV)-FA and CDDP treatment on JAR cells. Addi-tionally, the authors also reported that, when SWCNTPt (IV)-FAwas co-tethered with a uorophore, the presence of uorescentSWCNTs in endosomes was found to be greater in KB cells thanNTera-2 cells, further conrming the selectivity of this construct.Interestingly, monoclonal antibody specic for CDDP intrastrand1,2-d(GpG) cross-links was employed in an immuno-uorescencestudy to probe if the Pt releas

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