-
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.
-
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
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
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 1999
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 1999
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 1999
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2000
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2002
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2004
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2004
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2005
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2006
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2006
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2007
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2007
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2007
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2007
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2008
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2009
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2009
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2010
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2010
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2010
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2011
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2011
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. 2011
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]
1966B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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)
1967B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1968B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page) 1969B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1970B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1971B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
1972B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015gic
tro
ke& l
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]
(continued on next page)
1973B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1974B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1975B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1976B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1977B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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
Uptake & cytotoxicity inA549, HeLa & MCF-7
[84]
1978B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1979B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
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]
1980B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015gic
ro
ox
ox3
ox
e2
-
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
Uptake & cytotoxicity inA549, HeLa & MCF-7
[84]
(continued on next page)
1981B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1982B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1983B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1984B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1985B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1986B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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]
(continued on next page)
1987B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1988B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Carvedilol Pristine & oxidized MWCNTs Physical adsorption
[189]
AntioxidantsTPGS SWCNTs & MWCNTs Physical adsorption
[192]
QuercetinRutin
SWCNTs (pristine, hydroxylated & carboxylated) Physical
adsorption [193]
(continued on next page)
1989B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
Table 1 (continued)
Drug CNT system Drug loading Release control Targeting mechanism
Biological studies Ref
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]
1990B.S.W
ongetal./A
dvancedDrug
Delivery
Reviews65
(2013)1964
2015
-
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