13
Please cite this article in press as: Mehra, N.K., Palakurthi, S. Interactions between carbon nanotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http:// dx.doi.org/10.1016/j.drudis.2015.11.011 Drug Discovery Today Volume 00, Number 00 December 2015 REVIEWS Teaser The present review provides a novel platform to scientist for CNTs and their conjugation chemistry, interactions, conjugation, and their potential biological applications in drug delivery perspectives. Interactions between carbon nanotubes and bioactives: a drug delivery perspective Neelesh Kumar Mehra and Srinath Palakurthi Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A & M Health Science Centre, Kingsville, TX 78363, USA Applications of carbon nanotubes (CNTs) in the biomedical arena have gained increased attention over the past decade. Surface engineering of CNTs by covalent and noncovalent modifications enables site-specific drug delivery and targeting. CNTs are available as single-, double-, triple-, and multiwalled carbon nanotubes (SWCNTs, DWCNTs, TWCNTs, and MWCNTs, respectively) and have unique physicochemical properties, including a high surface area, high loading efficiency, good biocompatibility, low toxicity, ultra lightweight, rich surface chemistry, non-immunogenicity, and photoluminescence. In this review, we highlight current understanding of the different types of physical and chemical interaction that occur between therapeutics and CNTs, and the potential application of the latter in drug delivery and imaging. Such understanding will aid exploration of the utility of multifunctional CNTs as pharmaceutical nanocarriers, and potential safety and toxicity issues. Multifunctional CNTs: a new contour in drug delivery and targeting Carbon nanomaterials, including carbon nanohorns (CNHs), graphenes (GRs), carbon nanorods (CNRs), polyhydroxy fullerenes (PHF) and CNTs, represent safe and efficacious carrier systems for drug delivery and drug targeting because of their unique physicochemical properties. CNTs were first discovered by Roger Bacon in 1960, and were described fully by Sumio Iijima. CNTs are now the focus of many studies exploring their applications in drug delivery and drug targeting, as well as cosmetic products [1,2]. CNTs are ultra-light-weight, tubular, hollow monolithic structures, with a high surface:aspect ratio (length/diameter), rich functional surface chemistry and high drug-loading capacity. They are also biocompatible, nonimmunogenic, and photoluminescent, making them attractive nanocarriers for drug delivery and imaging. CNTs do not require any type of fluorescent labeling for detection because they can be detected directly because of their electron emission properties [3–6]. CNTs are available as SWCNTs, DWCNTs, TWCNTs and MWCNTs, with cylindrical graphitic layers [7–10]. Functionalization is a well-known approach for altering the surface of nanocarriers by attaching a variety of different bioactives. Functionalized CNTs (f-CNTs) have been used to Reviews KEYNOTE REVIEW Neelesh Kumar Mehra Dr Mehra is a research scientist in the Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, which is part of the Texas A & M Health Science Center. His research interests lie in the field of drug delivery and targeting for cancer theragnostics using carbon nanomaterials, quantum dots, nanoparticles, and dendrimers. Dr Mehra has (co)-authored more than >40 publications in international journals and has published six book chapters in the field of carbon- based nanomaterials. Srinath Palakurthi Dr Palakurthi is a professor and director of graduate studies in the Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, which is part of the Texas A & M Health Science Center. He gained his PhD from the Indian Institute of Chemical Technology. His current research interests encompass novel polymers for gene therapy, polymer-based nanoparticles for the targeted delivery of chemotherapeutics for the treatment of ovarian cancer, mucosal delivery of chemotherapeutics and antigens, and cellular trafficking of nanoconstructs. He has (co)-authored various publications in international journals related to his research interests Corresponding author: Palakurthi, S. ([email protected]) 1359-6446/ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2015.11.011 www.drugdiscoverytoday.com 1

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Page 1: 44 D DT Mehra Palakurthi

Reviews�KEYNOTEREVIEW

Drug Discovery Today � Volume 00, Number 00 �December 2015 REVIEWS

Teaser The present review provides a novel platform to scientist for CNTsand their conjugation chemistry, interactions, conjugation, and their

potential biological applications in drug delivery perspectives.

Interactions between carbonnanotubes and bioactives: a drugdelivery perspectiveNeelesh Kumar Mehra and Srinath Palakurthi

Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy,

Texas A & M Health Science Centre, Kingsville, TX 78363, USA

Applications of carbon nanotubes (CNTs) in the biomedical arena have

gained increased attention over the past decade. Surface engineering of

CNTs by covalent and noncovalent modifications enables site-specific

drug delivery and targeting. CNTs are available as single-, double-, triple-,

and multiwalled carbon nanotubes (SWCNTs, DWCNTs, TWCNTs, and

MWCNTs, respectively) and have unique physicochemical properties,

including a high surface area, high loading efficiency, good

biocompatibility, low toxicity, ultra lightweight, rich surface chemistry,

non-immunogenicity, and photoluminescence. In this review, we

highlight current understanding of the different types of physical and

chemical interaction that occur between therapeutics and CNTs, and the

potential application of the latter in drug delivery and imaging. Such

understanding will aid exploration of the utility of multifunctional CNTs

as pharmaceutical nanocarriers, and potential safety and toxicity issues.

Multifunctional CNTs: a new contour in drug delivery and targetingCarbon nanomaterials, including carbon nanohorns (CNHs), graphenes (GRs), carbon nanorods

(CNRs), polyhydroxy fullerenes (PHF) and CNTs, represent safe and efficacious carrier systems for

drug delivery and drug targeting because of their unique physicochemical properties. CNTs were

first discovered by Roger Bacon in 1960, and were described fully by Sumio Iijima. CNTs are now

the focus of many studies exploring their applications in drug delivery and drug targeting, as well

as cosmetic products [1,2].

CNTs are ultra-light-weight, tubular, hollow monolithic structures, with a high surface:aspect

ratio (length/diameter), rich functional surface chemistry and high drug-loading capacity. They

are also biocompatible, nonimmunogenic, and photoluminescent, making them attractive

nanocarriers for drug delivery and imaging. CNTs do not require any type of fluorescent labeling

for detection because they can be detected directly because of their electron emission properties

[3–6]. CNTs are available as SWCNTs, DWCNTs, TWCNTs and MWCNTs, with cylindrical

graphitic layers [7–10].

Functionalization is a well-known approach for altering the surface of nanocarriers by

attaching a variety of different bioactives. Functionalized CNTs (f-CNTs) have been used to

Neelesh Kumar Mehra

Dr Mehra is a research

scientist in the Department of

Pharmaceutical Sciences, Irma

Lerma Rangel College of

Pharmacy, which is part of the

Texas A & M Health Science

Center. His research interests

lie in the field of drug delivery

and targeting for cancer theragnostics using carbon

nanomaterials, quantum dots, nanoparticles, and

dendrimers. Dr Mehra has (co)-authored more

than >40 publications in international journals and

has published six book chapters in the field of carbon-

based nanomaterials.

Srinath Palakurthi

Dr Palakurthi is a professor

and director of graduate

studies in the Department of

Pharmaceutical Sciences, Irma

Lerma Rangel College of

Pharmacy, which is part of the

Texas A & M Health Science

Center. He gained his PhD

from the Indian Institute of Chemical Technology. His

current research interests encompass novel polymers

for gene therapy, polymer-based nanoparticles for the

targeted delivery of chemotherapeutics for the

treatment of ovarian cancer, mucosal delivery

of chemotherapeutics and antigens, and cellular

trafficking of nanoconstructs. He has (co)-authored

various publications in international journals related

to his research interests

Please cite this article in press as: Mehra, N.K., Palakurthi, S. Interactions between carbon nanotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.11.011

Corresponding author: Palakurthi, S. ([email protected])

1359-6446/� 2015 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.drudis.2015.11.011 www.drugdiscoverytoday.com 1

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REVIEWS Drug Discovery Today � Volume 00, Number 00 �December 2015

deliver both lipophilic (paclitaxel and docetaxel) [11,12], and

hydrophilic drugs (doxorubicin hydrochloride; DOX) [13–16].

CNTs readily cross different biological barriers, passing through

the plasma membrane and entering the cytoplasm through a ‘tiny

nanoneedle’ mechanism, which facilitates the transport and de-

livery of the cargo molecules or therapeutics into the target tissue

[2,6,17].

Purification, dispersion, and oxidation of CNTsThe physicochemical properties of CNTs, such as surface topog-

raphy, solubility, hybridization state, mechanical properties, ther-

mal conductivity, and structural and metallic or carbonaceous

impurities, need to be determined before they can be used in

pharmaceutical and biomedical applications [2,10,18]. There are

several factors (e.g., metal content, oxidation time, and oxidizing

agents and temperature used) that affect the purification efficiency

and yield of CNTs. The need for a mixture of strong acids (oxidiz-

ing agents) and corrosive solutions results in safety issues and,

therefore, appropriate precautions, such as the use of acid-resistant

gloves and adequate shielding, must be taken during their pro-

duction. Strong acid treatment not only removes any metallic

impurities, but also cuts the nanotubes into shorter pieces, gener-

ating oxygen-containing functional groups, such carboxylic (–

COOH) and hydroxyl (–OH), around the sidewalls and tips of

the tubes, where the curvature of the tubes results in a higher

strain on nanotubes structure [18–20]. The effect of oxidation on

the structural integrity of nanotubes was studied following acidic

(nitric acid and a mixture of sulfuric acid and hydrogen peroxide)

and basic (ammonium hydroxide/hydrogen peroxide) oxidation

processes. The increase in the number of surface oxygens per

chemical treatment (oxidation) followed the order: hydrochloric

acid (HCl) < ammonium hydroxide (NH4OH)/hydrogen peroxide

(H2O2) < piranha (H2SO4:HNO3) < refluxed nitric acid (HNO3).

Oxidation of CNTs with HNO3 under extreme conditions increases

the formation of defective CNTS because of shortening of the

length of nanotube [18].

Recently, Chajara and co-workers developed a fast, microwave-

assisted, organic solvent-free method for the efficient primary

purification of nanotubes [21]. The method dissociates and dis-

perses nonnanotube carbon in an organic solvent, resulting in

CNTs of high purity in few minutes, and with low few defects [21].

Alternatively, strong acids have also used to oxidize CNTs for

improving their dispersibility and purification [22]. The five meth-

ods used for the dispersion of hydrophobic nanotubes are: (i)

dispersion, reaction; (ii) dissolution, dispersion, precipitation;

(iii) dispersion, dispersion, precipitation; (iv) melt, powder, mix-

ing; and (v) no fluid mixing (reviewed in [23]). Methods (iv) and (v)

do not use any solvents [23].

The use of surfactants, such as sodium dodecyl sulfate (SDS) and

sodium dodecyl benzene sulfonate (SDBS), as coating agents to

improve the dispersibility of CNTs results in better long-term

stability. As an alternative, chitosan (CHI; a natural cationic

polysaccharide biopolymer obtained from the deacetylation of

chitin) can also be grafted onto the nanotube surface because of

its nontoxic, biocompatible and biodegradable properties [24,25].

CHI is an attractive way to encapsulate CNTs through hydrogen

bonding; for example, it enhanced the stability and sustained

release in vitro of DOX (degradation of chitosan and diffusion

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2 www.drugdiscoverytoday.com

through chitosan shell) from DOX-loaded CHI–folic acid conju-

gated CNTs (CHI-FA-CNTs) as a result of their hydrophilic and

cationic charges [24].

Horie and coworkers examined the cellular influences of chem-

ical or biological reagents, such as pluronic F-127 and F-68, 1,2-

dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, the pulmonary

surfactant preparation Surfactent1), bovine serum albumin (BSA),

and Tween 80 as dispersants of CNTs in an experiment with the

human lung carcinoma A-549 cell line [26]. The adsorbed disper-

sant on the surface of the nanotubes was shown to induce oxida-

tive stress in the cells [26]. The poly-L-lysine (PLL)–MWCNTs

aqueous solution after appropriate modification enhanced the

aqueous dispersibility. After sonication, the black MWCNT–PLL

aqueous solution was stable for up to 240 days at pH 5.0 (Fig. 1)

[27]. The dispersion of MWCNT–SDBS (453 nm) was better com-

pared with that of MWCNT–PLL (488 nm) and MWCNT–SDS

(758 nm); however, tangle and aggregates were seen in water with

unfunctionalized MWCNTs (899 nm). These results show clearly

that SDS, SDBS, and PLL improve the aqueous dispersibility of

MWCNTs, and that MWCNT–SDBS and MWCNT–PLL disperse

better than MWCNT–SDS. Galactosylated CHI-grafted oxidized

MWCNTs (O-CNTs-LCH-DOX) were synthesized for pH-depen-

dent sustained release and hepatic tumor targeting of DOX. The

particle sizes of O-CNTs, O-CNTs-LCH, and O-CNTs-LCH-DOX

were 176.1 � 2.4, 217.5 � 3.2, and 286.3 � 4.1, respectively, with

a polydispersity index (PDI) of 0.39 � 0.02, 0.35 � 0.06, and

0.31 � 0.01, respectively. A venous irritation study was performed

on New Zealand white rabbits after intravenous injection of O-

CNTs, O-CNTs-LCH, and O-CNTs-LCH-DOX in normal saline at a

5-mg/kg DOX dose for three consecutive days. The O-CNTs-LCH-

DOX formulation showed good biocompatibility, low toxicity,

higher cellular uptake, and higher antitumor activity, as well

decreased vascular irritation (Fig. 1), compared with O-CNTs, O-

CNTs-LCH, and free DOX in HepG2 cells [28].

The potential of biosurfactants to aid the effective dispersion of

nanotubes needs to be explored further to render the CNTs safer.

Only after obtaining a clear dispersion of CNTs using various

chemical functionalization strategies, will we begin to understand

the interactions of CNTs with therapeutics.

CNT interactions with theragnosticsCNTs can be considered as good adsorbents because of their ability

to interact with guest molecules via different mechanisms on their

surface. The adsorption of guest molecules into CNT bundles can

occur inside the tubes (internal sites), in the interstitial triangular

channels between the tubes, on the outer surface of the bundle

(external sites), or in the grooves (major and minor) formed at the

contacts between adjacent tubes. The influence of chemical mod-

ifications resulting from acid treatment, followed by triethylene-

tetraamine (TETA), has been studied at each stage of chemical

treatment using different analytical tools (Fig. 2) [22]. However,

unavoidable imperfections or vacancies, such as Stone–Wales

defects, pentagons, heptagons, and dopants, have a crucial role

in determining the adsorption properties of CNTs [29,30]. Apart

from these imperfections, five interactions (i.e., hydrogen bond-

ing, hydrophobic effects, covalent, electrostatic, and p–p stacking

interactions) could have a role in the attachment of biomolecules

[31]. In terms of the closed ends of CNTs, small molecules are easily

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

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DRUDIS-1717; No of Pages 13

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(a) (b) (c)(i) (i)

(ii)

(i)

(ii)

(ii)

(iii)

(v)

(iv)

0.2 µm 0.2 µm

0.2 µm 0.2 µm

CNTs O-CNTs O-CNTs-LCH O-CNTs-LCH-DOX

Drug Discovery Today

FIGURE 1

(a) Transmission electron microscopy (TEM) images of carbon nanotubes (CNTs): (i), O-CNTs (ii) , O-CNTs-LCH (iii) and O-CNTs-LCH-doxorubicin (DOX) (iv), anddispersion (v). (b) Unfunctionalized and functionalized multiwalled carbon nanotube (MWCNT) solutions after 6 h (i) and 30 days (ii), from left to right; MWCNTs,

MWCNT-sodium dodecyl sulfate (SDS), MWCNT-sodium dodecyl benzene sulfonate (SDBS), and MWCNT- poly-L-lysine (PLL). (c) MWCNT-PLL aqueous solutions at

different pH values after 2 days (i) and 240 days (ii), from left to right; pH 3, 5, 7, 9, and 11.Source: Reproduced, with permission, from [28] (a) and [27] (c).

RamanLight

scattering

Electronmicroscope

Elementalanalysis

FTIRXRD

NMR

DSC

XPS

TGA

CNTs

Drug Discovery Today

FIGURE 2

Characterization techniques for carbon nanotubes (CNTs). Abbreviations: DSC,differential scanning calorimetry; NMR, nuclear magnetic resonance

spectroscopy; TGA, thermo gravimetric analysis; XPS, X-ray photoelectron

spectroscopy; XRD, X-ray diffraction.

Reviews�KEYNOTEREVIEW

adsorbed most preferentially onto the external grooves and outer

walls of the nanotubes. The host–guest interaction depends on the

size of the guest molecules and interaction energy [32,33]. A few

studies have reported that CNTs are able to adsorb some toxic

substances, such as fluorides [34], dioxins, lead, and alcohols,

which are carcinogenic by-products of many industrial processes

[34–36]. This pioneering work established a new field of applica-

tions for CNTs as cleaning filters for many industrial processes

with hazardous by-products. CNTs could also be used as good

adsorbents for the removal of dichlorobenzene from wastewater

over wide range of pH. For example, 30 mg of organic molecule are

adsorbed per gram of CNT [37]. The nonspecific adsorption of

proteins on CNTs [38] is an interesting phenomenon, but repre-

sents a relatively less controllable mode of protein–nanotube

interaction.

Ji et al. investigated MWCNTs as potential adsorbents for the

removal of two sulfonamide antibiotics (sulfapyridine and sulfa-

methoxazole) from aqueous solutions. Both sulfonamide antibio-

tics are strongly adsorbed on to MWCNTs surfaces via p–p electron

interactions [39]. Since the discovery of CNTs, researchers have

been continuously exploring the interactions of CNTs with bio-

molecules (proteins, carbohydrates, and nucleic acids) for the de-

velopment of carbon nanocomposites for biomedical applications.

Hydrophobic interactionsThe hydrophobic nature of CNTs is best described by the prefer-

ential adsorption of different hydrocarbons (benzene, hexane,

and cyclohexane) over alcohols and organic molecules. There

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are problems associated with most anticancer drugs, including

taxol derivatives [paclitaxel (PTX), docetaxel (DTX)] [12,40,41],

and amphotericin B (AmB) [42], DOX [43,44], 10-hydroxycamp-

tothecin (HCPT) [45], etoposide (ETO) [46] and others, such as

small interfering (si)RNA [47], during formulation development,

but these can be ameliorated by conjugation through cleavable

disulfide bonds, hydrazone bonds with CNTs, and other nanos-

tructures [12,31].

p–p Stacking interactionCNTs can interact with organic molecules via p–p stacking inter-

actions. DOX interacts with the surface of CNTs to form supramo-

lecular complexes based on p–p stacking interactions, because

both have many p electrons [44,48]. Approximately 50–60%

(w/w) of DOX molecules can be attached to the surface of CNTs

via p–p stacking interactions. DOX molecules can also bind with

surface-modified CNTs noncovalently via hydrophobic and p–p

stacking interactions. The aromatic nature and relatively low

aqueous solubility of deprotonated DOX at basic pH conditions

can help it bind with nanotubes. Other aromatic molecules could

also be easily bound or wrapped with the backbone of the CNT and

released at the target site [44].

The p–p bonding interaction between organic molecules and

the surface of CNTs has been characterized by spectroscopy using

Raman, nuclear magnetic resonance (NMR), and fluorescence

techniques. Additionally, the p–p stacking interaction is affected

by the relative position of the benzene ring of organic molecules to

the hexagons present at the surface of the CNT. The p–p stacking

interaction is a noncovalent functionalization and allows con-

trolled release of the adsorbed drugs. Such a controlled release

approach has a significant impact on the development of nano-

pharmaceutical products in the treatment of cancer, HIV/AIDS,

and other diseases [24,31].

Linear poly (m-phenylenevinylene-co-2,5-dioctoxy-p-phenyle-

nevinylene) (PmPV) could wrap around CNTs or nanotube bun-

dles regardless of their diameter. Dendrimer–CNT electron donor–

acceptor monohybrids were also characterized after illumination

or by using different spectroscopic techniques. Several recently

published studies show that p–p interactions bind and wrap the

active theragnostics onto the engineered CNTs [6,13,24,34,41,

43,49–52].

Hydrogen-bonding interactionsHydrogen-bonding interactions also have a vital role in the

adsorption of numerous organic molecules and other chemicals

onto the surface of CNTs if functional groups, such as carboxylic

(–COOH), hydroxyl (–OH), amine (–NH2), and others, are present.

Surface-engineered CNTs can act as hydrogen-bonding acceptors

because of their aromatic nature, whereas carboxylic, hydroxyl,

phenolic, and amine groups act as hydrogen-bonding donors.

Therefore, surface-engineered nanotubes could bind with organic

chemical moieties via hydrogen-bonding interactions [12,31,44].

Covalent-bonding interactionsCovalent bonding can occur between organic reagents and active

functional groups containing nanotubes and are best illustrated

by different spectroscopic techniques, such as Fourier transform

infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy

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(XPS), small angle X-rays spectroscopy (SAXS), Raman spectrosco-

py, and NMR [3,51]. Covalent-bonding affinity to nanotubes is

higher compared with noncovalent-bonding interactions, which

can resist any desorption. Functionalization of CNTs can be

achieved with covalent modification via carboxylation [18], fluori-

nation, amidation, thiolation, and esterification [2]. Bioactive

molecules could be bound covalently to nanotubes via amide,

disulfide, ester, and carbamate bonds [2,31].

Electrostatic interactionsElectrostatic interactions are mainly related to the surface charge

potential of organic chemicals and CNTs. Electrostatic interac-

tions mainly occur when two oppositely charged molecules inter-

act, whereas electrostatic repulsion occurs between molecules

with the same charge. The pH-dependent adsorption of positively

charged (cationic) fluoroquinolone antibacterial agent (norfloxa-

cin) on CNTs was attributed to electrostatic interactions. Polyeth-

ylene imine (PEI), a cationic polyelectrolyte, was used to modify

acid-purified MWCNTs via electrostatic interactions between neg-

atively charged CNTs and PEI, and the physisorption process was

analogous to polymer wrapping [31,53].

Bioactives loading into surface-tailored CNTsPhysical loading of bioactivesSurface-engineered CNTs have unique properties facilitating their

role as nanocontainers wherein guest molecules could be filled

through the host–guest interaction mechanism depending upon

the diameter of the nanotube. Geometrical parameters have an

important role in determining the efficiency of host–guest mole-

cules interactions (i.e., the ratio of the internal diameter of the

nanotube to the size of the encapsulated molecule). The guest

molecule is unable to enter the nanotube if the diameter of the

latter is too small. If the diameter is too big, the host–guest

interaction might not be strong enough for the guest molecules

to be retained inside the nanotube. The most efficient host–guest

interaction mechanism is achieved when the van der Waals diam-

eter of the guest molecules matches the internal diameter of the

nanotubes. CNTs have an approximately 1.3-nm internal diameter

and fullerene C60 molecules, whose van der Waals diameter is

1.0 nm (an ideal geometric match), are referred to as having a

‘snug fit’. A snug fit ensures strong interactions between the guest

molecules and the sidewalls of the nanotubes in controlling the

chemical reactions of the encapsulated molecules as well as the

physicochemical properties of the nanotube itself [54].

From a chemist’s point of view, the most captivating property of

CNTs is their ability to load guest molecules inside their inner

cavity to achieve high loading efficiency, which is known as

‘endohedral filling’. Recently, researchers explored endohedral

filling via various interaction mechanisms. It is well known that

CNTs have many highly delocalized p electron structures, which

can be easily modified via p electron-containing moieties forming

p–p interactions [44].

The dual targeting of DOX to U87 human glioblastoma cancer

cells using folate-decorated magnetic MWCNTs has been reported

[24]. The DOX molecules became attached via p–p stacking and

hydrogen bonding, with a 96% loading efficiency when 0.5-mg FA-

MN-MWCNTs were loaded with <1 mg DOX (DOX:MWCNT < 2),

but decreased slightly to 92% with 1 mg DOX (DOX:MWCNT = 2).

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

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These novel folate-decorated MWCNTs showed enhanced cytotox-

icity toward U87 and human glioblastoma cells compared with

free DOX [52]. Similarly, a new family of folate-chitosan-decorated

SWCNTs showed the DOX-loading efficiency to be approximately

76% (plain SWCNTs) and approximately 91% (CHI-FA-SWCNTs)

via noncovalent p–p stacking interactions [24].

Cisplatin [cis-diaminedichloroplatinum (II, CDDP)], a US Food

and Drug Administration (FDA)-approved highly potent chemo-

therapeutic agent, was encapsulated into MWCNTs with a diame-

ter of approximately 13 nm via nano-extraction, resulting in a

final product that contained 21% CDDP [55]. In another report,

CDDP was encapsulated into MWCNTs based on the same princi-

ple of nano-extraction using ethyl acetate (EA) as an encapsulation

medium and capped with 1-octadecanethiol (ODT)-functionalized

gold nanoparticles (ODT-f-GNP), which constituted the ‘CNT

bottle’. The CNT bottle exhibited a higher amount of CDDP

loading (62.1%) over a shorter period of time (40 h) compared

with the previously published reports. The IC50 values of CDDP,

uncapped MWCNT-CDDP, and capped MWCNT-CDDP were

11.74, 12.92, and 7.74 MM, respectively [56]. Guven et al. evalu-

ated the anticancer activity of pluronic-F108 surfactant-wrapped

CDDP-encapsulated ultrashort SWCNTs (US-tube) against MCF-7

and MDA-MB-231 breast cancer cell lines. Cytotoxicities were

found to occur in both a dose- and time-dependent manner in

both the cell lines [57].

Loading of DOX into hybrid polyethylene-b-(polyethylene gly-

col) (PE-b-PEG)-CNTs at various pHs has also been reported [58].

The DOX-loading efficiency of these nanostructured hybrid car-

riers was 98% at pH 7.4, and approximately 42% and 61% at pH 2

and 10, respectively.

Based on these studies, we can conclude that surface-engineered

nanotubes have higher loading efficiency (up to 98%), which

could be exploited further in controlled and/or targeted drug

delivery. For example, thiol-modified siRNA cargo molecules were

linked to the amine functional groups on the sidewalls of phos-

pholipid–polyethylene glycol–SWCNTs (PL–PEG–SWCNTs) via

cleavable disulfide bonds. These disulfide linkages facilitate the

release of the cargo molecules from the SWCNT conjugate upon

cellular uptake [47].

Chemical conjugation of bioactivesOver the past two decades, several reports have been published

on drug–CNT conjugation and their application in drug delivery

and targeting. Covalent conjugation of a biomolecule (anticancer,

antifungal, antimalarial, and siRNA moieties) to the surface of

CNTs has been used to achieve the controlled release of the

conjugates either through hydrolytic cleavage and/or cleavable

disulfide bond linkages in a spatial and temporal release pattern. In

chemical conjugation, drug moieties with their chemical struc-

tures allow for conjugation while maintaining their inherent

geometry and hybridization in formulations. The conjugation

chemistry of nanotubes could provide new and exciting opportu-

nities in targeted and controlled drug delivery via cleavable bonds.

Chemical conjugation of AmB to the amino groups of engi-

neered CNTs using hydroxybenzotriazole (HOBT) and carbodii-

mide while preserving its high antifungal activity, was reported

in 2005 [42]. Benincasa and coworkers also synthesized two

conjugates with CNTs (SWCNTs and MWCNTs) with AmB to form

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f-CNT-AMB-1 and f-CNT-AMB-2 conjugates and tested them

against different strains of bacteria. f-CNTs-AMB-1 showed mini-

mum inhibitory concentration (MIC) values <10 mg/ml and dis-

played broad-spectrum activity against microorganisms (except

Candida famata SA550 strain, 20 g/ml). Moreover, f-CNTs 1 and

f-CNT 2 (without AmB) did not show any antifungal activity (MIC

80 g/ml) [59].

Dhar and coworkers conjugated platinum (IV) onto the surface

of SWCNTs. Initially, SWCNTs were sonicated with platinum (IV)-

PEG-NH2 for 1 h followed by centrifugation at 2.4 � 104 g for 16 h

to remove catalysts, whereas the large aggregates and free plati-

num (IV)-PEG-NH2 were removed by ultrafiltration. The platinum

(IV) complex (compound 1) and cisplatin [cis-dichlorodiammine-

platinum (II) or cis-DDP] were used as the controls in a cytotoxicity

study using the human nasopharyngeal epidermoid carcinoma

(KB), choriocarcinoma (JAR), and human testicular cancer (NTera-

2) cell lines. IC50 values of SWCNT-tethered 1 were 0.019 and 0.01

MM in FR(+) JAR and KB cells, respectively, whereas the IC50 values

of cis-DDP and compound 1 were 0.086 and 0.15 MM, respectively,

in KB cells. However, the IC50 value of SWCNT-1 increased to

0.0448 MM in folate receptor (FR–) NTera-2 cells, which demon-

strated targeted uptake through (FR+) cells. The platinum (IV)

attached PEG-NH2-SWCNTs improved cellular uptake of the drug

and achieved higher cell death rates [60].

HCPT was covalently attached to the outer surface of the

engineered MWCNTs via biocleavable ester bonds in the presence

of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylamino-

propyl)carbodiimide (EDC) as coupling agents [45]. In another

study, both methotrexate (MTX) (an anticancer agent) and fluo-

rescein isothiocyanate (FITC; an imaging agent) were attached on

to the sidewall of MWCNTs via the 1,3 dipolar cylcoaddition

reaction of azomethineylide and were shown to be internalized

by human Jurkat cells [61]. Gonadotrophin-releasing hormone

(GnRH), which is overexpressed in the plasma membrane of

several types of cancer cell, was covalently anchored onto the

surface of the oxidized MWCNTs via an amide linkage [62].

The covalent immobilization of an anticancer drug (DOX) onto

the surface of functionalized SWCNTs via hydrazone linkage

formed by the condensation of the C-13 ketone of DOX with a

hydrazine was reported by Gu and coworkers [63]. The DOX was

conjugated on to PEGylated SWCNT through hydrazone bonds.

Amine groups of PEGylated SWCNT were first coupled with the

COOH– terminal functional groups of hydrazinobenzoic acid

(HBA) to form hydrazine-modified SWCNT using EDC and NHS

as catalysts. Thereafter, DOX was conjugated to the SWNTs via

acid-sensitive hydrazone bonds (SWNT-HBA-DOX) between the

hydrazine moiety attached to the SWNTs and the ketone group

of DOX.

A few studies have reported paclitaxel (PTX) conjugation to

both types of CNT via cleavable ester bonds to form stable and

aqueous soluble conjugates. PTX was conjugated to the carboxyl

functional groups of poly citric acid (a highly functional polymer

with a large number of carboxylic groups) via cleavable ester bonds

to obtain MWCNT-grafted-PCA-PTX conjugates with a drug con-

tent of 38% w/w [12]. Similarly, Liu et al. also conjugated PTX at

the 20-OH position to the terminal amine group of branched

DSPE-PEG5000-4-arm-(PEG-amine) on SWCNTs via cleavable ester

bonds, which showed higher efficacy in suppressing tumor growth

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

www.drugdiscoverytoday.com 5

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EYNOTEREVIEW

compared with free PTX. These results suggest that higher con-

centrations of PTX were delivered to breast cancer cells using

SWCNT-PTX conjugates compared with using PTX alone [40].

Another taxol derivative, DTX was also conjugated with SWCNT

via p–p interactions, and further functionalized by the surfactant

polyvinylpyrrolidone K-30 (PVPk 30) and 1,2-distearoyl-sn-glycero-

3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]

(DSPEPEG 2000)-maleimide (DPM). The maleimide group at the

end of DPM covalently reacted with the cysteine in

CNGRRCK2HK3HK11 (C-containing sulfhydryl groups) [64].

Similarly, Chen et al. reported a novel strategy for engineering

functionalized SWCNTs. siRNA were bound to the amino func-

tional groups of the DSPE-PEG-amine via disulfide bonds to

achieve siRNA-mediated gene silencing in breast cancer cells [65].

PEGylation chemistry of CNTsPEG is a linear, uncharged, flexible organic molecule that is the most

widely used biocompatible polymer because of its nontoxicity,

Please cite this article in press as: Mehra, N.K., Palakurthi, S. Interactions between carbon ndx.doi.org/10.1016/j.drudis.2015.11.011

Tumor

NearIR

Tumorxenograft

Intratumoral injection(PEG-SWNTs or PBS) NIR irradiation

PBS

SWNT

Tumor growthDeath

Tumor destructionSurvival

O

O

OO

O

O OO

CN

H

P

SerumIn serum In P83

500 nm

2

1

2

6

(a)(f)

(g)

(h)

(b)

(e)

(c) (d)

FIGURE 3

Photothermal treatments for in vivo tumor ablation using polyethylene glycol (PE

procedure and results of PEG-SWCNT-mediated photothermal treatment of tumors

(KB) cells (70 mm3); (c) mouse after intratumoral injection of a PEG-SWCNT solution

3 min to tumor region showing the aqueous solution of PEG-SWCNTs. (e) Chemical schains of phospholipid-PEG on the sidewall of SWCNTs. (Inset) Photographs of fet

phosphate buffer saline (PBS, right). Atomic force microscope (AFM) image of indiv

SWCNTs for tumor obliteration. (f) Representative photographs of mice treated in

SWCNTs + NIR; II, untreated; III, PBS + NIR; IV, PEG-SWCNTs). (g) Four mice after 60dependent tumor growth curves of KB tumor cell xenografts.

Source: Reproduced, with permission, from Ref. [70].

6 www.drugdiscoverytoday.com

nonimmunogenicity, and excellent solubility in aqueous and or-

ganic solutions. PEG is approved by the FDA as a base for use in

pharmaceutical formulations. Conjugation of PEG to nanobioma-

terials (‘PEGylation’) has been a useful tool for certain carriers to

resist opsonization, and improve biocompatibility and solubility to

attain long blood circulation time in vivo. PEGylated nanotubes are a

novel class of targeted delivery system, capable pf efficiently deliv-

ering higher drug payloads to disease sites, for the treatment of

diseases such as cancer, TB, and leishmaniasis. PEGylation makes

the nanotubes hydrophilic and enhances their loading efficiency; it

also reduces their immunogenicity, antigenicity, and toxicity. The

PEGylation approach, depending upon the molecular weight (MW)

and surface density of the PEG chain, can overcome the problems of

first-generation nanotubes, such as their pulmonary and hemolytic

toxicities, and can also improve the stability kinetics, which is vital

for extended drug delivery [66]. PEGylation prevents nonspecific

phagocytosis by the reticuloendothelial systems (RES) [67,68]. For

a detailed review of the PEGylation of nanotubes, see [1].

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

Day

4 Day

0 Day

0 Day

I PEG-SWNTs + NIR II Untreated III PBS + NIR IV PEG-SWNTs

I PEG-SWNTs + NIR

3500

3000

2500

2000

1500

1000

500

0

0 5 10 15 20 25 30

Days

Mea

n t

um

or

volu

me

(mm

3 ) UntreatedPEG-SWNTsPBS + NIRPEG-SWNTs + NIR

Drug Discovery Today

G)-single-walled carbon nanotubes (SWCNTs). (a) Schematic view of the

in mice; (b) a mouse bearing human nasopharyngeal epidermoid carcinoma

(120 mg/l, 100 ml); (d) near-infrared (NIR) irradiation (808 nm, 76 W/cm3) for

tructure of PEG-SWCNTs, showing the strong adsorption of hydrophobic alkylal bovine serum (FBS, left), PEG-SWCNTs in fetal bovine serum (middle) and

idual PEG-SWCNTs deposited on a SiO2. In vivo photothermal effects of PEG-

different groups at various time points after each treatment (I, PEG-

days of photothermal treatment (I) from four independent sets. (h) Time-

Page 7: 44 D DT Mehra Palakurthi

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DRUDIS-1717; No of Pages 13

Reviews�KEYNOTEREVIEW

The intracellular distribution of FITC-labeled PEGylated

SWCNTs (FITC-PEG-SWCNTs) in living cells was investigated by

Cheng and coworkers [69]. FITC-PEG-SWCNTs entered the nucle-

us via energy-dependent processes. The FITC-PEG-SWCNTs did

not cause any discernible changes in the nuclear organization and

had no effect on the growth kinetics and cell cycle distribution up

to 5 days post-administration. Thus, the intracellular PEGylated

SWCNTs were highly dynamic and their cell penetration capacity

was found to be bidirectional [69].

Additionally, CNTs have photon-to-thermal abilities and are a

useful candidate for photothermal therapy because they generate

significant amount of heat by excitation of near-infrared light

(NIR). In vivo NIR-mediated tumor destruction by photothermal

effects using PEGylated SWCNTs was studied by Moon and co-

workers [70], who found that the PEGylated SWCNTs destroyed

solid malignant tumors completely in mice in a non-invasive

manner without any adverse effects (Fig. 3) [70].

Biodistribution and blood clearance of PEGylated SWCNTs as

drug delivery vehicles for cisplatin in mice has also been reported

[71]. Cheng and coworkers investigated the use of PEGylated CNTs

in multidrug resistant (MDR) cancer chemotherapy [72]. Based on

atomic force microscopy (AFM) examination, the authors found

the mean length of the PEGylated CNTs to be approximately

0.9 � 0.7 mm, with a median length of 0.55 mm, and an average

diameter of approximately 15 � 5.8 nm, and a median diameter of

14.5 nm. The PEGYlated CNTs were found to be efficient carriers

during chemotherapy treatment of MDR cancer [72].

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Dendrimers Micelles Nucleic acids Ang

Niosomes

Quantum dots

Carbon nanohorns

Liposomes Nanoparticles Proteins

FIGURE 4

Conjugation of carbon nanotubes (CNTs) using chemical and biological moieties, t

Abbreviation: HCPT, 10-hydroxycamptothecin.

A novel and simple ultraviolet-initiated ‘graft from’ polymeri-

zation method was introduced by Zhang and Henthor [73] to

synthesize PEGylated CNTs with significantly enhanced aqueous

dispersibility and long-term stability. Nie et al. functionalized

DWCNTs with two kinds of PEG azide (diazido-terminated PEG

and azido-terminated monomethylether; PEG800–DWNTs and

CH3O-PEG750–DWNTs, respectively) using [2 + 1] cylcoaddition,

which formed a highly stable suspension with good aqueous

solubility (0.36 and 0.37 mg/ml, respectively) [74]. PEG-grafted-

SWCNTs (PEG-g-SWCNTs) or PEG-grafted MWCNTs (PEG-g-

MWCNTs) was also synthesized by Lay and coworkers. PTX was

physically loaded onto CNTS, and the loading capacity (LD %) was

found to be 26% and 36% (w/w) for PEG-g-SWCNTs and PEG-g-

MWCNTs, respectively. However, PEGylated nanotubes reduced

the aggregation or agglomeration of nanotube and were nontoxic

in a mouse model [75]. Similarly, phospholipid-PEG-functional-

ized SWCNTs (PL-PEGs-SWCNTs) was synthesized by Hadidi et al.

using two types of PEG derivative (PL-PEG 2000 and PL-PEG 5000)

to improve their solubility in aqueous solution. Techniques, such

as D-optimal design and second-order polynomial equations, were

applied to investigate the effect of variables on PEGylation and the

solubility of SWCNTs. Optimization was performed for aqueous

solubility (found to be 0.84 mg/ml) and loading efficiency (nearly

98%) for PL-PEG 5000-SWCNTs conjugates and showed that the

increase in aqueous solubility is an essential criterion in the design

of a CNT-based drug delivery system. After the successful synthesis

of water-soluble nanotube conjugates, the authors evaluated the

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

iopep Antibodies Aptamers

GliotoxinCarboplatin

HCPT

Carbon nanotubes Oxaliplatin

Doxorubicin

Amphotericin B

O

OO

O

Pt

N

N

O

ON

N

OHS S

Drug Discovery Today

argeting ligands, nanocarriers, proteins, antibodies, nucleic acids, and drugs.

www.drugdiscoverytoday.com 7

Page 8: 44 D DT Mehra Palakurthi

REVIEWS Drug Discovery Today � Volume 00, Number 00 �December 2015

DRUDIS-1717; No of Pages 13

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EYNOTEREVIEW

effect of PEGylated nanotubes on the viability and proliferation of

cultured Jurkat cells. They concluded that the PEGylated SWCNTs

were substantially less toxic compared with SWCNTs and also

that the MW of PL-PEG had a role at higher concentrations, with

improved biocompatibility [76,77].

Angiopep-2 was successfully conjugated onto modified PEGy-

lated-oxidized MWCNTs (O-MWCNTs-PEG-ANG) for coopera-

tive dual-targeting to brain glioma using intracellular tracking

in vitro and fluorescence imaging in vivo [78]. Recently, Wu and

coworkers developed PEGylated MWCNTs as a sustained-release

drug delivery system using oxaliplatin (a third-generation plati-

num analog of 1,2-diaminocyclohexane). The loading of oxali-

platin into MWCNTs-PEG was found to be approximately 43.6%,

and only 34% of oxaliplatin was released into PBS medium

within 6 h. The authors claimed that the PEG-MWCNTs showed

a sustained release pattern with improved cytotoxicity of oxali-

platin on HT-29 cells [79]. The various chemical and biological

moieties, targeting ligands, nanocarriers, proteins, antibodies,

nucleic acids, and drug conjugations with functionalized CNTs

are shown in Fig. 4. Mehra and Jain recently discussed multi-

functional hybrid-CNTs with carbon nanohorns (CNHs), lipo-

somes, dendrimers, and nanoparticles in drug delivery and

targeting [4].

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COOH

COOH

EDC

CONH

CONH

FI

PEI

PEI

PEI

pH = 7.4pH = 5.8

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

00

0 10 20 30 40 50 60 70TIme (h)

(a)

(b) (c)

(d) (e)

0.5 1

Concentration (mg/L)DOX concentration ( µM)

2 4

DO

X r

elea

se (

%)

Cel

l via

blit

y (%

)

Cel

l via

bili

ty (

%)

0 1.25 2.5 5 10

PBS+HeL

MWCNT/

-FI-HA/DO

+HeLa

PBS+192

MWCNT/

-FI-HA/D

+1929

FIGURE 5

(a) Synthesis of multifunctional MWCNTs. (b) Release profile of DOX from MWCNConfocal microscopic images of HeLa and L929 cells treated with MWCNT/PEI-FI-HA

(d) free DOX and MWCNT/PEI-FI-HA/DOX complexes at DOX concentrations of 0–

concentrations of the complexes of between 1.25 and 10 mg/l.

Source: Reproduced, with permission, from Ref. [86].

8 www.drugdiscoverytoday.com

pH-responsive CNTsThe development of safe and effective nanomedicines by conju-

gating appropriate targeting ligands at the surface of engineered

CNTs is essential for binding to specific receptors that are over-

expressed at the desired target sites. Stimuli-responsive engineered

CNTs might offer interesting opportunities in targeted therapy

and could release bioactives in response to a specific stimulus, such

as light, temperature, and, most importantly, pH. Average extra-

cellular pH values at cancerous sites are 6.8. Such a low extracellu-

lar pH value is caused by hypoxia followed by the production of

lactate and protons in extracellular microenvironments, which

regulates glycolysis. The variation in pH values at cancer tissues

suggests new strategies to develop pH-sensitive targeted drug

delivery systems, such as pH-sensitive liposomes, nanoparticles,

and dendrimers, which could release bioactives at the endosomal

pH with minimum adverse effects and significantly enhance the

therapeutic efficacy of those bioactives [80].

The pH gradient has a pivotal role in the internalization of

nanocarriers via endocytosis and transport in endosomes and

lysosomes. Lysosomes (pH 4.5–5.0) and hydrolytic enzyme pH

is involved in the cleavage and/or degradation of biomolecules

in the cytosol. There are three factors that determine pH-respon-

sive drug release in the lysosomal pH microenvironment: (i)

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

CONH CONH

CONHCONH

FIFI

FIFI

EDC

HA-COOH

NHCO-HA

NHCO-HA

PEI

PEI

PEI

PEI

a

PEI

X

9

PEI

OX

MergedDOXFIBright fieldHocchst33342

Drug Discovery Today

T/PEI-FI-HA/DOX complexes (1 mg/ml) under different pH conditions. (c)/DOX complexes ([DOX] = 2 MM) for 2 h. MTT assay of HeLa cells treated with

4 MM for 24 h. (e) DOX-free MWCNT/PEI-FI-HA at corresponding DOX

Page 9: 44 D DT Mehra Palakurthi

Drug Discovery Today � Volume 00, Number 00 �December 2015 REVIEWS

DRUDIS-1717; No of Pages 13

Please cite this article in press as: Mehra, N.K., Palakurthi, S. Interactions between carbon nanotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.11.011

TABLE 1

Summary of the use of multifunctional CNTs after decoration with targeting, imaging, and biofunctional chemical moieties asnanomedicines for cancer treatment.

Conjugate

moieties/ligand

Receptors Chemotherapeutic

agent

Cell lines used In vivo model Outcomes Refs.

SWCNT

Angiopep-2 LDL receptor DOX BCEC and C6 cells Male Balb/C

mice

Median survival time of saline group, O-

MWNTs-PEG-ANG group, DOX group,

DOX-O-MWNTs-PEG group, and DOX-O-MWNTs-PEG-ANG group was 30, 33.5,

33.5, 36, and 43 days, respectively

[78]

Aptamer – – Molt-4 ad U266 cells Not performed Treatment of U266 cells with Dau-

aptamer-SWNT complex significantlyincreased viability to 78%, but did not

change viability of Molt-4 cells

[93]

Epidermal growthfactor (EGF)

EGF receptor Etoposide A-549 lung epithelialcancer cells

Not performed Cell death induced by EGF/CHI/SWCNTs-COOHs/ETO was 2.7 times

higher than due to ETO alone;

modified- and f-SWCNTs had only slight

cytotoxicity

[46]

SN-38 HCT 116 and HT 29 Not performed SN38 covalent conjugates with

SWCNTs25/py38 were cleave

enzymatically (carboxylesterase) and

showed controlled release with goodbiocompatibility

[94]

Folic acid Folate receptor DOX HCC SMMC-7721

cells

Female nude

Balb/C mice

DOX/FA/CHI/SWNTs shown negligible

toxicity to kidney and liver based on

histological examination; DOX/FA/CHI/SWNTs depressed growth of liver

cancer in nude mice compared with

free DOX

[13]

HER2 IgY HER-2 Not used MCF-7 and

SK-BR-3 cells

Not performed HER2 IgY-SWCNTs complex specifically

targeted HER2-expressing SK-BR-3 cells

but not receptor-negative MCF-7 cells

[89]

HA Hyaluronatereceptor

Salinomycin (SAL) CD 44++ Not performed SAL-SWNTs-CHI-HA selectivelyeliminated cancer stem cells (CSCs) in

AGS gastric cancer cells and inhibited

malignant behavior of gastric CSCs

[91]

NGR peptide CD 13 receptor 2-Methoxyestradiol MCF-7 cancercells

S180 tumorfemale Balb/C

mice

Inhibition ratio of NGR-SWCNTs-2MEwas approximately 88.2% at a

10.15 mg/ml dose after 72 h

[90]

Poly citricacid (PCA)

– PTX A-549 andSKOV3 cells

Not performed Cytotoxicity of MWNT-g-PCAPTX was13.3% higher at 5 nM concentration

compared with free PTX in SKOV3 cells

[12]

RGD Integrin avb3

receptor

DOX MCF-7 and

U87 cells

Not performed IC50 value for PL-SWCNTS-DOX (8 MM)

was higher than for free DOX (2 MM)

[44]

MWCNTDexamethasone

mesylate

Nuclear

receptor

DOX A-549 cells Not performed In vitro release of DOX was found

sustained release from the DOX/DEX-

MWCNTs at pH 5.5 with less hemolytic

[50]

Diameter

functionalized

– Platinum (IV) HeLa ad RAW

264.7cnells

Not performed Both Pt(IV)@CNT constructs were

poorly cytotoxic on macrophages and

induced negligible cell activation and

no proinflammatory cytokineproduction

[96]

Folic acid Folate

receptors

DOX MCF-7 cells Sprague-Dawley rats Median survival time of DOX/FA-PEG-

MWCNTs (30 days) compared with free

DOX

[92]

DTX MCF-7 cells Balb/C mice Tumor volume (mm3) at 30 days after

treatment with DTX/FA-PEG-MWCNTs

was 57.0 � 3.56 compared withnontargeted MWCNTs and free drug

[41]

Folic acid and

estrone

Folate and

estrogen

receptors

DOX MCF-7 cells Balb/C mice DOX/ES-PEG-MWCNTs showed

significantly longer survival span (43

days) than DOX/FA-PEG-MWCNs (42days), DOX/PEG-MWCNTs (33 days), or

free DOX (18 days)

[95]

www.drugdiscoverytoday.com 9

Reviews�KEYNOTEREVIEW

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REVIEWS Drug Discovery Today � Volume 00, Number 00 �December 2015

DRUDIS-1717; No of Pages 13

TABLE 1 (Continued )

Conjugate

moieties/ligand

Receptors Chemotherapeutic

agent

Cell lines used In vivo model Outcomes Refs.

Galactosylatedchitosan

Asialo-glycoproteinreceptor

DOX HepG2 cells Male nude mice Size of tumor in O-CNTs-LCH-DOX-treated mice smaller compared with

mice treated with free DOX

[28]

HA Hyaluronatereceptor

DOX HeLa andL929 cells

Not performed Percentage of HeLa cells uptakingMWCNT/PEI–FI–HA was seven times

more than that of L929 cells; they

exhibited less cytotoxicity compared

with HeLa cells

[86]

MWCNT – DOX – Not performed Sustained release pattern [11]

PEGylated MWCNT – Oxaliplatin HT29 cell Not performed Cytotoxicity of MWCNT-PEG-oxaliplatin

to HT-29 was less compared with

oxaliplatin alone and MWCNT-oxaliplatin

[79]

Vitamin E TPGS LDL receptors DOX MCF-7 cells Balb/C mice Higher fluorescence intensity in

qualitative and quantitative cell uptakeobserved for DOX/TPGS-MWCNTs

(78.72%) compared with DOX/MWCNTs

(62.46%) and free DOX (58.15%). DOX/

TPGS-MWCNTs nanoconjugate alsoshowed longer survival span (44 days,

P < 0.001) than DOX/MWCNTs (23

days) or free DOX (18 days)

[43]

Review

s�K

EYNOTEREVIEW

destabilization; (ii) dissociation via collapse or swelling (in the

case of liposomes); and (iii) pH-dependent changes in the parti-

tion coefficient between the drug and the carrier system [81].

The ultimate goal is to design, fabricate, and evaluate pH-re-

sponsive targeted delivery systems that are capable of releasing

the precise quantities of drug payload in the tumor microenvi-

ronment. Researchers are continuously trying to develop such

systems by engineering nanocarrier systems that are more sensitive

to releasing their payload under mild acidic conditions; that is, as

close to the lysosomal pH as possible to improve their overall

efficacy. Engineered PEGylated CNTs, known as ‘stealth’ CNTs,

are an alternative that could provide significant improvements in

cancer therapy. Additionally, CNTs can be engineered with appro-

priate moieties to target specific cell surface receptors, such as those

for transferrin, folate, epidermal growth factor, and so on.

The dispersion characteristics of various functionalized MWCNTs

at 0.05 mg/mL concentration in different pH (4.0, 7.0 and 9.0) media

have also been investigated [84]. The order for stability of dispersion

in deionized water was: mannosylated MWCNTs > carboxylated

MWCNTs > amine-modified MWCNTs > purified MWCNTs > pris-

pristine MWCNTs. The better stability of carboxylated MWCNTs

compared with the amine-modified MWCNTs was attributed to the

greater ionization at pH 7.4, whereas mannosylated MWCNTs

showed stable dispersion because of the enhanced hydrophilicity

of CNTs, which were also analyzed at different pH (4, 7.4, and 9)

[82,83]. DOX release from alginate-SWCNTs (ALG-SWCNTs) and

CHI/ALG-SWCNTs was pH triggered and stable in PBS at pH 7.4 at

37 8C, and appreciable release of DOX was observed over a 72-h

period in the reduced pH microenvironment of cancerous tissue [84].

Lu et al. studied pH-controlled DOX delivery using DOX-FA-

MN-MWCNTs at 37 8C in PBS at pH 5.3 and 7.4 and found a pH-

triggered response. DOX was released in a slow and controlled

manner at pH 7.4 to the extent of 14%, which was significantly less

than at pH 5.3 (71%) because of the weakening of the hydrogen

bonds between DOX and MWCNTs. The noncovalent attachment

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10 www.drugdiscoverytoday.com

of DOX to the surface-engineered MWCNTs involves hydrogen

bonds between the –COOH of MWCNTs and the –OH of DOX, and

between the –OH of MWCNTs and the –OH of DOX, and the

degree of hydrogen bond interaction is a function of pH. Finally,

the authors also suggested that FA-MN-MWCNTs are a promising

delivery vehicle for anticancer agents because of their high drug

loading and the pH-sensitive release of DOX [52].

Depan and Mishra described a transformative approach to CNT

synthesis and demonstrated the efficacy of a hybrid nanostructured

carrier using PE-b-PEG and SWCNTs. The hybrid nanostructured

carrier was characterized by DOX molecules anchored to disk-

shaped polymer crystals within the long axis of the nanotubes.

The release of DOX was greater at 40 8C compared with at 37 8C.

Temperature affects the segmental mobility of the PE-b-PEG chains,

resulting in enhanced drug release. The authors also suggested that

the difference in DOX release at pH 5.3 and 7.4 was related to the

difference in the swelling capability of the PE-b-PEG polymer at

those pH. The swelling and relaxation of polymeric macromolecular

chains was less under physiological pH and temperature conditions,

resulting in a low concentration of DOX release. By contrast, at

low pH (5.3), the hydrogen bonding interaction between DOX and

the PEG-b-PEG polymer weakened and a higher amount of DOX

was released [58]. Thus, it appears that CNTS enable the delivery

of bioactives in a pH-responsive and/or pH-triggered manner.

Several attempts have been made to develop safe and effective

CNT-based nanomedicines for cancer therapy as well diagnostic

and imaging purposes [85]. PEI-modified MWCNTs after conjuga-

tion with hyaluronic acid (HA)- and FITC-bearing DOX

(MWCNTs/PEI-FITC-HA/DOX) were developed and characterized

using different techniques and the complex was found to be stable

[86]. The schematic representation of synthesis of MWCNTs/PEI-

FITC-HA/DOX is shown in Fig. 5a, while Fig. 5b shows that the pH-

dependent in vitro DOX release rate from MWCNT/PEI-FI-HA/DOX

at pH 5.8 (i.e., tumor cell microenvironment) was higher than pH

7.4. Fig. 5c,d shows that the fluorescence signal within HeLa cells

anotubes and bioactives: a drug delivery perspective, Drug Discov Today (2015), http://

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Drug Discovery Today � Volume 00, Number 00 �December 2015 REVIEWS

DRUDIS-1717; No of Pages 13

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was significantly higher (seven times) than in normal L929 cells at

a 2 MM concentration. MWCNT/PEI-FITC-HA/DOX was internal-

ized through CD44-mediated endocytosis.

Table 1 summarizes the use of various multifunctional CNTs

after decoration with targeting, imaging, and biofunctional chem-

ical moieties after proper functionalization in oncology to develop

safe and effective nanomedicines. The various bioactives delivered

through functionalized CNTs for pharmaceutical and biomedical

applications were recently reviewed elsewhere [7].

Limitations to the use of CNTs as bioactive deliveryvehicleThe technical limitations in the use of CNTs in biomedical appli-

cation are their inherent hydrophobic nature, insolubility, and

bundling and/or aggregation behavior [87,88]. These major hur-

dles associated with pristine CNTs could be overcome by engineer-

ing or surface decoration; that is, functionalization makes CNTs

soluble and minimizes their inherent toxicities. Numerous studies

based on in vitro, ex vivo, and in vivo systems indicate that surface-

engineered CNTs have enhanced therapeutic efficacy in diseases

such as AIDS, malaria, cancer [50], leishmaniasis [83] and TB, but

only a few studies have reported on their potential outcomes in

clinical and preclinical use.

Concluding remarks and future perspectiveCNTs are promising, safe, and effective biomaterials for devel-

opment as nanomedicines. The progress of engineered CNTs

Please cite this article in press as: Mehra, N.K., Palakurthi, S. Interactions between carbon ndx.doi.org/10.1016/j.drudis.2015.11.011

toward clinical and preclinical trials will depend upon the

outcome of safety, efficacy, and toxicological studies of CNTs.

One major hurdle is that, in a 3-(4,5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide (MTT) assay, MTT itself binds

to CNTs (quenching its fluorescence), which could lead to the

mis-analysis of toxicological results [89]. The major physico-

chemical and toxicity limitations could be addressed by conju-

gating CNTs with targeting moieties, or PEG chains, which

render them more biocompatible and nonimmunogenic. In

our view, multifunctional CNTs will continue to received signif-

icant research attention because of their unique physicochemi-

cal properties in targeted and controlled drug delivery. Although

no CNT-based drug product is currently on the market, engi-

neered CNTs are now at the crossroads of the proof-of-principle

concept and are being developed as preclinical candidates for

a variety of biomedical applications. The presence of various

amorphous and metallic impurities of pristine CNTs limits

their clinical applications because of their inherent toxicity.

Researchers are continually exploring various options to over-

come such problems by using functionalization, including

PEGylation. Apart from their interactions with bioactives, the

physicochemical factors of CNTS, such as their length, diameter,

and type, also influence their safety and efficacy. The variety

of interactions between CNT and the bioactivates discussed

here could serve as a reference for the development of multi-

functional hybrid nanotubes as carriers for biomedical applica-

tions.

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