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MULTIFUNCTIONAL FULLERENE- AND
METALLOFULLERENE-BASED
NANOBIOMATERIALS
GAURAV LALWANI and BALAJI SITHARAMAN
*
Department of Biomedical Engineering
Stony Brook University
Stony Brook, New York 11794-5281
*
balaji.sitharaman@stonybrook.edu
Received 18 April 2013
Accepted 21 June 2013
Published 22 August 2013
Recent advances in nanotechnology have enabled the synthesis and characterization of nanoma-
terials suitable for applications in the ¯eld of biology and medicine. Due to their unique physico-
chemical properties, carbon-based nanomaterials such as fullerenes, metallofullerenes, carbon
nanotubes and graphene have been widely investigated as multifunctional materials for appli-
cations in tissue engineering, molecular imaging, therapeutics, drug delivery and biosensing. In this
review, we focus on the multifunctional capabilities of fullerenes and metallofullerenes for diagnosis
and therapy. Speci¯cally, we review recent advances toward the development of fullerene- and
metallofullerene-based magnetic resonance imaging (MRI) and X-ray imaging contrast agents,
drug and gene delivery vehicles, and photodynamic therapy agents. We also discuss in vitro and in
vivo toxicity, and biocompatibility issues associated with the use of fullerenes and metallofullerenes
for biomedical applications.
Keywords: Fullerene; metallofullerene; contrast agent; bioimaging; therapy; drug delivery; gene
delivery; toxicity; biodistribution.
1. Introduction
The development of tools such as electron and scan-
ning probe microscopes,
1,2
and the discovery of
nanomaterials such as fullerenes
3
and quantum dots
4
in the 1980s galvanized scienti¯c progress in the ¯eld
of nanotechnology. Today nanotechnology is an
exciting interdisciplinary ¯eld that could signi¯cantly
revolutionize technologies for diverse applications,
ranging from electronics and telecommunication,
to energy and healthcare. Nanomaterials, generally
in the range 1100 nm (in at least one dimen-
sion), exhibit unique mechanical, electrical and opti-
cal properties compared to micro- or macro-sized
materials. For example, carbon nanotubes possess a
Young's modulus of 1TPa(5 times greater than
steel), and are highly conductive with current den-
sities at 6107Acm2ð100 times greater than
metallic conductors such as copper).
57
Using various
\top-down" and \bottom-up" approaches such as
chemical vapor deposition (CVD),
8
chemical etch-
ing,
9
electrospinning,
10
self-assembly,
11
photolitho-
graphy,
12
electron beam lithography
13
and thin ¯lm
deposition;
14
nanomaterials can be synthesized from
metals,
15
ceramics,
16
semiconductors,
17
polymers,
18
organic carbon-based materials,
19
organic-inorganic
hybrid materials,
20
nanocomposites
21
and biological
Nano LIFE
Vol. 3, No. 3 (2013) 1342003 (22 pages)
©World Scienti¯c Publishing Company
DOI: 10.1142/S1793984413420038
1342003-1
nanomaterials
22
in a variety of morphologies such as
tubes, platelets, ribbons, ¯bers, wires, rods, horns,
shells, ¯lms and particles.
8,12,2326
Various covalent
and noncovalent functionalization strategies have
been formulated for the attachment of ligands and
molecules toward development of nanomaterials
suitable for biomedical applications such as molecular
imaging and targeted delivery, and to mitigate
nanomaterial toxicity.
27
Carbon nanostructures such as fullerenes, metal-
lofullerenes, carbon nanotubes and graphene are the
most widely researched class of materials. They show
remarkable physicochemical properties, and functio-
nalization capabilities that can be harnessed for the
next-generation biomedical applications. Indeed, they
have been extensively investigated for applications
such as imaging probes for cellular labeling and
tracking,
2831
reinforcing agents for polymeric
nanocomposites,
21,3234
substrates for cellular pro-
liferation and di®erentiation
3538
and drug and
gene delivery agents.
3942
The ¯rst studies investi-
gating the potential of carbon nanomaterials for bio-
medical applications were performed on fullerenes and
metallofullerenes (see Table 1for various biomedical
applications of fullerenes and metallofullerenes).
43
There are now multiple reviews that document
advances in the functionalization, physicochemical
properties and material science applications of either
fullerenes or metallofullerenes.
4447
Some recent
reviews also focus on biomedical applications of full-
erenes or metallofullerenes.
46,4851
This review seeks
to complement these existing publications, and focu-
ses on recent advances in the development of full-
erenes and metallofullerenes as multifunctional
platforms for diagnostic and therapeutic applications.
Speci¯cally, we review the published literature on
fullerenes and metallofullerenes as magnetic reson-
ance imaging (MRI) and X-ray imaging contrast
agents, drug and gene delivery vehicles and photo-
dynamic therapy (PDT) agents. Also, reviewed
are pertinent in vitro and in vivo toxicity and
biocompatibility issues related to these carbon nano-
materials.
We conducted a comprehensive search in the
database of US National Library of Medicine,
National Institutes of Health (PubMed, www.ncbi.
nlm.nih.gov). Relevant articles that focus on in vitro
and in vivo studies of fullerenes and metallofuller-
enes for imaging, drug and gene delivery and
therapeutic applications were identi¯ed using the
Table 1. Biomedical applications of fullerenes. Adapted from Ref. 46, (copyright °
cPartha and Conyers,
publisher and licensee Dove Medical Press Ltd.), with permission.
Type of fullerene (Keywords) Potential biomedical applications Reference
Gadofullerenes MRI contrast agent Sitharaman et al.
29,50,51
Toth et al.
59
Shu et al.
68
Iodinated fullerenes X-ray imaging contrast agents Wharton and Wilson
77
Amphiphilic fullerenes (buckysomes, PEBs) Drug delivery Partha et al.
96,97
Fullerene-paclitaxel Zakharian et al.
53
Fullerene-peptide conjugates Dermal drug delivery Rouse et al.
100,112
Fullerene-DOX conjugates Drug delivery and radical scavenging Injac et al.
92,93
Chaudhuri et al.
94
Liu et al.
95
Fullerenols Antioxidants and radical scavenging Gharbi et al.
125
Fullerene polyamine (tetra amino fullerenes) Gene delivery, transfection Isobe et al.
104,105
Nakamura et al.
106,108
Amino-fullerene adducts Nonviral gene delivery Sitharaman et al.
103
Hydrophobic or cationic fullerenes PDT Mroz et al.
81,86
Fullerene malonic acid adducts Rancan et al.
82
PEG modi¯ed fullerenes Tabata et al.
88
Porphyrin-C60 dyads Alvarez et al.
85
Milanesio et al.
84
C60-PEG-Gd 3þconjugate PDT and MRI Liu et al.
87
G. Lalwani & B. Sitharaman
1342003-2
following search algorithm:
\(fullerenes [All Fields]AND/OR gadofullerenes
[All Fields]AND/OR biomedical [All Fields]AND/
OR applications [All Fields]AND/OR MRI [All
Fields]AND/OR contrast [All Fields]AND/OR
agent [All Fields]AND/OR photodynamic [All
Fields]AND/OR therapy [All Fields]AND/OR
endohedral [All Fields]AND/OR fullerenes [All
Fields]AND/OR drug [All Fields]AND/OR deliv-
ery [All Fields]AND/OR gene [All Fields]AND/OR
delivery [All Fields]AND/OR biodistribution [All
Fields]AND/OR transfection [All Fields]AND/OR
vectors [All Fields], NOT nanotube [All Fields],
NOT nanotubes [All Fields])".
We systematically screened published articles
included in this review. The search resulted in 34
articles for MRI contrast agents, 8 for X-ray con-
trast agents, 75 for drug and gene delivery, 69 for
PDT and 72 for in vitro and in vivo toxicity. Full-
text experimental studies were considered and
evaluated independently by each author. Only
publications in English with an experimental group
sample size 5 were considered. The minimal group
size of n¼5 was chosen to avoid inclusion of studies
containing low sample sizes since studies were based
on varying methods and treatment conditions.
2. Bioimaging
Current medical imaging modalities such as MRI
and X-ray computed tomography (CT) possess
unique strengths and weaknesses. Signi¯cant
research has been devoted towards developing the
next generation of contrast agents for these mod-
alities. Contrast agents are compounds adminis-
tered to improve the sensitivity and diagnostic
con¯dence of these modalities for various pathol-
ogies and diseases. The contrast enhancement
mechanism for each of these modalities is di®erent.
The contrast seen in MRI is due to the concen-
tration and magnetic properties of water protons,
while CT contrast is due the attenuation of X-rays
transmitted through the patient. The multi-
functional capabilities of metallofullerenes have
been harnessed by encapsulating atoms within its
hollow interior space, which modulate water pro-
tons, the source of signal during MRI, or interact
with X-rays in CT. Furthermore, these nano-
particles can accumulate in the diseased tissue due
to enhanced permeability and retention properties,
and can be covalently or noncovalently func-
tionalized with suitable moieties to improve their
blood circulation half-life and tissue targeting
capabilities.
5053
2.1. MRI contrast agents
MRI is one of the central diagnostic technique used
for medical imaging in the clinic. Annually, contrast
agents are administered in 18 million MRI pro-
cedures worldwide to improve diagnostic con¯-
dence.
54
The two predominant types of MRI
contrast agents are T1and T2agents that decrease
longitudinal and transverse relaxation times, re-
spectively. Relaxivity (expressed in mM1s1Þis the
measure of contrast agent e±cacy that accelerates
the relaxation process of water protons. The most
widely used (99%of MRI procedures) clinical T1
contrast agents are metal ion-based chelate com-
plexes of transition elements such as Gd3þand
Mn2þ.
55,56
Recent theoretical and experimental
studies show that relaxivity values of these contrast
agents are suboptimal, and predict the development
of contrast agents with 50100 times greater
relaxivity.
50,57
Gd3þions possess a large magnetic moment
(2¼63 2
B), and symmetric electronic ground state
(
8
S7=2Þ, permitting Gd3þion-metal chelate com-
plexes to be used as MRI contrast agents. Over the
last decade, Gd3þ-based carbon nanostructure com-
plexes were developed that have signi¯cantly higher
relaxivity values than clinically used Gd3þ-based
contrast agents. Water-soluble endohedral metallo-
fullerenes encapsulating Gd3þions (a.k.a. gado-
fullerenes, see Fig. 1for representative structural
depictions) have relaxivity values 220 times
higher than clinically used gadolinium-based con-
trast agents.
29,50,5866
These increased relaxivity
values are attributed to interactions of a large num-
ber of water protons with Gd3þion via the fullerene
cage. Results suggest an entirely outer-sphere relax-
ation mechanism with largest outer-sphere relaxiv-
ities reported for a Gd3þ-based MRI contrast agent.
Additionally, it has been hypothesized that a full-
erene molecule that encapsulates Gd3þion shields
and limits in vivo dissociation of Gd3þion, thereby
signi¯cantly reducing toxicity concerns. Long-term
in vitro and in vivo stability studies are still needed to
test this hypothesis.
The propensity of water-soluble gadofullerenes to
aggregate in solution, and the dependence of this
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-3
aggregation on pH and salts present in biological
media are the main factors responsible for the large
variation in the relaxivity of gadofullerenes-based
MRI contrast agents.
59,65,6769
In vitro studies
show that decreased pH (from alkaline to acidic pH
values) of water-soluble gadofullerenes solutions
increases their aggregation. This increased aggre-
gation, in turn, increases the relaxivity of gadoful-
lerenes making them promising candidates for
monitoring and mapping pH changes in diseased
tissues (e.g., tumors) by MRI.
59
In vivo studies are
needed to validate the capabilities of gadofullerenes
as pH-responsive MRI contrast agents. A di±culty
in testing this hypothesis lies in the opposing e®ect
of biologically relevant salts on gadofullerene
relaxivity.
65,66
Studies show that the relaxivity of
gadofullerenes decreases (e.g., r1of Gd@C60(OH)x
decreases from 83.2 mM 1s1to 43.1 mM 1s1Þin
biological bu®ers such as phosphate-bu®ered saline
(which contains sodium, chloride and phosphate
ions) in comparison to salt-free gadofullerene sol-
utions.
65,66
This decreased relaxivity is attributed to
disaggregation of gadofullerene aggregates due to
the presence of salts.
65,66
In vitro studies were conducted to test the e±-
cacy of gadofullerenes as magnetic labels for cellular
MRI. Anionic gadofullerenes such as Gd@C60[C
(COOH)
2
]10, internalized by mesenchymal stem
cells (MSCs) within 28 h of incubation (see Fig. 2)
and intercalated within the cell membrane, main-
taining a stable intracellular Gd3þconcentration
(eliminating the possibility of Gd3þleaching).
29
In
vitro cellular toxicity and viability assays indicate
no cellular damage and 300%increase in the T1
weighted MRI signal intensity from gadofullerene-
labeled MSCs (compared to controls: MSCs labeled
with Magnevist
r
). These results suggest potential
application of gadofullerenes for in vivo stem cell
tracking applications via MRI, a highly desirable
property for tissue engineering and regenerative
medicine applications.
In vivo biodistribution of Gd@C60[C(COOH)
2
]10
tracked via MRI after administration to rodents
(35 mg/kg, tail vein) showed increased uptake of the
gadofullerene derivative in kidney, with minimal
uptake in liver.
64
MRI of kidneys showed 50%
increase in contrast within 16 min after injection. Ac-
cumulation and excretion of Gd@C60[C(COOH)
2
]10
began after 1 h of injection (via bladder) suggesting
clearance of Gd@C60[C(COOH)
2
]10.
Organophosphate functionalized endohedral
metallofullerene derivatives Gd@C82O
2
(OH)16(C
(PO
3
Et2Þ2Þ10 (referred to as TEMDP-EMFs), syn-
thesized via Bingel reaction, have also been devel-
oped as MRI contrast agents.
70
T1weighted MRI
phantom imaging showed that the organophosphate
Fig. 2. Cryo-transmission electron microscopy of a platelet
showing aggregates of gadofullerenes (marked-A), mitochon-
dria (M) and platelet granules (G). Presence of gadofullerenes
was con¯rmed by EDX spectroscopy. Adapted from Ref. 29
(copyright °
cJohn Wiley and Sons Ltd., 2007) with permission.
(a) (b) (c)
Fig. 1. Schematic representation of (a) Gd@C60(OH)x; (b) Gd@C60 [C(COOHyNa1yÞ2]10; (c) Gd-DTPA (Magnevist r). (a) and
(c) Adapted from Ref. 66 (copyright °
cAmerican Scienti¯c Publishers, 2005), and (b) adapted from Ref. 50 (copyright °
cAmerican
Scienti¯c Publishers, 2007); with permission.
G. Lalwani & B. Sitharaman
1342003-4
functionalization resulted in signi¯cantly increased
relaxivity values for TEMDP-EMFs (37 mM 1s1Þ
compared to clinically used Omniscan (Gd-DTPA
BMA, 5.7 mM1s1Þand carboxylated Gd@C82
(16 mM1s1Þat 0.35T. Furthermore, organopho-
sphate functionalization can impart bone targeting
abilities to fullerenes,
53
suggesting potential appli-
cability of TEMDP-EMFs as bone targeting MRI
contrast agents.
The development of a tri-metallic-nitride tem-
plating method to encapsulate multiple metal ions
by Dorn and coworkers led to a protocol for
encapsulation of multiple Gd3þions inside the full-
erene cage.
71
Referred to as `Hydrochalarones' (tri-
metallic nitride fullerene, Gd
3
N@C80Þ, these are
thermally and chemically stable derivatives of
gadofullerenes (cage decomposition 500C, re-
sistant to structural damage upon heating in con-
centrated nitric acid).
61
They exhibit high relaxivity
values (r1¼205 mM1s1Þdue to the presence of
multiple Gd3þions. Polyethylene glycol (PEG)
conjugated, polyhydroxylated derivatives of
Gd
3
N@C80 such as Gd
3
N@C80[DiPEG(OH)x] [see
Fig. 3] were synthesized by functionalization with
PEG of varying molecular weights (3005000
Da).
62
Gd
3
N@C80 functionalized with 350/750 Da
PEG derivatives exhibited high relaxivity values
(r1¼237=232 mM1s1,r2¼460=398 mM 1S1Þ
at a magnetic ¯eld of 2.4 Tesla. Gd
3
N@C80
[DiPEG350(OH)x], administered via convection
enhanced delivery to rat brain (0.0235 mM, infusion
time ¼180 min, rate ¼0:2L/min, total infusion
volume ¼36 L) showed a uniform distribution in
the tumor was observed after 3.5 h of adminis-
tration. MRI contrast signi¯cant for delineation of
tumor margins in vivo was observed seven days
post-administration illustrating the potential of
Gd
3
N@C80[DiPEG350(OH)x] as a MRI contrast
agent for long-term monitoring of tumors.
The ¯rst in vivo studies assessing the potential of
Gd@C82(OH)nas MRI contrast agent were per-
formed by Sinohara and coworkers in 2001.
63
Gd@C82(OH)nwas administered to female CDF1
mice (female, 8 weeks old) intravenously at a dose of
5mol Gd/kg, 20 times lower than the clinical dose
of Gd-DTPA (100 mol Gd/kg). T1weighted MRI
images showed localization of Gd@C82(OH)nin
reticulo-endothelial system (RES) organs such as
liver, spleen and lungs after 30 min of injection.
Biodistribution studies conducted to measure the
concentration of gadolinium ion (via ICP analysis)
in dissected organs matched the RES uptake trend,
suggesting that polyhydroxylated Gd@C82(OH)n
possess potential for development as RES-speci¯c
MRI contrast agent.
In another study, polyhydroxylated PEG-Gd
3
N@C60 administered via convection enhanced deliv-
ery to the extracellular space of rat brain tumors;
(glioma tumor models, female Fisher 344 rats)
demonstrated prolonged tumor retention via
T2-weighted MR imaging.
72
Compared to controls
(gadodiamide, commercial MRI contrast agent),
which are cleared from the injection site within 3 h of
administration, polyhydroxylated PEG-Gd
3
N@C60
show prolonged retention in the tumor (up to 5 days
post-administration). Due to the high relaxivity
values of polyhydroxylated PEG-Gd
3
N@C60,en-
hanced tumor uptake, and retention, their required
concentration can be signi¯cantly reduced (up to 40
fold reduction compared to gadodiamide) and still
display adequate contrast for clinical evaluations.
2.2. X-ray imaging contrast agents
X-ray imaging is one of the most commonly used
clinical imaging modalities. Image formation
depends upon the attenuation of X-rays from tissues
and organs, which is directly dependent upon tissue
density and thickness. Dense organs such as bone
can be easily visualized via radiography, but soft
tissues and organs with similar densities possess
similar attenuation properties, resulting in poor
X-ray contrast; this limits the use of X-ray imaging
to visualize soft-tissues or neo-vasculature in
Fig. 3. Schematic representation of Gd
3
N@C80[DiPEG(OH)x]
endohedral metallofullerene. Adapted from Ref. 62 (copyright
°
cAmerican Chemical Society, 2010) with permission.
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-5
regenerated/newly formed tissues and organs.
Therefore, contrast agents (generally iodine-based)
are administered to increase X-ray contrast of soft
or vascular tissues. Although iodinated contrast
agents are usually safe and reports indicating
serious adverse e®ects are rare (out of 61000
patients, 2.3% reported adverse events);
73
they still
have potential to cause adverse e®ects. Patients
have 2030% risk of contrast media-induced
nephropathy (due to tubular cell damage and
resultant reduction in renal perfusion) and allergic
reactions.
74,75
Thus, development of an X-ray con-
trast agent with superior performance and
decreased risk of nephrotoxicity would be bene¯cial.
Diamagnetic endohedral metallofullerenes en-
capsulating metal ions, for example, trimetallic
nitride fullerenes (Lu
3
N@C80Þ, synthesized via tri-
metallic nitride template method have been reported
as X-ray contrast agents.
76
In comparison to pristine
C60 containing Te°on blocks, X-ray imaging of
Te°on blocks deposited with Lu
3
N@C80 exhibit
sharp X-ray contrast. In another study, highly
iodinated fullerenes (containing six iodine atoms and
eight acetal-protected alcohols per fullerene cage)
were synthesized using a modi¯ed Bingel reac-
tion.
77,78
X-ray imaging of iodinated fullerenes at
concentrations of 150 mg I/mL, performed using
standard Kodak ¯lm (MIN R 2000), showed X-ray
attenuation comparable to commercially used com-
pound Iohexolr.
78
Polyhydroxylated endohedral
metallofullerenes encapsulating multiple heavy
metal ions (one or two ions of lutetium (Lu), erbium
(Er), dysprosium (Dy), europium (Eu) and gadoli-
nium (Gd) encapsulated into C82Þwere dissolved in
water at maximum stable concentrations and CT
was performed using a multidetector CT scanner.
79
The CT measurements (in Houns¯eld Units (HU))
were 23.3 HU for Lu
2
@C82(OH)40 , 111.5 HU for
Er@C82(OH)40, 56.0 HU for Dy@C82(OH)40, 100.9
HU for Eu@C82(OH)40 and 58.4 HU for Gd@C82
(OH)40. Although metallofullerenes encapsulating
heavy metal ions possess CT numbers comparable to
clinically used X-ray contrast agents, signi¯cant
work (for e.g., in vivo preclinical toxicity and bio-
distribution studies) still needs to be performed to
assess safety and e±cacy.
3. Therapeutics
Carbon nanoparticle-based therapeutics has several
advantages over conventional therapy. Here, intrinsic
physical properties of fullerenes and metallofullerenes
can be harnessed for various therapeutic appli-
cations. For example, fullerenes and metallofuller-
enes are strong absorbers of visible light, and
generate cytotoxic reactive oxygen species (ROS)
that induce cell death, a property exploited for
PDT applications. Furthermore, various therapeutic
agents can be attached via covalent and noncovalent
functionalization to the exterior carbon sheath of
fullerenes and metallofullerenes along with targeting
ligands to improve biocompatibility, and impart site-
speci¯c delivery capabilities. For example, fullerene
sheath can be chemically derivatized to bind to che-
motherapeutic drugs such as doxorubicin and cyclo-
phosphamide (CPA), and functionalized to increase
the water solubility by stacking.
39
Additionally,
fullerenes functionalized with cationic groups can
serve as vectors for nonviral gene delivery appli-
cations.
3.1. Photodynamic therapy
PDT employs nontoxic photosensitive molecules
capable of absorbing visible light to produce high-
energy triplet states for cellular therapy. In this
minimally invasive approach, photosensitizers in
the presence of visible light produce cytotoxic ROS
that impair cellular machinery causing cell death,
eventually leading to tissue destruction. Water-
soluble functionalized fullerenes, upon photo-
irradiation, become excited and transition to a
short-lived singlet electronic state (1C60Þ, lose
energy due to °uorescence and intersystem coup-
ling, and are converted to the long-lived stable tri-
plet electronic state (3C60Þ. The excited triplet state
interacts with molecular oxygen forming ROS such
as hydroxide (OH) and superoxide anion (O2)
radicals. Reaction of ROS with cellular components
such as proteins, nucleic acids and unsaturated
lipids causes oxidative damage, inducing cell death
via apoptosis.
80
Fullerenes have been used in PDT for the
destruction of viruses, microbes and cancer cells,
in vitro and in vivo.
81
Phototoxicity of dendritic C60
mono-adducts and malonic acid conjugated C60 tris-
adducts, studied on jurkat cells showed 19%
increase in cell death upon irradiation with UV light
after 2 weeks of incubation.
82
C60 tris-adducts
showed phototoxicity upon irradiation with visible
light whereas mono-adducts exhibited toxicity in
the absence of light. In another study, fullerenes
G. Lalwani & B. Sitharaman
1342003-6
inhibited the growth of HeLa cells upon irradiation
with low energy visible light (6-W °uorescent light
bulb, two times a day for 1 h each) by inducing
ROS-mediated cleavage at guanine bases in the
DNA.
83
Metal complexes of C60-porphyrin hybrids
(ZnP-C60Þproduce singlet oxygen species (
1
O2Þ
upon photoirradiation, inducing 80%cell death
(after 15 min of light exposure) upon incubation
with human larynx carcinoma cells (Hep-2) at
concentrations less than 1 M (see Fig. 4(a) for
representative structure of P-C60 complex).
84,85
The
generated singlet oxygen species induce caspase-3
activation, releasing caspase-activated DNase
(CAD) resulting in apoptosis via DNA fragmenta-
tion. C60-pyrrolidinium mono-adducts (compared to
C60-pyrrolidinium tris-adducts) exhibit increased
phototoxicity in murine cancer cells (CT26) after
24 h of incubation, measured by a °uorescence cas-
pase activity-based assay using an intracellular ROS
probe (H
2
DCFDA).
86
Cells incubated with C60-
pyrrolidinium mono-adduct (2 M in RPMI media
for 24 h) and H
2
DCFDA, subjected to photo-
irradiation by a 5 J/cm
2
405 nm laser, exhibit
increased °uorescence intensity compared to con-
trols incubated with H
2
DCFDA in the absence
of fullerene adducts (see Figs. 4(e) and 4(f) for
changes in the °uorescence intensity). These results
suggest high PDT e±cacy of C60-pyrrolidinium
complexes suitable for apoptotic death of cancerous
cells.
Several in vivo fullerene-based PDT studies have
been published.
87,88
Hybrid MRI-PDT systems
based on PEG conjugated C60-Gd 3þ-DTPA com-
plexes (diethylenetriaminepentaacetic acid) exhibit
increased PDT and improved MRI contrast (com-
pared to Magnevistrcontrols) following intrave-
nous administration to tumor bearing mice,
suggesting potential for theragnostic applications
that allow simultaneous monitoring of tumor PDT
[see Figs. 4(b)4(d)].
87
The MRI contrast for PEG-
conjugated C60-Gd3þ-DTPA complex reached its
peak after 3 h of administration and was sustained
up to 24 h. Magnevistrcontrols showed increased
contrast for only 1 h. In another study, prolonged
in vivo retention of PEG conjugated C60 resulted in
increased PDT activity and suppression of subcu-
taneous tumor growth.
88
C60-PEG conjugates were
injected intravenously (6 mg PEG-C60 conjugate at
424 g/kg C60 dose) and exposed to light (89.2 mW/
cm
2
, 400505 nm) 24 h post-injection. For com-
parison, aqueous glucose solution containing Pho-
tofrin (80 g/mL, 4 mg/kg injection, 72.5 mW/cm
2
,
610800 nm) was used as a positive control, and
PBS injection was used as a negative control.
Tumor volume ratio (ratio between the volume of
tumor before and after 11 days of light exposure)
was 10:22:23 for PBS controls and 0:54 0:05 for
C60-PEG conjugate, suggesting high e±cacy of C60 -
PEG conjugates for PDT of tumors. Histological
analysis showed tumor necrosis without damage to
the overlying skin tissue.
88
3.2. Drug and gene delivery
Fullerene-based systems for delivery of drugs and
genes possessing selective tissue targeting, and with
controlled release capabilities have been devel-
oped.
89,90
Water-soluble fullerenes can successfully
pass through the cell membrane and serve as
intracellular delivery agents.
90,91
To mitigate oxi-
dative damage to cells and tissues, poly-
hydroxylated fullerenes (a.k.a. fullerenols), with
radical scavenging ability, have been used to deliver
doxorubicin (DOX), a widely used chemother-
apeutic drug. Fullerenol [C60(OH)24], administered
in a rat colorectal cancer model (25 mg/kg/week,
50 mg/kg/week and 100 mg/kg/week for 3 weeks in
male Wistar rats) protected liver and heart from
DOX-induced oxidative damage (1.5 mg/kg/week
for 3 weeks) (see Fig. 5).
92
In another study, the
e®ects of chronic DOX toxicity were mitigated by
exploiting the radical scavenging abilities of full-
erenols [C60(OH)24 ]. Proof-of-principle in vivo
studies were performed on Sprague Dawley rats.
93
Fullerenols were injected at 100 mg/kg 30 min
before DOX injection (8 mg/kg). Fullerenols
reduced the elevated neutrophil count (from 58%
for DOX treatment groups to 52% for DOX-full-
erenol group). Additionally, results showed 37%
reduction in the level of LDH (a marker of cardio-
vascular damage), compared to free DOX group
past fullerenol administration.
Water-soluble, covalently crosslinked DOX-PEG-
C60 conjugates, capable of high therapeutic loading
of DOX and time-dependent DOX release, were
evaluated for cytotoxicity against metastatic human
breast cancer cells (MDA-MB-231), mouse mela-
noma cells (B16-F10) and mouse lung carcinoma
cells (LLCC1).
94
Results showed a time-dependent
increase in cytotoxicity of DOX-PEG-C60 con-
jugates against all cell lines (apoptosis due to G2-M
phase block). LC50 values for C60-DOX conjugates
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-7
(a) (b)
(c) (d)
(e) (f)
Fig. 4. (a) Schematic of a fullerene-porphyrin dyad. (b) MRI phantom imaging (T1weighted) after intravenous injections of
Magnevistrand (d) C60-PEG-Gd conjugate in mice (tumor mass is indicated by a circle). (c) Decline of in vivo tumor growth after
intravenous injections of C60-PEG-Gd conjugates and exposure to light (bars denote saline, C60 -PEG-Gd without light exposure and
with light exposure) for 1 h, 3 h, 6 h and 12 h, respectively. A signi¯cant decline in the tumor size is observed. (e) and (f) PDT of
CT26 cells incubated for 24 h with intracellular ROS probe (H
2
DCFDA), and probe þfullerenes. Increase in the °uorescence
corresponds to increased levels of caspase. (a) Adapted from Ref. 85 (copyright °
cElsevier, 2006), (b), (c) and (d) adapted from
Ref. 87 (copyright °
cElsevier, 2007), and (e) and (f) adapted from Ref. 86 (copyright °
cElsevier, 2007); with permissions.
G. Lalwani & B. Sitharaman
1342003-8
were 10 Mand12M (expressed in DOX concen-
tration) for melanoma and LLC cells after 24 h of
treatment, and 8 M for MDA-MB-231 cells after
72 h of treatment. To evaluate in vivo therapeutic
e±cacy, DOX-PEG-C60 conjugate was systemically
administered (6 mg/kg therapeutic dosage of free
DOX) into mice with melanoma tumors. Both
treatment groups showed similar tumor growth in-
hibition (90% reduction in tumor volume com-
pared to control group (mice injected with PBS));
however, DOX-PEG-C60 conjugate treatment resul-
ted in negligible hepato- and cardio- toxicity (deter-
mined by histopathological analysis) compared to
free DOX. Furthermore, in comparison to DOX-
PEG-C60 conjugate, free DOX showed a higher ac-
cumulation in tumor. In another study, cytoplasmic
accumulation of DOX-PEG-C60 was observed in
MCF-7 cells upon incubation with 230M of DOX-
PEG-C60 conjugate (expressed as DOX equivalent)
for 1 h (see Fig. 6).
95
Two-photon imaging of inter-
nalized DOX conjugate using a femtosecond pulsed
(900 nm) laser con¯rmed cellular uptake of DOX-
PEG-C60 conjugate. Since DOX can be excited using
two photons at wavelengths in the near infrared
region of the spectrum, DOX-PEG-C60 can be used
for cellular imaging as an intracellular two-photon
°uorescence probe.
C60-paclitaxel conjugates [see Fig. 7(a) for re-
presentative structure], forming stable liposomes
with dilauroylphosphatidylcholine (DLPC), suit-
able for aerosol-based delivery to lungs, exhibited
cytotoxicity against human epithelial lung carci-
noma cells (A549).
53
C60-paclitaxel-DLPC lipo-
somes (2.77 m in diameter, as measured by
dynamic light scattering) were incubated with A549
cells for 1 h at an equivalent paclitaxel concen-
tration of 12.5 M. Cell culture media was replaced
1 h after treatment and plates were incubated for an
additional 2 days. The mean IC50 value for C60-
paclitaxel-DLPC liposomes was 410 nM, whereas
paclitaxel-DLPC control was 253 nM, suggesting
similar drug delivery e±ciency of paclitaxel loaded
C60-DLPC liposomes compared to paclitaxel-DLPC
control.
Buckysomes, macroscopic structures of self-
assembled fullerenes containing dendritic and fatty
acid side chains, self-assemble to form nanometer-
sized vesicles. Paclitaxel embedded buckysomes
Fig. 5. Histological sections of rat heart and liver following treatment with DOX (a) and (b), DOX-C60 conjugate (c) and (d),
and normal saline (e) and (f), respectively. Presence of parenchymal degradation (D), vacuoles (V), necrotic monocytes and
hepatocytes (N), apoptotic myocytes (*) and interstitial edema was observed in DOX treated groups and mild parenchymal
degradation (D), no vacuoles and lesser necrotic hepatocytes (N) were present in groups treated with C60 -DOX conjugate. Cardiac
and liver sections of control rats had no lesions and parenchymal degradation. Adapted from Ref. 92 (copyright °
cElsevier, 2009)
with permission.
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-9
[PEBs, Fig. 7(b)] exhibit signi¯cantly increased
tumor drug delivery, leading to decreased infusion
times, thereby enhancing anticancer e±cacy of
paclitaxel. MCF-7 cells incubated with varying
concentrations of PEBs (28.6 ng/mL, 143 ng/mL
and 714 ng/mL of paclitaxel in PEBs) for 48 h and
72 h showed reduction in cell viability (compared to
empty buckysomes) as analyzed by Trypan Blue
assay. Cell viability for PEBs containing 714 ng/mL
paclitaxel was comparable to that of the clinical
chemotherapeutic agent Abraxane
r
. Preclinical
investigations using pig models are underway to
test PEBs as chemotherapeutic drug delivery
agents.
96,97
Fullerenes have also been investigated for the
delivery of drugs like CPA. Therapeutic e±cacy of
nitroxide methanofullerenes (NMFs) was evaluated
for leukemia P-388 in tumor bearing BDF
1
mice.
(a)
(b) (c)
Fig. 6. (a) Schematic of a C60-DOX conjugate. (b) and (c) Confocal images of MCF-7 cells treated with free DOX and PEG
conjugated C60-DOX complex. Nuclear localization of DOX is observed (image b), compared to the localization of DOX-PEG-
C60 conjugate in the cytoplasm (image c). Adapted from Ref. 95 (copyright °
cAmerican Chemical Society, 2010), with
permission.
G. Lalwani & B. Sitharaman
1342003-10
NMFs administered individually (50 mg/kg and
200 mg/kg, single dose, subcutaneous injection) did
not result in the suppression of tumor growth (death
of all 10 animals); however, administration in con-
junction with CPA (CPA þNMF ¼30 þ50 mg/kg
and 30 þ200 mg/kg, single dose, subcutaneous in-
jection) resulted in 30% and 70% survival rates for
each treatment groups, respectively.
98
These re-
sults suggest that NMFs can be administered with
chemotherapeutic drugs such as CPA to improve
e±cacy.
In addition to the delivery of chemotherapeutic
drugs as discussed above, few studies report the use
of fullerenes for potential drug delivery applications
(a)
(b)
Fig. 7. Schematic representation of (a) C60-paclitaxel conjugate and (b) amphiphilic fullerene (AF-1) capable of self-assembly to
form buckysomes. (a) adapted from Ref. 53 (copyright °
cAmerican Chemical Society, 2005), and (b) adapted from Ref. 96
(copyright °
cPartha et al., licensee Biomed Central Ltd); with permission.
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-11
to skin, heart and hydrophobic tissue such as
liver.
99102
Functionalized fullerenes have been
extensively investigated for exogenous DNA and
gene delivery applications. Using HirschBingel
chemistry, water-soluble derivatives of fullerenes
were synthesized, and reported as e±cient gene
delivery vectors.
103
Octa-amino derivatized C60
and dodeca-amino derivatized C60 were mixed with
GFP plasmid solution and incubated with NIH
3T3 mouse ¯broblasts and HEK 293 cells at 10 g/
mL DNA concentration for 2 h, 8 h, 24 h or 48 h.
Octa- and dodeca-derived amino-fullerenes exhib-
ited 28% and 31% transfection e±ciencies, re-
spectively, in NIH 3T3 cells. Cytotoxicity studies
show >50%cell viability for both octa-amino and
dodeca-amino fullerene complexes at 10 g/mL
treatment concentration.
Nakamura and coworkers performed pioneering
studies on the development of functionalized full-
erenes for in vitro and in vivo gene delivery.
104107
Exogenous GFP labeled fullerene DNA complexes
were internalized by COS-1 cells via phagocy-
tosis.
106
Fluorescence imaging after 2 days of incu-
bation displayed GFP expression, con¯rming the
stability and activity of DNA-fullerene conjugates
(see Fig. 8).
106
Transfection e±ciency for C60-DNA
complex was 26%, comparable to 33% achieved by
Transfectamr(commercially available transfection
agent). In another study, tetra-amino fullerenes
with strong DNA binding a±nity were investigated
as transfection vectors for mammalian COS-1
cells.
104
Plasmid vector DNA (440 kbp) loaded
onto C60 exhibited GFP expression even after 12
days of transfection. Compared to Lipofectinr
reagent which showed 0.039% transfection e±ciency
after 2 weeks of culture, C60-DNA conjugates
exhibited a transfection e±ciency of 0.73%, signi¯-
cantly greater than lipofection-based transfection.
Amino-fullerenes possess signi¯cant advantages
over other transfection vectors. For e±cient cellular
internalization, micro- and nano-aggregates of
amino-fullerene DNA complexes can be controlled
Fig. 8. Optical microscopy images of (a) aggregates of C60-DNA conjugates in DMEM media (without FBS), (b) C60 -DNA
aggregates in DMEM media containing 10% FBS, (c) C60-DNA aggregates after 1 h of transfection in COS-1 cells, and (d) C60-DNA
aggregates after 48 h of transfection in COS-1 cells using 4g GFP vector. Image (d) is a superimposed representation of °uorescence
micrograph (green) and di®erential interference contrast (DIC) micrograph (gray), containing spherical black lumps of endosomes
internalizing C60-DNA conjugates. Scale bar is 200 m for images (a) and (b) and 100 m for images (c) and (d). Adapted from
Ref. 106 (copyright °
cAmerican Chemical Society, 2006), with permission.
G. Lalwani & B. Sitharaman
1342003-12
by molecular design of amine groups. Following
cellular uptake, amino-fullerene DNA conjugates
lose the amine group to release bound DNA, a fea-
ture necessary for gene expression. Furthermore,
DNA binding ability of amino-fullerenes DNA
conjugates can be reduced by neutralization of
amine groups inside cells, assisting in the release of
bound DNA for e±cient transfection.
105
Cationic amino-fullerene derivatives such as tet-
rapiperazino fullerene epoxide (TPFE) are reported
to be resistant to endonuclease digestion and
therefore can release the bound DNA inside cells for
e±cient transfection.
108
In vitro gene delivery using
TPFE as an arti¯cial vector was examined by
adjusting various parameters such as fullerene/base
pair ratio, plasmid DNA concentration and trans-
fection time. TPFE vectors showed more than four-
fold increase in transfection e±ciency compared to
commercially available lipofectin-based delivery
systems. TPFE e±cacy for in vivo gene delivery
applications has been examined by delivering
enhanced green °uorescent protein (EGFP) to
pregnant ICR mice (intravenous injection, 24 g
plasmid DNA in 300 L injection volume).
108
When
animals were euthanized 24 h post-injections, TPFE
vectors exhibited distinct organ selection and
increased expression of EGFP in liver and spleen
(see Fig. 9). TPFE vectors were nontoxic to liver
and kidneys in comparison to lipofectin vectors, the
use of which resulted in increased BUN concen-
tration and liver enzymes (61%increase in AST
and 108% increase in ALT compared to TPFE
groups). Furthermore, TPFE vectors could also
successfully deliver insulin-2 gene to female C57/
BL6 mice, increasing plasma insulin levels and
reducing blood glucose levels (compared to naked
DNA controls) at 12 h post-injection (intravenous
injection, 24 g plasmid DNA in 300 L injection
volume).
4. Toxicology and Pharmacological
Studies (In Vitro and In Vivo)
Evaluation of cyto- and biocompatibility is necess-
ary to fully develop any new material for in vivo
biomedical applications. Additionally, in the future,
Fig. 9. Confocal microscopy images of lung, liver, kidney and spleen showing EGFP distribution after 24 h of injection. Red
°uorescence corresponds to cell nucleus and green corresponds to EGFP expression. Adapted from Ref. 108 (copyright °
cNational
Academy of Sciences, 2010), with permission.
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-13
use of these carbon nanomaterials for a wide range
of commercial materials science applications will
increase the risk of their release into the environ-
ment. Thus, evaluation of the in vitro cytotoxicity
and in vivo toxicology and pharmacology of fullerene
and metallofullerene formulations is necessary.
4.1. In vitro cytotoxicity
Table 2lists the LC50 values for various fullerene
derivatives examined for biomedical applications.
Di®erential cytotoxic responses were observed
depending on the type of cells and surface modi¯-
cations. Human U251 glioma cells, rat C6 glioma
cells and mouse L929 ¯broblast cells were incubated
with 1 g/mL and 1000 g/mL of pristine fullerenes
(nano-C60Þand polyhydroxylated fullerenes [C60
(OH)n], respectively.
109
After 24 h of incubation, 3
orders higher cytotoxicity was observed for all cells
treated with nano-C60 compared to C60(OH)n. ROS-
mediated cell death was observed in cells exposed to
nano-C60 whereas DNA fragmentation was observed
following C60(OH)nexposure. Addition of an anti-
oxidant (N-acetylcysteine) inhibited ROS-mediated
damage thereby reducing nano-C60 induced toxicity,
whereas addition of a caspase inhibitor (z-VAD-fmk,
0.05 M) inhibited caspase-mediated DNA frag-
mentation, reducing C60(OH)ninduced cytotoxicity
by 40% (compared to absence of z-VAD-fmk).
Appropriate surface functionalization strategies
can signi¯cantly reduce fullerene toxicity. In com-
parison to pristine fullerenes (C60Þ, water-
solubilized derivatives (C60(OH)24,Na
þ
23[C60O79
(OH)1215]ð23Þ and C3Þexhibit 7 fold decrease in
cytotoxic response upon incubation with human
dermal ¯broblast cells (HDF) at various concen-
trations, for 48 h, measured using LIVE-DEAD
and LDH assays (see Fig. 10).
110
The LC50 value
determined for HDF cells is 10000 ppb for C
3
,
40000 ppb for Naþ
23½C60 O79ðOHÞ1215ð23Þ and
>5 000 000 ppb for C60 (OH)24.
Toxicity of pristine fullerenes against human and
murine macrophages was assessed by measuring the
amount of nitric oxide (NO) released upon exposure
to nano-C60. Murine macrophages (J774), stimu-
lated by lipopolysaccharide (LPS), incubated for
24 h, 48 h and 72 h with nano-C60 at various con-
centrations (15 g/mL, 30 g/mL and 60 g/mL)
showed no signi¯cant increases in NO release
(compared to LPS stimulated positive controls).
111
Use of nano-C60 suspensions did not induce sig-
ni¯cant toxicity in human monocyte derived
macrophages (MDMs) at similar treatment con-
centrations after 1 h, 24 h and 48 h of incubation.
Compared to macrophages treated with graphite
and single walled carbon nanotubes, exposure to
fullerenes did not induce formation of elongated
microvilli-like structures and cell surface protrusions
(see Fig. 11), suggesting the absence of macrophage
activation and cellular damage.
Self-assembling supramolecular structures of
amphiphilic fullerene dendrimers (AF-1, a.k.a.
buckysomes) did not show any adverse e®ects on
cellular proliferation or membrane integrity of
macrophages, liver and kidney cells, compared to
PBS treated controls when evaluated using MTT
and LDH assays, for treatment concentrations
Table 2. LC50 values of various fullerene derivatives. Adapted from Ref. 60 (copyright °
cWorld Scienti¯c Publishing),
with permission.
Compound Cell type Exposure (h) LC50 Reference
Pristine C60 (in water) HDF 48 20 g/mL 110,135
Human neuronal astrocytes 48 2 g/mL 135
Human liver carcinoma cells 48 50 g/mL 135
Tris-malonic C60 HDF 48 10 mg/mL 110
Naþ
23[C60O79(OH)1215 ]ð23Þ HDF 48 40 mg/mL 110
C60(OH)24 HDF 48 >5 g/mL 110
C60/C70 extract in water
(C60 ¼79%, C70 ¼20%,
higher fullerenes ¼1%)
Mouse L929 ¯brosarcoma 24 0:25 mg/mL 109
Rat C6 glioma
U251 human glioma
C60(OH)nbased on C60 /C70 mixture
(nnot speci¯ed)
Mouse L929 ¯brosarcoma 24 0.81 g/L 109
Rat C6 glioma
U251 human glioma
G. Lalwani & B. Sitharaman
1342003-14
ranging from 0.022 mg/mL AF-1.
96
Stable in-
ternalization of buckysomes in cells was observed by
°uorescence microscopy. In another study, human
epidermal keratinocyte (HEK) cells were incubated
with fullerene-phenylalanine conjugates (fullerene-
based amino acid complexes, Baa) at 0.4 mg/mL,
0.04 mg/mL and 0.004 mg/mL concentrations for
24 h and 48 h.
112
MTT assay results indicated a
dose-dependent decrease in cell viability. Addition-
ally, for 0.4 mg/mL and 0.04 mg/mL treatment
groups, signi¯cant increases in the levels of proin-
°ammatory cytokines such as IL-1and IL-6 along
with cellular internalization of Baa were observed
after 24 h of exposure.
In vitro studies assessing cytotoxicity and cellular
internalization of fullerene derivatives such as [C61
Fig. 10. Schematic structural depiction, and cellular toxicity of nano-C60,C
3
,Na
þ
23[C60O79(OH)1215 ð23Þ and C60(OH)24 after
48 h of incubation in HDFs, showing live (green) cells and dead (red) cells. Adapted from Ref. 110 (copyright °
cAmerican Chemical
Society, 2004), with permission.
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-15
(CO
2
H)
2
] in COS-7 cells, and nano-C60(OH)2226 in
human retinal pigment epithelium (hRPE) and
human lens epithelium (HLE 3-B) cells were also
performed.
113115
COS-7 cells were incubated with
C61(CO
2
H)
2
(labeled with 14C,10 M) for 24 h. Post-
incubation, cells were harvested using trypsinization,
recovered using low-speed centrifugation (2000 g,
10 min) and homogenized. Cell lysate was subjected
to di®erential centrifugation and followed by radio-
active quanti¯cation. Maximum radioactivity was
recorded from the mitochondrial fraction suggesting
mitochondrial localization of C61(CO
2
H)
2
.In
another study, intracellular accumulation and cyto-
toxicity (25% cell viability) of hRPE cells was
observed upon exposure to >10 M nano-C60
(OH)2226 concentrations upon culture in the pre-
sence of light (8.5 J cm2).
115
However, in the
absence of light, 80% cell viability was observed up
to 50 M treatment concentrations. Singlet oxygen
measurements indicate that fullerenols produce
singlet oxygen species upon exposure to light with a
quantum yield of ¼0:05 in D
2
O, suggesting that
the observed cytotoxicity maybe a result of photo-
damage due to type I (free radical induced) or type II
(singlet oxygen induced) mechanisms.
Hemolytic potential of various water-soluble
fullerene derivatives was assessed by measuring
hemoglobin release after 30 min of RBC exposure.
116
Results indicate that bis-functionalized fullerene
derivatives (possessing carboxylic acid groups or
one cationic chain) did not induce hemolysis up to
treatment concentrations of 80 M whereas deriva-
tives with two cationic chains induced 4050%
hemolysis at treatment concentrations ranging
between 2060 M. Further, cytotoxicity of full-
erene derivatives was assessed using Hep-G2, LLC-
PK
1
and MCF-7 cell lines and MTT assay. Cationic
fullerene derivatives exhibited signi¯cant cytotox-
icity against all cell lines with LD50 values ranging
between 1 M and 20 M whereas anionic or neutral
derivatives did not induce any cytotoxic response
with LD50 values >80 M for all cell lines.
4.2. In vivo toxicity, and biodistribution
4.2.1. Oral administration
Biodistribution of water-soluble radiolabeled full-
erenes (14C), examined after 160 h of oral exposure
in mice showed clearance via feces within 48 h of
administration.
117
However, trace amounts were
observed in urine, suggesting that fullerenes possess
the ability to pass through mice gut walls. Oral
administration of water-soluble polyalkylsulfonated
fullerenes to rats (n¼6, two regimens: 50 mg/kg
Fig. 11. Surface morphology of human MDMs after 24 h of incubation with pristine fullerenes (a), (d), single walled carbon
nanotubes (b), (e), and graphite (c), (f), as depicted by scanning electron microscopy. Activation of macrophages, as evident by the
formation of elongated microvilli structures and extended cell surface protrusions, is moderate for cells treated with fullerenes and
carbon nanotubes whereas signi¯cantly higher for cells treated with graphite. Adapted from Ref. 111 (copyright °
cElsevier, 2006),
with permission.
G. Lalwani & B. Sitharaman
1342003-16
single dose and 50 mg/kg daily for 12 days) was
nontoxic.
118
Compared to healthy animals, sup-
pression of liver cytochrome P-450, cytochrome b5
and benzo(a)pyrene hydroxylase (liver marker
enzyme) were observed. The LD50 value for poly-
alkylsulfonated C60 was 50 mg/kg. In an acute tox-
icity study, fullerite (mixture of C60 and C70Þwas
administered to male and female Sprague Dawley
rats at high concentrations (2000 mg/kg) for
15 days.
119
This treatment resulted in survival of all
animals with normal weight gain (compared to
healthy controls, 0 mg/kg fullerite administration).
4.2.2. Dermal exposure
Toxicity studies using dermal exposure are import-
ant because of the use of fullerenes in cosmetics as
anti-wrinkle, anti-aging and skin-whitening
agents.
120
Skin penetrating ability of fullerenes is
dependent on the solvent phase used for dispersion.
In comparison to mineral oil, determined by trans-
mission electron microscopy, fullerenes dispersed in
chloroform, toluene and cyclohexane were localized
in the stratum corneum of pigs after 4 days of ap-
plication (200 g/mL, 500 L).
121
In another study,
C60 suspensions using squalane (LF-SQ) as the dis-
persing agent showed dermal penetration at high
exposure concentrations (223 ppm in LF-SQ).
122
Interestingly, dermal penetration was absent at lower
exposure concentrations (2.23 ppm and 22.3 ppm),
suggesting that fullerene penetration through the
epidermis is concentration dependent. Mechanical
compression and skin °exion increased passive dif-
fusion of fullerenes through epidermis.
100
In another
study, 30 volunteers were exposed to a patch of
Whatmann ¯lter paper (No. 3) saturated with water-
soluble fullerenes.
123
The results show absence of skin
irritation and allergies after 96h of exposure,
suggesting that fullerenes do not induce acute toxic
response in skin. However, long-term toxicity studies
need to be performed for a proper understanding of
fullerene toxicity via dermal exposure.
4.2.3. Intraperitoneal and intravenous
administration
Toxicity of C60-PEG conjugates administered intra-
peritoneally in tumor bearing mice was evaluated
using body weight as a marker after 15 days of
injections.
88
At low treatment concentrations (1.8
180 mg/kg injections), normal weight gain was
observed in treatment groups compared to the con-
trol group (no injection). However, transient weight
loss was observed for groups treated with higher
concentrations of PEG-C60 conjugate (1800 mg/kg).
Liver marker enzymes such as glutamic pyruvic
transaminase (GPT) and glutamate oxaloacetate
transaminase (GOT) were present at physiological
levels for groups with >1 mg/kg injections of C60-
PEG conjugates. Indicators of nephrotoxicity such as
blood urea nitrogen (BUN), GOT and GPT were
present at normal physiological levels (BUN ¼
20:62:5 mg/dL, GOT ¼35 4IU/L, GPT ¼
28 8 IU/L), suggesting that intraperitoneal injec-
tions of C60-PEG conjugate at 11.8 mg/kg in
mice are nontoxic. Histological analysis showed
tumor necrosis without damage to the normal
skin at a dose of 424 g/kg at irradiation power of
107 J/cm
2
.
In an acute toxicity study, fullerenol [C60O
5
(OH)18] was administered to mice via intraper-
itoneal injections (0.5 g/kg and 1 g/kg dose).
124
Animals receiving 0.5 g/kg and 1 g/kg dose of full-
erenol showed reductions in liver marker enzymes
such as cytochrome b5, NADPH-cytochrome P450,
benzo(a)pyrene hydroxylase and aniline hydroxyl-
ase, compared to animals receiving 0.01 g/kg and
0.1 g/kg dose. The LD50 value of fullerenol was
estimated to be 1.2 g/kg. In another study, e®ects of
pretreatment with C60 on acute carbon tetra-
chloride intoxication in rats were studied. In com-
parison to animals with CCl
4
intoxication and no
fullerene administration, histopathological examin-
ations show the absence of liver ¯brosis and in-
°ammation in rats injected with pristine fullerenes
(2 g/kg, single dose).
125
Furthermore, physiological
levels of alanine aminotransferase (100 IU/L) were
observed suggesting the absence of liver damage due
to fullerene administration.
Polyalkylsulfonated fullerene [C60((CH2Þ4SO
3
Na)46], administered intraperitoneally in rats at
sub-acute (60 mg/kg, 12 days) and acute (1000 mg/
kg, 24 h) levels resulted in death of 5 animals (out of
6) at 750 mg/kg and all animals at 1000 mg/kg
within 24 h of injections.
118
Although elimination of
[C60((CH2Þ4SO
3
Na)46] was observed via urine and
feces, accumulation was observed in liver and spleen
macrophages, along with suppression of cytochrome
P450 activity. Note, that such an acute toxicity
response was limited to animals receiving high doses
of fullerenes (1000 mg/kg), and was not observed for
lower treatment doses. In another study, a bis
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-17
(monosuccinimide) derivative of p p'-bis(2-ami-
noethyl-diphenyl C60Þ, MSAD-C60, did not elicit
acute toxicity (100% survival) when administered
at 15 mg/kg to Sprague-Dawley rats.
126
At high
treatment concentrations (25 mg/kg dose), short-
ness of breath, violent movements and eventual
death was observed within 5 min of injections.
4.2.4. Pulmonary administration (inhalation)
Water-based suspensions of C60(OH)24 and nano-
C60 were administered via intratracheal instillation
to mice at 0.2 mg/kg, 0.4mg/kg, 1.5 mg/kg or 3mg/
kg doses for 1 day to 3 months.
127
Normal physio-
logical levels of alkaline phosphatase (60120 IU/
L), lactate dehydrogenase (5075 IU/L) and neu-
trophils (15% of total cell count) (total cell
count ¼51067:5106cells) were observed in
bronchoalveolar lavage °uid (BAL) after 24 h of
administration. On the contrary, positive controls
showed a persistent increase in the number of neu-
trophils (2050% of total cell count), eventually
leading to an increase in lung weight due to par-
enchymal lung cell proliferation. Furthermore,
nano-C60 resulted in increased lipid peroxidases
levels (100% increase compared to controls
administered with water) in a dose-dependent
manner. Lung in°ammation and ¯brosis observed
in positive controls (see Fig. 12), were absent in
nano-C60 and C60(OH)24 treated groups (3 mg/kg
treatment for 3 months), indicating the absence of
nano-C60 and C60(OH)24 toxicity, contradictory to
previous reports.
109
C60 microparticles (2.35 mg/m
3
, 0.93 m diam-
eter) and nanoparticles (2.22 mg/m
3
, 55 nm diam-
eter) were administered via inhalation to Fisher 344
rats (10 week old) for 10 days (2 mg/m
3
C60 full-
erenes, 3 h everyday).
128
Compared to healthy con-
trols, rats administered with C60 nanoparticles
showed a minimal decrease in RBC (2:7%),
hemoglobin (2:9%) and packed cell volume
(2:6%) in the nanoparticle-exposed group. How-
ever, rats exposed to C60 microparticles showed
decreases in white blood cells (20:9%), monocytes
(72%), eosinophils (45%) and platelets (13%).
In comparison to healthy controls, rats exposed to
C60 nanoparticles also showed minimally increased
blood glucose levels (3:5%), while rats exposed to
Fig. 12. Histology of rat lung tissue after exposure (3 months, 3 mg/kg) to (a) DI water control, (b) Min-Usil (positive control),
(c) fullerene (nano-C60Þsuspensions in water, and (d) fullerenol [C60 (OH)24] suspensions. Normal lung architecture of alveolar ducts
(AD) and terminal bronchiole (TB) is observed in images (a), (c), and (d), suggesting that exposure of rat lungs to water-soluble
fullerenes does not elicit any adverse response. Image B depicts thickening of lung tissue (black arrow) and accumulation of
macrophages (white arrow). Adapted from Ref. 127 (copyright °
cAmerican Chemical Society, 2007), with permission.
G. Lalwani & B. Sitharaman
1342003-18
C60 microparticles showed increases in bile acids
(55%), creatinine kinase (33%) and decreased
albumin concentration (2%). Macrophages from
both exposure groups contained brown pigments
corresponding to intracellular localization of C60.
Lung half-lives for C60 micro and nanoparticles were
29 and 26 days, respectively. The lung deposition
rate and deposition fraction were 50% and 41%
higher in animals administered C60 nanoparticle
compared to animals receiving C60 microparticles.
In another study, C60-PBS suspensions (single
exposure, 0.05 mL) administered via intratracheal
instillation to female ICR mice at a ¯nal treatment
concentration of 625 g (euthanized after 0 min and
5 min of instillation) or 1000 g (euthanized after
1 h, 6 h, 24 h, or 7 days after instillation) resulted in
elevated levels of C60 in the circulation.
129
Capillary
lumens of pulmonary lymph nodes and lungs were
lined with aggregates of fullerenes and intracellular
accumulation of fullerenes in alveolar endothelial
and epithelial cells (via pinocytosis) was observed
by transmission electron microscopy imaging. These
results suggest that fullerenes have the potential to
permeate the air-blood barrier (ABB) and can be
used to administered therapeutics to organs via
blood circulation. These ¯ndings may not apply to
human exposure, as studies with mammalian species
do not report any fullerene translocation to blood
circulation via ABB.
Several studies assessing the genotoxicity and
reproductive toxicity of fullerenes do not show any
adverse e®ects.
119,130,131
However, for a complete
understanding of reproductive toxicity, additional
toxicity studies assessing the developmental e®ects
of fullerenes must be performed.
132,133
In general,
negligible in vitro and in vivo (mainly in small
animal such as mice or rats) toxicity of water-
soluble fullerene derivatives as determined by var-
ious cell culture and animal studies may not be
su±cient to answer all concerns related to fullerene
toxicity. Additional dose and time-dependent tox-
icity and pharmacology studies in other animal
models (preferably a large animal model), using the
various methods of administration, are necessary.
5. Summary and Future Perspective
Advancements in fullerene production led to
commercial ventures (such as Frontier Carbon
Corporation [Kitakyushu-shi, Japan], Nano-C Inc.
[Westwood, MA, USA]) to facilitate large-scale
production of fullerenes. Similar ventures for large-
scale production of endohedral metallofullerenes
have been limited. Companies such as TDA
Research Inc. [Wheat Ridge, CO, USA] have been
working since the late 1990s to scale up production
of metallofullerenes, but progress has been slow due
to limitations associated with the production pro-
cess such as low yields, batch to batch variability,
low purity (a batch of metallofullerenes typically
contains various types such as M@C60, M@C82, etc.)
and presence of impurities. For biomedical appli-
cations, products that utilize fullerenes ex vivo are
already available. For example, Nova C60TM,a
product of Nova C60 Skin Solutions Inc. (Markham,
Ontario, Canada [www.novaadvancedskincare.
com]) is an anti-aging, anti-wrinkle skin cream uti-
lizing the antioxidant properties of fullerenes to
reduce free radical induced damage in skin. Progress
toward development of fullerene or metallofullerene-
based products for in vivo biomedical applications
has been slow or has stalled. All the research and
development work on these products to date has
been at the preclinical stage. C-Sixty Inc. (now a
subsidiary of Arrowhead Research Corporation,
Toronto, Canada) is developing fullerene-based
drugs for AIDS and neurodegenerative diseases such
as Parkinson's, Alzheimer's and ALS (Lou Gehrig's
disease) with several products under preclinical in-
vestigations.
134
Luna nanoworks Inc. (division of
Luna Innovations Inc., Danville, VA, USA) is
developing metallofullerenes for diagnostic appli-
cations. Their products, such as Trimetaspherer
and Hydrochalaronerare derivatives of metallo-
fullerenes encapsulating three gadolinium ions
(Gd
3
N@C60Þ, are currently under preclinical inves-
tigation as MRI contrast agents.
Studies to date clearly demonstrate the multi-
functional capabilities of fullerenes and metal-
lofullerenes for diagnostic and therapeutic uses.
Despite signi¯cant progress, in vivo toxicity and
biodistribution of fullerenes and metallofullerenes is
not yet thoroughly understood. There is an emer-
ging consensus that in vivo toxicity of these carbon
nanoparticles is not only dose and time-dependent,
but also depends on a number of other factors such
as: type (e.g., C60,C
70, M@C60, M@C82Þ, functional
groups used to water solubilize these nanoparticles
(e.g., OH, COOH), and method of adminis-
tration (e.g., intravenous, intraperitoneal). There-
fore, pharmacology of every new fullerene- or
metallofullerene-based complex must be assessed
Multifunctional Fullerene- and Metallofullerene-Based Nanobiomaterials
1342003-19
individually as a di®erent compound. Additionally,
costs and time constraints associated with fullerene
production and processing (puri¯cation and sorting)
are barriers for some applications. However, these
costs are highly dependent on the speci¯c appli-
cation. In recent years, the focus has shifted toward
investigating the potential of other carbon nano-
particles such as carbon nanotubes and graphene for
biomedical applications. These shifts negatively
impacted biomedical research using fullerenes and
metallofullerenes resulting in decreased numbers of
scienti¯c publications about these nanoparticles.
Nevertheless, harnessing the multifunctional capa-
bilities of fullerene- and metallofullerene-based
compounds toward the development of biomedical
technologies represents a challenging, but poten-
tially rewarding opportunity towards the develop-
ment of next-generation biomedical products.
Acknowledgments
This work was sponsored by National Institutes of
Health (Grants No. 1DP2OD007394-01). The authors
acknowledge Renee Pessin and Jason Rashkow for
editorial assistance.
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