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Opportunities and Challenges of Fluorescent Carbon Dots in Translational Optical Imaging

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The fluorescent carbon dot (C-dot) is a new class of carbon nanomaterials. It has a discrete or quasispherical structure, typically measures less than 10 nm and contains sp2/sp3 carbon, oxygen/nitrogen-based groups and surface-modified functional groups. Compared with semiconductor quantum dots (QDs), C-dots offer much lower toxicity and a better biocompatibility profile. Their other favorable features include easy and inexpensive synthesis and surface modification potential. C-dots can be morphologically classified into graphene-based quantum dots (GQDs) and amorphous carbon nanodots (ACNDs). Numerous methods have been developed to synthesize C-dots, and are mainly divided into 'top-down' and 'bottom-up' routes. In the top-down route, C-dots (mostly GQDs) is derived from the separation of large carbon precursors. The 'bottom-up' method primarily involves the dehydration, polymerization and carbonization of small molecules to form the GQDs and ACNDs through thermal/hydrothermal synthesis, microwave irradiation, and solution chemistry. Potential applications of C-dots have been explored in a number of cellular and in-vivo Imaging approaches. However, some difficulties remain, including limited penetration depth and poorly controlled in-vivo pharmacokinetics, which depends on multiple factors such as the morphology, physiochemical properties, surface chemistry and formulation of C-dots. The exact mechanism of in-vivo biodistribution, cellular uptake and long-term toxicological effect of C-dots still need to be elucidated. An integrated multi-disciplinary approach involving chemists, pharmacologists, toxicologists, clinicians, and regulatory bodies at the early stage is essential to enable the clinical application of C-dots.
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Opportunities and Challenges of Fluorescent Carbon Dots in Translational Optical
Imaging
Junqing Wang1, Gang Liu2, Ken Cham-Fai Leung3, Romaric Loffroy4, Pu-Xuan Lu5* and Yì Xiáng J.
Wáng1,5*
1Department of Imaging and Interventional Radiology, Faculty of Medicine, The Chinese University of Hong
Kong, Prince of Wales Hospital, Hong Kong SAR, P.R. China; 2State Key Laboratory of Molecular Vaccinology
and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public
Health, Xiamen University, Xiamen, 361102, P.R. China; 3Department of Chemistry, Institute of Creativity, and
Partner State Key Laboratory of Environmental and Biological Analysis, The Hong Kong Baptist University,
Kowloon Tong, Kowloon, Hong Kong SAR P.R. China; 4Department of Vascular, Oncologic and Interventional
Radiology, University of Dijon School of Medicine, Bocage Teaching Hospital, 21079 Dijon Cedex, France;
5Department of Radiology, The Shenzhen No. 3 People’s Hospital, Guangdong Medical College, Shenzhen, P.R.
China
Abstract: The fluorescent carbon dot (C-dot) is a new class of carbon nanomaterials. It has a discrete or qua-
sispherical structure, typically measures less than 10 nm and contains sp2/sp3 carbon, oxygen/nitrogen-based groups and surface-modified
functional groups. Compared with semiconductor quantum dots (QDs), C-dots offer much lower toxicity and a better biocompatibility
profile. Their other favorable features include easy and inexpensive synthesis and surface modification potential. C-dots can be morpho-
logically classified into graphene-based quantum dots (GQDs) and amorphous carbon nanodots (ACNDs). Numerous methods have been
developed to synthesize C-dots, and are mainly divided into ‘top-down’ and ‘bottom-up’ routes. In the top-down route, C-dots (mostly
GQDs) is derived from the separation of large carbon precursors. The ‘bottom-up’ method primarily involves the dehydration, polymeri-
zation and carbonization of small molecules to form the GQDs and ACNDs through thermal/hydrothermal synthesis, microwave irradia-
tion, and solution chemistry. Potential applications of C-dots have been explored in a number of cellular and in-vivo imaging approaches.
However, some difficulties remain, including limited penetration depth and poorly controlled in-vivo pharmacokinetics, which depends
on multiple factors such as the morphology, physiochemical properties, surface chemistry and formulation of C-dots. The exact mecha-
nism of in-vivo biodistribution, cellular uptake and long-term toxicological effect of C-dots still need to be elucidated. An integrated
multi-disciplinary approach involving chemists, pharmacologists, toxicologists, clinicians, and regulatory bodies at the early stage is es-
sential to enable the clinical application of C-dots.
Keywords: Carbon Dots (C-dots), Photoluminescence (PL), Near-infrared (NIR), Quantum yield (QY), Optical Imaging, Iron oxide, Fe3O4.
1. INTRODUCTION
The rapid development of fluorescent nanomaterials over the
past three decades has led to increasing biological and medical
research into their potential applications [1]. Fluorescence nanopar-
ticles are colloidal semiconductor quantum dots (QDs) measuring
~5-10 nm in size [2]. Compared with organic fluorophores, QDs are
approximately 20 times brighter and 100 times more photostable
than standard fluorescent dyes [3]. Due to their high photolumines-
cence quantum yield (PL QY) and the extent to which their optical
properties can be modified by slightly altering their size, these
nanoparticles have been explored as a platform for imaging applica-
tions [4-7 ]. However, metal elements, such as cadmium, are widely
used in QDs (e.g. CdX; X= Te or Se or S), which makes clinical
application difficult as these metal elements can be toxic [8, 9].
Although the toxicity of these QDs can be partially mitigated by
embedding the toxic core with polymers or silica to form a core-
shell structure [1, 10], such encapsulation methods result in larger
hydrophilic QDs (~20 –30 nm). This would significantly limit in-
tracellular mobility and may hinder Förster resonance energy trans-
fer (FRET), as well as induce undesirable in-vivo behaviors [11].
Compared with semiconductor QDs, fluorescent carbon dots
*Address correspondence to these authors at the Department of Imaging and
Interventional Radiology, Faculty of Medicine, The Chinese University of
Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
SAR, P.R. China. E-mail: yixiang_wang@cuhk.edu.hk; Department of
Radiology, The Shenzhen No. 3 People’s Hospital, Guangdong Medical
College, Shenzhen, P.R. China; E-mail: lupuxuan@126.com
(C-dots) is perceived as lower toxic alternatives in the development
of optical imaging agents for various biomedical purposes [12-18].
In this review, we first present the composition and classifica-
tion of C-dots, and compare the various synthetic approaches. Then,
we provide an overview of optical performance, surface function-
alization and nanocomposites of C-dots. In-vitro/in-vivo studies and
toxicity profile of C-dots are then summarized, followed by a dis-
cussion of the requirements for clinical applications.
2. COMPOSITION AND CLASSIFICATION OF C-DOTS
C-dots commonly refers to a group of carbogenic fluorescent
nanoparticles that have a discrete or quasispherical structure and
typically measure less than 10 nm. They contain sp2/sp3 carbon,
oxygen/nitrogen based groups and surface-modified functional
groups [19]. They were discovered among the fragments of single-
walled carbon nanotubes (SWCNTs) during purification [20]. Over
the last decade, fluorescent C-dots have attracted much attention in
bio-applications because of their small size, tunable fluorescence,
inexpensive fabrication, and biocompatible features. Numerous
important discoveries have been made, particularly with regard to
improving their PL properties and methods to synthesize C-dots. To
date, various types of C-dots have been synthesized and they can be
morphologically classified into two major types, graphene-based
quantum dots (GQDs) and amorphous carbon nanodots (ACNDs).
In most cases, GQD s mainly consist of an sp2 carbonic nanocrystal-
line core with surface defects or passivation. Unlike GQDs, ACNDs
comprise a disordered carbon framework integrated by coexisting
Yi Xiang J. Wang
2 Current Pharmaceutical Design, 2015, Vol. 21, No. 00 Wang et al.
sp3 and sp2 hybridized carbon moieties with the possibility of sur-
face functionalization. This irregular configuration is probably
caused by relatively higher amounts of oxygen/nitrogen containing
moieties that disrupt the systematic carbonic framework though
covalent bonds [21]. In addition, both GQDs and ACNDs exhibit
excellent fluorescent characteristics including broad emission
wavelength (em), tunable PL, excitation-dependent behavior and
sometimes, multiphoton excitation properties. Their diversiform PL
property has been a curiosity for many years. Several principles
have been proposed including the possibility that the PL emissions
of C-dots are generated by synergistic effects including a quantum
confinement effect [22], th e free zig zag sites with triplet carbene at
particle edges [23], spontaneous emission of excitons in surface
energy traps through passivation [24], and band gap alterations
triggered by charge transfer effects [25].
3. COMPARISON OF APPROACHES TO SYNTHESIZE C-
DOTs
3.1. Top-Down Approaches
To date, numerous methods have been developed to synthesize
C-dots and these can be divided into ‘top-down’ and ‘bottom-up’
routes (Fig. 1). In the top-down route C-dots (mostly GQDs) is
derived from the separation of large carbon precursors, and usually
prepared from carbonic materials including carbon nanotubes [20],
carbon fibers [26], graphite powers [27], carbon black [28], and
even candle or tire soot [29, 30]. These carbonic materials with an
sp2 carbon structure are abundant, but have an infinite Bohr diame-
ter and lack an effective bandgap to produce luminesce on excita-
tion [31]. Thus, breaking down these large carbon sources into
nano-scale particles is an essential step to endow them with PL
through quantum confinement effects [22]This can usually be
achieved via arc-discharge [20, 32], combustion [30], electrochemi-
cal, or laser ablation [24, 27, 33].
The first C-dots was prepred using a top-down method by oxi-
dizing arc-discharge soot with 3.3N HNO3, followed by extraction
of the sediment with NaOH solution, resulting in a black suspension
[20]. A carboxyl-group-functionalized GQD was finally obtained
by gel electrophoresis separation from the black suspension. These
GQDs showed size-dependent PL properties (green-blue, yellow,
and orange) when excited by 366 nm ultra-violet (UV) irradiation.
Multicolor emission with increased water solubility of GQDs can
be achieved by this method, but the limited output and the fact that
the PL QY is less than 2% are major obstacles for its practical ap-
plication.
In comparison with the arc-discharge method, electrochemical
synthesis is an effective and scalable approach using various car-
bonic materials as precursors to synthesize C-dots without employ-
ing strong acid and purification procedures. It was initially per-
formed by Zhou et al. in a degassed acetonitrile solution with 0.1 M
tetrabutylammonium perchlorate (TBAP) as the supporting electro-
lyte, and multi-walled carbon nanotubes (MWCNT) were used as
the working electrode in an electrochemical cell consisting of a Pt
wire counter electrode and an Ag/AgClO4 reference electrode [34].
The GQDs was produced uniformly by cycling the applied potential
with the color change of the electrolyte solution from colorless to
dark brown, which emitted blue light when irradiated by a UV
lamp. Other electrochemistry methods also involve fragmentation
of MWCNT or graphite rod electrodes to form GQDs by using an
electric field to peel off nanoribbons from the anode through elec-
trochemical exfoliation of layers of graphite. The supporting elec-
trolytes include TBAP [34, 35], ethanol [36], ionic liquid [37],
NaH2PO4 [33], NaOH [38], and PBS/water [42,43]. GQDs synthe-
sized by electrochemical strategies generally range from 110 nm
in size with blue/green/yellow PL emission reaching a PL QY of
14% [33, 38].
In contrast to the above described top-down methods, laser
ablation pioneered by Sun et al. produces C-dots with multiple
colors and surface passivation [24, 27, 39-42]. Sun et al. synthe-
sized C-dots through laser ablation of a carbon target in the pres-
ence of water vapor with argon gas at 900 °C and 75 kPa, followed
by refluxing in HNO3 for 12 h and passivating the surface via heat-
ing mixture of acid-treated C-dots and simple organic species such
as PEG1500N (amine-terminated polyethylene glycol) or PPEI-EI
(poly(propionyl ethyleneimine-coethyleneimine)) at 120°C for 72 h
[24]. Finally the as-prepared C-dots gave tunable luminescence
emission, which can be controlled by specific surface passivation
(Fig. 2).
Recently, Hu et al. demonstrated a simp le one-step laser abla-
tion procedure that integrated synthesis and passivation. In this
procedure, a pulsed Nd:YAG laser was used to irradiate graphite
powders dispersed in three kinds of solvents (diamine hydrate, di-
ethanolamine, and poly(ethylene glycol), i.e. PEG200N) for ultra-
sonication [27]. After 2 h laser irradiation, centrifugation was used
Fig. (1). Schematic illustration of top-down and bottom-up approaches for C-dot preparation.
Opportunities and Challenges of Fluorescent Carbon Dots Current Pharmaceutical Design, 2015, Vol. 21, No. 00 3
to separate the black carbon precipitate and a colorful supernatant
containing C-dots. The as-synthesized C-dots measured 18 nm,
and gave blue/green fluorescence with PL QY varying from 3% to
8% [27]. Li et al. reported a simpler procedure to produce C-dots
[40]. Unlike earlier studies, they used nano-carbon materials (<50
nm) as the starting material and a simple solvent (such as eth anol,
acetone, or water) as the liquid medium [40]. Typically, 0.02 g of
nano-carbon material was dispersed in 50 mL of solvent for ultra-
sonication [40]. Subsequently, 4 mL of the suspension was dropped
into a glass cell for laser irradiation, followed by centrifugation to
obtain C-dots in the supernatant. The C-dots showed typical
blue/green luminescence w ith excitation-dependent features.
In general, GQDs can be readily synthesized via top-down
methods. This is because most carbon-based starting materials such
as graphite, carbon fibers and carbon nanotubes possess high gra-
phitic crystallinity. During preparation, different cutting/passivation
strategies may cause surface energy levels to change, thus resulting
in a series of emissive traps. It is suggested that more effective sur-
face oxidation or modification involving synergistic interactions
between multi-chemical groups and the carbon backbone may lead
to red-shifted PL emission of C-dots [19]. Nevertheless, there are
few reports of near-infrared (NIR) fluorescent C-dots being synthe-
sized using top-down methods. The relatively low PL QY (up to
20%) of C-dots with blue/green emission and synthesized using
top-down methods is a critical shortcoming [24, 42].
3.2. Bottom-Up Approaches
The ‘bottom-up’ method primarily involves the dehydration,
polymerization and carbonization of non-conjugated small mole-
cules (e.g. citric acid, amino acids, carbohydrates etc.) to form the
GQDs and ACNDs through thermal/hydrothermal synthesis, mi-
crowave irradiation, and solution chemistry. Typically, their size,
optical properties and PL QY can be tuned by altering the reaction
conditions, such as the composition of reagents, ratios, reaction
time and temperature.
Thermal/hydrothermal synthesis is a highly effective and low-
cost method as it directly leads to the formation and surface pas-
sivation of C-dots, whose surface chemical properties can be pre-
cisely designed by careful selection of the carbon source, solvent
and passivating agents. This approach provides better control over
the morphology and optical properties of C-dots. The first prepara-
tion of C-dots using thermal/hydrothermal synthesis was reported
by Giannelis et al. [43]. In their synthesis, C-dots were prepared by
single-step thermal carbonization of low-temperature-melting mo-
lecular precursors to form surface-passivated C-dots that were ei-
ther hydrophilic or organophilic [43]. They used two differen t pro-
cedures, both yielding monodispersed C-dots with an average size
of less than 10 nm. In the first procedure, organic ammonium citrate
salts were thermally decomposed, w ith the citrate unit serving as
the carbon source, and the organic ammonium moieties playing the
role of the surface passivating agent [43]. Organophilic C-dots were
prepared by directly pyrolyzing octadecylammonium citrate in air
at 300°C for 2 h and washing with acetone and ethanol [43]. Hy-
drophilic C-dots were prepared by heating diethylene glycolammo-
nium citrate hydrothermally in a teflon-lined stainless steel auto-
clave at 300°C for 2 h and washing with acetone [43]. The second
procedure involved the pyrolysis of 4-aminoantipyrine (4AAP) in
air at 300°C for 2 h. After pyrolysis, the raw product was dissolved
in bis(trifluoromethyl)methanol and precipitated by the addition of
water [43]. The first procedure provided a nearly uniform size (~7
nm) of C-dots, while 4AAP-derived C-dots were more variable in
morphology with sizes ranging between 5 and 9 nm. X-ray powder
diffraction (XRD) results revealed that all types of C-dots consisted
of highly disordered carbon, which can be referred to as ACNDs.
These as-prepared C-dots exhibited a wide fluorescence em, ex-
tending into to the NIR range. In later research, many small organic
molecules and polymers (citric acid, amino acids, carbohydrates
and polyethylene amines etc.) were employed for C-dot synthesis
using thermal/hydrothermal methods. These molecular precursors
always contained -OH, -COOH, -C=O and -NH2 groups, which can
dehydrate at high temperatures. Although a variety of C-dot synthe-
sis techniques based on thermal/hydrothermal methods have been
reported, there are few reports for the preparation of C-dots with a
high QY and high-output. Dong et al. reported the synthesis of
strong blue fluorescent carbon quantum dots (CQDs) with 42.5% of
QY produced using low temperature carbonization of branched
polyethylenimine (BPEI) and citric acid (CA) [44]. Zhu et al. pre-
pared highly luminescent (QY = 20.9%80.6%) N-doped C-dots at
large scale by using the hydrothermal reaction with ethylenedia-
mine (EDA) and CA as starting materials [21]. More recently, and
based on previous work reported by Zhu et al., Qu et al. improved
Fig. (2). Aqueous solution of C-dots passivated with PEG1500N. a) Excitation wavelength at 400 nm and picture obtained through band-pass filters of different
wavelengths as indicated, and b) excited at the indicated wavelengths and photographed directly. (Reprinted with permission from Ref. [24]).
4 Current Pharmaceutical Design, 2015, Vol. 21, No. 00 Wang et al.
the QY of GQD to 94%, which is the highest reported to date, by
optimizing the reaction time, temperature and ratios between ED A
and CA [45].
Microwave irradiation of organic molecules is faster than regu-
lar thermal/hydrothermal methods for C-dot synthesis [45-48]. In
the study by Yang et al. poly(ethylene glycol) (PEG-200) pas-
sivated C-dots were synthesized by a simple microwave-
hydrothermal reaction from the mixture of PEG200 and a saccharide
in aqueous solution [48]. In a recent study, Gu et al. synthesized
green luminescent C-dots (PL QY, 2.4%) through one-minute sin-
gle-step microwave irradiation with sucrose as the carbon source
and diethylene glycol (DEG) as the reaction medium [46]. These
DEG-stabilized C-dots dispersed well in water and could be in-
gested by C6 glioma cells, suggesting their potential use for tumor
cell labelling. Another study reported by Wei et al. demonstrated
the fabrication of a series of multicolor N-doped C-dots with QY up
to 69.1% through a microwave-assisted Maillard reaction within 35
minute from natural amino acids and glucose [45]. The size and em
of these C-dots depended on the type of amino acid used. Blue fluo-
rescent C-dots (diameter, 2.88 nm) were prepared from tryptophan,
green fluorescent C-dots (diameter, 3.78 nm) from leucine, and
yellow fluorescent C-dots (diameter, 4.93 nm) from aspartic acid.
Interestingly, increasing the diameter of the C-dots can result in a
longer em.
Solution chemistry is another bottom-up synthetic strategy. It
involves oxidative condensation of aryl groups to form the C-dots
[22, 49-54]. Although it has been demonstrated that intramolecular
oxidative cyclodehydrogenation was effective for the synthesis of
GQDs from polyphenylene precursors, some GQDs may suffer
from poor solubility with increasing size [22], and exhibit a ten-
dency to aggregate due to strong intergraphene  interaction [50].
Li and Yan reported the synthesis of stabilized GQDs of uniform
and tunable size by covalent binding of multiple 2’,4’,6’-trialkyl-
substituted phenyl moieties and edges of the graphene [22]. Three
different-sized GQDs containing 168, 132 or 170 conjugated carbon
atoms were obtained. More recently, Liu et al. prepared multico lor
GQDs (PL QY, 3.8%) with a uniform size of ~60 nm diameter and
2–3 nm thickness using Hexa-peri-hexabenzocoronene (HBC) as
the precursor through the process of pyrolysis and exfoliation, fol-
lowed by refluxing with oligomeric PEG1500N and finally reduc-
tion with hydrazine [50]. It was noticed that the morphology of the
GQDs was influenced by the pyrolysis temperature.
3.3. The Future of C-Dot Fabrication
Although the morphology and surface chemistry of C-dots are
controllable during the preparation or post-modification phase in
both top-down and bottom-up methods, several considerations in C-
dot preparation need to be taken into account: 1) C-dot formation
processes involving thermal/hydrothermal and microwave carboni-
zation are usually unmanageable due to the rapid and harsh reaction
conditions, which result in irregular morphology with polydisper-
sity, though this may be improved by carefully designing the mo-
lecular precursors and reaction conditions [45]. 2) Even though the
solution chemistry method is a powerful approach to produce uni-
form GQDs and to study the origin of the PL mechanisms of fluo-
rescent GQDs, these GQDs provide an extremely low PL QY as
compared with C-dots fabricated using other bottom-up methods.
3), Most C-dots synthesized from top-down methods suffer from an
insufficient PL QY. 4) Size control is important to obtain uniform
properties and to carry out mechanistic studies. Typically, post-
fabrication purification is required to separate different sized C-
dots, usually by using centrifugation, dialysis, and electrophoresis.
However, these procedures can be incomplete, time-consuming or
largely reduce the final yield of products. 5) Surface chemistry
properties are critical for solubility and in-vivo applications, and
need to be well controlled during preparation or post-treatment. The
features of representative fluorescent C-dots fabricated using the
different methods and their optical properties are summarized in
Table 1.
4. OPTICAL IMAGING PERFORMANCE
Despite their structure diversity, C-dots possess some common
features for absorption and PL. C-dots generally show strong opti-
cal absorption in the UV region, with a d escending tail extending
into the visible range. C-dots without surface passivation, typically
those synthesized from electrooxidation [34], laser abla-
tion/irradiation [27], and top-down microwave methods [65], have
an absorption band within the range of 260–320 nm. The absorption
peak in this range is associated with the * transition of aromatic
sp2 domains in the carbon core [66]. While the absorption band of
C-dots lies between 350–550 nm after surface passivation [67], an
absorption shoulder at around 300 nm, corresponding to n–* tran-
sition of C=O bonds or other connected organic groups, is observed
[66]. Several attractive optical features of C-dots, both from funda-
mental and clinical application aspects, are their potential resistance
to photobleaching [45, 68], excitation wavelength (ex )-dependent
emission and broad em [24, 69]. In one recent study, the emission
of individual GQDs appeared to be free of the intermittency and
bleaching effects that commonly appear in traditional organic dyes
and semiconductor QDs [68]. The results indicate the superior po-
tential of C-dots compared with traditional organic dyes and semi-
conductor QDs as ultra-compact fluorescent probes. As mentioned
above, the em of C-dots is usually wide with a larger Stokes shift
than that of organic dyes, and the emission peak of C-dots can be
tuned by changing ex. It has been speculated that the wide tunable
emissions of surface-passivated C-dots may result from the broad
size distribution of C-dots, their variable surface chemistry, or dif-
ferent edge defects/emissive traps on the surface [19, 70, 71]. How-
ever, the exact mechanism for ex-dependent emission behavior still
remains to be established, and the way to achieve optimal synergis-
tic interaction between the carbon backbone and surface state so as
to enhance PL emission is currently unknown. The wide em, and
ex-dependent PL properties can be used for multi-color fluores-
cence im aging, and their em can be extended into the NIR region
by raising the concentration of C-dots [52, 72]. This accessibility of
C-dots is a primary requirement particularly for in vivo bioimaging
applications because far-red and NIR light propagates for several
millimeters into tissue. em in this range can thus minimize tissue
absorbance, scattering, autofluorescence and consequently optimize
image quality (Fig. 3) [73-76].
Fig. (3). Penetration depth of light into tissue according to its wavelength.
Opportunities and Challenges of Fluorescent Carbon Dots Current Pharmaceutical Design, 2015, Vol. 21, No. 00 5
Table 1. Representative fluorescent C-dots fabricated using different methods and their optical properties.
Classifi-
cation
Method Carbon
Source
Passivating
Agent
Reaction
Conditions
Size
ex Emission
Color
PL QY
(%)
Surface
State
Refs.
GQDs Arc-
Discharge
(TD)
Arc soot of
SWNTs
HNO3 Boiled for 48 h ~ 18 nm 366 nm Green-
blue,
yellow,
orange
1.6% COOH [20]
GQDs Laser irra-
diation
Graphite
powders
Diamine
hydrate, di-
ethanolamine,
PEG200N
Irradiate at 6.0
106 W cm-2
for 2 h
18 nm 350,
420 nm
Blue, green
38% –COOH,
OH,
C–O–C
[27]
GQDs Electrooxi-
dation
Graphite — Electro-
oxidized at 3 V
~ 1.93.2
nm
330,
370 nm
Blue,
yellow
1.2% C=O [33]
GQDs Electrooxi-
dation
Graphite —
Electrolyzed at
80–200 mA
cm-2
510 nm 340-410
nm
Green,
yellow
14% O=C–
NH–NH2
[38]
GQDs Thermal
oxidation
Citric acid
Sodium 11-
amino-
undecanoate
Heated at
300 °C in air
for 2 h
~ 1020
nm
340 nm Full color
3% COO-
Na+
[55]
GQDs
Chemical
oxidation
Carbon
Fibers
H2SO4, HNO3
Heated at
120 °C for 24 h
14 nm 318,
331,
429 nm
Blue, green
yellow
–COOH,
OH, C–
O–C,
C=O
[26]
GQDs
Chemical
vapor depo-
sition
Polycrys-
talline
copper
foils
Heated at
1000 °C for
4050 min
515,
3050
nm
320,
350,
380,
410 nm
Blue
C–O–C,
C=O
[56]
GQDs
Microwave
pyrolysis
Amino
acids
Glucose Two steps:
heated at
125 °C for 30
min and 275 °C
for 5 min
~ 2.25.1
nm
350,
430 nm
Blue, green
yellow
3069% COOH,
C(=O)N
HR
[45]
GQDs Hydrother-
mal synthe-
sis
Citric acid L-cysteine Heated at
200 °C for 3 h
~ 59 nm
345 nm Blue 73% CSC, –
NHR,
COO-
[57]
GQDs Hydrother-
mal synthe-
sis
Citric acid Ethylenedia-
mine (EDA)
Heated at
160 °C for 4 h
~ 2.3 nm 34042
0 nm
Blue 94%
C(=O)N
HR,
COO-
[45]
GQDs Hydrother-
mal synthe-
sis
Fructose Sulphuric acid
Heated at
170 °C for 4 h
~ 5.2 nm 30057
5 nm
Full color 7.1% CSC,
OH,
C=O
[58]
GQDs Solution
chemistry
3-iodo-4-
bromoani-
line
Oxidative
condensation at
80°C
132170
conju-
gated
carbon
atoms
390,
550,
605,
645 nm
Full color CH,
C=C
[52]
GQDs
Solution
chemistry
4-
Bro-
mobenzoic
acid
1,3,5-trialkyl
phenyl moie-
ties
Oxidative con-
densation at
different
temperature
168 con-
jugated
carbon
atoms
390,
595,
740 nm
Full color CH,
C=C
[53]
6 Current Pharmaceutical Design, 2015, Vol. 21, No. 00 Wang et al.
(Table 1) Contd….
Classifi-
cation
Method Carbon
Source
Passivating
Agent
Reaction
Conditions
Size
ex Emission
Color
PL QY
(%)
Surface
State
Refs.
GQDs
Solution
chemistry
Dipheny-
lacetylene,
tetraphen-
ylcy-
clopentadi-
enone,
triethynyl-
benzene
Heated at
170 °C for 18 h
~ 1.2, 1.9,
814 nm
32044nm
Blue, green
yellow
3.68% CH,
C=C
[54]
ACNDs Laser abla-
tion
Carbon
soot
PEG1500N Heated at
900 °C and 75
kPa; 120 °C for
72 h
~ 5 nm 400 nm Full color
410% OH, C–
O–C
[24]
ACNDs Candle Soot
HNO3 Refluxed for
12 h
Combustion < 2 nm 315 nm Blue,
yellow,
orange
0.81.9%
OH,
COOH
[30]
ACNDs Microwave
pyrolysis
Saccharide
PEG-200 Heated in 500
W microwave
oven for 2–10
min
~ 2.83.7
nm
330, 380
nm
Blue, green
3.13.6%
COOH
[48]
ACNDs Thermal
pyrolysis
Citric acid 1-
hexadecy-
lamine (HDA)
Heated at
300 °C under
argon flow in
octadecene for
5 min–3 h
~ 47
nm
340560
nm
Blue, green
yellow
53% COOH,
C(=O)N
HR
[59]
ACNDs Thermal
pyrolysis
Citric acid Ethanolamine
(EA)
Heated at 180,
230, 300,
400 °C for 30
min
~ 8, 19
nm
275600
nm
Full color
1550% COOH,
C(=O)N
HR, OH
[60]
ACNDs Thermal
pyrolysis
Citric acid Diethylene-
triamine
(DETA)
Heated at
170 °C for 30
min
~ 35.5
nm
340500
nm
Blue, green
88.6% C(=O)N
HR, OH
[61]
ACNDs Hydrother-
mal synthe-
sis
Citric acid Ethylenedia-
mine (EDA)
Heated at 150,
200, 250 and
300 oC for 5 h
~ 26
nm
300420
nm
Full color
20.980.
6%
COOH,
C(=O)N
HR
[21]
ACNDs Hydrother-
mal synthe-
sis
Sodium
citrate
Ammonium
bicarbonate
(NH4HCO3)
Heated at
180 °C for 4 h
~ 1.6
nm
340 nm Blue
68%
OH,
COO-
[62]
ACNDs
Low tem-
perature
self-
polymeriza-
tion
2-
azidoimi-
dazole
Heated at
50,70, 100 °C
for 24 h
~ 2.15.4
nm
300500
nm
Blue, cyan,
green
914% OH, –
NHR
[63]
ACNDs Hydrother-
mal pyroly-
sis
Citric acid
or ethylene
glycol
Ethylenedia-
mine end-
capped poly-
ethylenimine
PEI-EC
Heated at
180 °C until the
color turned to
orange.
~ 112
nm
240695
nm
Full color ~ 6.519%
–NHR,
OH, C–
O–C
[64]
The PL QY of C-dots is a key parameter for their potential bio-
imaging application. The PL QY varies and depends on the synthe-
sis methods and surface chemistry involved. For unpas-
sivated/undoped C-dots, PL QYs range between 2% and 22.9% for
Opportunities and Challenges of Fluorescent Carbon Dots Current Pharmaceutical Design, 2015, Vol. 21, No. 00 7
those prepared via stepwise solution chemistry and microwave-
assisted acidic oxidation, respectively [52, 77]. Since GQDs com-
monly contain carboxylic and epoxide groups, which can act as
non-radiative electron hole recombination centers, the removal of
these oxygen-containing groups by either reduction or surface pas-
sivation may improve PL QY [78]. Shen et al. prepared GQDs-PEG
with QY as high as 28%, which was twice that of GQDs (~13.1%)
[79]. This improvement probably originates from the stabilization
effect of excitons in the GQDs after passivation by PEG [70, 80].
Moreover, of PL QY can be dramatically enhanced by thermal or
hydrothermal approaches, by choosing a specific carbon source and
a doping/passivating agent [45].
In addition to Stokes shift type fluorescence, anti-Stokes type
PL is another useful optical property of C-dots, or namely upcon-
version or multi/two-photon PL [39, 69, 81, 82]. Anti-Stokes type
PL often refers to the non-linear sequential absorption of two or
more photons and leads to the emission of light at a shorter wave-
length than the excitation wavelength [83]. The upconversion PL of
C-dots has received increasing attention in recent years. In 2008,
Sun et al. reported that C-dots were strongly emissive in the visible
region with excitation via either argon-ion laser (458 nm) or femto-
second pulsed two-photon laser in the NIR (800 nm) range [39].
Also, in-vitro evidence showed that C-dots could be potentially
used for cell imaging with two-photon luminescence microscopy.
Liu et al. demonstrated that biocompatible nitrogen-doped GQDs
were efficient two-photon fluorescent probes for cellular and tissue
imaging [82]. They prepared N-GQDs by a one-pot solvothermal
approach using DMF as the solvent and nitrogen source. The N-
GQD exhibited a two-photon absorption cross section as high as
48000 Göppert-Mayer (GM) units (1 GM is 1050 cm4 s photon1).
The imaging penetration depth of 1800 μm achieved by N-GQD in
tissue phantom significantly extended the imaging depth limit of
two-photon microscopy (Fig. 4). It is anticipated that the upconver-
sion PL of C-dots will provide new opportunities for two-photon
luminescence imaging microscopy.
To conclude, in the current state of the art, the fact that C-dot
emission wavelength is related to tissue penetration is one of the
major weaknesses compared with QDs, fluorescent protein and
indocyanine green (Table 2). GQDs shows relativ ely lower PL
efficiency than ACNDs, and both C-dots exhibit high emission
peaks ranging from the blue to green region of the spectrum. In
addition, the PL QY of C-dots tends to fall in serum, and autofluo-
rescence generates background noise that interferes with imaging
quality.
5. C-DOT SURFACE FUNCTIONALIZATION AND NANO-
COMPOSITES
Surface modification is an effective method to tune the surface
properties of C-dots for selected applications. C-dots prepared from
CNTs by the arc-discharge method lead to oxygen-containing
groups at their surface [20]. Other methods include electrooxidation
from graphite [84] and hydrothermal carbonization from organic
molecules [44, 85-87], which showed similar results with surface
functionalization using carboxyl groups or amines. The presence of
these organic groups alters the physicochemical properties of C-
dots including their water solubility, biostability, functionality and
PL properties. Typically, the binding of amine-containing mole-
cules (fo r example, ethanolamine, PEG1500N), through the amide
linkages is used for the surface passivation of C-dots for biological
applications. As mentioned earlier, some single-step preparations of
C-dots allow the direct incorporation of surface functionality by
selecting appropriate starting materials [43, 44, 55, 85-87]. Chandra
et al. synthesized green-PL-colored C-dots by microwave irradia-
tion of sucrose with phosphoric acid [88]. Fluorescein, rhodamine B
and -naphthylamine were covalently functionalized onto the C-
dots via EDC condensation, with improved fluorescence and re-
duced cytotoxicity [88]. In another study, folic acid was conjugated
onto the C-dots, and such functionalization can be applied to target
cancer cells [89].
More recently, attention has been drawn to the preparation of
novel hybrids comprised of C-dots and inorganic nanoparticle cores
(e.g., iron oxide [90, 91], gold [90], silica [92], and titanium [36,
91]). With C-dots based on organic compounds, surface function-
alization, doping, or multifunctional nanocomposites for specific
biomedical applications should select appropriate non-toxic ele-
ments or molecules for the configuration of C-dots, as their toxicity
profile may influence the overall safety of C-dot nanocomplexes.
Fe3O4@C-CDs nanocomposites were synthesized by a one-pot
solvothermal method [91]. In this experiment, H2O2 is present as an
oxidizing agent in the reaction medium, thus abundant small-sized
fluorescent C-dots can be formed in situ in the porous carbon shell
from the oxidation and deep decomposition of the precursor fer-
Fig. (4). (a) Diagram showing the setup used for two-photon fluorescence imaging of N-GQDs (nitrogen doped GQDs) in tissue phantom with thickness rang-
ing between 0 and 1800 μm. (b, c and d) shows upconversion cell imaging under a bright field, 800 nm excitation and the overlay image (Reproduced with
permission from ref. [82]).
8 Current Pharmaceutical Design, 2015, Vol. 21, No. 00 Wang et al.
rocene. In addition to the magnetic responsive properties and MRI
ability (r*
2 = 674.4 mM1 s
1) of the Fe3O4 nanocrystal core, the
synthesized Fe3O4@C-CDs nanocomposites also exhibit attractive
optical properties from CDs with strong and upconversion fluores-
cence (PL QY, ~6.8%), excellent photostability, and a NIR pho-
tothermal effect [91]. These results demonstrated that Fe3O4@C-
CDs nanocomposites have combined the biocompatible iron oxide
nanoparticles with carbon materials, thus providing potential for use
in multimodality imaging and therapy. More recently, Zhou et a l.
developed multifunctional hybrid nanocomposites (Fe3O4@PC-
CDs-Au) that integrated magnetic Fe3O4 nanocrystals, fluorescent
C-dots and Au nanocrystals into a porous carbon matrix [90]. It was
obtained via the synthesis of core–shell structured Fe3O4@C-CDs
template nanoparticles, followed by loading and in situ reduction of
Ag+ ions, and a final replacement of Ag with Au nanocrystals via a
galvanic reaction [90]. in vitro evaluation results indicated that the
nanocomposites can enter intracellular regions and light-up mouse
melanoma B16F10 cells with laser scanning confocal microscopy
[90]. By taking the advantages of the combined photothermal ef-
fects of the carbon dots and the Au nanocrystals embedded in the
carbon matrix, the NPs can not only serve as efficient NIR pho-
tothermal therapeutic agents to kill cancer cells, but also control the
release rate of the loaded drug by NIR irradiation [90].
6. IN-VITRO AND IN-VIVO STUDIES
C-dots provide properties in photostability comparable to those
in traditional semiconductor QDs, but with much lower cytotoxicity
for long-term cell labeling and tracking. As an example, Xiong
et al. reported GQDs derived from single-walled carbon nanotubes
(SWNTs) which exhibited very low cytotoxicity towards HeLa
cells with the LC50 over 5 mg mL-1 for 24 h [93]. In comparison,
semiconductor QDs exhibit cytotoxic effects in much lower con-
centrations varying from 62.5 μg mL1 to 400 μg mL1 depending
on the type, size or biocompatible shell and the type of cell lines
tested [94]. In our recent studies, we prepared ACNDs by hydro-
thermal carbonization of citric acid and polyethylenic amine (PEA)
analogs (ethylen ediamine (EDA), diethylenetriamine (DETA) and
triethylenetetramine (TEPA)). These nitrogen-doped ACNDs dem-
onstrated low cytotoxicity and bright fluorescence in HeLa cells.
The resu lts of the cell viability test demonstrated that these ACNDs
did not exert any obvious cytotoxicity as cell viability was over
80% even at a relatively high concentration (800 μg mL1) of
ACNDs (Fig. 5). The confo cal fluorescence images of HeLa cells
incubated with three types of ACNDs at a concentration of 200 μg
mL1 and cell nucleuses stained with blue DAPI (4',6-diamidino-2-
phenylindole) revealed high-contrast fluorescent signals form mul-
ticolor ACNDs around each nucleus (Fig. 6). The results obtained
indicate that nitrogen-doped ACNDs can be used for high-contrast
multicolor cell labeling.
Fig. (5). In-vitro cytotoxicity testing results of three types of nitrogen doped
ACNDs with increased concentrations against HeLa cells from an MTT
assay. The cell viability steadily decreased to around 80% when concentra-
tions reached 800 μg mL1. (Reproduced with permission from ref. [95]).
As compared with fluorescence cell labeling, in-vivo fluores-
cence imaging is more challenging for C-dots. As we mentioned in
the sections above, a number of criteria for C-dots need to be con-
sidered, especially concerning the optical penetration in tissues and
safety profiles. Yang et al. reported in-vivo optical imaging using
C-dots [42]. In their experiment, PEGylated C-dots in an aqueous
solution were injected subcutaneously into mice, and the fluores-
cence images at different excitation wavelengths were obtained
with sufficient contrast in both green and red channel [42]. Moreo-
ver, they also performed sentinel lymph node imaging of ZnS salt
doped C-dots following intradermal injection. The observation
revealed that C-dot migration along the arm to the axillary lymph
node was slower than that of semiconductor QDs. One possible
reason could be the smaller sizes of the carbon dots (around 5 nm)
and/or the surface PEGylation [42]. The longer time taken by C-
dots to migrate to lymph nodes could result in a prolonged surgical
procedure if they are used in fluorescence-guided surgery (Fig. 7).
Tao et al. applied GQDs with the same protocol to nude mice
and obtained comparable results. In addition, they found the best
fluorescence contrast with 595 nm excitation [96]. Recently, we
studied three types of nitrogen-doped ACNDs for fluorescence
imaging in mice following subcutaneous injection. The fluores-
cence im aging of the mice was captu red with ex and em at 535 nm
and 695–770 nm, respectively. Our results demonstrated that NIR
fluorescence signals can be readily visualized following the injec-
tion of 50 μL of 0.05, 0.5 and 5 mg ACNDs into a nude mice (Fig.
8). The 5 mg dose of CD-EDA(2/1) showed the highest signal inten-
sity, above 1105. The ROI fluorescence signals of Both CD-
DETA(2/1) and CD-TEPA(2/1) show ed lower inten sities than that of
CD-EDA(2/1) at corresponding doses. It should be pointed out that
autofluorescence was the major problem and significantly de-
creased the signal-noise ratios when the concentration of ACNDs
was below 0.05 mg per 50 μL. Overall, the results suggested that
non-labeled N-doped ACNDs might potentially serve as optical
contrast agents for near tissue fluorescence imaging with a concen-
tration no less than 0.05 mg per 50 μL.
Yet another attractive biomedical application of C-dots is their
ability to serve as a nanocarrier for drug delivery (such as
siRNA/DNA and chemotherapy drugs) [18, 87, 97-99]. Sun et al.
demonstrated in-vivo that C-dots was able to deliver anticancer
drugs by using functionalized amino groups [87], which was syn-
thesized via amide bonding between oxidized oxaliplatin (Oxa
(IV)-COOH) and the surface group of C-dots. The in vivo results
demonstrated that the distribution of oxaliplatin can be tracked by
monitoring the fluorescence signal of CD-Oxa (C-dots-oxaliplatin
nanocomplexes) [87]. Additionally, it has been reported that C-dots
can be used as photodynamic therapy (PDT) agents by generating
reactive oxygen species including singlet oxygen under absorption
wavelengths, and kill cancer cells by inducing oxidative stress [17,
100].
7. TOXICITY PROFILE OF C-DOTS
A notable advantage of C-dots is their biocompatible elemental
composition. The in-vitro cytotoxicity of C-dots has been studied
by various research groups, which revealed that C-dots appear to
have low toxicity to numerous cell lines [33, 85, 88, 101-107].
Zhang et al. investigated th e cellular internalization, uptake mecha-
nism, and cytotoxicity of GQDs in human gastric cancer (MGC-
803) and breast cancer (MCF-7) cell lines [108]. The GQDs were
synthesized via the photo-Fenton reaction of graphene oxide (GO)
[109-111]. The cytotoxicity of GQDs was lower than that of gra-
phene oxide sheets, which was proven by cell viability, internal
cellular reactive oxygen species levels, damage to mitochondrial
membrane potential, and cell cycle. In addition, they also found that
the GQDs were internalized primarily through caveolae-mediated
endocytosis [108]. Wang et al. studied the cytotoxicity of C-dots
synthesized by various combinations of passivation molecules on
Opportunities and Challenges of Fluorescent Carbon Dots Current Pharmaceutical Design, 2015, Vol. 21, No. 00 9
C-dot precursors, and showed that the cytotoxicity of C-dots was
dependent on the selection of surface passivation molecules [112].
In another study, Chandraet et al. evaluated the cytotoxicity of C-
dots to healthy human blood cells by measuring the hemolysis rate
[88]. C-dots with carboxyl groups at the surface exhibited some
toxic effects, but the surface modifications by organic molecules
through the amide bonds resulted in a significant reduction of cyto-
toxicity [88].
The in vivo evaluation of C-dots has also been studied in recent
years. Sun et al. investigated in vivo safety and imaging optical
performance of PEG1500N surface functionalized C-dots in reference
to CdSe/ZnS QDs [113]. The quantification of C-dots in various
organs in dissected mice was investigated via isotope-ratio mass
spectroscopy, by using 13C-enriched PEGylated C-dots [113]. The
results indicated that these C-dots had low levels of accumulation in
the liver, spleen and kidneys according to the experimentally de-
termined 13C/12C isotope-ratios. Liver and kidney functions were
Fig. (6). Confocal fluorescence microscopy images of HeLa cells observed under bright field, blue, green, red, and merged channels. Cells were incubated
with 200 μg mL1 of different ACNDs (CD-EDA(2/1), CD-DETA(2/ 1) and CD-TEPA(2/1) ) in PBS buffer for 4 h and cell nuclei were stained with DAPI (4',6-
diamidino-2-phenylindole), see blue channel. Three types of ACNDs were prepared through hydrothermal carbonization of ethylenediamine
(EDA)/diethylenetriamine (DETA)/triethylenetetramine (TEPA) and citric acid (CA) using a fixed molar ratio, i.e. EDA/DETA/TEPA: CA = 2 : 1. (EX =
excitation wavelength, EM = emission wavelength of bandpass filter) (Reproduced with permission from ref. [95]).
Fig. (7). Intradermal injection of CZnS salt doped C-dots: (a) bright field, (b) as-detected fluorescence, and (c) color-coded images. Pictures inserted below
show the dissected (in the circled area) axillary lymph node (LN). (Reproduced with permission from ref. [42]).
10 Current Pharmaceutical Design, 2015, Vol. 21, No. 00 Wang et al.
also evaluated by serum biochemistry assays. Alanine amino trans-
ferase (ALT) and aspartate amino tran sferase (AST), uric acid
(UA), blood urea nitrogen (BUN), and creatinine (Cr) levels were
similar to those in control groups. Moreover, histopathological
examinations showed normal liver, spleen, and kidneys [113]. Fur-
ther toxicological studies according to regulatory guidelines will be
very useful.
Tao et al. investigated in vivo fluorescence imaging, biodis-
tribution and toxicity of carbon nanotube-derived C-dots (C-dots-
Ms) [96]. C-dots-Ms were radioactively labeled with 125I for phar-
macokinetic and biodistribution evaluations in mice. The blood
circulation of 125I-C-dots-Ms was fitted by a two-compartment
model, with first- and second-phase circulation half-lives of ap-
proximately 1 and 2 h, respectively [96]. The urine and feces of
mice were collected after injection, and high radioactivity was de-
tected in the urine and feces samples, suggesting that some of the
C-dots had possibly been eliminated through both renal and fecal
excretions. 125I-C-dots-Ms mainly accumulated in the reticuloendo-
thelial system (RES) organs, including the liver and spleen at post-
intravenous injection [96]. Systematic chemical analysis of the
blood over time, complete blood counts and histological studies
demonstrated the safety of C-dots in female Balb/c mice over 3
months following an injected dose of 20 mg kg-1 [96]. Collectively,
in-vivo evidence has shown that C-dots are mainly taken up by
reticuloendothelial system (RES) organs, such as liver and spleen,
or eliminated by the renal excretion pathway after intravenous in-
jection [42, 96, 114, 115].
Fig. (8). In-vivo fluorescence imaging of nude mice following subcutaneous injection of increasing doses CD-EDA(2/1), CD-DETA(2/1) and CD-TEPA(2/1) at
three injection sites (blue arrow). (ai, bi & ci) The true color fluorescent composite images of C-dot fluorescence and autofluorescence from mice. (aii, bii &
cii) unmixed true color images of C-dot fluorescence. (aiii, biii & ciii) Fluorescence intensities measured in ROIs (region of interest) (Reproduced with per-
mission from ref. [95]).
Opportunities and Challenges of Fluorescent Carbon Dots Current Pharmaceutical Design, 2015, Vol. 21, No. 00 11
Even though the currently available in vitro/in vivo toxicity data
shows an encouraging toxicity profile of C-dots, many safety con-
siderations still need to be addressed before they can be used in
clinical practice. For clinical biomedical imaging purposes, the best
delivery route is intravenous injection, as this can ensure the distri-
bution of C-dots throughout the body. The final hydrodynamic
diameter (HD) of C-dots in the bloodstream is thus a critical factor
for systemic clearance. Once the C-dots enter the bloodstream,
various plasma proteins could non-specifically bind the C-dots due
its non-neutral surface charges, which consequently in creases th e
final HD and obstructs the clearance pathway, since previous stud-
ies have shown that the major clearance of C-dots is through the
renal and fecal pathways [96]. Renal clearance is mediated by the
slit diaphragm of the glomerular basement membrane (GBM). The
physiological pore size of the slit diaphragm is ~ 5 nm in diameter
[116, 117]. The physicochemical properties of C-dots, including
HD, dispersity, shap e, flexibility, and surface charge will determine
whether the C-dots can be filtered by the GBM [118-120]. In par-
ticular, C-dots that have an overall HD size exceeding 8 nm after
interaction with plasma proteins, such as albumin, may never be
filtered at all [118]. This circumstance would result in much longer
exposure time to C-dots in the body. These C-dots would eventually
be metabolized in the liver and/or be taken up by the reticuloendo-
thelial system (RES) [121, 122]. In this regard, C-dots degenerated
by the liver into clearable components and excreted into the bile
and feces would be a desirable fate. However, C-dot uptake by the
RES, involving phagocytic cells, primarily monocytes and macro-
phages, located in the reticular connective tissue of the liver, spleen,
and bone marrow would be unclearable and result in long residence
times in the body. In addition, surface modification of C-dots using
biologically stable, hydrophilic, and neutral polymers, such as PEG
can increase half-lives in the blood and uptake by the RES. Nota-
bly, large chemically stable or non-biodegradable nanoparticles
could accumulate in these RES rich organs for long periods of time,
and potentially cause immunotoxicity, reproductive risks, and car-
cinogenic effects [123]. It was also reported that nanoparticles may
evade detection by the body's immune system, and under rare cir-
cumstances cross the brain blood barrier (BBB) [124]. Moreover,
even for C-dots that contain non-toxic elements, their heterogeneity
with regard to size, agglomeration ( interaction) and sample
purity is a major obstacle for standard evaluations. For clinical ap-
plications of C-dots, “Choi Criteria” were proposed as a guide for
the use of nanoparticles for clinical biomedical imaging. These
criteria include b iodegradability, minimal non-specific tissue uptak e
and smaller than 5 nm with renal clearance [13].
8. DESIGN CONSIDERATIONS FOR THE CLINICAL AP-
PLICATION OF C-DOTS
8.1 Sensitivity: Signal to Background Ratios
Like any other optical contrast agents, C-dots must have the
desired characteristics to be detected and visualized in target tis-
sues. Therefore, as discussed in previous sections, optical imaging
in the NIR window (700-900 nm) offers the greatest tissue penetra-
tion. Optical contrast agents for tissue imaging should be designed
so that ex and em are in the NIR window. NIR imaging can also
minimize light scattering in tissue and signal interference from
autofluorescence. In addition, to achieve a su fficient signal in target
tissue, the following optical properties in a physiological environ-
ment must be considered: light absorptivity of contrast agents, PL
QY, photostability, thermodynamic stability and fluorescence
quenching [73]. Unfortunately, given the current research status of
C-dots, only a few reports have shown success in preparing fluores-
cent C-dots that have em extended into the NIR range, and these
have a very limited PL QY [24, 64, 125].
8.2. Stability and Safety: Biophysicochemical Interaction at the
Nano-Physiological Interface
In designing contrast agents, one of the main considerations is
the stability of nanoparticles in vivo and in vit ro [12]. Biophysico-
chemical influences such as final HD, shape, surface area, surface
charge, hydrohphilicity/lipophilisity, surface functional groups/
Table 2. The physicochemical properties of C-dots versus QDs, fluorescent protein (FP) and indocyanine green (ICG).
Properties C-dots QDs FP ICG
Typical diameter 110 nm 510 nm ~ 7 nm ~ 1 nm
Final HD ( SF & NSPB ) 50 nm 50 nm 35 nm 20 nm
Stability in serum +/ +/ + +
Morphological homogeneity +/ + +
Structural flexibility +/ +/ +
em range/TP 400650 nm/ 4501200 nm/++ 470650 nm/+ 820 nm/++
Photostability + + +
PL QY +/ + +/
Potential toxicity (in vivo) +/ + +
SF = surface functionalization, NSPB = non-specific protein binding, HD = hydrodynamic diameter, em = emission wavelength, TP = Tissue penetration, PL QY = photolumines-
cence quantum yield, ‘+’ = positive, ‘’ = negative.
12 Current Pharmaceutical Design, 2015, Vol. 21, No. 00 Wang et al.
ligands are key factors to determine the interactions between
nanoparticles and biological systems, which consequently affect the
in vivo biocompatibility and toxicity of nanoparticles (Table 2)
[126]. Most nanoparticles consisted of multiple organic and/or in-
organic components, each of which has its own efficacy and toxi-
cology profile, which requires individualized investigations [127].
Problems may arise if the nanoparticles are not readily excreted,
biodegraded, or metabolized, or eliminated, and thus accumulate in
organs/tissues for long periods. Additional investigations in a pre-
clinical setting are required to clarify possible adverse outcomes. It
is also necessary to fully evaluate the consequences of long-term
retention of nanoparticles in vivo according to regulatory agency
guidelines. Designing nanoparticles for rapid clearance is the one
important prerequisite to avoid prolonged preclinical testing [13,
127]. A final HD less than 5.5 nm is the key criterion for the rapid
elimination of nanoparticles via renal filtration after intravenous
injection. In light of the “Choi Criteria” and current knowledge of
C-dots, the safety profile of nanoparticles needs to be characterized
before clinical translation. The profile will include morphology
control (such as size/final HD, shape and uniformity), surface
charge, solubility or dispersibility, biodegradability, oxidative stress
or free radical generation, interactions between nanoparticles and
serum protein, and short and long-term stability in various physio-
logical environments. Finally, we would suggest that the main con-
cerns for safety should focus on improvements in the homogeneity
of the size and shape of C-dots, as well as their surface chemistry,
because uneven surface chemistry may result in agglomeration or
aggregation through  interactions, ionic bonding, hydrogen-
bonding, and Van der Waals interactions between C-dots and C-
dots/biomolecules, which can result in unpredictable biological
fates. Moreover, product purity also needs to be addressed for stan-
dard evaluations and further applications.
8.3. Specificity: Passive and Active Targeting
The specificity of optical contrast agents is the ability to selec-
tively accumulate or attach to target organ/tissue sites so as to im-
prove imaging resolution by raising the signal to noise ratio [12].
Therefore, it is essential to design contrast agents with selective
binding to the target sites, a reasonable half-life in the blood, and
efficient elimination from the biological system before their appli-
cation in clinical practice. Active targeting often refers to the con-
jugation of contrast agents with targeting ligands (such as small
molecules, peptides, antibodies, and aptamers), which specifically
interact with molecular targets, including receptors and enzymes
[13, 128]. While passive targeting is associated with organ and
tumor-specificity, and is governed by the physiochemical properties
of contrast agents, or pathophysiological characteristics of irregular
tumor vessels (EPR effects). To develop efficient targeted contrast
agents, both the targeting ligands and physiochemical properties of
contrast agents should be carefully designed to balance target and
organ-specificity [129]. In the case of C-dots, some reports have
demonstrated that they can be conjugated with various ligands
mainly through the amide linkage for targeted imaging [18, 87,
130], or via surface passivation with neutral molecules for passive-
targeted imaging [29, 42, 131]. Because of the limited in-vivo evi-
dence of the efficacy of targeted imaging by C-dots, it is as yet
difficult to judge the sensitivity and specificity of C-dots for bio-
medical imaging.
9. CLINICAL PERSPECTIVES
The field of biomedical optical imaging has been developing
for more than two decades and it already achieved some success in
patient care, especially in image-guided surgery [132-134], which
potentially provides an effective way to protect normal tissues (such
as blood vessels and nerves) during surgical resection procedures
(such as tumors and lymph nodes). In contrast to visible light, NIR
fluorescence imaging has the advantage of penetrating relatively
deeply in to tissue to provide real-time, quantitative, and inexpen-
sive imaging with a high signal/noise ratio at depths < 1 cm [135-
137]. Since most biomolecules have minimal light absorption in the
NIR window, and the naked human eyes is unable to detect light in
the NIR range, a specific NIR contrast agent and specific imaging
systems are required to facilitate NIR imaging in the field of surgi-
cal. Now, several NIR fluorescence image-guided surgery systems
are commercially available. One organic-based NIR fluorescent
agent, indocyanine green (ICG), has been approved by the FDA and
European Medicines Agen cy (EMA ) for surgical guidance of lym-
phatic mapping [138-140], hepatic tumors, metastases [141], and
the fluorescence-aided endoscopic examination of superficial gas-
tric tumors [142, 143]. Methylene blue can be used as a fluores-
cence agent in the 700 nm range and has been applied clinically in
NIR fluorescence imaging [144-146]. Another small molecule, 5-
aminolevulinic acid (5-ALA) is a non-fluorescent prodrug which
induces the generation and accumulation of the fluorescent mole-
cule protoporphyrin IX (PpIX) in epithelial and neoplastic tissues
[147-149]. 5-ALA has been used for fluorescence cystoscopy in the
detection of bladder cancer and in the removal of cancerous tissue
from the bladder [150-152]. Although Methylene blue, ICG and 5-
ALA-induced PpIX have been assessed in some clinical p roof-of-
principle studies in several types of surgery, they exhibit some
shortcomings including poor photostability, non-specific uptake in
normal tissues and organs which limited their uses. QDs process
NIR absorption and emission properties with relatively high PL QY
and better photostability, which result in high signal intensity, thus
enabling long-term detection at lower concentrations compared
with organic fluorophores, but their toxicity is a serious concern. In
this regard, C-dots, given its good biocompatibility, may become a
potential alternative to QDs. To achieve this goal, the early in-
volvements of multi-disciplinary teams that combine chemists,
pharmacologists, toxicologists, clinicians, pharmacists, and regula-
tory authorities, as well as imaging equipment manufacturers, is
essential [153-156].
CONCLUSION
Given their organic and biocompatible nature, tunable PL, and
versatile surface functionalization, C-dots is an attractive lumines-
cent nanomaterial that may enable the development of a viable
optical imaging platform. A major limitation of C-dots is tuning the
PL into the NIR region and simultaneously achieving a high PL
QY. Long-wavelength excitation is essential for NIR fluorescence
imaging in order to obtain more effective tissue penetration and
simultaneously increase the resolution. Thus a systematic investiga-
tion of PL mechanisms for different types of C-dots is required.
Recent research in C-dots has been done in proof-of-concept ex-
periments, and uncovered various physicochemical properties of C-
dots. These properties are relevant for biological imaging [73, 153].
Although encouraging findings have been reported with regard to
the applications of C-dots, their exact m echanism of cellular uptake
and their long-term toxicological effects remain to be elucidated.
This is a challenge because the pharmacokinetics and biodistribu-
tion of C-dots depend on many factors such as their morphology,
physiochemical properties, surface chemistry and formulation. Fur-
ther research is likely to continue, which will be beneficial for th e
overall development for C-dots, which will eventually become a
more effective and less expensive alternative to conventional semi-
conductor QDs.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of
interest.
ACKNOWLEDGEMENTS
This study was funded by the Shenzhen municipal government
project No. JCYJ20130401164750006, and The Chinese University
of Hong Kong Direct Grant for Research 2014. No. 4054087. The
Opportunities and Challenges of Fluorescent Carbon Dots Current Pharmaceutical Design, 2015, Vol. 21, No. 00 13
authors thank Philip Bastable at University of Dijon School of
Medicine, France, for English Language editing.
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Received: July 13, 2015 Accepted: September 16, 2015
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