Biomaterials 53 (2015) 117e126

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Fluorescent porous carbon nanocapsules for two-photon imaging,

NIR/pH dual-responsive drug carrier, and photothermal therapy
Hui Wang a, Yubing Sun b, Jinhui Yi a, Jianping Fu b, Jing Di c,
Alejandra del Carmen Alonso c, Shuiqin Zhou a, *
a
b
c

Department of Chemistry of College of Staten Island and The Graduate Center, The City University of New York, Staten Island, NY 10314, USA
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
Department of Biology of College of Staten Island, The City University of New York, Staten Island, NY 10314, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 21 September 2014
Received in revised form
13 February 2015
Accepted 19 February 2015
Available online

An efcient nanomedical platform that can combine two-photon cell imaging, near infrared (NIR) light
and pH dual responsive drug delivery, and photothermal treatment was successfully developed based on
uorescent porous carbon-nanocapsules (FPC-NCs, size ~100 nm) with carbon dots (CDs) embedded in
the shell. The stable, excitation wavelength (lex)-tunable and upconverted uorescence from the CDs
embedded in the porous carbon shell enable the FPC-NCs to serve as an excellent confocal and twophoton imaging contrast agent under the excitation of laser with a broad range of wavelength from
ultraviolet (UV) light (405 nm) to NIR light (900 nm). The FPC-NCs demonstrate a very high loading
capacity (1335 mg g
1) toward doxorubicin drug beneted from the hollow cavity structure, porous
carbon shell, as well as the supramolecular p stacking and electrostatic interactions between the
doxorubicin molecules and carbon shell. In addition, a responsive release of doxorubicin from the FPCNCs can be activated by lowering the pH to acidic (from 7.4 to 5.0) due to the presence of pHsensitive carboxyl groups on the FPC-NCs and amino groups on doxorubicin molecules. Furthermore,
the FPC-NCs can absorb and effectively convert the NIR light to heat, thus, manifest the ability of NIRresponsive drug release and combined photothermal/chemo-therapy for high therapeutic efcacy.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Hollow nanostructure
Carbon dots
Two-photon imaging
Responsive drug release
Photothermal therapy

1. Introduction
The ability of a single nano-object to perform multiple functions
has gained signicant momentum in biomedicine areas because
practical applications often require detection, drug and photothermal treatments, and monitoring of therapeutic efcacy simultaneously [1e3]. For this purpose, carbon nanomaterials have been
recently explored because of their good biocompatibility, high
surface area, large pore volume for drug storage, and excellent
optical properties for imaging, and biocompatibility [4e9]. For
example, small sized carbon dots (CDs) with high photostability,
tunable emission spectra, and low toxicity have widely been used
as bioimaging agent and sensor [10,11]. However, the application of
CDs as drug carrier for chemotherapy is still limited due to their
solid nanostructure [12e17]. Hollow or mesoporous nanomaterial
is an ideal drug-delivery platform which cannot only immensely
* Corresponding author. Tel.: 1 718 982 3897; fax: 1 718 982 3910.
E-mail address: shuiqin.zhou@csi.cuny.edu (S. Zhou).
http://dx.doi.org/10.1016/j.biomaterials.2015.02.087
0142-9612/ 2015 Elsevier Ltd. All rights reserved.

enhance the drug loading capacity and transport drugs through the
cell membrane, but also efciently release the drug to kill tumor
cells [18e20]. Recently, hollow CDs (Diameter ~ 6.8 nm with cavity
hole ~ 2 nm) has been synthesized for both bioimaging and drug
delivery [21]. However, the small size and low surface areas of the
hollow CDs limited their loading capacity for DOX drug (only ~ 6 wt
%). In addition, the small sized hollow CDs can be easily cleared
through kidney excretion in the body, which limits their circulation
time [22]. It is known that large particle drug carriers tend to
accumulate in reticuloendothelial system and can be also cleared
quickly [22e24]. To improve the circulation times and targeting
ability, a worthwhile synthetic challenge is to develop a kind of
hollow CDs with suitable size and large capacity, which cannot only
be used as optical imaging agent, but also as a drug carrier for high
therapeutic efcacy.
Conventional chemotherapeutic drugs are nonspecically
combined with tumor cells and randomly distributed in the body
[25]. In order to improve therapeutic efcacy and reduce side
effects of drugs, a key attribute of drug carrier is their ability to

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H. Wang et al. / Biomaterials 53 (2015) 117e126

regulate the drug release in response to external stimulus [26e30].

Two ways can be applied to regulate the release of chemotherapeutic agents from the drug carrier: endogenous and exogenous
activation [31,32]. In general, the pathological processes in tumor
tissue and intracellular endosome/lysosome are accompanied with
local pH decrease by 1e2.5 pH units in comparison with that (pH
7.4) of blood and normal tissues [33,34]. Thus, endogenous activation based on pH-responsive drug delivery for cancer therapy is
possible to reduce the side effects of drugs to the human body. On
the other hand, the exogenous activation by employing external
stimuli may provide a complementary approach to regulate the
drug release. Near-infrared (NIR) light, with low energy absorption
and deep penetration for human tissue, is an excellent external
stimulus that can provide a great opportunity to release a drug at a
desirable site and time. NIR light has been considered crucial to
improve therapeutic efcacy in cancer treatment while minimizing
side effects [35e37]. Recently, drug carriers based on nanodiamond
complex or mesoporous carbon nanospheres that can effectively
regulate the release of the loaded drugs in response to a pH change
have been developed [28,29]. NIR-controlled drug release from the
polymer functionalized graphene oxides or single-walled carbon
nanotube has also been demonstrated [38e41].
In this manuscript, we develop uorescent porous carbon
nanocapsules (FPC-NCs) with uorescent CDs buried in the porous
carbon shell for a potential nanomedical platform that can combine
cell imaging, responsive drug delivery and NIR photothermal
therapy simultaneously. As shown in Fig. 1, the hollow structured
FPC-CNs as drug carriers can combine the functions from each
components. The CDs embedded in the shell with stable, excitation
wavelength (lex) tunable, and upconversion uorescence (a type of
uorescence excited in the higher wavelength region with emission
in the lower wavelength region) are designed for optical imaging
contrast agent in laser confocal and two-photon uorescence cell
imaging covering a broad excitation wavelength range from ultraviolet (UV) light to NIR light. The CDs can also effectively absorb and
convert the NIR light to heat for regulated drug release and photothermal treatment. The hollow cavity structure and porous carbon shell of the FPC-CNs are designed to provide high loading
capacity for drugs. Furthermore, the surface carboxyl and hydroxyl
groups and the p rings of the FPC-CNs can be used to interact with
specic functional groups of drug molecules to further improve the
drug loading capacity and trigger the drug release in response to an
environmental change [42]. With Doxorubicin (DOX) as a model
anti-cancer drug, the newly developed FPC-NCs have demonstrated
a very high loading capacity (1335 mg g
1) and a responsive DOX
release upon a pH decrease to acidic condition or a NIR light irradiation. The laser confocal and two-photon imaging studies using
the DU145 human prostate cancer cells as a model indicate that the

small sized FPC-NCs can overcome cellular barrier and enter the
intracellular region to light up the cells in multicolor forms with
excellent photostability. The drug-free FPC-NCs can also effectively
kill the DU145 human prostate cancer cells under NIR irradiation
due to their excellent photothermal conversion ability. Such
nanostructured FPC-NCs demonstrate a great promise toward the
advanced nanoplatform for simultaneous imaging diagnostics and
high therapeutic efcacy.
2. Materials and methods
2.1. Materials
All chemicals were purchased from Aldrich (Atlanta, GA, USA). Ferrocene
(Fe(C5H5)2, 98%), hydrogen peroxide (H2O2, 30%), acetone (C3H6O), ethanol
(C2H6O), HCl (37%), Doxorubicin hydrochloride, disodium hydrogen phosphate
(Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were used as received
without further purication. The water used in all experiments was of Millipore
Milli-Q grade.
2.2. Synthesis of magnetite@carbon coreeshell NPs
The coreeshell template NPs were synthesized using the reported method [43].
Briey, 0.10 g ferrocene dissolved in 30 mL acetone was intensively sonicated (EW08891-21 sonicator, Cole-Parmer) for 30 min at room temperature (~25  C). Afterward, 2.5 mL of 30% H2O2 solution was slowly added into the solution, which was
then vigorously stirred for 30 min with a magnetic stirring apparatus. The precursor
solution was then transferred to a 50.0 mL Teon-lined stainless autoclave. After
sealing it, the autoclave was heated to and maintained at 200  C to allow the reaction going for 48 h. The autoclave was then cooled naturally to room temperature.
After intense sonication for 15 min, the products from the Teon-lined stainless
autoclave were magnetized for 10 min using a 0.20 T neodymium rare earth magnet
with a size 5 cm  5 cm  2.5 cm (model N50, Amazon). The supernatant was
discarded under a magnetic eld. The obtained precipitate was then washed with
acetone three times to remove excess ferrocene. Finally, the black product was dried
at room temperature in a vacuum oven.
2.3. Synthesis of FPC-NCs
The FPC-NCs were typically synthesized by the in situ dissolution of the magnetic
core (Fe3O4) under the assistance of HCl from the magnetite@carbon coreeshell
template NPs. Briey, 0.01 g magnetite@carbon coreeshell NPs was added into 10 ml
deionized water. After intense sonication for 30 min to disperse the template NPs,
20 ml HCl solution (37%) was slowly added into the above solution and the sonication
was continued for 30 min. The precursor solution was transferred to a 50.0 mL
Teon-lined stainless autoclave. After sealing it, the autoclave was heated to and
maintained at 100  C for 24 h. The autoclave was then cooled naturally to room
temperature. The solution was centrifuged three times at 11,405 g force (30 min,
Thermo Electron Co. SORVALLRC-6 PLUS superspeed centrifuge) with the supernatant discarded and the precipitate redispersed into 30 mL deionized water. The
resultant FPC-NCs with a volume of 30 mL was further puried to remove the HCl by
3 days of dialysis (Spectra/Pormolecular porous membrane tubing, cutoff
12,000e14,000) against very frequently changed water at room temperature
(~22  C).
2.4. Drug loading into and release from the FPC-NCs
The drug loading was performed by adding 1.0 mg FPC-NCs into a phosphate
buffered saline (PBS) solution (10 ml, 0.005 M, pH 7.4) containing DOX (2 mg)

Fig. 1. Schematic illustration of multifunctional FPC-NCs. The uorescent CDs embedded in the porous carbon shell cannot only be used for confocal and two-photon imaging
contrast, but also convert the NIR light to heat effectively. In addition, the large hollow cavity and porous carbon shell can provide a high loading capacity for anti-cancer drug (DOX)
through the supramolecular p stacking, hydrogen bonding, and electrostatic interactions between DOX and FMC-NCs. Thus, the FPC-NCs can combine photothermal/chemotherapy
into a single nano-object to provide high therapeutic efcacy.

H. Wang et al. / Biomaterials 53 (2015) 117e126

under magnetic stirring at 37  C for 24 h. Then the mixture was centrifuged at
10,000 rpm for 30 min at 37  C. In order to remove unloaded DOX, the precipitate
was washed with the PBS solution of 0.005 M and pH 7.4 and then redispersed in
10 mL of the same PBS solution. The precipitating/washing purication process was
further repeated for ve times until the separated solution is clear. All the supernatant and washed solution was collected to determine the drug loading content.
The unloaded DOX in the collected solution was quantied by a UVeVis spectrophotometer at 480 nm. The DOX loading content in the FPC-NCs was calculated by
(M0
Mt)/MN  100%, where M0 and Mt are the total mass of DOX dissolved in the
initial solution and the mass of DOX remained in the supernatant solution,
respectively. MN is the mass of the FPC-NCs used in the loading process.
The in vitro NIR/pH-responsive release of DOX from the DOX-loaded FPC-NCs
was evaluated by a dialysis method. The puried DOX-loaded FPC-NCs were redispersed in 25 ml PBS solution (0.005 M, pH 7.4). To examine the NIR lightresponsive release, two dialysis bags lled with 5 mL DOX-loaded FPC-NCs were
respectively immersed into 50 mL 0.005 M PBS solutions of pH 7.4 at 37  C with
one of them exposed to a NIR lamp of an output power of 1.5 W/cm2 for 5 min. The
NIR light was provided using a Philips infrared reector lamp with a power density
of 1.5 W/cm2 and a lter to block the ultravioletevisible light. The wavelength
spectrum of the NIR lamp covers 780 nme3000 nm with a pronounced peak at
approximately 1000 nm. To examine the pH-responsive release, three dialysis bags
lled with 5 mL DOX-loaded FPC-NCs were respectively immersed into 50 mL
0.005 M buffer solutions with different pH (7.4, 6.2 and 5.0). The released DOX
outside of the dialysis bag was sampled at dened time period and assayed by
UVeVis spectrometry with the molar extinction coefcient of DOX being
10,400 M
1 cm
1 at 480 nm. Cumulative release is expressed as the total percentage
of drug released through the dialysis membrane over time.

119

clearance organs and cerebellum. Samples were submitted pathology assay 5 days
after intravenous administration of drug-free FPC-NCs (n 3) with a concentration
0.1 mg/mL and compared to both mice receiving no injection (n 3).
2.8. Characterization
Transmission electron microscopy images were obtained on a FEI TECNAI
transmission electron microscope at an accelerating voltage of 100 kV. Highresolution transmission electron microscopy images were aquired on JEM 2100
with an acceleration voltage of 200 kV. Energy-dispersive X-ray analysis was obtained with an EDAX detector installed on JEM 2100. The Raman spectrum was taken
on a LABRAM-HR Confocal Laser Micro-Raman spectrometer using an Ar laser
with 514.5 nm at room temperature. The FT-IR spectrum of the sample was recorded
with a Nicolet Instrument Co. MAGNA-IR 750 Fourier transform infrared spectrometer. The UVeVis absorption spectra were obtained on a Thermo Electron Co.
Helios b UVeVis Spectrometer. Nitrogen adsorptionedesorption measurements
were carried out on a Micromeritics ASAP 2020 instrument. The photoluminescence
spectra of the FPC-NCs aqueous dispersions were obtained on a JOBIN YVON Co.
FluoroMax-3 Spectrouorometer equipped with a Hamamatsu R928P photomultiplier tube, calibrated photodiode for excitation reference correction from 200
to 980 nm, and an integration time of 1 s. The photothermal experiments were
conducted using a Philips infrared reector lamp with a power density of 1.5 W/cm2
and a lter to block ultraviolet light.

3. Results and discussion

3.1. Composition and structure of FPC-NCs

2.5. Cell imaging ability of the drug-free FPC-NCs

DU145 human prostate cancer cells were employed to evaluate the two-photon
imaging function of the FPC-NCs that were endocytosed by the cells. The stock cells
are cultured using tissue culture asks and passaged every 4e5 days. DU145 cells
were tested mycoplasma free when obtained from ATCC. We have previously proven
the successful endocytosis of the magnetite@carbon coreeshell template NPs used
to synthesize the FPC-NCs, based on the top-down Z-scanning confocal uorescence
images of the cells incubated with the NPs [43]. Considering the same size and
surface properties of the FPC-NCs with their precursor coreeshell NPs, the FPC-NCs
should have the same endocytosis ability to enter the intracellular region. Briey,
DU145 human prostate cancer cells were seeded into collagen coated glass bottom
dishes (MatTek) at a density of 4000 cells/cm2. The cells were cultured for 24 h in
RPMI 1640 medium supplied with 10% fetal bovine serum (Life technology) before
incubating with the FPC-NCs. The FPC-NCs were suspended in cell culture media by
extensive ultrasonication for 30 min with a nal concentration of 0.5 mg/mL before
they were mixed into the culture medium containing cells. The cells were then
incubated with the drug-free FPC-NCs overnight (12 h) at 37  C before imaging. Livecell two-photon uorescent images were obtained using a Leica SP8_2-Photon/FLIM
confocal microscope equipped with an environmental chamber to maintain the
experimental environment at 37  C. The excitation wavelength is 900 nm for twophoton imaging. The laser confocal live-cell images were obtained using an
Olympus Revolution XD Spinning Disk confocal microscope equipped with an
environmental chamber to maintain the experimental environment at 37  C. Diode
lasers with wavelength of 405 nm, 488 nm and 561 nm were used for the excitation.
2.6. In vitro cytotoxicity of drug-free and DOX-loaded FPC-NCs with or without NIR
irradiation
In this study, DU145 human prostate cancer cells were cultured in the 96 wells
microplate (Corning) in 100 mL medium containing about 2000 cells seeded into
each wells. After an overnight incubation for attaching, the medium was removed
and another 100 mL medium containing the drug-free or DOX-loaded FPC-NCs was
added to make the nal exact concentration of 150 mg/mL, 100 mg/mL and 50 mg/mL,
respectively. Wells with the normal culture medium without FPC-NCs were used as
control. For photothermal treatments, the cells incubated with the FPC-NCs were
irradiated with 1.5 W/cm2 NIR light for 5 min. After being incubated for 24 h, 10 mL of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) solution
(5 mg/mL in PBS) is added into the wells and incubate in a humidied environment
of 5% CO2 and 37  C for 2 h. The medium was removed after 2 h and 100 mL of
dimethyl sulfoxide (DMSO) solution is added. The plates were gently agitated until
the formazan precipitate was dissolved, followed by measurement of optical density
(OD) value by spectrophotometer at 570 nm and 690 nm.
2.7. Histopathological evaluation
After an intracardial perfusion of buffered 10% formalin, whole organs (liver,
kidney, spleen, and cerebellum) of C57BL/6 mice were removed through necropsy
and post xed in the same xative for 48 h and embedded in parafn processed for
histology, sliced into 5 mm sections, and stained with hematoxylin and eosin (H&E)
according to standard clinical pathology protocols. A veterinary pathologist was
then consulted to evaluate if any signs of acute toxicity were present in these

The magnetite@carbon coreeshell nanoparticles (NPs)

(Supplementary Information Fig. S1) were synthesized following
the reported procedure [43]. The hollow structured FPC-NCs were
then synthesized by dissolving the magnetic (Fe3O4) core of the
template NPs. The size and size distributions, in terms of the hydrodynamic diameters (Dh) obtained from photon correlation
spectroscopy measurements, of the coreeshell template NPs and
the resulted hollow FPC-NCs were compared (see Fig. S2). Both the
average Dh and the polydispersity increased slightly after the
dissolution of the Fe3O4 nanocrystals embedded in the core area,
which may indicate some shell disruption and slight degree of
interparticle connection during the core dissolution process. Fig. 2a
and b shows the typical transmission electron microscopy (TEM)
images of the as-synthesized FPC-NCs, which obviously demonstrate a hollow morphology with an average outer diameter of
~100 nm. The clear diffraction rings from the selected-area electron
diffraction (SAED) pattern (Fig. 2c) of a single FPC-NC indicate the
polycrystalline nature of the as-obtained FPC-NCs. The crystallinity
was conrmed by the Raman spectrum of the FPC-NCs (Fig. S3). The
peak at 1588 cm
1 (G-band) is associated with the E2g mode of the
graphite and is related to the vibration of sp2-hybridized carbon
atoms in a two-dimensional hexagonal lattice [44]. The D band
around 1358 cm
1 is ascribed to the vibrations of carbon atoms
with dangling bonds in the termination plane of disordered
graphite or glassy carbon [45]. The low intensity ratio of D-band to
G-band (ID/IG) suggests that the FPC-NCs exhibit an appreciable
degree of graphitization. Fig. S4 shows the typical high-resolution
TEM image of a sectional carbon shell of the FPC-NCs. Some
nanocrystals with a size range about 3e8 nm embedded in the
amorphous carbon shell were clearly observed, indicating the existence of uorescent CDs. The 2D lattice fringes of the small
nanocrystals demonstrate an interplanar distance about 0.327 nm,
which corresponds to the (002) lattice planes of graphitic (sp2)
carbon [46,47]. The energy dispersive spectrum (EDS) of the single
FPC-NCs (Fig. 2d) shows the existence of C, O and Cu elements.
While the Cu is from the copper grid for TEM sample preparation,
the C should be attributed to the carbon shell of FPC-NC and the
carbon lm coated on copper grid. The O should be from the surface
carboxyl/hydroxyl groups of the FPC-NCs (see FT-IR spectrum in
Fig. 3a and analysis). Combining all these analytical results, it is
reasonable to conclude that the as-synthesized FPC-NCs are

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H. Wang et al. / Biomaterials 53 (2015) 117e126

Fig. 2. (a) Low and (b) high magnied TEM images of FPC-NCs. (c) The SAED pattern and (d) EDS of single FPC-NC with elements of C, O and Cu being identied at different energy
level. Cu is from the copper grid for TEM sample preparation. The C is from the carbon shell of FPC-NC and the carbon lm coated on copper grid. The O should be from the surface
carboxyl/hydroxyl groups of the FPC-NCs.

composed of hollow cavity in the center and carbon shell

embedded with uorescent CDs.
3.2. Optical properties of FPC-NCs
Fig. 3a shows the FT-IR spectrum of the FPC-NCs. An obvious
absorption peak of the eOH group at 3409 cm
1 and a C]O
stretching mode at 1709 cm
1 of the carboxylic acid groups conjugated with condensed aromatic carbons were observed, respectively [48]. The peak at 2928 cm
1 should come from the stretching
vibration of CeH in the products. The peak around 1626 cm
1 could
be attributed to the C]C stretch of the aromatic carbon systems of
the FPC-NCs. The presence of the surface carboxyl and hydroxyl
groups of the porous carbon shell endows the FPC-NCs excellent
dispersibility and stability in aqueous media (Fig. S5), which is
critical for biomedical applications. The UVeVis absorption spectrum of the as-obtained FPC-NCs (Fig. 3b) shows a sharp absorption
at about 240 nm, which is ascribed to the pep* transition of aromatic domains in the porous carbon shell [49,50]. Fig. 3b also shows
the excitation spectrum of the FPC-NCs obtained under the selection of an emission wavelength at 620 nm and an excitation
wavelength range of 250e600 nm, which shows an obvious peak at
468 nm. When the aqueous dispersion of the as-obtained FPC-NCs
was exposed to a UV lamp with a light wavelength of 365 nm, green
light was emitted and easily observed (Fig. S5). Almost no color
change and bleaching of the sample were observed after a
continuous exposure to the UV light of 365 nm for 24 h, indicating
that the FPC-NCs have excellent photostability.
To further explore the optical properties of the FPC-NCs, a
detailed PL study was carried out under different excitation
wavelengths (lex). As shown in Fig. 3c, when the lex increases from
240 nm to 560 nm, the emission peak gradually red-shifts to longer
wavelengths and the PL intensity gradually increases and then

decreases, which indicate a distribution of the different surface

energy traps of the CDs [51]. The maximum PL emission located at
520 nm was obtained with lex 460 nm. The PL quantum yield of
the FPC-NCs was determined to be 8.8% by using rhodamine B as a
standard. To further evaluate the photostability of the FPC-NCs, the
time dependence of the maximum PL intensity of the FPC-NCs at
520 nm obtained with the excitation wavelengths of 460 nm was
quantied. As shown in Fig. S6, only a slight change (a decrease
~4.6%) of the PL intensity was observed after 2 h continuous
exposure of the sample solution to the excitation light of
lex 460 nm in a uorospectrometer. More importantly, the FPCNCs exhibit excellent upconversion PL properties besides their
strong luminescence in visible-to-NIR range. Fig. 3d shows the PL
spectra of the FPC-NCs excited by long-wavelength light from 980
to 660 nm, which clearly demonstrate upconverted emissions from
545 to 500 nm. The upconversion PL property of the FPC-NCs can be
attributed to the multiphoton active process similar to that of the
previously reported CDs [52]. These results indicate that FPC-NCs
not only demonstrate lex tunable emissions with excellent photostability against light illumination, but also exhibit upconverted PL
properties.
3.3. Cell internalization and optical imaging ability of drug-free
FPC-NCs
After conrming the strong uorescence and tunable emission
properties (Fig. 3c) of the FPC-NCs, DU145 human prostate cancer
cells were selected as a model to evaluate the optical cellular imaging function of the FPC-NCs. Fig. 4aec shows the laser scanning
confocal images of the DU145 cells incubated with the FPC-NCs
under the irradiation of the laser with different wavelengths of
405 nm (a), 488 nm (b), and 561 nm (c), respectively. Obviously, the
FPC-NCs with uorescent CDs embedded in the carbon shell

H. Wang et al. / Biomaterials 53 (2015) 117e126

121

Absorbance
Excitation

%Transmittance

Absorbance (a.u.)

240 nm

468 nm

3409
1709

c 10.0M

PL Intensity

8.0M
6.0M
4.0M

3500

240
260
280
300
320
340
360
380
400
420

3000 2500 2000

Wavelength (nm)

1500

200

1000

440
460
480
500
520
540
560

600

650

600

660 nm
700 nm
740 nm
780 nm
820 nm
860 nm
900 nm
940 nm
980 nm

200k

0.0
450
500
550
wavelength (nm)

500

300k

100k

400

400
Wavelength (nm)

500k

2.0M

350

300

400k

PL Intensity

4000

1626

400

450

500
550
600
650
wavelength (nm)

700

750

Fig. 3. (a) Typical FT-IR spectrum (b) UVeVis absorption and excitation spectra of the FPC-NCs; (c) Photoluminescence (PL) and (d) Upconverted PL spectra of the FPC-NCs obtained
with different excitation wavelengths.

produced bright uorescence that can light up the DU145 cells in

multicolor forms, which possibly originates from the different sizes
of the uorescent CDs embedded in the carbon shell. It should be
mentioned that the confocal images of DU145 cells did not show
uorescent signal change after a continuous irradiation with the
excitation laser at 488 nm for 30 min (Fig. S7), which further indicates that the FPC-NCs have excellent photostability and can be
used for long-term cellular imaging. This stable multicolor uorescent imaging ability of the FPC-NCs provides a great potential for
multicolor bioimaging applications.
Two-photon uorescence microscopy has attracted much
attention because of its application in direct observation of cellular
structure and biological process with the advantages of deep
penetration in biological tissues, low photobleaching and weak
autouorescence since its invention by Webb et al., in 1990
[53e56]. Given the obvious upconverted PL properties of the FPCNCs (Fig. 3d), we expect that the FPC-NCs should have twophoton uorescence imaging ability. The average s value (its
large two-photon absorption cross-section) of the FPC-NCs at
900 nm is estimated to be z36,000 GM (GoepperteMayer unit,
with 1 GM 10
50 cm4 s photon
1) by comparing the two-photon
luminescence intensities of the FPC-NCs and a reference under the
same experimental conditions [52]. In addition to the similar s
value with other high-performance two-photon luminescent
quantum materials, the carbon-based FPC-NCs should exhibit

lower cytotoxicity [57]. After being taken up by the DU145 human

prostate cancer cells, the FPC-NCs demonstrate strong two-photon
uorescence (Fig. 4e) upon excitation by femtosecond infrared laser
pulses of 900 nm. These results imply that the FPC-NCs could serve
as a transmembrane drug delivery carrier to release the drug
molecules inside the tumor cells for effective chemotherapy at tumor site.
3.4. Porous property and drug loading of FPC-NCs
Full nitrogen sorption isotherm was measured to obtain information about the specic surface area and pore sizes of FPC-NCs.
Fig. 5a shows typical N2 sorptionedesorption isotherm of the
FPC-NCs. The determined BrunauereEmmetteTeller (BET) surface
area and total pore volume of the FPC-NCs are 239.5 m2 g
1 and
0.63 cm3 g
1, respectively, which are obviously larger than those of
the corresponding magnetite@carbon coreeshell NPs (94 m2 g
1
and 0.29 cm3 g
1) [43]. The signicant increase in the total pore
volume and surface area per unit mass of the FPC-NCs should result
from the dissolution of the magnetite Fe3O4 nanocrystal clusters in
the core zone, which not only signicantly increase the number of
pores (originally occupied by the Fe3O4 nanocrystals) in the core
zone, but also reduce the density of the materials (only porous
carbon with no Fe3O4 in the FPC-NCs). The porosity of the carbon
shell of FPC-NCs are also demonstrated in the HRTEM image

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H. Wang et al. / Biomaterials 53 (2015) 117e126

Fig. 4. Laser scanning confocal microscopy images of DU145 human prostate cancer cells incubated with drug-free FPC-NCs at a nal concentration of 0.5 mg/mL in the cell culture
medium for 12 h at 37  C, obtained under different lex of (a) 405 nm; (b) 488 nm; (c) 561 nm, respectively. (d) Transmission, (e) two-photon uorescence, and (f) overlaid images of
DU145 human prostate cancer cells incubated with the FPC-NCs. Excitation laser wavelength is 900 nm.

(Fig. S4a). The hollow cavity, porous carbon shell and quite large
surface area of the FPC-NCs are suitable for drug carrier application.
Here, DOX was selected as a model to test the drug loading capacity
of the FPC-NCs by simply mixing the FPC-NCs into the phosphate
buffered saline (PBS) solution of DOX. Result shows that the DOX
molecules can be readily loaded into the FPC-NCs and the drug
loading capacity of the FPC-NCs for DOX reaches as high as
1335 mg g
1. The appearance of the characteristic UVeVis absorption peak of the DOX at around 480 nm (Fig. 5b) conrms the
successful loading of the DOX molecules into the FPC-NCs, because
the peak shape and positions of the absorption spectrum from the
DOX-loaded FPC-NCs are nearly the same as those from free DOX in
the higher wavelength region (>480 nm). At the wavelengths
below 480 nm, the absorption spectrum of the DOX-loaded FPCNCs demonstrates the additive effect from the absorption of drugfree FPC-NCs and DOX molecules due to the signicantly high
absorption intensity of the drug-free FPC-NCs. The high drug
loading capacity of the FPC-NCs should be attributed to two factors.
First, the DOX molecules can associate with the drug host FPC-NCs
through several types of interactions, including the supramolecular
p-stacking between the conjugated rings of DOX molecules and the
aromatic rings in the carbon shell of FPC-NCs, the hydrogen
bonding between the hydroxyl/amine groups of DOX and the surface hydroxyl/carboxyl groups of the FPC-NCs, and the electrostatic
attractions between the protonated amine groups of DOX and the
dissociated surface carboxylate groups of the FPC-NCs [42,58].
Second, the unique structure with large central hollow cavity and
pores in carbon shell of the FPC-NCs provide plenty space for drug
storage. Thus, the DOX drug molecules diffusing into the FPC-NCs
can be retained and stored in the FPC-NC carriers. It should be
mentioned that the average hydrodynamic diameter of the FPC-NCs
only increases very slightly from 176 nm to 186 nm after loading the
DOX molecules for 15 h, which indicates no signicant aggregation

was detectable within experimental errors. The polydispersity of

the FPC-NCs also has no obvious change after loading drug (Fig. S2).
3.5. pH and NIR responsive drug release of FPC-NCs
The release behavior of DOX from the FPC-NCs was investigated
using a dialysis membrane against PBS solution. It has been reported that the pH value of releasing medium can trigger the
releasing rate of DOX from different polymeric NP drug carriers
containing carboxylic acid moieties [59e61]. Considering the pHsensitive surface carboxylic acid moieties in the FPC-NCs carrier
and amine moieties on the DOX molecules, we expect that the
release of DOX from the FPC-NCs will have similar pH-responsive
behavior. Fig. 6 shows the DOX releasing kinetics from the FPCNCs dispersed in buffer solutions of different pH values (5.0, 6.2
and 7.4, respectively) at 37  C. At a physiological pH 7.4, only
38.1% DOX was released from the FPC-NCs after 120 h. In contrast,
53.2% and 68.1% of DOX were respectively released at pH 6.2 and 5.0
after 120 h, which means that the DOX can be released at a much
faster rate from the FPC-NCs in acidic medium. This pH-responsive
drug releasing behavior from the FPC-NCs is very important
because the microenvironments of extracellular tissues of tumors
and intracellular lysosomes and endosomes are acidic, potentially
facilitating active drug release from the drug carriers [33,34]. The
faster releasing rate at pH 6.2 and 5.0 than at pH 7.4 is likely
due to the following reason. The DOX is a weak amphipathic base
with pKa 8.3 [60]. At pH 7.4, part of the DOX molecules are
protonated with positive charge and the surface carboxylic acid
groups anchored on the FPC-NCs are nearly completely dissociated
to carboxylate with negative charges. Thus, the DOX drug molecules have strong electrostatic interactions with the carrier FPCNCs. At pH 5.0, the carboxylic acid moieties on the FPC-NCs
will be much less dissociated. The less negative charge of the

H. Wang et al. / Biomaterials 53 (2015) 117e126

500
Surface area: 239.54 cm2/g

400
3

300

Pore volume:0.63 cm /g

200
100
0
0.0

0.2
0.4
0.6
0.8
Relative Pressure (P/Po)

1.0

Absorbance (a.u.)

FPC-NCs
FPC-NCs+DOX
DOX

further understand the drug release process, the drug release rates
in Fig. 6 were also expressed as the square root time dependence
(Fig. S8). Fig. S8 shows that the drug release rate at initial stage
strongly depends on the pH, with higher release rate observed at
lower pH value. After about 16 h, the drug release rate linearly
depends on the square root of time. More interestingly, the three
linear lines obtained at different pH values of 5.4, 6.2, and 7.4 exhibits similar slope, which indicates that the DOX release is diffusion controlled process with no pH dependence after 16 h. All these
results suggest that the DOX release can be described as two-step
process with fast pH-controlled desorption of DOX followed by
slow but steady diffusion through the porous structure of capsules.
It should be mentioned that the UVeVis absorption spectrum of the
released DOX solution (Fig. S9) is almost the same as that of the
original DOX solution (Fig. 5b) based on the peak positions and
shape, indicating no chemical change occurred on the DOX molecules during the loading and releasing process.
Fig. 7a shows the photothermal effect of aqueous dispersion of
the FPC-NCs with different concentrations. When exposed to a NIR
light with a power density of 1.5 W/cm2 for 5 min, the aqueous
dispersions of the FPC-NCs demonstrated a signicant temperature
increase by 35, 25 and 16  C for concentration of 150, 100 and
50 mg/L, respectively. In contrast, the temperature change of water

55
50

Temperature ( C)

Quantity Adsorbed (cm3/g)

400

440

480
520
Wavelength (nm)

560

600

Fig. 5. (a) N2 adsorption and desorption isotherm of the FPC-NCs. (b) Typical
UVevisible absorption spectra of the free DOX, drug-free FPC-NCs, and DOX-loaded
FPC-NCs.

123

water
50
100
150

45
40
35
30
25
20

100

15

7.4

50

100

6.2

80

5.0

150
200
Time (S)

250

300

b 100

60

0 h 10 h

40 h

76 h

80
Drug release (%)

Drug release (%)

40
20
0
0

20

40

60
Time (h)

80

100

120

Fig. 6. The pH-dependent release of DOX from the DOX-loaded FPC-NCs dispersed in
buffer solutions of different pH values (7.4, 6.2 or 5) at 37  C. Error bars indicate the
standard error of the mean for N 3 independent experiments.

FPC-NCs will weaken the drugecarrier electrostatic interaction.

Meanwhile, the complete protonation of amine groups on DOX
molecules will signicantly increase the hydrophilicity or solubility
of DOX molecules in aqueous media. Thus, DOX molecules can be
released from the FPC-NCs more easily at pH 5.0 than pH 7.4. To

60
40
Without NIR
With NIR

20
0
0

20

40

60
80
Time (h)

100

120

Fig. 7. (a) The photothermal curves of water (control) and aqueous dispersions of the
FPC-NCs with different concentration of 50, 100 and 150 mg/L under NIR irradiation for
5 min, respectively. (b) Releasing proles of DOX from the DOX-loaded FPC-NCs in PBS
of pH 7.4 at 37  C, irradiated with/without 1.5 W/cm2 NIR light for 5 min at the
indicated time points. Error bars indicate the standard error of the mean for N 3
independent experiments.

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H. Wang et al. / Biomaterials 53 (2015) 117e126

(control) was much less signicant (~5.0  C) under the same NIR
light irradiation condition. This result indicates that the FPC-NCs
possess excellent photothermal conversion ability in response to
a NIR light irradiation. Fig. 7b compares the DOX releasing kinetics
from the FPC-NCs dispersed in PBS solution of pH 7.4 at 37  C, in
the absence and presence of 5 min NIR light irradiation at certain
releasing time points of 0, 10 h, 40 h, 76 h, respectively. Without NIR
radiation, the release of DOX from the FPC-NCs is slow with only
38.1% of loaded DOX released after 120 h. In contrast, the 5 min
irradiation of NIR light at different time points of 0, 10 h, 40 h, 76 h
signicantly speeds up the release of DOX from the FPC-NCs with
about 68% of loaded DOX released after 120 h. Such a signicantly
enhanced DOX releasing rate is clearly induced by the NIR light
irradiation. The releasing curve also indicates that the DOX release
could return to its slow non-NIR light-assisted releasing rate when
the NIR light was turned off. The enhanced DOX releasing rate upon
NIR light irradiation can be attributed to the local heat produced by
the efcient photothermal conversion of the uorescent CDs
embedded in the carbon shell, which could weaken the
drugecarrier interactions of DOX with the surface carboxyl/hydroxyl groups of the porous carbon shell and increase the mobility
of DOX at elevated temperatures. When the NIR irradiation was
turned off, the CDs in the FPC-NCs could not produce local heat,
thus the DOX release returned to its slow rate. It is expected that the
combination of high drug loading capacity and NIR-responsive drug
releasing behavior of the FPC-NCs will provide high therapeutic
efcacy. While the FPC-NCs can act as a regular drug carrier for
basal chemotherapy, they can also offer fast-acting dosage under
NIR photo-activation when necessary.
3.6. Cell viability of in vitro chemo, photothermal, and chemophotothermal treatments
Fig. 8 shows the in vitro cytotoxicity of the drug-free and DOXloaded FPC-NCs against DU145 cells at different concentrations,
without and with the 5 min treatment of 1.5 W/cm2 NIR light,
respectively. The DU145 cells were incubated with the drug-free
FPC-NCs or DOX-loaded FPC-NCs for 24 h with the 5 min NIR
irradiation introduced when the incubation time reached 12 h.
Compared to the medium control, the drug-free FPC-NCs demonstrate very similar cell viability, indicating that the FPC-NCs as drug
carrier are non-toxic to DU145 cells after 24 h treatment in a

Cell Viability (%)

100
Control
FPC-NCs
FPC-NCs+DOX
FPC-NCs+NIR
FPC-NCs+DOX+NIR

80
60
40
20
0
50

100
Concentration (ug/mL)

150

Fig. 8. In vitro cytotoxicity of drug-free and DOX-loaded FPC-NCs at different concentrations without/with 1.5 W/cm2 NIR irradiation for 5 min, respectively. The DU145
cells were incubated with the drug-free FPC-NCs or DOX-loaded FPC-NCs for 24 h with
the 5 min NIR irradiation introduced when incubation time reaches 12 h. Error bars
indicate the standard error of the mean for N 3 independent experiments.

concentration up to 150 mg/mL. Meanwile, the results from the

in vivo experiments by injecting the drug-free FPC-NCs solution
(0.1 mg/mL) into the mice body show that no appreciable abnormalities were observed in the cell types analyzed (Fig. S10). The
results suggest that the drug-free FPC-NCs were well-tolerated at
the doses evaluated in this study and no evidence of toxicity was
observed in any of the clearance organs. The non-toxicity of the
FPC-NCs is a critical characteristic for future biological applications.
In contrast, the cell viability dramatically decreased when the cells
were incubated with the DOX-loaded FPC-NCs under the same
conditions, indicate that the FPC-NCs can successfully release DOX
drug to kill cancer cells for chemotherapy. To investigate the photothermal effect, the DU145 cells being incubated with the drugfree and DOX-loaded FPC-NCs were irradiated with 1.5 W/cm2
NIR light for 5 min at a certain time point. The DU145 cell viability
results show that the 5 min NIR irradiation can signicantly kill the
tumor cells in the presence of FPC-NCs in the cell culture medium.
In contrast, without the addition of FPC-NCs into the cell culture
medium, the 5 min NIR irradiation has negligible effect on the
DU145 cell viability (Fig. S11). Clearly, the NIR photothermal
treatment efcacy can only be observed in the presence of FPC-NCs,
which indicates that the FPC-NCs can serve as a highly efcient
photothermal therapeutic agent. The combination of the 5 min NIR
irradiation with the released DOX treatment could kill the cancer
cells even more drastically in comparison with the treatments by
either DOX-loaded FPC-NCs alone (chemotherapy) or 5 min NIR
irradiation alone (photothermal therapy using FPC-NCs) under the
same conditions.
The possible chemo- and combined chemo-photothermal
treatment process of the FPC-NCs for cancer cells is illustrated in
Fig. 9. The DOX-loaded FPC-NCs with optimal size and surface
properties could enter into the DU145 cells and accumulate in the
cytoplasm of cancer cells (see two-photo uorescence images in
Fig. 4e). When the DOX-loaded FPC-NCs transported into the lysosomes, the DOX molecules could be quickly released due to the
acidic microenvironments in the intracellular lysosomes and
endosomes. The released DOX molecules then entered the nuclei
and killed the tumor cell (Fig. 9 left). In the presence of NIR light,
the CDs embedded in the carbon shell of FPC-NCs can absorb and
convert NIR light to local heat, which can further enhance the
release rate of DOX (Fig. 9 right). The more DOX molecules are
released, the more tumor cells can be killed. In addition, the local
temperature enhancement induced by NIR irradiation can also kill
the tumor cells. Therefore, the FPC-NCs as drug carriers can facilitate a combined photothermal/chemotherapy to provide high
therapeutic efcacy.
4. Conclusions
In summary, hollow structured porous carbon nanocapsules
with uorescent CDs embedded in the carbon shell can be synthesized by acid-dissolution of the magnetite core from a magnetite@carbon coreeshell NPs. Such prepared FPC-NCs with size
about 100 nm demonstrated a great potential to combine multiple
functions into a single nano-object for simultaneous two-photon
cell imaging, responsive drug delivery, and photothermal therapy.
The confocal and two-photon uorescence imaging results
conrmed that the CDs embedded in the carbon shell of FPC-NCs
with tunable emissions, upconversion uorescence and excellent
photostability can light up tumor cells under a broad range of
excitation wavelengths from UV to NIR light. The central hollow
cavity, porous structure of the condensed aromatic carbon shell,
and the surface carboxyl/hydroxyl groups provide the FPC-NCs
with extraordinarily high drug loading capacity (1335 mg g
1) for
DOX beneted from both the large drug storage volume and

H. Wang et al. / Biomaterials 53 (2015) 117e126

125

Fig. 9. Schematic illustration of the chemotherapy and combined chemo-photothermal treatment of the DOX-loaded FPC-NCs in the presence of NIR irradiation.

specic drugecarrier interactions. More importantly, the release of

DOX from the FPC-NCs exhibits a faster rate in acidic pH media than
in neutral pH environments. Furthermore, the FPC-NCs manifest
NIR-responsive drug release and combined photothermo/chemotherapy for high therapeutic efcacy because the uorescent CDs
can effectively absorb and convert the NIR light to heat. Such a
nanostructured carbon nanocapsules provide a great promise
toward the advanced nanoplatform for simultaneous imaging
diagnostics and high efcacy therapy.
Acknowledgments
We gratefully acknowledge the nancial support from the
American Diabetes Association (Basic Science Award 1-12-BS-243)
and PSC-CUNY Research Award (64511-00 42).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2015.02.087.
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