Novel graphene capsule-aminoporphyrin nanohybrids: Preparation and application in photodynamic therapy of cancer

Article abstract of DOI:10.1039/c7ra08308d

In this work, we firstly prepared new sealed graphene capsules (GCs) that were about 300 nm in size and had different morphologies using a simple reduction method. The quality of the graphene oxide (GO) sheets and the concentration of hydrazine hydrate are proposed to be influencing factors for the formation of sealed graphene capsules. GCs are covalently functionalized with 5,10,15,20-tetra-(4-aminophenyl)porphyrin, forming dispersible GC-aminoporphyrins in aqueous solution. The observed fluorescence quenching of the GCs by the aminoporphyrin indicated that the GCs are electron donors and electron transfer occurs from the GCs to the aminoporphyrin. This F?rster Resonance Energy Transfer (FRET) between the GCs and aminoporphyrins is probably the reason for the enhancement of ¹O₂ generation of the GC-aminoporphyrins. More importantly, the as-prepared GC-aminoporphyrin nanohybrids as novel photosensitizers (PSs) with a short excitation wavelength can be used as PDT reagents for the treatment of malignant melanoma A375 with high efficiency. We expect that GC-aminoporphyrins will be promising PSs in future cancer therapy.

Full text of DOI:10.1039/c7ra08308d

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PAPER
Novel graphene capsule–aminoporphyrin
nanohybrids: preparation and application in
Cite this: RSC Adv., 2017, 7, 56062
photodynamic therapy of cancer†
Guoli Liu, Hongying Lv, Jing Zhao, Shen Zhang and Shuqing Sun *a
a
b
a
a
In this work, we firstly prepared new sealed graphene capsules (GCs) that were about 300 nm in size and
had different morphologies using a simple reduction method. The quality of the graphene oxide (GO)
sheets and the concentration of hydrazine hydrate are proposed to be influencing factors for the
formation of sealed graphene capsules. GCs are covalently functionalized with 5,10,15,20-tetra-(4-
aminophenyl)porphyrin, forming dispersible GC–aminoporphyrins in aqueous solution. The observed
fluorescence quenching of the GCs by the aminoporphyrin indicated that the GCs are electron donors
and electron transfer occurs from the GCs to the aminoporphyrin. This F o¨ rster Resonance Energy
Transfer (FRET) between the GCs and aminoporphyrins is probably the reason for the enhancement of
Received 27th July 2017
Accepted 15th November 2017
1
2
O generation of the GC–aminoporphyrins. More importantly, the as-prepared GC–aminoporphyrin
nanohybrids as novel photosensitizers (PSs) with a short excitation wavelength can be used as PDT
reagents for the treatment of malignant melanoma A375 with high efficiency. We expect that GC–
aminoporphyrins will be promising PSs in future cancer therapy.
DOI: 10.1039/c7ra08308d
rsc.li/rsc-advances
14
been fabricated by layer-by-layer (LbL) assembly. Recently, Qu
et al. prepared branched graphene nanocapsules on which
1
. Introduction
Graphene has attracted tremendous research interest due to its there are abundant edges and defects, which largely favored the
outstanding physical and chemical properties in various elds lithiation/delithiation reaction and resulted in a high Li-ion
18
such as electronic devices, photonic devices, energy conversion storage level. However, these processes are complex and
1–12
and storage, nanocomposites, catalysis, and biosensing.
To time consuming. Therefore, it is important to develop simple,
date, many research works have focused on graphene sheets. practical, and effective methods for the fabrication of sealed
Obviously, graphene with different morphologies, such as 3D graphene capsules to take advantage of its properties.
graphene networks, graphene capsules or graphene tubes, etc.,
is of signicant importance in the areas of supercapacitors, minimally invasive nature, fewer side effects, cost-effectiveness,
PDT is a promising cancer treatment modality owing to its
13–15
catalysis and drug delivery.
rials with different morphologies, only a few reports are avail- to avoid the disadvantages of conventional modalities.
Among these graphene mate- and its nondamaging effects on healthy tissue, which may help
19–22
PDT
able in the literature about the preparation and application of has been used in extensive investigations and has emerged as
graphene capsules that were prepared by assembling low- a disease site specic treatment modality. In PDT, aer the
dimensional graphene sheets. Hong et al. reported that gra- administration of a tumor-localizing photosensitizer, the
phene sheets were deposited onto the surface of sacricial tumors are irradiated under light with a specic wavelength to
colloidal particles using a layer-by-layer assembly method, activate the PS. Then cancerous cells are locally killed by reac-
1
1
3
leading to the formation of the graphene capsules. The tive oxygen species (ROS), especially singlet oxygen ( O ), which
2
23
capsule-embedded reduced graphene oxide (rGO) was synthe- is generated by the PS. In the past few decades, Hu et al. have
sized by photocatalytic reduction in the presence of mono- published several important works on the PDT effects of
16,17
24–29
dispersed peanut shaped a-Fe
2
O
3
particles.
Novel composite graphene-based nanohybrids.
For nanometer carbon mate-
GO/poly(allylamine hydrochloride) multilayer capsules have rials, green-light-emitting carbon quantum dots (CQDs) have
been conjugated with Ce6 to improve their biocompatibility and
light emission intensity and this composite has a PDT anti-
cancer effect. In addition, it was reported that graphene
a
30
Department of Chemistry, College of Science, Tianjin University, Tianjin, 300072,
China. E-mail: sunshuqing@tju.edu.cn
b
quantum dots (GQDs) passivated with polyethylene glycol
Chinese Academy of Medical Sciences, Institute of Radiation Medicine, Tianjin,
1
derivatives could generate O2 upon irradiation with blue
3
†
1
00192, China
31
light.
Electronic supplementary information (ESI) available. See DOI:
0.1039/c7ra08308d
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Porphyrins are a class of macrocyclic compound that 2.2 Preparation of graphene capsules
RSC Advances
have p-electron conjugated frameworks, which can be linked
Graphene oxide sheets were prepared using a modied
Hummers’ method. In a typical procedure, 1.5 g of graphite
powder and 48 mL of concentrated sulfuric acid were added
into a 250 mL ask. The mixture was stirred vigorously for
onto the surfaces of carbon nanomaterials through
3
2
covalent and noncovalent interactions. The extensive 2D
1
8p-electron porphyrins and porphyrin-modied acceptor
3
3
nanoparticles exhibit good optoelectronic properties.
3
0 min. Next, 1.8 g of NaNO
3
was added, followed by cooling of
Porphyrin-based functional carbon materials have been
extensively researched because of their structural rigidity,
thermal stability, and photochemical and photophysical
properties. Research indicates that the extensive 18p-electron
conjugated tetraphenyl porphyrins can be used for the func-
tionalization of carbon nanotubes, graphene and nanohorns
ꢀ
the mixture to 0 C using an ice water bath. Aer continuous
stirring for 1 h, 4.5 g of KMnO was added slowly over a period
4
ꢀ
of 1 h. Subsequently, the system temperature was kept at 35 C
for 30 min. Then the reaction was quenched by adding 0.18 L of
ice water and 15 mL of H
centrifuged and washed with plenty of water until no precipi-
tation of BaSO occurred in the presence of the aqueous BaCl
2 2
O (30%). The resultant GO was
3
4
on the basis of p–p interactions between them, and in these
nanohybrids it resulted in the uorescence emission of
porphyrin being quenched and the electron-transfer occurring
from porphyrin to the carbon nanotubes, graphene and
4
2
solution and the chloride test with AgNO
3
was negative. The
ꢀ
resultant product was dried at 50 C for 24 h in a vacuum oven.
The GCs were formed from the GO sheets using hydrazine
hydrate as the reducing agent. Typically, 0.1 g of the GO sheets
was treated for 2 h by ultrasonication in a 250 mL mixture of
water and ethanol with a ratio of Vwater/ethanol ¼ 4/1 to obtain
a uniform brown solution. Then, the required quantity of
hydrazine hydrate was slowly added to the brown dispersion,
which gradually became gray-black.
3
5
nanohorns, respectively.
Until now, GC–porphyrins have not yet been reported. In this
work, a simple liquid phase reaction method was developed to
prepare new sealed GCs and this is our novel work. Then, the
GCs were covalently functionalized with 5,10,15,20-tetra-(4-
aminophenyl)porphyrin, forming dispersible GC–amino-
porphyrin nanohybrids in aqueous solution. The observed

uorescence quenching of the GCs by the aminoporphyins
2
.3 Synthesis of 5,10,15,20-tetra-(4-aminophenyl)porphyrin
Synthesis of 5,10,15,20-tetra-(4-nitrophenyl)porphyrin: 7.34 g of
-nitrobenzaldehyde was mixed with 20 mL of acetic anhydride,
indicated that the GCs are electron donors and electron transfer
occurs from the GCs to the aminoporphyrin. Subsequently, the
photoactivity of the GC–aminoporphyrin nanohybrids is higher
than that of the aminoporphyin nanorods (NRs). Furthermore,
the GC–aminoporphyrin nanohybrids were used as a photo-
sensitizer to kill cancer cells in vitro and to inhibit the growth of
tumors in vivo.
4
then the mixture was added into 250 mL of propionic acid at
ꢀ
140 C. Pyrrole (3.36 mL) was added dropwise to the hot solu-
tion and the resultant mixture was reuxed for 0.5 h. The
resulting solution was allowed to cool and stand in air for 2 h.
Filtration of the tar-like mixture afforded a black solid which
was washed with methanol (6 ꢁ 50 mL) and dried in vacuo. The
residue was added into pyridine (60 mL) and the mixture was
2. Experimental section
ꢀ
reuxed at 120 C for 1 h. The resulting mixture was allowed to
cool to room temperature and then to stand in a fridge (4 C) for
2.1 Materials and instruments
ꢀ
Natural graphite akes (325 mesh, 99.8%) were purchased from
Alfa Aesar. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT), phosphate citrate buffer, tetrahydrofuran
1
4 h. Next, the mixture was ltered to remove the pyridine under
vacuum. The residue was washed with acetone and a purple
solid was obtained (1.3 g).
(
THF) and dimethyl sulfoxide (DMSO) were purchased from
Sigma-Aldrich. RPMI 1640 culture medium, fetal bovine serum
FBS), Dulbecco’s phosphate buffered saline (PBS), and the
Synthesis of 5,10,15,20-tetra-(4-aminophenyl)porphyrin:
2 2
a solution of SnCl $2H O (6.52 g) in 14 mL of concentrated
hydrochloric acid was added to a mixture of 5,10,15,20-tetra-(4-
nitrophenyl)porphyrin (1.43 g) and concentrated hydrochloric
acid (70 mL) drop by drop. The system was heated to 70 C and
kept for 0.5 h and then cooled at 4 C. 140 mL of ammonia water
(
antibiotic agents were purchased from Invitrogen (USA). The
Singlet Oxygen Sensor Green (SOSG) reagent was ordered from
Life Technology. High-purity water with a resistivity greater
than 18.4 MU cm was collected from an in-line Millipore RiOs/
Origin water purication system. All chemicals were used as
supplied without further purication.
TEM images were captured on a Jeol JEM-2100F trans-
mission electron microscope operated at 100 keV. FT-IF spectra
were recorded on a Magna-560 spectrophotometer using a KBr
pellet. Raman analyses of the GO sheets and GCs were carried
out by micro-Raman spectroscopy with confocal conguration
ꢀ
ꢀ
(25%) was added into the system and was stirred for 1 h. Next,
the suspension was ltered and the residue was washed with
water and dried in vacuo. Finally, the raw product was puried
by Soxhlet extraction with acetone and chloroform to gain violet
5
,10,15,20-tetra-(4-aminophenyl)porphyrin.
2
.4 Preparation of CG–aminoporphyrin nanohybrids
(NT-MDT NTEGRA Spectra, with a 532 nm laser). Photo- 40 mg of 1-ethyl-3-(3-dimetylaminopropyl)carbodiimide
luminescence (PL) spectra were measured using a Cary Eclipse hydrochloride (EDC, sigma) and 52 mg of N-hydrox-
EL06063917 uorescence spectrophotometer with a xenon arc ysulfosuccinimide sodium salt (sulfo-NHS, sigma) were dis-
lamp as the light source.
solved in 2 mL of deionized water. Then the solution was
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ꢂ1
added into a 10 mL graphene capsule suspension (1 mg mL
)
2.8 In vivo animal experiments
to react for 15 min. Next, 8 mg of 5,10,15,20-tetra-(4-amino-
phenyl)porphyrin dissolved in 1 mL of THF was gradually
added. The mixture was stirred at room temperature for 2 h
and then transferred to a membrane bag for dialysis against
pure water to remove unreacted molecules (the cutoff
molecular weight was 12 000).
All animal experiments were conducted at the Institute of
Radiation Medicine Chinese Academy of Medical Science and
were approved by the China Committee for Research and
Animal Ethics in compliance with the law on experimental
animals. PDT treatments were performed on 4 to 6 week-old
3
nude mice aer inoculation of the about 2–4 mm melanoma
A375 tumor pieces. The xenograed mice were randomly
divided into 6 groups (ve mice per group). When the tumor
volume reached about 100 mm PBS, the aminoporphyrin NRs,
2
.5 Preparation of the aminoporphyrin NRs
3
To prepare aminoporphyrin NRs, a reprecipitation method is
used. Typically, a 400 mL (0.5 mg mL ) aliquot of the amino-
porphyrin solution in THF is added into 5 mL of milli-Q water
under vigorous stirring at room temperature for 10 min. Aer
the mixture is stirred for another 30 min, THF is removed by
nitrogen blowing.
ꢂ1
and the GC–aminoporphyrins were administered respectively
ꢂ1
by intratumoural injection at a dose of 2 mg kg once every
other day. Then, light treatment was performed 2 h aer
injection by irradiating the tumour regions with white LED light
for 15 min. The changes in the tumor volumes in the mice were
then monitored once every other day. The mice were sacriced
and their tumors were immediately removed and weighed aer
2
.6 Singlet oxygen detection
14 days of treatment.
1
The
2
O production of the GCs, aminoporphyrin NRs, and
GC–aminoporphyrin conjugates was studied using SOSG as an 2.9 Haematoxylin- and eosine-staining
ꢂ1
indicator. Specically, 60 mL of the SOSG solution (1 mg mL
)
Paraffin-embedded tumors and organs were cut into 7 mm
sections and the sections were transferred to slides and dried
was mixed with 3 mL of the dispersion containing 100 mg of
the GCs, aminoporphyrin NRs or GC–aminoporphyrin
conjugates. The mixtures were irradiated with white light
overnight at room temperature, followed by drying in an oven at
ꢀ
4
3 C. Then, the sections were stained and dehydrated accord-
ꢂ2
(
100 mW cm ) for different durations. Fluorescence spectra
ing to the following method: the sections were incubated for 3
minutes with xylene, 1 minute with 100% ethanol, 2 minutes
with 95% ethanol, 1 minute with 70% ethanol, 3 minutes with
distilled water, 7 minutes with haematoxylin, 3 minutes with
distilled water, and 30 seconds with eosin. Next, the sections
were rinsed with distilled water and incubated again with an
increasing gradient of ethanol (2 minutes of 70%, 95%, and
100% ethanol, respectively) and nally 1 minute with xylene.
Finally, the sections were mounted, covered with a cover slip
and dried overnight. Microscopy was performed to take images.
of the irradiated samples were then measured with an excita-
tion wavelength of 504 nm. The oxidized SOSG in the irradi-
ated samples was quantied by its uorescence intensity
1
based on the O
2
amount. The experiments for each group
were run in triplicate.
2.7 In vitro cytotoxicity evaluation
In vitro cytotoxicities of the GCs, aminoporphyrin NRs and GC–
aminoporphyrin conjugates were evaluated using the standard
3
-(4,5-dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide
2.10 Statistical analysis
(MTT) assay in the Hela cell line. The Hela cells were seeded on
9
6-well plates in Dulbecco’s modied Eagle’s medium (DMEM) All data are presented as the mean ꢃ standard deviation (S.D.).
containing 10% fetal bovine serum (FBS) under an atmosphere The signicant differences between groups were evaluated with
of 5% CO . Aer growing overnight, the original medium in Tukey’s method aer analysis of variance (ANOVA). In all
2
each well was removed and 200 mL of DMEM containing the statistical analyses, p < 0.05 was considered to be statistically
designated concentrations of the GCs, the aminoporphyrin NRs signicant.
or the GC–aminoporphyrin dispersion was added to the planed
ꢀ
wells. Aer 12 h of incubation in the dark at 37 C, the cells
3
. Results and discussion
incubated with the GCs, the aminoporphyrin NRs or the GC–
aminoporphyrins were irradiated under a xenon lamp with
a power of 100 mW cm for 15 min. The cells were then The GCs were prepared using a simple reduction method. In
incubated at 37 C for another 12 h and then the medium in this method, the GO sheets and hydrazine hydrate were used as
3.1 Preparation of the graphene capsules
ꢂ2
ꢀ
each well was removed. Next, 180 mL of DMEM and 20 mL of the precursors of the graphene capsules and reducing agent,
ꢂ1
MTT solution (5 mg mL in PBS) were added and incubated for respectively. The TEM images of the GCs show their
4
h. Subsequently, the medium containing MTT was completely morphology features as shown in Fig. 1. Differently shaped GCs
removed, followed by the addition of 200 mL of DMSO to each were formed during the reduction of the GO sheets and they are
well. Aer that, a micro-plate reader was used to measure the about 300 nm in size (Fig. 1a and c). These GC are sealed
absorbance at 490 nm to determine cell viability. The cells because they are bellied under E-beam impact during TEM
incubated with a serum-supplemented medium represent 100% measurement. This type of GC was not observed earlier by self-
cell survival. To avoid random errors, ve replicate wells were assembly processes. Previously, researchers used sacricial
run for each concentration and control.
2 3
colloidal particles or a-Fe O particles as the cores and then the
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Fig. 1 TEM images of the graphene capsules with different morphologies (a and c); TEM image of GC–aminoporphyrin nanohybrids (b); TEM
image of many graphene capsules (d).
GO or rGO sheets were covered on the surfaces of these cores to were functionalized with –OOH, –OH and –O– on the surfaces
form shells. Aer the cores were removed, the shells became and edges of the GO sheets. When the hydrazine hydrate was
13,16,17
GCs.
Obviously, GCs formed by self-assembly processes added into the dispersion of the GO sheets it reduced some
2
are not sealed. Therefore, it can be concluded that the sealed –OOH, –OH and –O– groups, resulting in the increase of sp
GCs are rstly achieved by our simple reduction method. It is domains. As a result, the hydrophobicity of the surface of the
worth noting that all graphene capsules are single without GO sheet would increase and the GO sheet would shrink to
linking (Fig. 1d). However, when the graphene capsules and decrease its surface area in the aqueous solution. The forma-
aminoporphyrins formed the GC–aminoporphyrin nanohybrids tion of the graphene capsules initiated aer the concentration
through a series of reactions, the two graphene capsules joined of hydrazine hydrate reached 0.5% (Fig. 2b). In this case,
together appear as shown in Fig. 1b. Due to too small a size of a larger hydrophobicity of the surface of the rGO sheets due to
the aminoporphyrin molecule compared with the graphene the increase of the degree of reduction of the GO sheets tended
capsules they cannot be seen in the TEM image. The DLS to make the surface area minimize in the aqueous solution,
measurement (see ESI, Fig. S1†) gives a hydrodynamic diameter leading to the rGO sheets gradually diffusing and forming
of about 303 nm for the graphene capsules and 353 nm for the sealed GCs. Interestingly, the reduction rates on the different
GC–aminoporphyrin nanohybrids.
surfaces of the GO sheets are different. The surfaces with
Two factors may result in the formation of the sealed GCs. larger reduction rates are inward and the surfaces with smaller
Firstly, the precursors of the GCs are large-sized, at and thin GO reduction rates are outward. Otherwise, the GCs cannot form.
sheets as shown in Fig. 2a. The TEM image reveals that the GO When the concentration of hydrazine hydrate was below 0.5%,
sheets are transparent under the electron beam, suggesting that the GO sheets were reduced to become rGO sheets. When the
the single-layer or few-layer graphene sheets were exfoliated from concentration of hydrazine hydrate was over 0.8%, the GO
graphite. In addition, the GO sheets are shown to be large-sized sheets were overly reduced, resulting in open graphene
and they have lateral dimensions of over 3 mm. Secondly, the capsules with bigger holes (see ESI, Fig. S2†).
concentration of the hydrazine hydrate was in an appropriate
Porphyrin-based functional carbon materials have been
range from 0.5% to 0.8%. It is well known that some C atoms extensively researched and the research indicates that
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Fig. 2 TEM images of the GO sheets (a) and the single graphene capsule (b) with the arrow between the two images indicating that the GO
sheets were reduced with hydrazine hydrate to form the graphene capsule. Raman spectra of the GO sheets (c) and the graphene capsules (d).
porphyrins can be used to functionalize carbon nanotubes, occurred from the porphyrin to the carbon nanotubes, gra-
graphene and nanohorns on the basis of p–p interactions phene and nanohorns, respectively. So far, no study has been
between them. In the nanohybrids formed, the electron-transfer
Scheme 1 Synthesis reaction scheme of the conjugate of the graphene capsule and 5,10,15,20-tetra-(4-aminophenyl)porphyrin.
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reported for the functionalization of GCs to build nanohybrids
with porphyrins with GCs as the carbon nanomaterial.
3.2 Preparation of the GC–aminoporphyrins
The GC–aminoporphyrin nanohybrids were synthesized
through a series of reactions as described in Scheme 1.
To conrm the formation of the amide bond which linked
the GCs and the aminoporphyrin, FT-IR spectroscopy was per-
formed on the GCs, aminoporphyrin and GC–aminoporphyrin.
In the FT-IR spectrum of the GCs (Fig. 3a), the absorption bands
ꢂ1
at 3000–3500 cm are assigned to the stretching vibrations of
–
OH groups, and the absorption intensity of the C]C peak at
ꢂ1
1
549 cm is stronger so that the C]O absorption becomes
ꢂ1
a shoulder peak. In addition, the absorption peaks at 2923 cm
and 1071 cm are attributed to C–H and –O–, respectively. For
ꢂ1
aminoporphyrin, a series of peaks found in the FT-IR spectra in
ꢂ1
the range of 700 to 1700 cm were attributed to the typical
modes of the pyridine cycles, including the sharp peaks at
ꢂ1
ꢂ1
ꢂ1
1
599 cm , 794 cm and 720 cm , which are attributed to the
C]C and C]N stretching vibrations, 4-monosubstituted ring
C–H deformation vibrations and 4-monosubstituted ring
deformation vibrations, respectively (Fig. 3b). Comparing the
typical FT-IR spectra of the GC–aminoporphyrin and those of
the GCs and aminoporphyrin as controls, it could be seen that
ꢂ1
a new peak appeared at 1640 cm , which is attributed to the
amide bond. This indicated that the aminoporphyrin had been
covalently attached to the surface of the GCs through the amide
bond (Fig. 3c).
Appropriate concentrations of hydrazine hydrate (ranging
from 0.5% to 0.8%) were used to reduce the GO sheets to form
GCs. It is well known that some C atoms were functionalized
with –OOH groups on the surfaces and edges of the GO sheets.
When hydrazine hydrate was added to the dispersion of GO
sheets it reduced some –COOH groups, but there were some
excess –COOH groups on the surface of the GCs. This can be
conrmed by the fact that the GCs are hydrophilic and can be
evenly dispersed in water. When the concentration of hydra-
zine hydrate was over 0.8%, the GO sheets were fully reduced,
resulting in the GCs becoming hydrophobic and a black
precipitate falling to the bottom of the vials. In addition,
Fig. 3 FT-IR spectra of the graphene capsules (a), 5,10,15,20-tetra-(4- in the FT-IR spectrum of the GCs, there are absorption bands
ꢂ1
aminophenyl)porphyrin (b) and the GC–aminoporphyrin (c).
at 3000–3500 cm
of –OH groups and the absorption
Fig. 4 Wide scan XPS of the GC–aminoporphyrin (a); thermogravimetric analysis of the GC–aminoporphyrin powder (b).
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Fig. 5 Fluorescence spectra of the SOSG solution incubated with GC–aminoporphyrins, aminoporphyrin NRs or GCs over increasing irradiation
time (a–c). The evolution of the fluorescence intensity of SOSG at 525 nm over different irradiation times ((d), the fluorescence intensities are
calculated by peak area).
ꢂ1
ꢀ
ꢀ
ꢂ1
2
shoulder peak of C]O groups at about 1700 cm , which from 30 to 700 C at 10 C min in a N atmosphere on
indicate that there are –COOH groups on the surface of the a thermogravimetric analyzer to determine the mass loss of
ꢀ
GCs. In the presence of EDC and sulfo-NHS, the reactivity the sample. An obvious weight loss appears from 320 C to
ꢀ
between the –COOH groups on the surface of the GCs and the 700 C, which corresponds to the mass loss of the mixture
–
NH
noporphyrins are covalently linked to the graphene capsules carried out to evaluate the composition of the surface of the
by amidation. GC–aminoporphyrin nanohybrid. A wide scan XPS was ob-
2
groups of the aminoporphyrins is high and the ami- containing aminoporphyrin, EDC and sulfo-NHS. XPS was
The existence of aminoporphyrin in the GC–amino- tained for the sample and is shown in Fig. 4a, displaying the
porphyrin nanohybrid was conrmed by thermogravimetric characteristic C 1s, N 1s, and O 1s core-level photoemission
analysis. The TG curves of the samples were determined peaks at 285, 399, and 532 eV, respectively. The carbon atom is
directly by lling a GC–aminoporphyrin powder into a ther- dominant in the GC–aminoporphyrin nanohybrid with an
mogravimetric analysis sample pan and then it was heated atomic percent of 57.8%. The atomic percent of N is 20.6%
Fig. 6 Excitation spectrum of aminoporphyrin and emission spectrum of the graphene capsules (a). PL spectra of the graphene capsules,
ꢂ1
aminoporphyrin and GC–aminoporphyrins under a 365 nm excitation wavelength (b) (aminoporphyrin in chloroform, 0.5 mg mL ; graphene
ꢂ1
ꢂ1
capsules in water, 5 mg mL ; GC–aminoporphyrin in water, 5 mg mL ).
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which is higher than that of the GCs due to the existence of
aminoporphyrin in the GC–aminoporphyrin nanohybrid
(
see ESI, Fig. S3†). The load of porphyrin in the nanohybrid
was determined via absorption measurements and the value
is 6.5%.
3.3 Singlet oxygen detection
Aminoporphyrin NRs were prepared using a reprecipitation
method and their morphology and size were investigated using
SEM (see ESI, Fig. S4†). The singlet oxygen generation of the
GCs, aminoporphyrin NRs and GC–aminoporphyrins was eval-
uated with the standard SOSG probe. Upon the reaction
1
between SOSG and
2
O , the intramolecular electron transfer
within SOSG is prohibited, which recovers the quenched green
36,37

uorescence with high selectivity.
As shown in Fig. 5, when
the mixture of SOSG and GCs, aminoporphyrin NRs or GC–a-
minoporphyrins is irradiated with visible light, the uorescence
intensity of SOSG at 525 nm exhibits a time-dependent increase,
1
indicating the gradual generation of
2
O . Under white light
1
2
irradiation, the generation of O of GC–aminoporphyrins is
highest among the three PSs (Fig. 5a–c). Fig. 5d illustrates the
changes in SOSG’s uorescence intensity when irradiating the
mixtures of SOSG with GCs, aminoporphyrin NRs, and GC–
1
aminoporphyrins, respectively. The lower O generation of the
2
aminoporphyrin NPs may be due to aggregation quenching
resulting from p–p stacking of the planar molecules. For the
GC–aminoporphyrins, F ¨o rster Resonance Energy Transfer
between the GCs and aminoporphyrins is probably the reason
1
for the enhancement of
O
2
generation of GC–amino-
porphyrins. These results demonstrate good potential for the
application of GC–aminoporphyrins as PS agents.
Fluorescence spectroscopy is a useful tool for assessing the
degree of electronic communication in the excited states of the
nanohybrids. Fig. 6b shows the PL spectra of the GCs, amino-
porphyrins and GC–aminoporphyrins. For the GCs, the domi-
nant uorescence emission peak is at 503 nm upon 365 nm
excitation. The aminoporphyrin sample exhibits a main uo-
rescence peak at 650 nm with the same excitation wavelength.
The uorescence emission of the photoexcited GCs at 490 nm is
signicantly quenched in the covalently associated GC–amino-
porphyrins, conrming that the porphyrin moieties are in close
proximity to the GCs in the nanohybrids. FRET between the GCs
Fig. 7 Hela cell viability at different concentrations of graphene
and aminoporphyrins occurred. Very interestingly, unlike other capsules (a), aminoporphyrin NRs (b), and GC–aminoporphyrin (c).
carbon materials including fullerenes, carbon nanotubes and
graphene as electron acceptors, in our results, the GCs were
found to be electron donors. Excitation and emission spectra of
with only irradiation but without any PSs or only PSs without
the aminoporphyrins (acceptor) and the GCs (donor) are shown
irradiation. As presented in Fig. 7, the three PSs including the
in Fig. 6a.
GCs, aminoporphyrin NRs and GC–aminoporphyrins with irra-
diation show dose-dependent toxicity while the control groups
exhibit no toxicity. This suggests that the irradiation itself and the
3.4 Cell cytotoxicity assay
To evaluate the PDT efficiency of the GC–aminoporphyrins, we PSs in the dark exhibit no cytotoxicity. As shown in Fig. 7, when
ꢂ1
measured the viability of treated Hela cell lines using a 3-(4,5- the doses of the PSs are 6.25, 12.5, 25, 50 and 100 mg mL
,
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) respectively, the cell viability induced by the GC–aminoporphyrins
assay. Hela cells were individually incubated for 24 h with the is lower than that induced by the aminoporphyrin NRs. On
GCs, aminoporphyrin NRs and GC–aminoporphyrins (with or average, the former accounted for 77% of the latter. The GC–a-
without irradiation). The control group corresponds to the cells minoporphyrins have the highest cytotoxic efficacy upon
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1
irradiation as a consequence of the higher generation of O
2
from control groups. These results conrm that the GC–amino-
1
them, which leads to reactions between O and the tumor cells porphyrins are excellent PSs. Photographs of the sacriced mice
2
and to the subsequent killing of cancer cells by chemical and the tumors from the sacriced mice are shown in Fig. 8a
oxidation.
and b. These results indicate that the present GC–amino-
porphyrin dose has predominant tumor growth inhibitory effi-
cacy. Usually, in PDT, absorption in the long wavelength region
is desired, however, PSs with a short excitation wavelength can
also be used as PDT reagents for the treatment of skin cancer or
lesions. In our work, GC–aminoporphyrins as PSs against the
human malignant melanoma A375 are highly efficient.
H&E staining was performed on tissue sections of the
malignant melanoma A375 tumors from each group aer
treatment as shown in Fig. 9. It is found that most tumor cells
were severely damaged or destroyed in mice treated with the
GC–aminoporphyrins and irradiated with a Xe lamp with
a 400 nm lter. Compared to the four control groups, obvious
cell apoptosis can also be observed for the tumor treated with
aminoporphyrin NRs under Xe lamp illumination. In the other
groups, there was no tumor cell death, suggesting the successful
destruction of tumor cells by GC–aminoporphyrins and ami-
noporphyrin NRs with the irradiation of white light. This result
indicates that the PDT effect of the GC–aminoporphyrins
against the malignant melanoma A375 tumor is better than that
of the aminoporphyrin NRs.
3.5 In vivo animal experiment and biosafety
1
Being aware of the high light-induced O
2
generation capability,
we nally carried out in vivo PDT treatment researches using
female nude mice with melanoma A375 tumor pieces as xeno-
gras. Seven days aer inoculation, the average volume of
tumors reached ꢄ100 mm . A total of 30 tumor-bearing mice
3
were randomly divided into 6 groups (n ¼ 5 per group) as
follows: (1) control group by PBS injection only; (2) control
group by white light exposure only; (3) aminoporphyrin NR
injection only without light exposure; (4) GC–aminoporphyrin
injection only without light exposure; (5) aminoporphyrin NR
injection plus white light exposure; (6) GC–aminoporphyrin
injection plus white light exposure. At 2 h post-injection, the
tumor regions were irradiated with white LED light. We then
assessed the photodynamic effects of each group by monitoring
the tumor volumes over a period of 14 days. At day 15, all test
mice were sacriced and the corresponding tumors were
excised for further studies. The tumor volumes in mice
receiving GC–aminoporphyrins with white light irradiation
were signicantly inhibited (Fig. 8c). In contrast, no signicant
differences in tumor volumes were found among the four
To evaluate the biosafety of the GC–aminoporphyrins as PSs
in vivo, aer 14 days of treatments with the GC–
Fig. 8 In vivo antitumor activity of the aminoporphyrin NRs and GC–aminoporphyrins: (a) photographs of mice after various treatments of the
PBS injection (1), light exposure (2), aminoporphyrin NR injection (3), GC–aminoporphyrin injection (4), aminoporphyrin NR injection plus light
exposure (5) and GC–aminoporphyrin injection plus light exposure (6); (b) corresponding images of excised tumors at the time of sacrifice from
the nude tumor-bearing mice after 14 days of treatment; (c) time-dependent tumor growth curves after different treatments.
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Fig. 9 Qualitative histological analysis of tumors with representative hematoxylin- and eosin-stained histological sections (scale bar upper row
00 mm, lower row 50 mm).
1
Fig. 10 H&E staining images of major organs (lung, liver, spleen, kidney and heart) of the tumor-bearing mice in groups of the control and the
GC–aminoporphyrins (scale bar 50 mm).
aminoporphyrins and the PBS buffer, healthy organs including shapes. GC–aminoporphyrins are formed by covalent combi-
lung, liver, spleen, kidney and heart were excised from the nation between the GCs and 5,10,15,20-tetra-(4-aminophenyl)
tumor-bearing mice. The hematoxylin and eosin (H&E) staining porphyrin. Based on the uorescence quenching of the GCs by
images of these organs from the tumor-bearing mice in aminoporphyrin, the GCs are electron donors and electron
different groups, respectively, are shown in Fig. 10. In the group transfer occurred from the GCs to the aminoporphyrin. The
of the GC–aminoporphyrin treatment, neither noticeable organ prepared GC–aminoporphyrins possess good biocompatibility
damage nor inammation lesions were found as compared to and can efficiently generate singlet oxygen. Importantly, the as-
the control groups, suggesting that there was no obvious lung, prepared GC–aminoporphyrin nanohybrids as a novel PS with
liver, spleen, kidney and heart dysfunction in mice that was a short excitation wavelength can be used in PDT for the
induced by the GC–aminoporphyrin with white light treatment of melanoma A375 with high efficiency.
irradiation.
4
. Conclusions
Conflicts of interest
In summary, we rstly developed a simple method to fabricate
sealed GCs that were about 300 nm in size and had different There are no conicts to declare.
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1
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Acknowledgements
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