Non-covalent hydrophilization of reduced graphene oxide used as a paclitaxel vehicle

Article abstract of DOI:10.1039/C6RA04349F

Graphene oxide (GO) and reduced graphene oxide (RGO) are attractive materials due to their potential biomedical applications, especially as drug delivery vehicles because of their huge specific surface area. In the current study, GO and RGO were prepared. In addition, a perylene derivative, phosphorylcholine oligomer grafted perylene (Perylene-PCn), was synthesized following the atom transfer radical polymerization procedure. In order to determine their structures, the products were characterized carefully with Fourier transform infrared spectra, proton nuclear magnetic resonance, X-ray photoelectron spectroscopy, wide angle X-ray diffraction, thermogravimetric analysis, and atomic force microscopy etc. The properties including fluorescence and biocompatibility were also evaluated. Utilizing the p–p stacking interaction of RGO and the perylene moiety, water dispersible RGO/Perylene-PCn composites were fabricated and then investigated as a vehicle for anti-tumor paclitaxel (PTX). The anti-tumor effects of free PTX and PTX@RGO/Perylene-PCn were compared with an in vitro cytotoxicity assay. The results demonstrated that RGO/Perylene-PCn can be used as an anti-tumor agent delivery vehicle for potential oncology applications.

Full text of DOI:10.1039/C6RA04349F

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Page 1 of 29
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Non-covalent hydrophilization of reduced graphene oxide
used as paclitaxel vehicle
Yu Liu1, Han Zhong1, Yu Qin1, Yan Zhang1,2, Xinfeng Liu3, Tao Zhang1,2,*
1. College of Engineering and Applied Sciences, Nanjing University, Nanjing 210093, China
2. Nanjing Excellence Technology Center for Interventional Medical Devices, Nanjing 210093,
China
3. Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Nanjing
210003, China
Abstract
Graphene oxide (GO) and reduced graphene oxide (RGO) are attractive materials due to
their potential biomedical applications, especially as drug delivery vehicles because of their
huge specific surface area. In the current study, GO and RGO were prepared. In addition, a
perylene derivative, phosphorylcholine oligomer grafted perylene (Perylene-PCn), was
synthesized following the atom transfer radical polymerization procedure. In order to
determine their structures, the products were characterized carefully with Fourier transform
infrared spectra, proton nuclear magnetic resonance, X-ray photoelectron spectroscopy,
wide angel X-ray diffraction, thermogravimetric analysis, and atomic force microscope etc.
The properties including fluorescence and biocompatibility were also evaluated. Utilizing the
*
To whom correspondence should be addressed. E-mail: ztnj@nju.edu.cn

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π-π stacking interaction of RGO and perylene moiety, a water dispersible RGO/Perylene-PCn
composites were fabricated and then investigated as a vehicle of anti-tumor paclitaxel (PTX).
The anti-tumor effects of free PTX and PTX@RGO/Perylene-PCn were compared with an in
vitro cytotoxicity assay. The results demonstrate that RGO/Perylene-PCn can be used as an
anti-tumor agent delivery vehicle for potential oncology applications.
Introduction
Since firstly reported in 2004, graphene has drawn a great deal of attentions due to its
extraordinary electrical, optical, catalytic and mechanical properties 1-4. Graphene is
consisted of layers of π-conjugated structures of six-carbon rings, which can be conceptually
viewed as a planar aromatic macromolecule. This planar structure offers an excellent
capability of immobilizing a large number of substances, including metals, drugs,
biomolecules, fluorescent probes and cells 5-10. Therefore, it is not surprising that graphene
have been extensively investigated for its nanomedicine and biomedical applications,
including but not limited to drug delivery platform for anti-cancer/gene delivery, biosensing,
bioimaging, antibacterial applications, cell culturing and tissue engineering 6, 7, 9-13
.
However, the applications of graphene were limited due to the insolubility of graphene in
water and the tendency of agglomerating irreversibly or even restacking into graphite
through Van der Waals interactions 14. Thus, the modifications of graphene turned out to be
of great importance for their potential biomedical applications. Usually, there are two
methods to prepare the soluble graphene, namely chemical modifications and non-covalent

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functionalization. The chemical modification, which usually means covalent modifications on
graphene, may result in the changes of their electronic structure by creating sp3 centers,
leading to the alteration of its physical properties 15-17. On the other hand, it was believed
that the non-covalent functionalization, which mainly relies on Van der Waals interactions or
π-π stacking interactions, normally impacted little on structures and properties of graphene.
Stankovich et al. reported the first example of non-covalent functionalization of graphitic
nanoplatelets using poly(sodium 4-styrenesulphonate) 18. Liu et al. designed a series of
pyrene terminated polymers to functionalize reduced graphene oxide (RGO) via π-π stacking
interaction 19, 20. Feng et al. functionalized graphene oxide (GO) by PEI polymers utilizing
non-covalent electrostatic interactions, which produced GO-PEI complexes with strong
21
positive charges, high stability in physiological solutions and reduced cytotoxicity to cells
.
Yang et al. constructed a nanosuperamolecular assembly using folic acid-modified
β-cyclodextrin and GO non-covalently, which were linked by an adamantane-grafted
porphyrin through π-π stacking interaction 22. Depan et al. prepared a novel folate-decorated
and graphene-mediated drug delivery system by attaching doxorubicin to GO via strong π-π
stacking interaction, followed by encapsulation of GO with folic acid conjugated chitosan to
enhance the stability of the nano-carrier system in aqueous medium 23
.
In this work, perylene-3,4,9,10-tetracarboxylic acid bisimide (PBI) connected with
phosphorylcholine oligomers has been employed to modify graphene by π-π stacking
interaction. PBI derivatives are highly fluorescent, chemically and photochemically stable
molecules which have been used as pigments and dyes. In previous work, Kang et al.
designed perylene containing poly(glyceryl acrylate) derivatives to modify reduced graphene

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oxide. The low cytotoxicity of the hybrids was shown by treating 3T3 fibroblasts with the
hybrids in vitro 24. Phospholipids are components of lipid bilayers and plasma membranes 25
.
It is believed that polymers containing a phospholipid structure are highly biocompatible as
a result of their characteristic ability to mimic biomembranes 26; however, phospholipid
molecules cannot bond covalently and, as a result, are highly mobile. A simple bilayer
membrane containing a phospholipid structure cannot provide the physical and chemical
properties necessary to maintain stability 27. To obtain stable phospholipid systems, several
28
phospholipid-analogous molecules with polymerizable groups have been synthesized
.
Among them, 2-methacryloyloxyethyl phosphorylcholine (MPC) is one of the most effective
compounds. It has been proven to be capable of significantly reducing protein absorption as
well as platelet and cell adhesion to materials 29-31. Soon after its debut, MPCs have been
introduced into many materials by grafting onto a functionalized surface to enhance the
material’s biocompatibility 32-34
.
Initially, Perylene-PCn was synthesized with atom transfer radical polymerization (ATRP)
method from MPC as monomer and PBI derivative as an initiator. Graphene, or more
accurately, the reduced graphene oxide (RGO), was obtained from chemical reduction of GO
by N2H2·H2O. Then, Perylene-PCn was allowed to self-assemble onto the RGO nanosheets via
π-π stacking interactions to achieve non-covalent functionalization of graphene. In the
present study, paclitaxel loading RGO/PBI-PCn were fabricated by self-attachment in ethanol
solution at room temperature with ultrasonication. Paclitaxel (PTX) is one of the most widely
used anticancer agents, that shows activities against various solid tumors 35. The current
study outlines the characterizations of RGO/PBI-PCn, in vitro toxicity of paclitaxel loading

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RGO/PBI-PCn against L929 murine fibroblasts and human gastric cell lines SGC-7901.
Experimental
Materials
The natural graphite powder (C > 99% with particle size about 5 ꢀm) was purchased from
Shanghai Huayuan Graphite Co., Ltd. (Shanghai, China). KMnO4, H2SO4 (98%), H2O2 (30%
solution in water) and Hydrazine hydrate (N2H4·H20, 80%) were purchased from Nanjing
Reagent Co. Ltd. (Nanjing, China). N,N,N’,N”,N’’-Pentamethyldiethylenetriamine (PMDETA,
99%), CuBr (99%), 2-Bromoisobutyryl bromide (99%) and Perylene-3,4,9,10-tatracarboxylic
dianhydride (PTCDA) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China).
Inhibitor-free 2-Methacryloyloxyenthyl Phosphorylcholine (MPC, > 96%) was supplied by
Joy-Nature Institute of Technology (Nanjing, China). Paclitaxel (PTX) were offered by Jiangsu
Yew Pharmaceutical Co., Ltd. (Jiangsu, China). All other reagents and solvents were
purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China) and used as received.
Human umbilical vein endothelial cell lines (HUVEC), mouse leukaemic monocyte
macrophage cell lines (RAW264.7), mouse fibroblast cell lines (L929) and human gastric
cancer cell lines (SGC-7901) were obtained from Zhongyuan Co., Ltd (Beijing, China).
Dulbecco’s modified Eagle’s medium (DMEM, with 4500 mg/L-glucose and L-glutamine) was
purchased from Thermo Fisher Scientific Inc. (Beijing, China). Fetal bovine serum (FBS) was
purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou,
China), which was heat inactivated for 30 min at 56°C and then stored at -20°C before use.
0.25% trypsin-EDTA (1X) with phenol red was purchased from Life Technologies Co.

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(Shanghai, China). Penicillin/streptomycin (100×) was purchased from Life Technologies Co.
(Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular
Technologies, Inc. (Shanghai, China).
Instruments
Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on a Bruker ARX500
NMR Spectrometer (Bruker, German) using D2O as the solvent. Fourier transform infrared
spectra (FTIR) were recorded on a PE GX spectrometer (Perkin-Elmer, USA) at room
temperature on KBr pellets with sample concentrations of ~1% from 4000 to 400 cm-1 with a
resolution of 4 cm-1. Thermogravimetric Analysis (TGA) were recorded on a Netzsch
STA409PC (Selb, Germany) in N2 atmosphere at the heating rate 20 °C·min-1 from room
temperature to 700 °C. Wide angle X-ray diffraction (XRD) measurements were performed
on a Rigaku ULTIMA-3 setup with Mar 345 image plate as detector and the Cu Kα was used
as the source (wavelength = 0.1542 nm). The recorded region of 2θ was from 3° to 45° with
scanning speed 2°·min-1. A tapping mode atomic force microscope (AFM) (NanoScope IIIa,
Digital Instrument, Inc.) was utilized to observe the micromorphology of samples. A LabRAM
Aramis Raman Spectrometer (LabRAM HORIBA Jobin Yvon, Edison, NJ, USA) excited by the
532 nm coherent line of a Nd:YAG laser was used to determine the carbon structure of the
samples at room temperature, the samples were prepared by casting suspensions onto the
silicon substrates and drying at 60 °C for 24 h. X-ray Photoelectron Spectroscopy (XPS)
measurements were recorded on a Thermo Scientific K-Alpha Photoelectron Spectrometer
equipped with a monochromatic Al Kα X-ray source. XPS PEAK software (v4.1) was utilized to
analyze and deconvolute the XPS peaks. Peak deconvolutions were performed using optional

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Lorentzian-Gaussian components after
a
Shirley background subtraction. The
Ultraviolet-visible absorption spectra were obtained from a MAPADA UV-1800PC
spectrophotometer (Shanghai, China). Fluorescence experiments were carried out on a
Horiba Fluorolog-3-22 spectrofluorometer (Horiba Scientific, Japan) with 373 nm excitation
wavelength (20 nm band pass for both entrance and exit). The cells used in the cytotoxicity
evaluations were cultivated in a Heracell model 150i CO2 incubator (Thermo Scientific, USA),
and the evaluations were carried out with an RT-6000 micro-plate reader (Rayto Ltd.,
Shenzhen, China) at a wavelength of 450 nm. The confocal fluorescent images were taken
with an Olympus Fluoview confocal microscope (Olympus, Japan).
Preparation of GO and RGO
GO was prepared from natural graphite powder according to the modified Hummers
method 36. In detail, 2 g graphite powders were added into 50 mL cooled (0 °C), stirring
concentrated H2SO4. Then, 6 g KMnO4 was charged gradually below 5 °C. After being stirred
below 5 °C for 1 h, the mixture was heated to 35 °C and kept stirring for 30 min. In order to
dilute the reaction system, 100 mL distilled water was dropped slowly and the temperature
was maintained no more than 100 °C. At last, 15 mL 30% H2O2 was added. The crude GO was
filtered off, washed with 5% HCl solution and distilled water for at least 3 times, and then
2-
dialyzed for at least 48 h until SO4 could not be determined by BaCl2 solution. GO was
filtered out again and vacuum dried under 60 °C to obtain the dried target products.
In a 250 mL round bottom flask, 100 mg GO and 100 mL water were added to obtain an
inhomogeneous yellow-brown dispersion. The dispersion was ultrasonicated till clear

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without any visible particles. 3 mL hydrazine hydrate was then added and the solution was
heated in an oil bath at 90 °C for 24h, over which the RGO gradually precipitated out as a
black solid37, 38. The products were isolated by filtration over a medium fritted glass funnel,
washed copiously with water, and dried on the funnel at 60 °C over night.
Synthesis of Perylene-PCn
Perylene-PCn was synthesized via ATRP, with PBI derivative as initiator and MPC as monomer
in 3 steps illustrated in Figure 1.
Step 1. Preparation of PBI-OH: PTCDA (400 mg, 1 mmol) and ethanol amine (7.92 g, 40 mmol)
were introduced into a 50 mL Teflon lining hydro-thermal reaction kettle. The mixture was
ultrasonicated for 10 min and sealed, reacted at 150 °C for 24 h. The unreacted ethanol
amine was removed by filtration, washing with copious amount of water and vacuum drying.
N,N’-bis(oxethyl)perylene-3,4,9,10-tetracarboxylic acid bisimide (PBI-OH) was obtained as a
dark red solid 39, 40
.
Figure 1. Synthesis routes of Perylene-PCn

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Step 2: Preparation of PBI-Br: PBI-OH (430 mg, 1 mmol) was suspended in dry CH2Cl2 (150
mL) containing triethylamine (Et3N, 0.43 mL, 3 mmol). After been cooling to 0 °C,
2-bromoisobutyryl bromide (0.69 g, 3 mmol) in dry CH2Cl2 was added dropwise with
continuous stirring over a period of 1 h. Then, the reaction mixture was stirred for another
24 h at room temperature. The produced (CH3)3N·HBr and unreacted PBI-OH were removed
by filtration. The target products, PBI-Br remained in CH2Cl2 solution was retrieved by drying
at 60 °C as dark red solid.
Step 3: Preparation of Perylene-PCn: PBI-Br (40.3 mg, 0.05 mmol), MPC (1 g, 3.4 mmol),
CuBr (14.4 mg, 0.01 mmol) and tetrahydrofuran (THF, 15 mL) were introduced into a 25 mL
glass flask and the solution was degassed with N2 for 20 min. After PMDETA (17.3 mg, 0.1
mmol) was added quickly, the flask was sealed and the reactants were kept stirring for 24 h
at 60 °C, then the reaction mixture was cooled and filtrated to remove the copper catalyst,
followed by drying in a vacuum oven for 24 h 24
.
Non-covalent hydrophilization of RGO
In an ice-water bath, 15 mg RGO were dispersed in 20 mL distilled water under an ultrasonic
cell disruption system (with power of 600W) for 10 min. After that, 45 mg Perylene-PCn
aqueous solutions were added, followed by an additional 10 min ultra-sonication. The
mixture was centrifuged at relative centrifugal force (RCF) of 11000 g to obtain the
RGO/Perylene-PCn composites. The resultant RGO/Perylene-PCn composites were
re-dispersed in ethanol with concentration of 0.5 mg/mL.
Paclitaxel loading on RGO/Perylene-PCn

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A series of PTX-ethanol solutions with different concentrations were added to 10 mL
RGO/Perylene-PCn solutions. After 30 min sonication, the mixtures were filtered by 0.22 μm
membrane to obtain the PTX loading RGO/Perylene-PCn composites, which was recorded as
PTX@RGO/Perylene-PCn.
The drug loading content and encapsulation efficiency of PTX loading RGO/Perylene-PCn
were analyzed by an Agilent high performance liquid chromatography (HPLC) system with a
Zorbax C18 column (150 mm × 4.6 mm, 5 μm, Agilent Technologies, USA). The mobile phase
was acetonitrile/water (52/48, v/v). The retention time for PTX was 5.0 min at 227 nm
detective wavelength. The drug loading content (DL) was defined as the mass percentage of
PTX present in PTX@RGO/Perylene-PCn composites, and encapsulation efficiency (EE) was
expressed as the percent of added drug that was entrapped in RGO/Perylene-PCn.
Cell cultivation
HUVEC, RAW264.7, L929 and SG7901 were grown and maintained in DMEM medium
supplemented with 15% FBS, 100 μg/mL streptomycin and 100 μg/mL penicillin in a culture
flask and were incubated in a 5% CO2 atmosphere at 37 °C.
Cytotoxicity assay
Viable cells were plated at a density of 5×105/mL in 96-well plates and cultivated until the
cell monolayer became confluent. RGO/Perylene-PCn was dispersed in cell culture medium
by ultrasonication at a concentration of 1 mg/mL. The samples were diluted down to 5
separate concentrations: 10, 25, 50, 75 and 100 μg/mL. According to the drug loading results,
PTX loading RGO/Perylene-PCn and free PTX were dispersed in cell culture medium by

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ultrasonication at a concentration of 60 μg/mL. Then, the samples were diluted down to 5
separate concentrations: 0.3, 1.5, 3.0, 6.0 and 15.0 μg/mL. A total of 100μL of the
dispersions were added to each well and cultured for another 24 h at 37°C. Next, 20 μL of
CCK-8 (5 mg/mL in culture medium) was added, and the samples were incubated for
another 1 h at 37°C. The absorbance of the solution at 450 nm was recorded with a Rayto
RT-6000 microplate reader. Every experiment was performed at least in sextuplicate.
Results and Discussion
Preparation and characterizations of GO and RGO
Firstly, GO and RGO were prepared and FTIR, XRD and XPS were used to characterize the
target products. The results were shown in Figure 2. Figure 2A shows the FTIR spectra of
graphite, GO and RGO. For GO, oxidation in sulfuric acid provides a typical carboxylic group
absorption peak at 1716 cm-1 and also provided the absorbance at approximately 1055 cm-1,
which corresponds to the other side groups containing C-O bonds such as epoxy, hydroxyl
etc. The huge absorption peak at about 3500 cm-1 is obviously from the hydroxyl groups. For
RGO, most of the side groups on GO have been eliminated in reduction reactions, especially
the carboxylate groups. But because the excess usage of hydrazine, which created some
defects in the RGO, there are still a few side groups bonded on the GO sheets reflected in
FTIR.
Normally, the formation of GO was the result of the expansion of the graphite, which led to
the increasing of the inter-lamellar spacing of pristine graphite that could be investigated
with XRD. Figure 2B showed the diffraction peaks of pristine graphite, GO and RGO samples.

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Compared to the graphite's inter-lamellar spacing of 0.34 nm calculated from Bragg’s
equation with determined diffraction angle (2θ = 26.4°), GO had a much higher value of 0.78
nm (2θ = 12.3°), due to the formation of oxygen-containing function groups on carbon basal
planes. The result agreed with the literatures and proved the successful preparation of GO 41,
42. After being reduced by Hydrazine Hydrate, the incisive peak of GO disappeared, a weak
and broad peak appeared at 2θ = 24.2°, indicating the restack of graphene sheets.
400
RGO
8.0k
(A)
(B)
Graphite
GO
200
0.34 nm
Graphite
0
6.0k
4.0k
2.0k
0.0
0.78nm
GO
10
20
30
2θ (°)
RGO
RGO
3800
3000
2200
1400
600
10
20
30
Wavenumber (cm-1)
2θ (°)
40k
20k
0
Survey Scan
C1s
O1s
300k (C)
150k
0
(D)
C1s
GO
GO
60k
30k
0
C1s
300k
150k
O1s
RGO
RGO
0
750
600
450
300
150
293
289
285
281
Binding Energy (eV)
Binding Energy (eV)
Figure 2. Spectrum characterization of GO and RGO. Where (A), FTIR; (B), XRD of graphite,
GO and RGO; (C), XPS survey scan of GO and RGO; and (D), XPS intensive scan for C1s of GO
and RGO
The XPS survey scan and C1s spectra of GO nanosheets were shown in Figure 2C and 1D. The
C1s spectrum of GO sheets can be fitted with three peak composites with binding energies

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at about 284.8, 286.6 and 289.0 eV, attributable to the C-C, C-O and O-C=O species,
respectively 43. Figure 2C also showed the survey scan spectra of RGO nanosheets from
chemical reducing. Comparing to the spectra of the GO nanosheets, the O1s signal of RGO
nanosheets at the binding energy of about 533eV decreased significantly, suggesting that
most of the oxygen-containing functionalities in GO have been reduced or removed. The XPS
C1s spectrum of the RGO nanosheets (Figure 2D) can be curve-fitted into two peak
components with binding energies at about 284.2 and 285.7eV, which belongs to the C-C
and C-N species, respectively 43
.
Chemical, physical and endocytosis properties of synthesized Perylene-PCn
Figure 3A shows the FTIR spectra of PTCDA and Perylene-PC. The spectra of the PTCDA used
by us is fully compatible with the standard spectra in HR Aldrich FTIR collection Edition II
(Spectra No. 11350). On the Perylene-PCn curve, the peak at 3400 cm-1 corresponds to the
hydroxyl group (-OH), while the double peaks at 3034 and 2956 cm-1 are associated with the
transformation of the methylene carbon. The absorbance at 964 cm-1 was assigned to a
tertiary amine group while the peak at approximately 1247 cm-1 is due to the synergistic
effect of the phosphate and the tertiary amine groups; the peak at 1489 cm-1 belongs to the
P-O-alkyl and the amide groups 44
.
1
The H-NMR spectra of Perylene-PCn in D2O was shown in Figure 3B. The incisive peak at
4.70 ppm is the resonance of H2O. The chemical shifts in the ranges of 0.86, 1.05 and 1.11
ppm belong to the methyl protons of the MPC and 2-bromoisobutyryl bromide, respectively.
The peak at 3.20 ppm is the methyl protons of the quaternary amine that comes from

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phosphorylcholine. The typical signals of Perylene at 8.0-8.5 ppm persist in the polymer
products, indicating that the Perylene moieties have been successfully attached to the
polymers. The number-average molecule weight of oligomeric PCn was determined by
integrating the area of peaks at 3.20 ppm and 8.0-8.5 ppm. Using the methyl protons of the
quaternary amine and protons on perylene core as the standards, the degree of
polymerization n is about 32, indicating that the number-average molecular weight is about
9800 g/mol.
(A)
(B)
PTCDA
9.0 8.5 8.0 7.5
ppm
Perylene-PCn
3800
3000
2200
1400
600
5
4
3
2
1
0
Wavenumber (cm-1)
Chemical Shift (ppm)
400
300
200
100
0
Perylene-PCn in water
1.00 mg/mL
Excited in water at 373 nm
0.5 mg/mL
(C)
(D)
1.2
0.9
0.6
0.3
0.0
0.75 mg/mL
1.0 mg/mL
0.50 mg/mL
2.5 mg/mL
5.0 mg/mL
400
500
600
700
800
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Figure 3. Characterizations of Perylene-PCn. Where (A). FTIR; (B). 1H-NMR spectra of
Perylene-PCn; (C). UV-Visible spectra; and (D). fluorescence excitation spectra of
Perylene-PCn in water
The optical properties of Perylene-PCn were investigated by UV-visible absorption spectra

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and fluorescence spectroscopy, as shown in Figure 3C. Three different concentrations of
Perylene-PCn water solution were investigated by UV-visible absorption, including 0.50, 0.75
and 1.00 mg/mL. The UV-visible absorption spectrum of Perylene-PCn in H2O shows the
presence of three adsorption bands at 475, 501 and 543 nm, corresponding to the 0→2,
0→1 and 0→0 electronic transiꢀons of perylene moiety 45. The maximum absorption is at
the wavelength of 501 nm which is the λ0-1 vibration band, whereas the λ0-0 adsorption band
at 543 nm is relatively weak, suggesting that Perylene-PCn aggregates in water to some
degrees. Obviously, the absorption is also related to the concentration itself, which increased
with the increasing of concentration. By using the Beer-Lambert law (A=εlC), the molar
absorption coefficient of Perylene-PCn at 501 nm can be calculated as 11418 L·mol-1·cm-1.
The concentration-dependent fluorescence behavior of the Perylene-PCn in water at the
excitation wavelength λex = 373 nm was also investigated and the results were shown in
Figure 3D. The photo luminescent properties of perylene was retained in Perylene-PCn. A
well-resolved vibronic pattern was shown. The fluorescence intensity and the patterns of
Perylene-PCn in aqueous solution varied a lot at different concentrations, due to the
self-quenching of perylene dye at high concentration. At the concentration of 5.0 mg/mL,
the fluorescence intensity was less than 200 a.u. and showed single fluorescence
wavelength at about 600 nm. When the concentration was diluted to 2.5 mg/mL, the
fluorescence intensity at 600 nm became stronger and a weak fluorescence-emission
appeared at about 550 nm. By further diluting the concentration to 1.0 mg/mL, two
fluorescence-emission bands with nearly equal intensity presented and both of the bands
were stronger than the ones observed at higher concentration. By even diluting the

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concentration further to 0.5 mg/mL, the emission at about 550 nm was even stronger than
that at 600 nm and the fluorescence excitation band showed obvious red shifting, which was
similar to the previous reports 24
.
Figure 4. Cytotoxicity assay (A) and confocal fluorescent image of endocytosis of
Perylene-PCn in HUVECs (B)
The in vitro cytotoxicity of Perylene-PCn were investigated to evaluate its biomedical
applications. The studies were performed against three different cell lines, HUVEC,
RAW264.7 and L929 at a series of concentrations for 24 h. The results were shown in Figure
4A. The relative cell proliferations data were determined by CCK-8.
Among the three different cell lines, after exposure to Perylene-PCn HUVECs showed
relatively lower extent of proliferation than that of L929 and RAW264.7. However,
comparing to the blank control group, even after raising the Perylene-PCn concentrations
from 10 μg/mL to 200 μg/mL, the relative proliferations of all three cells exposure to
Perylene-PCn remained higher than 80% and show no statistical difference. The results
suggest that HUVEC is the most sensitive cell lines among the three studied here to the

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exotic substance, but even for HUVEs Perylene-PCn showed very low cytotoxicity after a
24-hour exposure.
As shown in Figure 3D, Perylene-PCn was presented with a high photoluminescence
quantum yield with recognizable green fluorescence, therefore can be potentially used as a
fluorescent probe. Based on this observations, we investigated the fluorescent images of
HUVECs exposure to Perylene-PCn for 24 h with a confocal microscopy. A typical photograph
was shown in Figure 4B. As shown in Figure 4B, Perylene-PCn permeated through the cell
membrane and distributed in cytoplasm, emitting intensive green fluorescence. However, in
the nucleus, no fluorescence was observed, which indicated that Perylene-PCn cannot enter
cell nucleus because of its macromolecular properties.
Non-covalent hydrophilization of RGO with Perylene-PCn
Figure 5A shows the Raman spectra of graphite, GO, RGO and RGO/Perylene-PCn
composites, which was roughly identical with the previous report 46. In the Raman spectra,
the original graphite has a weak D band at 1350 cm-1 and an incisive G band at 1580 cm-1 47
.
However, The D band of GO and RGO increased significantly. In addition, the G band shifted
to higher frequencies due to graphite amorphization. Both of the observations confirmed
the lattice distortions caused by oxidation and reduction processes. Furthermore, the 2D
band at 2700 cm-1 is highly sensitive to stacking of graphene sheets 48. The Raman spectrum
of graphite shows an incisive band relating to its multilayer structure. After oxidation and
reduction, GO and RGO showed an decreased and broadened peak at around 2700 cm-1,
which indicated a mixed single and few-layer structure 48. The new peak at about 2940 cm-1

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(D+G) also proved the disorder-induced combination mode 49. Moreover, The GO layers will
agglomerate while using excess amount of hydrazine and creates defects in the RGO. Raman
spectra of RGO showed that the defect content in RGO is increased as compared to GO. The
sp3 hybridization of GO should change to sp2 in the RGO resulting lower defects. However,
the present data shows that the defect in RGO is higher as compared to GO. It may be
attributed to the use of excess hydrazine37. After been compounded with Perylene-PCn, the
D band of RGO at 1350 cm-1 transformed to a double peak at 1290 and 1369 cm-1, which
indicated the further disordering of carbon bonds in the composite caused by the
introduction of Perylene-PCn polymeric structures. The changes in Raman spectra proved
the successfully synthesis and preparation of RGO/Perylene-PCn composites.
10k
RGO/Perylene-PCn
(A)
(B)
8k
6k
Graphite
N1s
393
409
405
292
401
288
397
GO
40k
20k
0
RGO
C1s
280
RGO/Perylene-PCn
1000
1500
2000
2500
3000
3500
296
284
Raman shift (cm-1)
Binding Energy (eV)
400
300
200
100
0
Excited in water at 373 nm
(C)
(D)
100
75
50
25
0
RGO
RGO/Perylene-PCn
Perylene-PCn 0.5 mg/mL
GO
Perylene-PCn
RGO/Perylene-PCn
570 620
150
300
450
600
520
670
720
Temperature (oC)
Wavelength (nm)
Figure 5. Characterizations of RGO/Perylene-PCn composites. Where (A). Raman spectra; (B).

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XPS intensive scanning of C and N in RGO/Perylene-PCn; (C). TGA analysis; and (D).
Fluorescent spectra of composite and its compositions.
As shown in Figure 5B, the XPS C1s spectrum of the resulting RGO/Perylene-PCn composites
could be curve-fitted into three peak components with binding energies at 284.2, 286.0 and
288.7eV, attributable to the C-C, C-O/C-N and O-C=O species, respectively. The increase in
relative intensity of the C-O peak component and the appearance of the C-N peak
component comparing to RGO XPS spectra shown in Figure 2D, and N1s signal shown in
Figure 5B were consistent with the successful composition of RGO and Perylene-PCn. The
XPS N1s core-level spectrum of the resulting RGO/Perylene-PCn composites could be
curve-fitted into two major peak components with binding energies at about 399.4 and
402.3 eV, attributable to the bisimide group and the quaternary amine species, indicating
that both phosphorylcholine groups and perylene bisimide core were introduced.
Figure 5C shows the TGA curves of GO, RGO, Perylene-PCn and RGO/Perylene-PCn
composites at a heating rate of 20 °C/min in nitrogen atmosphere. For GO, the 12% weight
loss below 120 °C should due to the evaporation of the residual adsorbed and combined
water which occupied the spaces between the GO layers. By continuing heating up to 300 °C,
the labile oxygen-containing functional groups decomposed gradually. The fasted
decomposition temperature was recorded at about 220 °C, at which about 30% weight loss
was observed. After that, the slowly weight loss was due to the decomposition of the carbon
backbone which continued until 700 °C, when 30% mass remained. RGO exhibited better
thermal stability. It was stable over a wide temperature range, with only about 10% mass
lost due to the removal of the residual water at temperature below 120 °C and only about 5%

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mass lost beyond 120 °C in a wide temperature range due to the decomposition of the
residual oxygen functional groups. The TGA curve of the RGO/Perylene-PCn composites
showed a major weight loss between 300-400 °C due to the decomposition of Perylene-PCn,
which was consistent with the decomposition of Perylene-PCn itself as shown on the TG
curve. The consistency indicated that the interaction of RGO and Perylene-PCn was π-π
stacking rather than covalence bonds. The main residual component in the
RGO/Perylene-PCn composites at 700 °C was RGO with a weight content of 50%, indicating
that the ratio of RGO to Perylene-PCn was about 5:2.
The fluorescence spectrum of the RGO/Perylene-PCn composites in water obtained at an
excitation wavelength λex=373 nm was shown in Figure 5D. The fluorescence emission of the
RGO/Perylene-PCn composites was completely quenched due to the charge or energy
transfer, consistent with the presence of strong π-π stacking interactions between the
perylene moieties and the RGO nanosheets.

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Figure 6. Water dispersion photographs (A), AFM images (B), Thickness distribution in AFM
(C), and π−π stacking model (D) of RGO/Perylene-PCn composite.
Figure 6A shows the photographic images of aqueous dispersions of Perylene-PCn, GO, RGO,
and
RGO/Perylene-PCn.
With
modifications
by
the
water-soluble
poly(2-methacryloyloxyethyl phosphorylcholine) segments, the insoluble PTCDA dissolved in
water homogenously. GO was also presented as a pseudo-homogeneous yellow-brown
dispersion for the hydrophilic oxygen containing groups’ modification, but RGO completely
precipitated after been reduced. In the RGO/Perylene-PCn system, it was obvious that RGO
was solubilized by Perylene-PCn. The RGO/Perylene-PCn composites remained stable and
uniformly dispersed in the aqueous solution for at least three days, indicating the successful
π-π stacking interaction between RGO nanosheets and perylene core.

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The AFM images of GO and RGO/Perylene-PCn, as shown in Figure 6B, further confirmed the
interaction between RGO sheets and Perylene core. The original GO sheets that were
spin-coated on a mica substrate showed an ultra-flat like slice, had smooth edges and had a
thickness of 1.0 nm. For the samples of RGO/Perylene-PCn, due to the agglomeration and
the attachment of Perylene-PCn onto RGO surface, the surface became rough and had
various bulges with average heights of 40 nm. Some ultra-flat sheets with a thickness of 1.0
nm were also seen in the images, indicating that some surface was not covered by
Perylene-PCn. Figure 6C shows the AFM thickness distribution of GO and RGO/Perylene-PCn
composites studied at different areas of the substrates. It shows that the thickness of ~70%
of GO sheets is in the 0.9-1.3 nm range and the thickness of ~18% of GO sheets is in the
1.3-1.5 nm range. After being composed as RGO/Perylene-PCn, the thickness of ~78% of
composites is in the 15-60 nm thickness. Thus, few layer graphene sheets were formed
during the synthesis of GO, while most of the RGO sheets become much more thick because
of the agglomeration and the covering by Perylene-PCn.
In summary, RGO interacted with Perylene-PCn through π-π stacking as shown in Figure 6D.
As for GO, after been reduced by hydrazine, most of the oxygen containing groups on
graphene nanosheets were removed and their blocking effects no longer exist, the graphene
nanosheets then tended to restack due to the strong Van de Waals force. When
Perylene-PCn solution was added into the RGO dispersion, with a slight ultrasonic treatment,
the perylene core was attached to RGO surface through π-π stacking interaction. Once
enough Perylene-PCn attached onto the RGO nanosheets, due to the excellent water
solubility of PCn segments, the RGO/Perylene-PCn formed a stable dispersion in water again.

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Paclitaxel loading on RGO/Perylene-PCn
Figure 7A shows the FTIR spectra of PTX, RGO/Perylene-PCn composites and PTX loaded
RGO/Perylene-PCn composites (PTX@RGO/Perylene-PCn). Several characteristic FTIR
absorption peaks of PTX appeared in the spectra of PTX@RGO/Perylene-PCn, including the
broad absorption at 3400-3500 cm-1, the enhanced peak at 1720 cm-1 related to the
combination of carbonyl groups which exist in both PTX and RGO/Perylene-PCn, the peak at
1650 cm-1 related to the amido linkage in PTX molecule, and the peak at 712 cm-1 related to
the mono-substituted benzene structure in PTX. The result indicated the successful loading
of PTX on RGO/Perylene-PCn.
The PTX loading content and encapsulation efficiency were assessed by HPLC. As shown in
Figure 7B, the drug loading content increased with the increasing of weight ratio of PTX to
RGO/Perylene-PCn, and reached as high as about 25% at the ratio of 3. Considering both the
economic aspect and efficiency, to add 1 equivalent of PTX to 1 equivalent of
RGO/Perylene-PCn composites should be the best choice. The loading capacity of PTX on
RGO/Perylene PCn is about 15% at this ratio.
100
(A)
(B)
PTX
80
60
40
20
0
PTX Loading Efficiency
PTX Loading Content
RGO/Perylene-PCn
PTX@RGO/Perylene-PCn
3800
3000
2200
1400
600
0
1
2
3
Wavenumber (cm-1)
Mass Ratio of PTX:RGO/Perylene-PCn
Figure 7. (A). FTIR of PTX, RGO/Perylene-PCn and PTX@RGO/Perylene-PCn; (B). Drug loading

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efficiency and contents of PTX on RGO/Perylene-PCn composites
Cytotoxicity assay of RGO/Perylene-PCn, PTX and PTX@RGO/Perylene-PCn
In our previous work, we demonstrated that GO showed cytotoxicity with a dose dependent
manner. Because of the huge specific surface area and hydrophobic conjugated aromatic
structure of GO, many cell membrane biomolecules, such as peptides, proteins,
polysaccharides and other molecules with hydrophobic moiety can be attracted and
adsorbed by GO sheets, which in turn wrapped the cells by aggregation and then affected
the normal function of cell membrane44. After covering the RGO surface with Perylene-PCn,
it was highly likely that the surface of RGO/Perylene-PCn was covered by hydrophilic
phosphorylcholine oligomer which hindered the interaction between the conjugated
aromatic structure and cell membrane molecules, thus improve the biocompatibility of
RGO/Perylene-PCn. In order to verify the reduction in cytotoxicity and also to evaluate the
anti-tumor efficiency of PTX@RGO/Perylene-PCn system, in vitro cytotoxicity assays were
carried out and the results were shown in Figure 8.
SGC7901
RGO/Perylene-PCn
for 24 h at 37 °C
L929
Free PTX
PTX@RGO/Perylene-PCn
Free PTX
PTX@RGO/Perylene-PCn
(A)
(B)
(C)
150
100
50
n=6, * p<0.05
n=6, * p<0.05
L929
SGC7901
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
0
10 50 100 200 400
0
3
6
15 30 60
0
3
6 15 30 60
Dose (ꢀg/mL)
Figure 8. Cytotoxicity assay of RGO/Perylene-PCn, PTX and PTX@RGO/Perylene-PCn. Where
A. Cell compatibility assay of RGO/Perylene-PCn against L929 and SGC1901 cells; B. L929

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cytotoxicity of free PTX and PTX@RGO/Perylene-PCn; and C. anti-tumor comparison of free
PTX and PTX@RGO/Perylene-PCn using SGC7901 as model cells.
Figure 8A shows the relative cell proliferation of SGC7901 and L929 cell lines after being
exposed to RGO/Perylene-PCn composites for 24 h at the dosages ranged from 10 to 400
μg/mL. Under all of the doses, both cell lines proliferated well and the relative cell
proliferations showed no statistical differences comparing to the control group, proving the
good cell-compatibility of RGO/Perylene-PCn composites.
The in vitro cytotoxicity of PTX@RGO/Perylene-PCn was further evaluate using SGC7901 cell
line. L929 was used as a comparison. The result indicated the anti-tumor properties. Figure
8B and 7C shows the relative proliferations of two cell lines after being exposed to PTX and
the PTX@RGO/Perylene-PCn for 24 h. The dose range was 10 to 60 μg/mL. In
PTX@RGO/Perylene-PCn, the dosage was calculated as the loaded free PTX (with PTX
content 15%). For L929 cell lines, the exposure to free PTX led to significant cell cytotoxicity,
even at the lowest dose of 3 μg/mL. However, as the dose was raised, the cell apoptosis did
not increase accordingly. After PTX was loaded onto RGO/Perylene-PCn, cytotoxicity can only
be observed at the concentration as high as 60 μg/mL. For SGC7901, a typical gastric
carcinoma cell line, the introduction of PTX inhibited the cell proliferation remarkably. With
a low PTX concentration of 6 μg/mL, the relative cell proliferation was reduced to less than
50%. A dose dependence decreasing was observed. The same trend was also observed in
the assay of SGC7901 being exposed to PTX@RGO/Perylene-PCn, but relatively higher
proliferation was observed. The result suggested that the wrap of PTX by RGO/Perylene-PCn
could reduce the cytotoxicity of free PTX. It is beneficial for the situations where the

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reduction of the side effect of PTX is necessary.
Conclusions
A water-soluble and biocompatible Perylene-PCn was synthesized by a typical ATRP
procedure, while GO and RGO was prepared according to Hummer’s method.
RGO/Perylene-PCn composite was fabricated through π-π stacking interactions between
RGO and Perylene moiety. The properties of the composites were extensively investigated
and the structures were proved. Furthermore, PTX was successfully loaded in
RGO/Perylene-PCn. Using the typical tumor cell line SGC7901 as a model system, the in vitro
cytotoxicity assay of PTX@RGO/Perylene-PCn presented the similar anti-tumor efficacy
comparing to free PTX. The results highlighted RGO/Perylene-PCn as an anti-tumor agent
delivery vehicle for potential oncology applications.
Acknowledgements
The authors acknowledge the support of Science and Technology Support Program (Society
Development) of Jiangsu Province (No. BE2012735), the National Basic Research Program of
China (No. 2011CB933400), the Foundation of Jiangsu Provincial Key Laboratory for
Interventional Medical Devices (No. JR1504). The works are also a part of the Project Funded
by the Priority Academic Program Development of Jiangsu Higher Education Institutions
(PAPD).
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116x61mm (300 x 300 DPI)