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Preparation of millimetre-sized mesoporous carbon spheres as an effective

bilirubin adsorbent and their blood compatibility

Article  in  Chemical Communications · October 2010

DOI: 10.1039/c0cc02060e · Source: PubMed

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COMMUNICATION
Preparation of millimetre-sized mesoporous carbon spheres as an
effective bilirubin adsorbent and their blood compatibilityw
Limin Guo,a Jiamin Zhang,b Qianjun He,a Lingxia Zhang,a Jinjin Zhao,a Ziyan Zhu,b
Wei Wu,a Jing Zhangc and Jianlin Shi*a
Received 24th June 2010, Accepted 19th July 2010
DOI: 10.1039/c0cc02060e
Millimetre-sized mesoporous carbon spheres (MMCSs) with
smooth surface and penetrating mesoporous channels have been
Published on 20 August 2010 on http://pubs.rsc.org | doi:10.1039/C0CC02060E
successfully prepared by an emulsion-EISA technique, and are
Downloaded by Shanghai Institute of Ceramics, CAS on 10 April 2012
found to be a much better bilirubin adsorbent than commercial
activated carbon spheres. Hemolysis and coagulation assays of
MMCSs indicate that they have negligible hemolysis effect and
do not induce blood coagulation.
Mesoporous carbon materials have attracted considerable
attention since the first report on the synthesis of CMK-1 in
the late 1990s for their remarkable structural properties, such
as high specific surface area, large pore volume, chemical
inertness and good mechanical stability.1–4 A variety of potential
applications of mesoporous carbon materials has also been
reported, such as energy storage,5,6 catalyst support,7,8 drug
delivery9 and adsorption and separation of organic molecules.10
Recently, carbon nanomaterials have received considerable
attention for biological applications:9,11,12 Hurt’s group
synthesized size-tunable carbon nanoparticles without metal
impurities. These nanoparticles cause no observable cytotoxicity
after 72 h and show rapid internalization with preferential
perinuclear location.11 Lin’s group has found mesoporous
carbon nanoparticles are fairly biocompatible in vitro and
could serve as a transmembrane carrier.9 Recently, our group
has found that mesoporous carbon materials have excellent
adsorption properties for bilirubin,13,14 which is one of the
most important pathogenic substances in human blood.15
If mesoporous carbon materials could be used as the clinical
adsorbents for blood purification, a new and meaningful
application of mesoporous carbon materials might be
achieved. Fig. S1 in Electronic Supplementary Information
(ESI) is the typical scheme of the practical blood perfusion and
the adsorbents are added into the one-off adsorption column.
In order to make sure that the blood cells around ten micron in
size can conveniently and safely travel through the adsorption
column during the therapy process, diameters in millimetre
a
State Key Laboratory of High Performance Ceramics and Superfine
Microstructure, Shanghai Institute of Ceramics,
Chinese Academy of Science, Shanghai 200050
People’s Republic of China. E-mail: jlshi@sunm.shcnc.ac.cn;
Fax: +862152413122; Tel: +862152412712
b
Shanghai (Red Cross) Blood Center, Shanghai Institute of Blood
Transfusion, Shanghai 200051, P. R. China
c
Laboratory of Nanotechnology,
Shanghai Nanotechnology Promotion Center, Shanghai 200237
P. R. China
w Electronic supplementary information (ESI) available: The experimental
section, SEM images, typical scheme of the practical blood perfusion
and UV-vis adsorption spectra. See DOI: 10.1039/c0cc02060e
This journal is c The Royal Society of Chemistry 2010
View Online / Journal Homepage / Table of Contents for this issue
www.rsc.org/chemcomm | ChemComm
level of carbon spheres is the one of the preliminary demands.
So activated carbon absorbents currently used in clinics for blood
perfusion are millimetre sized spheres (Fig. S2). However, the
activated carbon materials have some well-known drawbacks,
such as slow mass transport of molecules because of space
confinement imposed by small pore sizes mostly less than 1 nm,
incapable of adsorbing large target molecules and collapse of
porous structures during high-temperature treatments.1,10 The
mesoporous carbon materials can well conquer the above
mentioned drawbacks, so the preparation of millimetre-sized
mesoporous carbon spheres (MMCSs) will be the key to solve
the problem. Although many mesoporous carbon materials
with different morphologies and structures such as hollow
mesoporous carbon spheres,16,17 mesoporous carbon spheres
with tunable size18,19 and hierarchical porous carbon materials,
have been synthesized,20 no report on the preparation of
MMCSs can be found up to now.
Here we report an emulsion-EISA (evaporation-induced
self-assembly) strategy, which is illustrated in Fig. 1, to prepare
MMCSs. At first, the precursor ethanol solution, containing
triblock copolymer F127 and carbon precursor polymer oligomers
(phenolic resols), was dispersed into a hot oil medium as
emulsion droplets. With the thermal evaporation of ethanol,
which progressively increased the concentration of F127 and
oligomers in emulsion droplets, the spontaneous self-assembly
took place, leading to the formation of mesophase hybrids in a
microscopic process. Afterwards the primary droplets underwent
continuous coalescence into larger spherical polymer particles
by thermal polymerization under stirring. After filtration and
Fig. 1 The scheme of the synthesis procedure of MMCSs: Step 1:
EISA-emulsion process to millimetre sized copolymer spheres which
includes the microscopic self-assembly process (step 1a) to form
mesophase hybrids and the macroscopic coalescence process (step 1b)
to form millimetre sized spheres by corresponding mesophase hybrids.
Mesostructure was formed during solvent evaporation; Step 2:
Filtration of the mixture to obtain millimetre sized polymer particles;
and Step 3: Carbonization of the polymer particles into millimetre
sized mesoporous carbon spheres (MMCSs).
Chem. Commun., 2010, 46, 7127–7129 7127

Published on 20 August 2010 on http://pubs.rsc.org | doi:10.1039/C0CC02060E
Downloaded by Shanghai Institute of Ceramics, CAS on 10 April 2012
Fig. 2 (a) Macroscopic image of MMCSs; (b) TEM image of ground
MMCSs; (c) low resolution SEM surface image of MMCSs (the
arrowed substrate is conductive adhesive) and (d) high resolution
SEM surface image of MMCSs.
carbonization, mesoporous carbon spheres of millimetre size
were obtained. The synthesis and characterization details are
shown in the ESI experimental section.
Fig. 2a is a digital picture of as-prepared MMCSs, which are
spherical and millimetre level in diameter. The diameters
are distributed in a range of 1.08–1.90 mm as determined by
randomly measuring 100 MMCSs. Then the MMCSs were
ground into fine powder for TEM observation and the represent-
ative TEM image (Fig. 2b) demonstrates that MMCSs have
worm-like mesoporous channels. SEM was used to observe the
surface of MMCSs. A low resolution SEM image of MMCSs
(Fig. 2c) shows a smooth surface of MMCSs, meanwhile in a
high resolution SEM image of MMCSs (Fig. 2d) relatively
uniform but randomly distributed mesopores can be clearly
observed. The mesopore can be further clearly observed in a
magnified image in Fig. S3. According to above testing results of
MMCSs, we found that the as-prepared MMCSs have macro-
scopical smooth surface, millimetre level diameter, and worm-like
mesochannels. Furthermore, the mesoporous channels are penet-
rating through the whole MMCSs, which facilitate the molecular
diffusion and mass transport. Nitrogen sorption isotherms were
recorded to investigate the pore properties of the MMCSs.
Fig. 3 shows the nitrogen sorption isotherms of MMCSs and
the isotherms are type w with a marked leap on the adsorption
branch at P/P0 = 0.6–0.9, which is typical of mesoporous
materials. The BJH pore size distribution (Fig. 3 inset) exhibits
a narrow mesopore distribution between 5 nm and 11 nm,
consistent with the results of high resolution SEM imaging
(Fig. 2d and Fig S3). The BET surface area is measured to be
555.3 m2 g 1 and BJH pore volume is 0.60 cm3 g 1.
Hemolysis and coagulation assays were conducted to evaluate
the blood compatibility of MMCSs. Here, commercial activated
carbon spheres for clinical blood purification were used as a
reference (R-ACS). For hemolysis assays, the red blood cells
(RBC) suspensions (0.3 mL for each sample) were mixed with:
(a) 1.2 mL of PBS suspensions as a negative control; (b) 1.2 mL
7128 Chem. Commun., 2010, 46, 7127–7129
View Online
Fig. 3 N2 adsorption and desorption isotherm of MMCSs and
corresponding pore size distribution curve.
deionized water as a positive control; (c) 0.03 g MMCSs and
1.2 mL PBS solution; and (d) 0.03 g R-ACS and 1.2 mL PBS
solution. For (a) and (b), the mixtures were vortexed and then
allowed to rest for 2 h at room temperature, and for (c)
and (d), the mixtures were vortexed every 15 min in order to
ensure sufficient interactions between ACSs or MMCSs and
RBC, and the total treatment time is also 2 h. After above
treatments, the samples were centrifuged. During the hemo-
lysis assay experiments, hemoglobin will be released into the
solution by hemolysis, and the resulting solution become
visually red. The denser red color of corresponding solution
means the higher hemolytic activity, therefore, the intensity of
absorbance at 541 nm can be used to determine the quantity
of hemoglobin released by hemolytic effect in solution. The
result of hemolysis assay in the supernatant is shown in Fig. 4.
No visible hemolysis effect can be observed visually for both
ACSs and MMCSs. The RBC hemolyzed effect was further
determined by measuring the absorbance of supernatants at
541 nm (hemoglobin) by UV-visible spectroscopy (Fig. S4).
From the above results, we can find that MMCSs have very
low, i.e., negligible hemolytic activity.
Conventional clinical blood coagulation assays, prothrombin
time (PT) and activated partial thromboplastin time (APTT) were
employed to test the blood coagulation effects of MMCSs and
R-ACSs (as a reference). PT was used to evaluate the extrinsic
and common coagulation pathway and APTT was used to
evaluate the intrinsic and common coagulation pathway.21,22
Data were taken from three parallel tests for each sample and
are shown in Table 1. There is no significant statistical difference
among the PT values in fresh human platelet poor plasma (PPP),
Fig. 4 Hemolysis assay for ACSs and MMCSs, using PBS as a
negative control (left) and water as a positive control (right). The
mixtures were centrifuged to detect the presence of haemoglobin in the
supernatant visually.
This journal is c The Royal Society of Chemistry 2010

Table 1 The PT and APTT values of PPP, R-ACSs and
MMCSs samples
Samples PT/s APTT/s
PPP 17.2  0.1 35.7  0.2
R-ACSs 17.1  0.3 34.6  0.4
MMCSs 16.8  0.2 35.0  0.5
Published on 20 August 2010 on http://pubs.rsc.org | doi:10.1039/C0CC02060E
Downloaded by Shanghai Institute of Ceramics, CAS on 10 April 2012
Fig. 5 (a) The change of bilirubin concentration as a function of time
using MMCSs as adsorbent and (b) bilirubin equilibrium adsorption
isotherms of MMCSs.
MMCSs and R-ACSs. The results indicate that both MMCSs
and ACSs do not induce any significant coagulation.
MMCSs were used as adsorbents to remove bilirubin from
as-prepared bilirubin PBS solutions. In order to measure
the bilirubin concentration changes as a function of time,
MMCSs were added into 250 mg L 1 bilirubin PBS solution
and Fig. 5a is the corresponding adsorption results. It can be seen
that the bilirubin concentration decreases rather quickly at the
initial stage and then decreases further but slowly afterwards.
Equilibrium adsorption is achieved in approximately 120 min
and the equilibrium bilirubin concentration is 64.2 mg L 1.
Under the same adsorption conditions, the equilibrium
adsorption of ACSs is achieved up to 300 min and the
equilibrium concentration is up to 110.3 mg L 1.14 To determine
the bilirubin adsorption capacity of MMCSs, equilibrium adsorp-
tion experiments were introduced. Fig. 5b shows the equilibrium
adsorption curve of bilirubin on MMCSs at room temperature.
The adsorption capacities of MMCSs increase with the increasing
equilibrium concentrations. However, up to a certain equilibrium
concentration, no more bilirubin can be adsorbed on MMCSs.
Therefore, it can be estimated that the adsorption capacity of
MMCSs for bilirubin is 148.4 mg g 1, as compared to 70 mg g 1
of ACSs.14 This indicates that MMCSs have much higher
bilirubin adsorption rate and capacity than commercial ACSs.
Bilirubin is a tetrapyrrole dicarboxylic acid and a kind of
lipophilic endotoxin, and there is hydrogen bonding between
This journal is c The Royal Society of Chemistry 2010
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bilirubin molecules.23,24 So MMCSs with much larger pore size
(5–11 nm) than ACSs, which are dominated by micropores, can
provide a much more expeditious pathway and available pore
volume for bilirubin transfer and storage than ACSs.
In summary, we have successfully prepared millimetre-sized
mesoporous carbon spheres (MMCSs) by an emulsion-EISA
technique. The MMCSs have high surface area (555.3 m2 g 1)
and pore volume (0.60 cm3 g 1). Furthermore, the mesoporous
channels are penetrating through the whole MMCSs. Hemolysis
and coagulation assays of MMCSs indicate that they have
negligible hemolysis effect and do not induce blood coagulation.
MMCSs are found to be a much better bilirubin adsorbent
than commercial ACSs with much enhanced adsorption rate
and capacity, and therefore are very promising for applications
in practical blood perfusion.
The authors gratefully acknowledge the support by National
Nature Science Foundation of China (Grant Nos. 20633090
and 50823007), National 863 High-Tech Program (Grant No.
2007AA03Z317), Shanghai Rising-Star Program (Grant No.
07QA14061, 08QA14074), Shanghai Nano-Science Project
(Grant No. 0852nm03900) and CASKJCX Projects (Grant
Nos. KJCX2-YW-M02 and KJCX2-YW-210).
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