C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9

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Purification and separation of carbon nanocapsulesas a magnetic carrier for drug delivery systems

Sunghoon Kim*, Etsuro Shibata, Ruslan Sergiienko, Takashi Nakamura

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 1,1 Katahira, 2-Chome, Aobaku, Sendai 980-8577, Japan

A R T I C L E I N F O

Article history:

Received 11 March 2008

Accepted 1 May 2008

Available online 28 June 2008

0008-6223/$ - see front matter � 2008 Elsevidoi:10.1016/j.carbon.2008.05.027

* Corresponding author: Fax: +81 22 217 5214E-mail address: stelmo@mail.tagen.tohok

A B S T R A C T

Nanoparticles of iron carbides wrapped in multilayered graphitic sheets (carbon nanocap-

sules) were synthesized by electric plasma discharge in an ultrasonic cavitation field in

liquid ethanol. The as-prepared material contained carbon nanocapsules but also included

impurities such as amorphous carbon, graphite balls and flakes. These impurities must be

removed to allow the development of applications for the carbon nanocapsules and, it is

necessary to develop a suitable purification method to obtain samples of monodispersed

carbon nanocapsules. Two purification methods were used. The first used a hydrogen per-

oxide solution and acid etching to remove the amorphous carbon and iron particles. These

impurities were easily oxidized and dissolved in the solution and the graphite layers of the

carbon nanocapsules were not damaged. The second used magnetic separation using per-

manent magnets to remove the graphite balls and flakes. Here, centrifugation was applied

to separate the purified carbon nanocapsules around 100–200 nm in size.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Magnetic nanoparticles of pure metals such as Fe, Co and Ni

are used as materials in various fields of magnetism. The

main difficulty using pure metals arises from their instability

towards oxidation in air, which become easier as the metal

size becomes smaller. One approach to chemical stabilization

is the formation of protective shells around the nanoparticle

surface, which thus prevent the reaction of oxygen with the

surface atoms [1,2].

Indeed, magnetic nanoparticles coated with protective

shells are widely used in technological applications such as

in the transport of anticancer drugs [3], and in magnetic

recording materials [4] and have great potential in in vivo bio-

medical applications such as in the contrast enhancement of

magnetic resonance imaging (MRI) [5–9]. The carbon coating

of metals presents an effective protection method against

environmental degradation and has excellent adhesive bond-

ing with the surface of the metal particles. Such carbon shells

er Ltd. All rights reserved

.u.ac.jp (S. Kim).

are airtight and protect the entrapped materials from oxida-

tion, and they are generally known as ‘‘carbon nanocapsules’’.

Such nanocapsules are proposed as a new approach of the

magnetic carriers for the administration of drugs and vac-

cines. One of the prospective medical applications of carbon

nanocapsules can be the drug delivery to the specified targets

which is controlled by a magnetic field [11–13]. The sizes of

carbon nanocapsules are generally smaller than those of cells

(10–100 lm), viruses (20–450 nm), proteins (5–50 nm) or genes

(2 nm wide and 10–100 nm long) [10]. This means that these

nanocapsules can ‘get close’ to a biological entity of interest.

The targeted drug delivery using magnetic particles and an

external magnetic field imposes several requirements on

the adsorbent particles. The particles themselves must be

biologically inert and biodegradable. Moreover, they must also

have a high sorption capacity for the drug, and the rate of

drug desorption in an organism needs to be slow so that the

high concentration of the cytostatic drug can be maintained

in the tumor area for a prolonged period of time. Since the

.

1524 C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9

particles must be electively controlled by the applied mag-

netic field, both their magnetic properties and their dispersity

and agglomeration degree are important.

In this study, the carbon nanocapsules prepared by electric

plasma discharge in an ultrasonic cavitation field of liquid

ethanol, usually include impurities of the exposed metals

and other forms of carbon such as amorphous carbon, graph-

ite balls and flakes. Also, the electric plasma discharge meth-

od synthesizes particles with a wide size distribution. Thus,

the synthesized carbon nanocapsules are required to be puri-

fied to efficiently remove the impurities and control the size

distribution of the nanoparticles by a size-selection process

to be effective for further technological applications.

One of the conventional techniques for removing amor-

phous carbon involves the thermal or plasma cleaning with

Ox (x � 2) [13], while another technique concerns the treat-

ment with a toxic gas such as halogen [14]. However, these

techniques attack the graphite shell as well as amorphous

carbon, and may also require the disposal of hazardous sub-

stances. So, in this study, we have conducted a selective-oxi-

dation method using hydrogen peroxide, which is one of the

most safest and effective methods [15,16] for removing amor-

phous carbon, followed by acid treatment to remove the me-

tal impurities. Then, magnetic separation was conducted in

order to remove graphite balls and flakes, and centrifugation

was used to separate carbon nanocapsules around 100–

200 nm in size.

Fig. 1 – Diagram of the modifie

2. Experimental

The principle of the present method of electric plasma dis-

charge in organic liquids has previously been demonstrated

in experiments using liquid benzene and ethanol [17–21].

We modified the experimental apparatus (see Fig. 1) for the

synthesis of carbon nanocapsules. Plasma discharge was gen-

erated between one Fe electrode–anode (Ø 4 mm, purity

99.9%) and the bottom of the iron tip (Ø 18 mm, purity

99.9%) fixed on the top of a titanium ultrasonic horn which

served as a cathode.

An ultrasonic homogenizer (Nissei, US-600NCVP) was used

at 600 W and 20 kHz in the experiments to irradiate 300 ml of

liquid ethanol (Wako, S-grade). During the ultrasonic irradia-

tion, the voltage between the electrode–anode and iron tip-

cathode was kept at 55 V using a regulated DC power supply

(PAS 60-18), and the current of the power unit was set at

3.0 A. The distance between the iron electrode–anode and

the bottom of the iron tip-cathode was automatically ad-

justed using a motorized Z-axis precision stage (Sigma Koki

Co., Ltd., SGSP80-20ZF) connected to a programmable stage

controller (SHOT-602) for driving a stepping motor. Plasma

discharge occurred between the iron tip and electrode–anode

at a distance of about 100 lm or less. However, without ultra-

sonic irradiation in the electrode gap, no plasma was gener-

ated using this low electric power supply. During the

experiments, the Fe electrode–anode was mainly consumed

d experimental apparatus.

C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9 1525

by thermal evaporation. The glass vessel was cooled in a

water bath and an argon gas flow was directed into the vessel

to maintain an inert atmosphere.

After the experiment, the synthesized carbonaceous pow-

der was separated from the liquid ethanol by centrifugation

and vacuum evaporation. A 200 mg sample of the as-prepared

powder was treated in 200 ml of 25% hydrogen peroxide

(Wako) solution refluxed at 90 �C for 40 h. After the hydrogen

peroxide treatment, the purified powder was separated by

centrifugation from the remaining hydrogen peroxide and

etched in 15% HCl solution for 4 h at room temperature to re-

move iron oxide particles. Finally, the purified carbon nano-

capsules were washed with distilled water five times, and

dried at 40 �C for 24 h in a vacuum oven.

The purified sample was dispersed in 50 ml of ethanol

solution by ultrasonic homogenization for 10 min. The graph-

ite balls and flakes were easily removed by magnetic separa-

tion [22,23]. The magnetic separator design includes the

application and travel of a permanent magnet near the wall

Fig. 2 – (a) TEM image of the as-prepared sample after arc-

discharge synthesis in ethanol liquid and (b) SEM image of

the as-prepared sample.

of a test tube along its length to cause aggregation of the car-

bon nanocapsules. For magnetic separation, the sample after

the oxidation and etching was dispersed in the test tube with

10 ml ethanol solvent and positioned such that the bottom of

the tube lay between the poles of a 1.3 T permanent magnet.

After that, the tube was moved by a precise motorized stage

(Sigma Koki Co., Ltd., SGSP 20-85) between the poles of the

permanent magnet. After a few hours, aggregation of black

material was observed adjacent to both poles of the perma-

nent magnet and the further accumulation of particles was

clearly seen over a period of one day.

After magnetic separation, a centrifuge (Kobuta 6200) was

used to separate the particles of different size. The solution

was placed into a small tube, which was centrifuged for

10 min at 1000 rpm. The liquid part of the solution after cen-

trifugation was moved to another centrifuge tube and then

centrifuged again for 10 min at 2000 rpm. In the same man-

ner, the liquid part was re-centrifuged step by step at four dif-

ferent rotor speeds such as 1000, 2000, 3000 and 4000 rpm.

Fig. 3 – (a) and (b) TEM and SEM images of carbon

nanocapsules with graphite impurities after H2O2 selective

oxidation.

1526 C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9

After centrifugation, each sample was put into a vacuum oven

at 40 �C for 4 h.

The structure, morphology and size distribution of the

purified and separated carbon nanocapsules were evaluated

by 300 kV transmission electron microscopy (TEM) (JEOL,

JEM-3010) and field emission scanning electron microscopy

(FE-SEM) (JEOL, JSM-7000 F). The TEM and FE-SEM observa-

tions allowed us to determine the structure of the carbon

nanocapsules and the overall morphology of the samples.

Some of the observed nanocapsules were examined by means

of selected area electron diffraction (SAED) patterns and an

Fig. 4 – (a, b) TEM and SEM images of carbon nanocapsules afte

impurities (d) the corresponding electron diffraction pattern of

graphitic carbon (e) XRD patterns of powder samples: as-prepar

15% HCl solutions.

energy-dispersive X-ray spectroscopy (EDX) attached to the

electron microscope. A soft imaging system (analySIS) was

applied to treat the digitized SEM images to obtain the size

distribution of the carbon nanocapsules in each sample after

centrifugation.

3. Results and discussion

Carbon encapsulated iron carbide nanocapsules have been

synthesized by a new method in which ultrasonic cavitation

permits an electric plasma discharge to occur, with the appli-

r magnetic purification, (c) TEM image of the carbon

the carbon impurities. Miller indices in (d) is for the

ed and after treatment in 25% H2O2 and etching in

C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9 1527

cation of a relatively low level of electric power even in insu-

lating ethanol [19,20]. TEM and SEM images revealed interest-

ing structural characteristics of the synthesized carbon

nanocapsules. Fig. 2a indicates that the spherical carbon

nanocapsules of the as-prepared sample ranged from as

small as 5 nm to as large as 1000 nm in diameter as already

shown in previous works [19,20]. A raw material, as shown

in Fig. 2a, was surrounded by amorphous carbon as impuri-

ties, graphite balls and also smaller carbon nanocapsules.

These amorphous carbon and graphite impurities came from

the thermal decomposition of ethanol by arc discharging. Fig.

2b shows a typical SEM image of raw material shown in Fig.

2a. Most of the carbon nanocapsules were covered by amor-

phous carbon impurities.

The morphological changes induced by the hydrogen per-

oxide and acid etching treatment are evident in the TEM and

SEM images, as shown in Fig. 3a and b. A comparison between

Figs. 2 and 3 show that the amorphous carbon and exposed

iron particles were mostly removed after the first purification

treatment with the hydrogen peroxide and acid solution. This

purification technique using hydrogen peroxide may have the

advantages of limited damage to the graphite shell and softer

reactions when compared with the previous acid purification

methods [23]. The TEM and SEM images in Fig. 3a and b re-

vealed other impurities such as large graphite balls and

graphite flakes, because this reaction did not remove graphite

Fig. 5 – (a) SEM image of carbon nanocapsules after centrifuge f

2000 rpm, (c) centrifuged for 10 min at 3000 rpm and (d) centrifu

structures. After this treatment, the size-distribution of car-

bon nanocapsules changed from 30 to 1000 nm. From the

TEM observations in Fig. 3a, it seems that the carbon nano-

capsules sized under 30 nm were also damaged and removed

by the treatment due to the weak bonding of the graphite

shell. The previous TEM investigations [20] revealed that car-

bon layers of small (<30 nm in size) carbon nanocapsules are

incomplete and cores not uniformly closed (or covered). For

these reason the iron carbide cores of small carbon nanocap-

sules could be removed by H2O2 and HCl solutions and mainly

large tightly covered by graphite shells cores (>100 nm in size)

remained after treatment and etching.

Fig. 4a and b reveal the typical morphologies of the mag-

netically purified carbon nanocapsules consisting of a sphere

core surrounded by a graphite shell. The purified samples, as

shown in Fig. 4a, indicate that the carbon nanocapsules were

composed of a metallic core and a graphite shell, where the

diameters of the carbon nanocapsules ranged from 30 to

1000 nm, having a relatively wide size distribution. The re-

moved graphite impurities remained in solution after the

magnetic purification method (Fig. 4c). These impurities could

not be removed by the H2O2 oxidation method, but they were

removed efficiently by magnetic separation. An electron dif-

fraction (Fig. 4d) analysis of a group of impurities revealed

an ordered graphitic arrangement, especially with respect to

the (100) plane. In addition, the outer rings also suggest that

or 10 min at 1000 rpm, (b) centrifuged for 10 min at

ged for 10 min at 4000 rpm.

Fig. 6 – TEM image of carbon nanocapsules after centrifuge

for 10 min at 4000 rpm.

1528 C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9

the impurities are graphite. Careful high-resolution TEM

investigations of these impurities revealed lattice fringes with

Fig. 7 – (a) Particle size distribution of carbon nanocapsules after

at 2000 rpm, (c) centrifuged for 10 min at 3000 rpm and (d) cent

a spacing between them of 3.38 A, a value which is close to

that of the lattice associated with the graphite (002) planes.

The results of the TEM observations and SAED measurements

indicate that the graphite impurities were efficiently removed

from the sample by magnetic separation. Fig. 4e shows the

XRD patterns of the samples at different conditions: as-pre-

pared and after treatment in 25% hydrogen peroxide solution

and in 15% HCl solution. The XRD profile of the as-prepared

powder shows the one broad peak centered at about 44.5�.The small size of the carbon nanocapsules and some of their

iron carbide cores being in an amorphous state could result in

peak broadening in the as-prepared sample. The sharp peaks

in the XRD pattern of the carbon nanocapsules after treat-

ment in 25% H2O2 and etching in 15% HCl solutions suggest

that, treated and etched carbon powder sample consist

mainly of large (>100 nm in size) carbon nanocapsules includ-

ing iron carbides cores. Additionally, the graphite peak at

2h = 26� (corresponding to d002 = 0.342 nm) was well defined

in the treated and etched sample.

Fig. 5a–d shows the SEM images of the carbon nanocap-

sules separated by different centrifuge speeds. It is obvious

that the sizes of the carbon nanocapsules and their distribu-

tions decreased with increasing centrifuge speeds. The nearly

monodispersed carbon nanocapsules sized around 100–

150 nm can be observed in the TEM image (Fig. 6). These

centrifuge for 10 min at 1000 rpm, (b) centrifuged for 10 min

rifuged for 10 min at 4000 rpm.

C A R B O N 4 6 ( 2 0 0 8 ) 1 5 2 3 – 1 5 2 9 1529

carbon nanocapsules are covered with a graphite shell around

5–8 nm in thickness and these purified nanocapsules are con-

sidered to be enough for use in further medical applications.

Fig. 7a–d shows the size distributions of the purified carbon

nanocapsules after each change in centrifuge speed. They

were measured and estimated from the SEM images as shown

in Fig. 5a–d. Centrifugation at 1000 rpm resulted in the widest

particle size distribution from 200 nm to 1200 nm. With

increasing centrifuge speed, the average size decreased and

the size distribution became narrower, as already confirmed

by the SEM images. After centrifuging for 10 min at

4000 rpm, the size of the carbon nanocapsules was around

100–200 nm, and the size distribution became sharper as

shown in Fig. 7d.

4. Conclusions

Carbon encapsulated iron carbide nanocapsules have been

synthesized by a new method in which ultrasonic cavitation

permits an electric plasma discharge to occur, with the appli-

cation of a relatively low level of electric power even in insu-

lating ethanol. The carbon encapsulated iron carbide

nanoparticles, ranging from 5 to 1000 nm in diameter, were

produced using an arc-discharge apparatus. Their crystalline

cores are spherical in shape, and surrounded by tightly

wrapped graphitic layers. Amorphous carbon impurities were

removed by H2O2 treatment, while the exposed iron particle

impurities were eliminated by 15% HCl solution. The graphite

impurities were easily removed by magnetic separation. After

magnetic separation, a centrifuge was used to obtain nearly

monodispersed particles around 100–200 nm in size after cen-

trifuging for 10 min at 4000 rpm. Finally, this study presents

for the first time a novel results to purify and separate carbon

nanocapsules of around 100–200 nm in size.

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