C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9

. sc iencedi rec t .com

avai lab le at www

journal homepage: www.elsev ier .com/ locate /carbon

In vitro and in vivo behaviors of dextran functionalizedgraphene

Shuai Zhang 1, Kai Yang 1, Liangzhu Feng 1, Zhuang Liu *

Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials,

Soochow University, Suzhou, Jiangsu 215123, China

A R T I C L E I N F O

Article history:

Received 27 February 2011

Accepted 30 May 2011

Available online 2 June 2011

0008-6223/$ - see front matter � 2011 Elsevidoi:10.1016/j.carbon.2011.05.056

* Corresponding author: Fax: +86 512 6588284E-mail address: zliu@suda.edu.cn (Z. Liu)

1 These authors contributed equally to this

A B S T R A C T

Development of biocompatible surface coating is critical to engineer various functional

nanomaterials for biomedical applications. Here, we present a new surface chemistry of

graphene by covalently conjugating graphene oxide (GO) with dextran (DEX), a biocompat-

ible polymer widely used for surface coating of biomaterials. Compared with GO, the

graphene–dextran (GO–DEX) conjugate shows reduced sheet sizes, increased thickness

and significantly improved stability in physiological solutions. Cellular experiments

uncover that DEX coating on GO offers remarkably reduced cell toxicity. We further label

GO–DEX with a radioactive isotope, 125I, for in vivo tracking in animal studies. It is found

that GO–DEX accumulates in the reticuloendothelial system (RES) including liver and

spleen after intravenous injection, and importantly, shows obvious clearance from the

mouse body within a week without causing noticeable short-term toxicity to the treated

animals. Our results suggest that this DEX coating method on GO may potentially be useful

to the further development of novel graphene-based bioconjugates for various biomedical

applications.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Graphene with unique two-dimensional structures, fascinat-

ing electronic, physical and chemical properties has shown

promise in a wide range of fields including electronic devices,

solar cells, nano-catalysts, as well as chemical and biological

sensors [1–11]. Recently, tremendous attentions have been

paid to the graphene based nanomedicine for biological

detection, drug delivery, and cancer therapies [12–15]. In the

past three years, numerous publications have reported

graphene-based novel biosensors by utilizing the interesting

chemical, optical, electrical, and electrochemical properties

of graphene for detection of various biomolecules with high

sensitivities [11,14,15]. Monolayer graphene with all atoms

exposed on its surface exhibits a huge surface area (theoreti-

cally 2600 m2/g), which can be utilized for ultra-efficient

er Ltd. All rights reserved

6..

work.

loading of aromatic molecules such as anti-cancer drugs,

useful for applications in drug and gene delivery [16–21]. In

a recent work by our group, polyethylene glycol (PEG) func-

tionalized nano-graphene with strong optical absorption in

the near-infrared region (NIR) and was used for in vivo photo-

thermal therapy of cancer, showing excellent tumor ablation

effect in a mouse model [7]. Although still at its infant stage,

the graphene-based nanomedicine appears to have great

potential in a number of different directions.

It is well known that the surface chemistry of nanomateri-

als is highly important to their behaviors and applications in

biological systems. Several latest reports uncovered that as-

prepared GO exhibited dose-dependent toxicity in vitro to

cells [21]. After intravenous injection, GO would dominantly

accumulated in lungs of mice over long periods of time,

inducing obvious pulmonary toxicity [22,23]. Coating of

.

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9 4041

biocompatible polymers on graphene may help to circumvent

these problems. Our recent work showed that PEGylated

nano-graphene sheets with sizes smaller than 50 nm (10–

30 nm) could be cleared out from the mouse body after intra-

venous injection, without rendering noticeable toxicity to the

treated animals. Although PEG has been used to functionalize

GO for several in vitro and in vivo studies [7,8,24,25], efforts

are still needed to develop new biocompatible surface coat-

ings of graphene for different applications in biomedicine.

DEX is a hydrophilic natural polymer extensively used to

functionalize many nanomaterials such as iron oxide, quan-

tum dots, single walled carbon nanotubes and nanodiamond,

to improve their stability, biocompatibility, pharmacokinetics,

and thus biomedical functions [26–31]. Different from PEG

with linear structure, DEX in aqueous phase exhibits spheri-

cal shape which may help to better coat nanomaterials. More-

over, DEX is a natural polymer that can be fully degraded in

living biological systems [32–35]. Herein, we report a new sur-

face coating of GO by functionalized DEX, and test the solubil-

ity of the yielded GO–DEX in physiological solutions in

comparison to non-functionalized GO. It is found that GO–

DEX is able to enter cells, without showing significant inter-

ference to the cell growth. Using radioactive 125I-labeled GO–

DEX (125I–GO–DEX), we further study the in vivo pharmacoki-

netics and biodistribution of GO–DEX in mice, observing high

uptake of GO–DEX in the mouse liver and spleen together

with neglectable accumulation in the lung. Both radiolabel-

ing-based biodistribution data and liver slice images indicate

the significant clearance of graphene from the mouse organs,

strongly favorable for future biomedical applications.

2. Experimental section

2.1. Chemical materials

Graphite powder was purchased from Hua Dong Graphite Man-

ufactory, China. Dextran (Mw = 8000–12,000 Da) was obtained

from SERVA. Sodium periodate, 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide

(DMSO), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide

hydrochloride (EDC) and sodium cyanoborohydride were pur-

chased from Sigma–Aldrich. Cy5-NHS was purchased from

Fanbo Chemicals Co. Ltd. Ethanediamine and sodium azide

were obtained from Sinopharm Chemical Reagent Co. Ltd.

Na125I was obtained from Chengdu nuclear isotope Qualcomm

Inc. Ultra-filter tubes with 100 kD and 10 kD molecular weight

cut off (MWCO) were purchased from Millipore.

2.2. Synthesis of GO

GO was prepared from graphite following a modified Hum-

mers’ method [7,36]. The as-prepared material was stirred

in the presence of sodium hydroxide for 2 h at 50 �C. After

adjusting the solution pH to 1 by hydrochloric acid, the sus-

pension was centrifuged at 8000g for 5 min and washed by

distilled (DI) water several times to remove excess acid and

salts. The GO suspension became rather stable afterwards

and was then centrifuged at 10,000g for 5 min to remove

any insoluble aggregates, leaving based treated GO (GO-b) in

the supernatant for future use.

2.3. Synthesis of dextran amine (DEX-NH2)

DEX with a molecular weight of 8000–12,000 Da was dissolved

in DI water at a concentration of 0.2 g/ml. Sodium periodate

was then added in dark to break the glycol C–C bond to two

aldehyde groups. The mole ratios of sodium periodate to dex-

tran monomers were 2.5%, 5%, 10% and 20%. After dialysis

against water for 48 h to remove excess sodium periodate,

the oxidized dextran was frozen dried. To introduce amino

groups into dextran, oxidized dextran (0.2 g) in 15 mL DI water

was mixed with excessive ethanediamine (48 mg) and 0.5 mL

sodium cyanoborohydride (caution!) (5 mol/L) alkaline solu-

tion at pH 14 for 24 h. After removing small molecules by dial-

ysis, the final product was lyophilized and stored at �20 �C.

Oxidized dextran and DEX-NH2 were characterized to

determine the aldehyde and amino contents, respectively.

The content of aldehyde in oxidized dextran was measured

by a classical redox titration method. In this method, excess

sodium bisulfate was reacted with aldehyde groups on dex-

tran, with leftover free sodium bisulfate expended by iodime-

try. One gram of sodium bicarbonate was then added into the

solution to release bisulfate conjugated to aldehyde groups.

Iodimetry was used again to measure the concentration of so-

dium bisulfate, which was equivalent to that of aldehyde

groups. The nitrogen concentration of DEX-NH2 was mea-

sured by a standard Kjeldahl assay to determine the ratios

of amino groups in various DEX-NH2 samples [37,38].

2.4. Synthesis of GO–DEX

The protocol to synthesize GO–DEX was similar to that of

PEGylated GO synthesis reported earlier [7]. 1 mL GO solution

(2 mg/mL) dispersed in 4 mL DI water was first mixed with

�15 mg DEX-NH2 in 400 lL DI water and sonciated for 5 min.

1 mg EDC dissolved in 100 lL DI water was then added into

the suspension instantly. After 30 min reaction under sonica-

tion, another 2.5 mg EDC was added afterwards. After 6 h

reaction under stirring, the suspension was filtrated through

a MWCO 100 kDa centrifugal filter (Millipore) several times

to completely remove excess EDC and dextran amine. Finally,

the prepared GO–DEX was dispersed in DI water at the con-

centration of 2 mg/mL.

2.5. Fluorescent labeling of GO–DEX

To fluorescently label GO–DEX, 50 lg Cy5-NHS in 20 lL DMSO

was added into 500 lL GO–DEX (2 mg/mL) in phosphate buf-

fered saline (PBS) solution and stirred for a few seconds.

The amine reactive N-hydroxysuccinimide (NHS) ester in

Cy5-NHS would react with the leftover amino groups on the

DEX-NH2 coating of the GO–DEX conjugate. After 12 h reac-

tion in dark, excess fluorescent dyes were removed by filtra-

tion via 100 kDa centrifugal filters.

2.6. Cellular experiments

HeLa cells were cultured in standard Dulbecco’s modified ea-

gle medium (DMEM) supplemented with 10% fetal bovine ser-

um (FBS) and 1% penicillin–streptomycin solution under

37 �C. For cell proliferation measurement, HeLa cells were

4042 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9

seeded into 6-well plates at the density of 2 · 105 per well. Dif-

ferent concentrations (10 mg/L, 50 mg/L, 200 mg/L) of GO and

GO–DEX were then added into the cells. Doxorubicin (DOX) at

the concentration of 20 mg/L was used as the positive control.

Cells were detached from the plates and collected for cell

number counting after 24, 48, and 72 h of incubation. Dead

cells were excluded by trypan blue staining.

For confocal fluorescence imaging experiments, HeLa cells

cultured in 35 mm dishes were added with 10 lL Cy5 labeled

GO–DEX (2 mg/ml) and incubated at different temperature

(4 �C and 37 �C) for 2 h. After washing with PBS for several

times, cells were then imaged by a laser scanning confocal

fluorescence microscope (Leica TCS SP5). The excitation

wavelength was 633 nm and the emission band is between

660 and 680 nm. All the images were taken under the same

instrumental condition.

The Calcein AM/propidium iodide (PI) staining experiment

was carried out following a classic method. Briefly, Hela cells

were seeded in 35 mm culture dishes overnight. GO and GO–

DEX solutions at concentrations of 50 mg/L and 200 mg/L (fi-

nal concentrations in the cell medium) were added into cells.

Doxorubicin (DOX) at the concentration of 20 mg/L was added

as the positive control. After incubation for various periods of

time, the cells were washed with PBS and stained with 1 mL

PBS solution containing Calcein-AM (0.3 mg/L) and PI

(0.5 mg/L). Confocal fluorescence images were taken 20 min

after staining.

2.7. Histology examination

Healthy female Balb/c mice were injected with 200 ll of 2 mg/

ml GO–DEX per mouse (a dose of 20 mg/kg) and sacrificed at

various time points after injection (1 day, 3 and 7 days). Major

organs were then taken and fixed in 4% neutral buffered for-

malin, processed routinely into paraffin, sectioned at 8 lm,

stained with hematoxylin and eosin (H&E) and examined by

a digital microscope (Leica QWin). Examined tissues include

liver, spleen, kidney and lung.

2.8. Radioactive labeling of GO–DEX

The radioactive labeling of GO–DEX was carried out by a chlo-

ramine-T oxidation method [25,39]. 500 lL GO–DEX solution

(2 mg/mL), �800 lCi Na125I and 100 lL chloramine-T (4 mg/

mL) were mixed together in a phosphate buffer (pH �7.5). Ex-

cess Na125I and chloramine-Twere removed by filtration with

100 kDa centrifugal filters. The suspension was washed 4–6

times with DI water until no detectable gamma radioactivity

in the filtrate. The radiolabeling yield was about 40–45%. As

a control experiment, GO–DEX and Na125I were mixed to-

gether without chloramine-T. Almost no gamma radioactivity

signal could be detected in the GO–DEX suspension after the

same washing procedures.

2.9. Radiolabeling stability test

10 lL 125I labeled GO–DEX was added into 200 lL mouse plas-

ma (collected from a healthy Balb/c mouse) and incubated in

a 37 �C water bath. After incubation for different periods of

time, 10 lL of the mixture was added into 1 mL DI water.

The solution was filtered by a centrifugal filter (MWCO =

10 kD) and washed with water. The filtrate was collected

and measured by a Gamma counter (Science and Technology

Institute of China in Jia Branch Innovation Co., Ltd.) to detect

the released radioactive iodine.

2.10. Blood circulation and biodistribution of radioactivelabeled GO–DEX

To study the blood circulation and biodistribution of GO–DEX,125I–GO–DEX was injected via tail veins into healthy female

Balb/c mice. Blood was drawn at different time points after

injection and measured by the Gamma Counter. For biodistri-

bution study, mice were sacrificed 4, 24, 72 and 168 h after

injection. Major organs were collected and wet weighted for

radioactivity measurement.

3. Results and discussion

The procedure of converting dextran to DEX-NH2 and conju-

gating GO to DEX-NH2 was shown in Fig. 1 To synthesize

DEX-NH2, the glycol bond in a dextran monomer was oxidized

by sodium periodate, giving two aldehyde groups which were

available for conjugation to ethanediamine by reductive ami-

nation. Excess ethanediamine was added into the solution of

oxidized dextran, forming a Schiff base between the amino

group in ethanediamine and the aldehyde group in oxidized

dextran. The Schiff base could then be reduced by a middling

reducing agent, sodium cyanoborohydride, transforming the

C@N bond into a more stable C–N bond. The measured aver-

age numbers of aldehyde and amino groups per oxidized dex-

tran and DEX-NH2, respectively, in the four samples were

presented in Table 1.

We then conjugated DEX-NH2 with different amine ratios

to GO. It was found that the synthesis yield of GO–DEX (after

reaction and the followed centrifugation to remove unstable

aggregates) increased as the raise of nitrogen contents in

DEX-NH2 (Fig. 2a), and reached to the optimized yield when

the DEX-NH2 with 1.20% N was used. Further increase in the

nitrogen content of DEX-NH2 resulted in slightly decreased

GO–DEX yield, likely due to the heavy oxidization that dam-

aged the dextran structure. The DEX-NH2 sample with 1.20%

nitrogen composition (4.28 amino groups per dextran) was

thus chosen for the following experiments. Compared to

non-coated GO which precipitated in the presence of salts

and was unstable in various biological solutions including sal-

ine, serum and cell medium, GO–DEX was rather stable in the

above solutions without obvious agglomeration (Fig. 2b). The

UV–vis absorption spectra of GO, base treated GO (GO-b),

and GO–DEX were recorded (Fig. 2c), showing a similar

absorption peak at 230–240 nm, which was originated from

the p-plasmon of carbon [40,41]. Interestingly, the optical den-

sity of GO-b and GO–DEX in the NIR region significantly in-

creased by 3.76 and 18.6 folds at 800 nm, compared to the

as-prepared GO. We attributed the increase of absorbance to

the base treatment and DEX conjugation, which would de-

crease the surface flaw by breaking epoxide and ester groups,

releasing the local strain and increasing the p-plasmon of car-

bon, that would raise the absorbance of GO-b and GO–DEX in

the visible-NIR range [8,42,43].

Fig. 1 – A reaction scheme of converting dextran to DEX-NH2 (a), conjugating DEX-NH2 to GO (b), and fluorescent labeling of

GO–DEX (c).

Table 1 – Oxidation ratios in dextran oxide and nitrogen contents in DEX-NH2. The numbers of aldehyde groups and aminogroups were estimated by redox titration measurement and Kjeldahl assay, respectively.

Sodium periodatebatch ratio (%)

Effective oxidationratio (%)

Aldehyde group numberin one dextran oxide

Nitrogencontent (%)

Amino group numberin one DEX-amine

2.5 2.19 2.63 0.64 2.285 4.33 5.20 0.75 2.6810 9.06 10.87 1.20 4.2820 17.71 21.25 1.70 6.07

Fig. 2 – Characterization of GO–DEX. (a) Stability of GO–DEX with different nitrous concentration. All samples were added into

9% NaCl solutions with the same beginning concentration (1 mg/mL). The final concentrations of mixtures were measured by

UV–vis spectra after centrifugation at 10,000g for 10 min. (b) The stability of GO and GO–DEX in different solvents. The GO

concentration in all solutions was 0.15 mg/mL. (c) UV–vis spectra of as prepared GO, GO after NaOH treatment, and GO–DEX.

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9 4043

4044 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9

We further characterized our products by atomic force

microscope (AFM, Vecco Multimode V). The graphene sheet

sizes showed an obvious decrease from GO (�100–500 nm)

to GO–DEX (�50–100 nm), likely due to the sonication steps in-

volved in the dextran conjugation procedure (Fig. 3a and b). It

was also observed that the average thickness of GO–dextran

sheets was �2.8 nm, compared to �0.9 nm for single layer

GO sheets (Fig. 3d). The increased sheet thickness was attrib-

uted to the dextran polymer coating on graphene. The sizes

and thicknesses of GO–DEX were considerably larger than

that of PEGylated GO shown in our earlier work (10–30 nm

in size, �1 nm in thickness) [7,24]. To confirm that DEX was

covalently conjugated to GO instead of non-covalent adsorp-

tion, we repeated the DEX conjugation step however without

the addition of EDC. The yielded product was not stable in sal-

ine (data not shown), indicating that DEX was not coated on

GO under such a condition. The GO + DEX sample was also

characterized by AFM, showing no significant increase in

sheet thickness but obvious decrease in sheet sizes (Fig. 3c

and d). The successful dextran conjugation in GO–DEX was

further evidenced by infrared (IR) spectra and thermal gravi-

metric analysis (TGA) (Supplementary information Figs. S1

and S2). The TGA data of GO and GO–DEX suggested that

approximately a half of the GO–DEX weight was contributed

by the polymer coating.

The GO–DEX was then used for biological studies. To test

the potential cytotoxicity of GO–DEX, we incubated HeLa cells

with plain GO and GO–DEX at a series of concentrations for

24 h, 48 h and 72 h. The cell proliferation data determined

Fig. 3 – Atomic force microscope (AFM) characterization. (a–c) Typ

(d) Thickness distribution of GO, GO–DEX and GO–DEX without

by cell counting showed that as-prepared GO was able to de-

lay the cell growth by a dose dependent manner (Fig. 4a–c). In

contrast, the cell proliferation inhibition effect by GO–DEX

was notably less significant (Fig. 4a–c). Calcein-AM/PI double

staining was employed to further study the cell toxicity of

GO and GO–DEX. It was found that although GO did not in-

duce obvious cell death even at high concentrations up to

200 mg/L (Fig. 4d), the densities of GO treated cells were much

lower than that of untreated cells. On the other hand, cells

incubated with GO–DEX showed densities comparable to con-

trol untreated cells (Fig. 4d). The cell growth inhibition by GO

was reported by be related to the GO–induced cell cycle phase

harvest [9,44]. Our data here clearly demonstrate that proper

surface coating could remarkably improve the biocompatibil-

ity of GO.

To study the interaction of GO–DEX with cells, we incu-

bated Cy5 labeled GO–DEX with HeLa cells at different tem-

peratures for 2 h. Successful Cy5 labeling was evidenced by

UV–Vis absorption spectra (Supplementary information

Fig. S3). Laser scanning confocal microscope images showed

obvious GO–DEX cellular uptake when cells were incubated

with GO–DEX at 37 �C but not for those incubated at 4 �C(Fig. 5) [45]. Although this observation is consistent to the en-

ergy dependent endocytosis mechanism, further evidences

are still required to fully understand the cell entry pathways

of GO–DEX.

Radiolabeling has been an effective method for in vivo

tracking and detection of molecules and nanomaterials [46].

To investigate the in vivo behaviors of dextran coated

ical AFM images of GO, GO–DEX and GO + DEX without EDC.

EDC determined by AFM measured sheet heights.

Fig. 4 – In vitro cell proliferation and toxicity assays. (a–c) The proliferation of HeLa cells in the presence of 10 mg/L (a), 50 mg/L

(b) and 200 mg/L (c) of GO or GO–DEX. DOX at 20 mg/L was used as the positive control. The relative cell proliferation was

determined by cell counting. Standard deviations were based on five parallel samples. (d) Confocal fluorescence images of

Calcein AM/PI stained HeLa cells 24, 48 and 72 h after various treatments indicated. Live and dead cells were stained by

Calcein AM (green fluorescence) and PI (red fluorescence), respectively.

Fig. 5 – In vitro cellular experiments. (a) Confocal fluorescent images of HeLa cells incubated with Cy5 labeled GO–DEX for 2 h

at 4 �C and 37 �C.

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9 4045

Fig. 6 – In vivo behaviors of GO–DEX. (a) The blood circulation curve of 125I–GO–DEX. The pharmacokinetics of GO–DEX

followed a two-compartment model. (b) Time-dependent biodistribution of 125I–GO–DEX in female Balb/c mice. Error bars in

both two graphs were based on three mice per group.

4046 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9

graphene, we labeled GO–DEX with 125I by the chloramine-T

oxidization method, a similar strategy previously used to la-

bel oxidized carbon nanotubes and PEGylated nano-graphene

[22,25,39]. Excess 125I was thoroughly removed by repeated fil-

tration. Control experiments showed that simply mixing of

Fig. 7 – Excretion of GO–DEX from mice. (a and b) H&E stained live

7 days p.i. (b). Black spots, which were associated with GO aggreg

numbers of black dots per image field in the mouse liver 1 day

slices per each sample. A remarkable decrease in the density of

clearance of GO–DEX from the mouse liver. (d) Distribution of 12

metabolism cages. The egesta was wet weighed and measured

GO–DEX and Na125I in the absence of chloramine-T offered

no appreciable 125I labeling on graphene, suggesting that io-

dine was covalently conjugated to GO–DEX instead of physical

adsorption. The DEX polymer itself could not be labeled by 125I

under this condition as we tested. Similar to the 125I labeling

r images from mice injected with GO–DEX at 1 day p.i. (a) and

ates, were pointed by white arrows. (c) Statistics of averaged

and 7 days after GO–DEX injection. Error bars were based 10

black spots was noticed 7 days after injection, indicating the5I–GO–DEX in urine and feces of Balb/c mice collected by

by a gamma counter.

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9 4047

of oxidized carbon nanotubes [25,39], 125I was likely conju-

gated to the defect sites and dangling bonds of GO. The stabil-

ity of 125I labeled GO–DEX was tested in Balb/c mouse serum,

showing only a small degree of 125I detachment within 7 days

at 37 �C (Supplementary information Fig. S4).

To study the in vivo pharmacokinetics of GO–DEX, we

intravenously injected 100 ll of 125I–GO–DEX (4 mg/kg,

20 lCi) into mice. Blood was drawn at different time points

post injection (p.i.) and measured by a gamma counter.

(Fig. 6a) The blood circulation curve showed that the pharma-

cokinetics of GO–DEX followed a two-compartment model,

with first and second phases of half-lives at 0.19 ± 0.03 h

and 1.81 ± 0.17 h, respectively. Mice were sacrificed at 4, 24,

72 and 168 h for biodistribution study. It was found that125I–GO–DEX distributed in several organs including liver,

spleen, stomach, lung, kidney and intestine at 4 h, and

showed dominated RES uptake in liver and spleen at later

time points with neglectable lung accumulation (Fig. 6b). A

low thyroid uptake was observed only at 4 h and not detect-

able at later time points, suggesting the decent stability of

the 125I radiolabeling in the mouse body. Decreased signals

with elapsing time indicated the possible clearance of GO–

DEX in body. Interestingly, the excretion of GO–DEX appears

to be slightly faster compared to that of GO–PEG observed in

our previous study [25]. While �4% ID/g and �6% ID/g of

Fig. 8 – Microscopy images of H&E stained organ slices collected

DEX at the dose of 20 mg/kg. No noticeable abnormality was ob

lung.

125I–GO–PEG were detected in the liver and spleen 7 days after

injection, respectively[25], the 125I–GO–DEX levels in these two

organs were lower than 2% ID/g at 7 days p.i. (Fig. 6b).

To confirm the decrease of radioactivities in organs was

due to the clearance of GO–DEX instead of the detachment

of radioisotope 125I from the labeled graphene, we examined

the hematoxylin and eosin (H&E) stained liver slices (Fig. 7).

Black spots, which were likely aggregates of GO–DEX, could

be found in liver of mice at 1 day p.i. but not in control un-

treated mouse liver (Fig. 7a and b). Interestingly, the number

of the black spots remarkably dropped 7 days after injection,

suggesting that the clearance of GO–DEX could indeed hap-

pen (Fig. 7c). Again, the excretion speed of GO–DEX deter-

mined by counting GO black aggregates in liver slices

appeared to be faster than that of GO–PEG observed in our

previous study, in which nearly complete clearance was not

noticed until 20 days after injection [25]. However, direct

side-by-side comparison may be needed in future studies to

carefully investigate how different surface coatings affect

the GO excretion in animals.

In order to study the excretion mechanism of GO–DEX, the

urine and feces of four mice injected with 125I–GO–DEX were

separately collected by metabolism cages every day for a

week. High radioactivity was detected in both feces and urine,

suggesting that the clearance of GO–DEX after intravenous

from mice sacrificed at 1 day and 7 days post injection of GO–

served in major organs including liver, spleen, kidney, and

4048 C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9

injection could take place in both renal and fecal pathways

(Fig. 7d). Since GO–DEX has a wide size distribution, a small

fraction of very small GO–DEX sheets may pass the glomeru-

lus for renal excretion, while the majority of GO–DEX

accumulated in RES organs could be cleared out in feces via

the biliary pathway. It worth noting that compared with

GO–PEG (sheet size 10–50 nm) in our previous work [25], nota-

bly higher portion of GO–DEX was excreted by feces than ur-

ine, likely owing to the increased sheet sizes of GO–DEX (50–

100 nm).

Mice injected GO–DEX at the dose of 20 mg/kg were sacri-

ficed at 1 day and 7 days p.i. Major organs were harvested and

sectioned into thin slices for H&E staining (Fig. 8). No obvious

abnormality was found in various organs including the lung

of the GO–DEX injected mice, in marked contrast to the obvi-

ous pulmonary toxicity induced by the as-prepared GO re-

cently observed in earlier studies [23]. Although further

careful long-term studies are needed to fully illustrate the

toxicology profiles of GO–DEX, our preliminary data here

showed that the dextran coating could offer GO improved bio-

compatibility. However, future experiments are still required

to understand the long-term fate, excretion pathways, and

toxicology of dextran functionalized GO in animals.

4. Conclusions

We develop a new graphene surface coating method using a

biocompatible polymer, dextran. After covalent conjugation

of dextran, the GO–DEX shows excellent stability in physio-

logical solutions. Compared to plain unfunctionalized GO,

GO–DEX exhibits remarkably improved in vitro biocompatibil-

ity with notably reduced cell growth inhibition effects. Radio-

labeling of GO–DEX by 125I enables tracking and detection of

graphene in animals. After being intravenously injected into

mice, GO–DEX is cleared from the circulating blood in a few

hours and accumulates in RES organs including liver and

spleen, in which the GO–DEX levels show a gradual decrease

over time with the majority excreted within a week. Different

from as-prepared GO, which is primarily trapped in the lung

after intravenous injection and causes obvious pulmonary

toxicity [22,23], neglectable lung uptake and no noticeable

in vivo short-term toxicity is found in GO–DEX treated mice.

Our work provides an alternative functionalization method

to produce biocompatible graphene bioconjugates for poten-

tial biomedical applications. However, the in vivo long-term

fate and toxicology of dextran coated graphene still require

further investigations. Using of GO–DEX for actual bio-appli-

cations such as drug delivery and bio-sensing is currently un-

der-going in our laboratory.

Acknowledgements

This work was partially supported by the National Natural

Science Foundation of China (51002100), a National ‘‘973’’ Pro-

gram of China (2011CB911002), and A Project Funded by the

Priority Academic Program Development of Jiangsu Higher

Education Institutions. We thank Dr. Jianmei Wan and Prof.

Youjiu Zhang in the Medical School of Soochow University

for their great help on radioactive experiments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.carbon.2011.05.056.

R E F E R E N C E S

[1] Geim AK. Graphene: status and prospects. Science2009;324(5934):1530–4.

[2] Allen MJ, Tung VC, Kaner RB. Honeycomb carbon: a review ofgraphene. Chem Rev 2010;110(1):132–45.

[3] Hong WJ, Bai H, Xu YX, Yao ZY, Gu ZZ, Shi GQ. Preparation ofgold nanoparticle/graphene composites with controlledweight contents and their application in biosensors. J PhysChem C 2010;114(4):1822–6.

[4] Tang LAL, Wang JZ, Loh KP. Graphene-based SELDI probe withultrahigh extraction and sensitivity for DNA oligomer. J AmChem Soc 2010;132(32):10976–7.

[5] He QY, Sudibya HG, Yin ZY, Wu SX, Li H, Boey F, et al.Centimeter-long and large-scale micropatterns of reducedgraphene oxide films: fabrication and sensing applications.Acs Nano 2010;4(6):3201–8.

[6] Mao S, Lu GH, Yu KH, Bo Z, Chen JH. Specific protein detectionusing thermally reduced graphene oxide sheet decoratedwith gold nanoparticle-antibody conjugates. Adv Mater2010;22(32):3521–6.

[7] Yang K, Zhang SA, Zhang GX, Sun XM, Lee ST, Liu ZA.Graphene in mice: ultrahigh in vivo tumor uptake andefficient photothermal therapy. Nano Lett2010;10(9):3318–23.

[8] Sun XM, Liu Z, Welsher K, Robinson JT, Goodwin A, Zaric S,et al. Nano-graphene oxide for cellular imaging and drugdelivery. Nano Research 2008;1(3):203–12.

[9] Hu WB, Peng C, Luo WJ, Lv M, Li XM, Li D, et al. Graphene-based antibacterial paper. Acs Nano 2010;4(7):4317–23.

[10] Qi XY, Pu KY, Li H, Zhou XZ, Wu SX, Fan QL, et al.Amphiphilic graphene composites. Angew Chem Int Ed2010;49(49):9426–9.

[11] He SJ, Song B, Li D, Zhu CF, Qi WP, Wen YQ, et al. A graphenenanoprobe for rapid, sensitive, and multicolor fluorescentDNA analysis. Adv Func Mater 2010;20(3):453–9.

[12] Song YJ, Qu KG, Zhao C, Ren JS, Qu XG. Graphene oxide:intrinsic peroxidase catalytic activity and its application toglucose detection. Adv Mater 2010;22(19):2206–10.

[13] Wang Y, Li ZH, Hu DH, Lin CT, Li JH, Lin YH. Aptamer/graphene oxide nanocomplex for in situ molecular probing inliving cells. J Am Chem Soc 2010;132(27):9274–6.

[14] Mohanty N, Berry V. Graphene-based single-bacteriumresolution biodevice and DNA transistor: interfacinggraphene derivatives with nanoscale and microscalebiocomponents. Nano Lett 2008;8(12):4469–76.

[15] Gulbakan B, Yasun E, Shukoor MI, Zhu Z, You M, Tan X, et al.A dual platform for selective analyte enrichment andionization in mass spectrometry using aptamer-conjugatedgraphene oxide. J Am Chem Soc 2010;132(49):17408–10.

[16] Yang XY, Zhang XY, Liu ZF, Ma YF, Huang Y, Chen Y. High-efficiency loading and controlled release of doxorubicinhydrochloride on graphene oxide. J Phys Chem C2008;112(45):17554–8.

[17] Yang X, Wang Y, Huang X, Ma Y, Huang Y, Yang R, et al. Multi-functionalized graphene oxide based anticancer drug-carrierwith dual-targeting function and pH-sensitivity. J MaterChem 2011;21(10):3448–54.

[18] Feng L, Zhang S, Liu Z. Graphene based gene transfection.Nanoscale 2011;3(3):1252–7.

C A R B O N 4 9 ( 2 0 1 1 ) 4 0 4 0 – 4 0 4 9 4049

[19] Zhang LM, Xia JG, Zhao QH, Liu LW, Zhang ZJ. Functionalgraphene oxide as a nanocarrier for controlled loading andtargeted delivery of mixed anticancer drugs. Small2010;6(4):537–44.

[20] Zhang L, Lu Z, Zhao Q, Huang J, Shen H, Zhang Z. Enhancedchemotherapy efficacy by sequential delivery of siRNA andanticancer drugs using PEI-grafted graphene oxide. Small2011;7(4):460–4.

[21] S A, Z ZX, Ye F, He QY, Chen GCK, Soo J, et al. Interfacing livecells with nanocarbon substrates. Langmuir2010;26(4):2244–7.

[22] Wang K, Ruan J, Song H, Zhang J, Wo Y, Guo S, et al.Biocompatibility of graphene oxide. Nanoscale Res Lett2010;6(1):8.

[23] Zhang XY, Yin JL, Peng C, Hu WQ, Zhu ZY, Li WX, et al.Distribution and biocompatibility studies of graphene oxidein mice after intravenous administration. Carbon2010;49(3):986–95.

[24] Liu Z, Robinson JT, Sun XM, Dai HJ. PEGylated nanographeneoxide for delivery of water-insoluble cancer drugs. J AmChem Soc 2008;130(33):10876–7.

[25] Yang K, Wan JM, Zhang S, Zhang YJ, Lee ST, Liu Z. Invivo pharmacokinetics, long-term biodistribution andtoxicology of PEGylated graphene in mice. ACS Nano2011;5(1):516–22.

[26] Goodwin AP, Tabakman SM, Welsher K, Sherlock SP, PrencipeG, Dai HJ. Phospholipid–dextran with a single coupling point:a useful amphiphile for functionalization of nanomaterials. JAm Chem Soc 2009;131(1):289–96.

[27] Hogemann D, Josephson L, Weissleder R, Basilion JP.Improvement of MRI probes to allow efficient detection ofgene expression. Bioconjugate Chem 2000;11(6):941–6.

[28] Harris LA, Goff JD, Carmichael AY, Riffle JS, Harburn JJ, PierreSt TG, et al. Magnetite nanoparticle dispersions stabilizedwith triblock copolymers. Chem Mater 2003;15(6):1367–77.

[29] Moore A, Weissleder R, Bogdanov A. Uptake of dextran-coated monocrystalline iron oxides in tumor cells andmacrophages. Jmri-Journal Magnetic Reson Imaging1997;7(6):1140–5.

[30] Sonvico F, Mornet S, Vasseur S, Dubernet C, Jaillard D,Degrouard J, et al. Folate-conjugated iron oxidenanoparticles for solid tumor targeting as potential specificmagnetic hyperthermia mediators: synthesis,physicochemical characterization, and in vitro experiments.Bioconjugate Chem 2005;16(5):1181–8.

[31] Wilson R, Spiller DG, Beckett A, Prior IA, See V. Highly stabledextran-coated quantum dots for biomolecular detection andcellular imaging. Chem Mater 2010;22(23):6361–9.

[32] Van Tomme SR, Hennink WE. Biodegradable dextranhydrogels for protein delivery applications. Expert Rev MedDevic 2007;4(2):147–64.

[33] Hiemstra C, van der Aa LJ, Zhong ZY, Dijkstra PJ, Feijen J.Rapidly in situ-forming degradable hydrogels from dextranthiols through michael addition. Biomacromolecules2007;8(5):1548–56.

[34] Bachelder EM, Beaudette TT, Broaders KE, Dashe J, FrechetJMJ. Acetal-derivatized dextran: an acid-responsivebiodegradable material for therapeutic applications. J AmChem Soc 2008;130(32):10494–5.

[35] Perez JM, Asati A, Nath S, Kaittanis C. Synthesis ofbiocompatible dextran-coated nanoceria with pH-dependentantioxidant properties. Small 2008;4(5):552–6.

[36] Hummers WS, Offeman RE. Preparation of graphitic oxide. JAm Chem Soc 1958;80:1339.

[37] Silverstein RM, Robert perthel J. Kjeldahlmicrodetermination. Anal Chem 1950;22(7):949–50.

[38] Concon JM, Soltess D. Rapid micro Kjeldahl digestion ofcereal grains and other biological materials. Anal Biochem1973;53(1):35–41.

[39] Wang HF, Wang J, Deng XY, Sun HF, Shi ZJ, Gu ZN, et al.Biodistribution of carbon single-wall carbon nanotubes inmice. J Nanosci Nanotechnol 2004;4(8):1019–24.

[40] Attal S, Thiruvengadathan R, Regev O. Determination of theconcentration of single-walled carbon nanotubes in aqueousdispersions using UV–visible absorption spectroscopy. AnalChem 2006;78(23):8098–104.

[41] Reed BW, Sarikaya M. Electronic properties of carbonnanotubes by transmission electron energy-lossspectroscopy. Phys Rev B 2001;64(19):195404.

[42] Li D, Muller MB, Gilje S, Kaner RB, Wallace GG. Processableaqueous dispersions of graphene nanosheets. NatureNanotechnol 2008;3(2):101–5.

[43] Stankovich S, Dikin DA, Dommett GHB, Kohlhaas KM,Zimney EJ, Stach EA, et al. Graphene-based compositematerials. Nature 2006;442(7100):282–6.

[44] Zhang YB, Ali SF, Dervishi E, Xu Y, Li ZR, Casciano D, et al.Cytotoxicity effects of graphene and single-wall carbonnanotubes in neural phaeochromocytoma-derived PC12cells. Acs Nano 2010;4(6):3181–6.

[45] Kam NWS, Liu ZA, Dai HJ. Carbon nanotubes as intracellulartransporters for proteins and DNA: an investigation of theuptake mechanism and pathway. Angew Chem Int Ed2006;45(4):577–81.

[46] Liu Z, Tabakman SM, Chen Z, Dai HJ. Preparation of carbonnanotube bioconjugates for biomedical applications. NatureProtocols 2009;4(9):1372–82.