Accepted Manuscript

Fluorescent Carbonaceous Nanospheres As Biological Probe for Noninvasive

Brain Imaging

Jun Qian, Shaobo Ruan, Xi Cao, Xingli Cun, Jiantao Chen, Shun Shen, Xinguo
Jiang, Qin He, Jianhua Zhu, Huile Gao

PII: S0021-9797(14)00616-X
DOI: http://dx.doi.org/10.1016/j.jcis.2014.08.059
Reference: YJCIS 19794

To appear in: Journal of Colloid and Interface Science

Received Date: 24 July 2014

Accepted Date: 28 August 2014

Please cite this article as: J. Qian, S. Ruan, X. Cao, X. Cun, J. Chen, S. Shen, X. Jiang, Q. He, J. Zhu, H. Gao,
Fluorescent Carbonaceous Nanospheres As Biological Probe for Noninvasive Brain Imaging, Journal of Colloid
and Interface Science (2014), doi: http://dx.doi.org/10.1016/j.jcis.2014.08.059

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Fluorescent Carbonaceous Nanospheres As Biological Probe for Noninvasive Brain

Imaging

Jun Qian a,1, Shaobo Ruan b,1, Xi Cao b, Xingli Cun b, Jiantao Chen b, Shun Shen a, Xinguo

Jiang a, Qin He b, Jianhua Zhu a,* and Huile Gao b,*

a
Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education; School

of Pharmacy, Fudan University; 826 Zhangheng Road, Shanghai, 201203, China.

b
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of

Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu,

610041, China.

1
contributed equally to this work.

* Corresponding author: Jianhua Zhu: Fax: 86-21-51980099. E-mail: jhzhu@shmu.edu.cn.

Huile Gao: Fax: 86-28-85502532. Tel: 86-28-85502575. E-mail: gaohuile@scu.edu.cn

1

Abstract:

Fluorescent carbonaceous nanospheres (CDs) have generated much excitement in

bioimaging because of their impressive fluorescent properties and good biocompatibility.

In this study, we evaluated the potential application of CDs in noninvasive brain imaging. A

new kind of CDs was prepared by a heat treating method using glutamic acid and glucose

as the precursors. The hydrated diameter and zeta potential of CDs were 101.1 nm

(PDI=0.110) and -22.4 mV respectively. Palpable emission spectrum could be observed

from 400 nm to 600 nm when excited at corresponding wavelength, suggesting CDs could

be used as a noninvasive bio-probe for in vivo imaging. Additionally, several experiments

indicated that CDs possess good serum stability and hemocompatibility with low

cytotoxicity. In vitro, the CDs could be efficiently taken up by bEnd.3 cells in a

concentration- and time- dependent manner. In vivo, CDs could be used for noninvasive

brain imaging due to its high accumulation in brain region, which was demonstrated by in

vivo imaging and ex vivo tissue imaging. Moreover, the fluorescent distribution in tissue

slice showed CDs accumulated in brain with high intensity. In conclusion, CDs were

prepared using a simple one-step method with unique optical and good biological

properties and could be used for noninvasive brain imaging.

Key-words: Fluorescent carbonaceous nanospheres, brain imaging, biological probe.

2

1. Introduction

Fluorescent carbonaceous nanospheres (CDs) are gaining intense interest because of

their unique optical and novel properties, such as strong and tunable photoluminescence,

low photobleaching, no optical blinking and high levels of brightness and photostability

[1-6]. Compared with traditional quantum dots and organic dyes, carbon-based

nitrogen-doped CDs are promising alternatives due to not only their distinctive optical

characteristics but also low toxicity and good biocompatibility. These properties enable

CDs with potential application for noninvasive bio-imaging [7-12].

Currently, most researches focused on the methods to develop CDs with excellent

properties via different ways using various materials, including cocoon silk, chitosan,

glucose and amino acids[13-16]. However, the short excitation and emission wavelengths

of most CDs restricted their potential in vivo application. Therefore, it is essential to

prepare CDs with relatively longer excitation and emission wavelengths through a simple

one-step method[17, 18].

Although it has been reported that CDs could be used for imaging/tracking in biological

systems, the interaction of CDs with biological systems mostly stayed at the cellular

level[19]. The in vivo compatibility and in vivo behavior was far from well-known, which

was crucial for in vivo bio-imaging of CDs in normal and diseased tissues. For biological

applications, the stability and safety of CDs was a major concern[20, 21]. However,

reports focused rarely on their stability and safety, despite cytotoxicity was often evaluated

in previous studies[7, 22]. Thus the stability and safety of CDs were evaluated in this

study.

3

The brain is one of the most important organs for human being. Correspondingly,

brain-related diseases are huge threats for human being[23]. Normally, brain imaging was

performed using traditional methods, such as computed tomography scan (CT scan),

Positron emission tomography (PET), magnetic resonance imaging (MRI) and

angiography[24]. However, the potential of CDs in brain imaging was not clear as far as

we know.

In this study, we developed a modified method to prepare CDs with relatively long

excitation and emission wavelength using glutamic acid and glucose as the co-precursors

(Fig.1). The serum adsorption, hemocompatibility and cytotoxicity were carried out to

evaluate the stability and safety of CDs. bEnd.3 cells were used to elucidate the cellular

uptake behavior of CDs. In vivo and ex vivo imaging and tissue distribution were

performed to evaluate the potential brain imaging effect of CDs.

2. Materials and methods

2.1 Materials

The Glutamate and Glucose were obtained from Chengdu Kelong Chemical Co., Ltd

(Chengdu, China). 4',6-diamidino-2-phenylindole (DAPI) and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from

Beyotime (Haimen, China). Plastic cell culture dishes and plates were obtained from Wuxi

NEST Biotechnology Co., Ltd (Wuxi, China). Dulbecco’s Modified Eagle Medium (high

glucose) cell culture medium (DMEM) and FBS were obtained from Life Technologies

(Grand Island, NY, USA). bEnd.3 cell line was obtained from institute of Biochemistry and

Cell Biology, Shanghai institutes for Biological Sciences, Chinese Academy of Scienses

4

(Shanghai, China). Male nude mice (20 ± 2 g) were purchased from Dashuo Bio

technology Co., Ltd, (Chengdu, China) and maintained under standard housing conditions.

All animal experiments were carried out in accordance with guidelines evaluated and

approved by the ethics committee of Sichuan University.

2.2 Preparation and characterization of CDs

CDs could be possessed from directly pyroprocess treatment of glutamate and glucose.

Briefly, all the glass wares were immersed in the potassium dichromate sulfuric acid

solution, rinsed with high-purified deionized water for several times and then dried. 100

mL round-bottomed flask was heated to 250°C, then 1 g glutamic acid was added into the

flask. When the glutamate melted to nearly half, 0.1 g glucose was introduced rapidly into

the reaction system and the temperature was maintained till the glutamate and glucose

melted completely. After 30 second’s cooling, 5 mL deionized water was added into the

flask. All the suspension was directly extruded with 0.22 μm aqueous filter.

The hydrated diameter and zeta potential were determined by a Malvern Zetasizer

(Malvern, NanoZS, UK). The morphology was captured by transmission electronic

microscopy (TEM) (JEM 100CX, JEOL, Japan). Fluorescence spectroscopy was

evaluated by a Shimadzu RF-5301PC spectrofluorophotometer. UV-Vis spectra of CDs

dispersed in water were determined on a Varian cary 100 conc UV-Vis

spectrophotometer.

2.3 Stability of CDs

The stability of CDs was evaluated in PBS with different concentrations of human plasma.

CDs were suspended in 0%, 10%, 50% plasma and incubated in a 37 °C incubator. The

5

absorption at 570 nm was determined by a microplate reader (Multiskan MK3, Thermo,

USA) at 0, 1, 2, 4, 8, 12 and 24 h.

2.4 Hemocompatibility of CDs

Whole blood was collected from mice using heparin as anticoagulant. After centrifugation

at 3500 rpm for 5 min, the red blood cells were resuspended in PBS at a final density of

2%. Different concentrations of CDs were added into cell suspension and incubated at

37 °C for 0, 1, 2, 4, 8 and 12 h. The absorption at 450 nm was determined by a microplate

reader (Multiskan MK3, Thermo, USA). PBS was used as the negative control while 1% of

Triton X-100 was used as positive control.

2.5 Cytotoxicity of CDs

bEnd.3 cells were seeded into 96-well plate at a density of 5×103 cell/mL and allowed to

grow until 60% confluent. Each well was added with CDs at a series of concentrations and

incubated for 24 h. Then 20 μL of MTT solution (5 mg/mL in PBS) was added into each

well and cells were further incubated for 4 h under 37 °C. Then cells were dissolved by

150 μL dimethyl sulfoxide after removing the media. The absorbance was measured by a

microplate reader (Multiskan MK3, Thermo, USA) at 490 nm.

2.6 In vitro bEnd.3 cells uptake study

bEnd.3 cells were seeded into 12-well plates at a density of 1×105 cell/mL. After 2 days

incubation, cells were treated with different concentrations of CDs in FBS-free DMEM for

2 h under 37 °C. Meanwhile, cells were treated with equivalent concentration of CDs in

FBS-free DMEM for different periods of time at 37 °C. Then cells were washed with PBS

twice, harvested and suspended in 300 μL of PBS for analysis using FC-500 flow

6

cytometer (Beckman Coulter, Fullerton, CA, USA).

bEnd.3 cells were seeded onto 12 mm coverslips in 6-well plate at a density of 1×105

cell/mL and allowed to grow until 60% confluent. Cells were treated with different

concentrations of CDs in FBS-free DMEM for 1 h under 37 °C. The cells were also treated

with CDs in FBS-free DMEM for 15 min, 1 h, 4 h at an equivalent concentration under

37 °C. Cells were then washed with PBS twice and fixed with fresh 4% paraformaldehyde

for 20 min at room temperature, and the nuclei were stained by 0.5 μg/mL DAPI for 5 min.

The coverslips were mounted on glass microscope slides with a drop of antifade mounting

media to reduce fluorescence photobleaching. The fluorescent distribution in cells was

visualized by a confocal microscope (LSM710, Carl Zeiss, Germany).

2.7 In vivo imaging and tissue distribution

Male mice were i.v. administered 100 mg/kg of CDs through the tail vein. Then the whole

body fluorescent distribution was observed via an in vivo imaging system (IVIS Spectrum,

Caliper, USA) at 0.25, 0.5, 1 and 2 h after injection. Then, the mice were heart perfused

with saline and then sacrificed. The brains were sampled and subjected to ex vivo

fluorescent imaging. After fixed with 4% paraformaldehyde for 24 h at 4 °C, brain tissues

were further dehydrated by 15% sucrose for 24 h, then followed with 30% sucrose for

another 24 h. Consecutive frozen sections of 10 μm thicknesses were prepared. Brain

slices were stained with 0.5 μg/mL of DAPI under a procedure established previously[25].

Then the fluorescent distribution was visualized by a confocal microscope (LSM710, Carl

Zeiss, Germany).

2.8Statistical analysis

7

Statistical comparisons were performed by one-way ANOVA for multiple groups, and p

value<0.05 and <0.01 were considered indications of statistical difference and statistically

significant difference, respectively.

3. Result and discussion

3.1 preparation and characterization

The preparation methods for CDs have been extensively explored, including Laser

ablation, passivation, microwave, ultrasonic synthesis, thermal and hydrothermal

oxidation[26]. Among which, heat-treatment method was widely used because of the

simple and controllable procedure. In this contribution, heat-treatment method was used

to prepare CDs using glutamic acid and glucose as the materials. The hydrated particle

diameter of prepared CDs was 107.2 nm with a polydispersity index of 0.161 (Fig. 1A).

TEM showed CDs was spherical with uniform size and well dispersed (Fig. 1B). Zeta

potential of CDs was -21.7 mV, which was mainly due to the carboxyl units in the surface

of CDs.

The CDs have special optical properties mainly owing to the quantum confinement effect.

There was obvious absorption peaks at 213 nm, 328 nm and 457 nm in the UV-Vis

spectra of CDs (Fig. 1C), which was the typical properties of fluorescent carbonaceous

nanospheres. When the excitation wavelength was increased from 350 nm to 430 nm with

a step length of 10 nm, the emission spectroscopy was elevated accordingly (Fig. 1D).

During the 350 nm to 420 nm, the emission spectroscopy demonstrated a long-stokes

shift according to the excitation wavelength, suggesting the CDs could be excited by

different wavelengths of source. The maximum emission wavelength was 458 nm when
8

the CDs were excited by 395 nm. The maximum excitation wavelength has a long-stokes

shift than our previously prepared fluorescent carbon dots from spider silk[16], which was

attributed to the synergistic effect of the core and the surface of CDs. The fluorescent

properties enabled CDs with potential application in bio-imaging.

3.2 Plasma stability

For potential in vivo application, the particle should be stable in the blood circulation.

Protein absorption is one of the major concerns that needed to be introduced since protein

coronas were often formed on the surface of nanoparticles and altered their stability and

in vivo behavior[27]. Thus plasma stability was evaluated by incubating CDs with different

concentrations of plasma for different periods of time (Fig. 2A). The adsorption of protein

onto CDs at 560 nm was obviously increased with the prolongation of incubation time,

indicating that the CDs could considerably adsorb plasma protein and lead to increasing

absorption at a time-dependent manner. The result also demonstrated that the interaction

of CDs with serum protein was in a concentration-dependent manner. The protein

adsorption is normal phenomenon when particles contracted with serum protein, resulting

in modification of in vivo behaviors of nanoparticles, which needed to be further

explored[27].

3.3 Hemocompatibility

9

Hemocompatibility is another concern for in vivo application because poor

hemocompatibility could lead to acute toxicity to bodies. The hemocompatibility was

carried out using 2% fresh blood red cells. Increasing the incubation time of CDs with

blood red cells and the concentration of CDs could significantly elevate the hemolysis rate

(Fig. 3A), suggesting the hemocompatibility was time- and concentration-dependent. The

hemolysis rate of CDs could increase to 24.5% when incubated with blood red cells for 12

h at a concentration of 5 mg/mL. However, for traditional quantum dots, such as CdTe,

obvious hemolysis (19.4%) could be observed after 4 h incubation with blood red cells at a

concentration of 120 wg/mL[28]. Additionally, the microscopy images also showed the

integrity of blood red cells after 8 h incubation at a concentration of 1.66 mg/mL (Fig. 3B).

These results indicated that the hemolysis of CDs was relatively low, which was

consistent with previous study and the CDs could be further used for in vivo application[29,

30].

3.4 Cytotoxicity

To evaluate the safety of CDs in biological systems, MTT assay was performed to

elucidate the toxicity to cell. bEnd.3 cells were incubated with a series of concentrations of

CDs for 24 h. The cell viability displayed no apparent change between cells treated with

CDs from the concentration of 0.002 mg/mL to 1.66 mg/mL (Fig. 2B). The cell viability

could approach to approximately 80% even at a concentration of 5 mg/mL, which was

much higher than the concentration used in cell imaging, suggesting that the cytotoxicity

10

of CDs was relatively low. In comparison, the cytotoxicity of traditional quantum dots was

considerably higher, which was consistent with previous study[16, 19].

3.5 In vitro bEnd.3 cells uptake study

The intensity increased as the incubated concentration increased (Fig. 4A), suggesting

that the uptake of CDs by bEnd.3 cells was concentration-dependent. Further expanding

the incubation time at an equivalent concentration of 0.33 mg/mL, the intensity showed a

gradual enhancement (Fig. 4B), indicating that the uptake procedure was also

time-dependent. These results were consistent with previous study[30].

To determine the potential application of CDs in cell imaging, the interaction of CDs with

bEnd.3 cells was further evaluated using a confocal microscope. The fluorescent

distribution in bEnd.3 cells was observed (Fig. 5). Increasing the concentration of CDs and

extending the incubation time could significantly enhance the fluorescent intensity,

suggesting that CDs could be used for cell imaging and the uptake behavior of CDs was

concentration- and time-related. Although it has showed that CDs could be taken up

efficiently by bEnd.3 cells, the internalized behavior of CDs with bEnd.3 cells remained

unclear. As far as we know, endosome-mediated pathway existed in most cases of the

cellular internalization of nanoscale particles, which accounted for the main reason why

CDs could be taken up efficiently into bEnd.3 cells. Furthermore, Receptor-mediated

endocytosis could promote the uptake of CDs by bEnd.3 cells. It has been reported that

glucose receptors and glutamate receptors were both expressed on the cell surface,

which might be the cause that CDs could be taken up by bEnd.3 cells efficiently.[31]

11

3.6 In vivo and ex vivo imaging

After intravenous injection of CDs, epi-fluorescent distribution showed a relatively high

intensity at 5 min in brain region (Fig. 6A), which increased gradually to highest at 1 h and

got weaker as time prolonged to 2 h, suggesting that CDs could be delivered quickly and

sufficiently to brain with a relatively quick systemic elimination. Ex vivo imaging of brain

further demonstrated the CDs could be used for noninvasive brain imaging. Normally, the

rate of nanosized particles accumulated and eliminated in brain was probably related with

particle size[32, 33]. These results demonstrated that the nanoscale CDs could efficiently

penetrate the blood brain barrier (BBB) and accumulate in brain, which was useful for

brain imaging and needed to be explored[30, 34]. In most of the situations, it was difficult

to remain in biological blood circulation without modification with PEG and traverse the

BBB without the mediation of ligand for normal nanoparticle. However, CDs has

demonstrated its superior ability in traversing BBB and brain imaging, which was possibly

owing to the expression of glucose and glutamate receptors on brain capillary endothelial

cells. Meanwhile, the CDs were prepared by organic materials and consisted of simple

elements, which might reduce the rejection from BBB towards exogenous materials. All

these reasons might contribute to the main possible mechanisms of targeting and

traversing the BBB. Further modification of PEG and specific ligands on CDs could

improve the targeting efficiency, which enabled CDs to be a superior alternative in

traversing and bioimaging in brain[35, 36]. Current application of CDs was mainly focused

on the tracking and diagnosing of lesion location, such as glioma, meningitis and

adeno-associated virus (AAV)[37]. Therefore, it was interesting and necessary to explore

12

the potential application of CDs in biological system.

3.7 Tissue distribution

Tissue slices were prepared to elucidate the distribution of CDs in different brain regions.

In the cortex of brain, obvious fluorescent intensity could be observed in the edge, and the

intensity was also high in hippocampus and ventricle, which indicated that the CDs could

traverse the BBB and accumulate in brain efficiently even in the deep brain region. The

result demonstrated that CDs could be used as the noninvasive brain imaging probe with

low cytotoxicity to brain tissue.

4. Conclusion

In this study, a new kind of CDs was developed with designed long excitation/emission

wavelengths through simple heat treatment of glutamic acid and glucose. The CDs

showed good hemocompatibility, low plasma adsorption and low cytotoxicity, indicating its

good stability and safety. In vitro, CDs could be taken up by bEnd.3 cells in a

concentration- and time-dependent manner. In vivo, CDs could efficiently distributed into

brain with a high intensity, suggesting that CDs could be used for noninvasive brain

imaging.

Acknowledgements:

The work was granted by National Basic Research Program of China (973 Program,

2013CB932504), National Natural Science Foundation of China (81373337) and the

Sichuan University Starting Foundation for Young Teachers (2014SCU11044).

13

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ϭϳ͘ z͘>ŝƵ͕͘ͲLJ͘>ŝƵ͕͘ͲLJ͘ŚĂŶŐ͘:ŽƵƌŶĂůŽĨDĂƚĞƌŝĂůƐŚĞŵŝƐƚƌLJ͘ϭ;ϮϬϭϯͿϰϵϬϮͲϰϵϬϳ͘
ϭϴ͘ >͘tĂŶŐ͕^͘Ͳ:͘ŚƵ͕,͘Ͳz͘tĂŶŐ͕^͘ͲE͘YƵ͕z͘Ͳ>͘ŚĂŶŐ͕:͘Ͳ,͘ŚĂŶŐ͕Y͘Ͳ͘ŚĞŶ͕,͘Ͳ>͘yƵ͕t͘,ĂŶ͕
͘zĂŶŐ͘^ŶĂŶŽ͘ϴ;ϮϬϭϰͿϮϱϰϭͲϮϱϰϳ͘
ϭϵ͘ ^͘ZƵĂŶ͕:͘tĂŶ͕z͘&Ƶ͕<͘,ĂŶ͕y͘>ŝ͕:͘ŚĞŶ͕Y͘ŚĂŶŐ͕^͘^ŚĞŶ͕Y͘,Ğ͕,͘'ĂŽ͘ŝŽĐŽŶũƵŐĂƚĞ
ĐŚĞŵŝƐƚƌLJ͘;ϮϬϭϰͿ͘
ϮϬ͘ W͘ ZƵĞŶƌĂƌŽĞŶŐƐĂŬ͕ :͘D͘ ŽŽŬ͕ ͘d͘ &ůŽƌĞŶĐĞ͘ :ŽƵƌŶĂů ŽĨ ŽŶƚƌŽůůĞĚ ZĞůĞĂƐĞ͘ ϭϰϭ ;ϮϬϭϬͿ
ϮϲϱͲϮϳϲ͘
Ϯϭ͘ <͘Z͘ sĞŐĂͲsŝůůĂ͕ :͘<͘ dĂŬĞŵŽƚŽ͕ :͘͘ zĄŹĞnj͕ ͘D͘ ZĞŵƐďĞƌŐ͕ D͘>͘ &ŽƌƌĞƐƚ͕ E͘D͘ ĂǀŝĞƐ͘
ĚǀĂŶĐĞĚĚƌƵŐĚĞůŝǀĞƌLJƌĞǀŝĞǁƐ͘ϲϬ;ϮϬϬϴͿϵϮϵͲϵϯϴ͘
ϮϮ͘ z͘zĂŶŐ͕:͘Ƶŝ͕D͘ŚĞŶŐ͕͘,Ƶ͕^͘dĂŶ͕z͘yŝĂŽ͕Y͘zĂŶŐ͕z͘>ŝƵ͘ŚĞŵŝĐĂůŽŵŵƵŶŝĐĂƚŝŽŶƐ͘ϰϴ
;ϮϬϭϮͿϯϴϬͲϯϴϮ͘
Ϯϯ͘ ,͘'ĂŽ͕͘WĂŶŐ͕y͘:ŝĂŶŐ͘WŚĂƌŵĂĐĞƵƚŝĐĂůƌĞƐĞĂƌĐŚ͘ϯϬ;ϮϬϭϯͿϮϰϴϱͲϮϰϵϴ͘
Ϯϰ͘ t͘͘ WĞŶŶLJ͕ <͘:͘ &ƌŝƐƚŽŶ͕ :͘d͘ ƐŚďƵƌŶĞƌ͕ ^͘:͘ <ŝĞďĞů͕ d͘͘ EŝĐŚŽůƐ͕ ^ƚĂƚŝƐƚŝĐĂů WĂƌĂŵĞƚƌŝĐ
DĂƉƉŝŶŐ͗dŚĞŶĂůLJƐŝƐŽĨ&ƵŶĐƚŝŽŶĂůƌĂŝŶ/ŵĂŐĞƐ͗dŚĞŶĂůLJƐŝƐŽĨ&ƵŶĐƚŝŽŶĂůƌĂŝŶ/ŵĂŐĞƐ͘
ϮϬϭϭ͗ĐĂĚĞŵŝĐWƌĞƐƐ͘
Ϯϱ͘ ,͘'ĂŽ͕:͘YŝĂŶ͕^͘ĂŽ͕͘zĂŶŐ͕͘WĂŶŐ͕^͘WĂŶ͕>͘&ĂŶ͕͘yŝ͕y͘:ŝĂŶŐ͕Y͘ŚĂŶŐ͘ŝŽŵĂƚĞƌŝĂůƐ͘
ϯϯ;ϮϬϭϮͿϱϭϭϱͲϱϭϮϯ͘
Ϯϲ͘ z͘yƵ͕D͘tƵ͕z͘>ŝƵ͕y͘͘&ĞŶŐ͕y͘͘zŝŶ͕y͘t͘,Ğ͕z͘<͘ŚĂŶŐ͘ŚĞŵŝƐƚƌLJͲƵƌŽƉĞĂŶ:ŽƵƌŶĂů͘
ϭϵ;ϮϬϭϯͿϮϮϳϲͲϮϮϴϯ͘

14

Ϯϳ͘ ,͘'ĂŽ͕Y͘,Ğ͘džƉĞƌƚŽƉŝŶŝŽŶŽŶĚƌƵŐĚĞůŝǀĞƌLJ͘ϭϭ;ϮϬϭϰͿϰϬϵͲϰϮϬ͘
Ϯϴ͘ z͘Ͳ&͘>ŝƵ͕:͘Ͳ^͘zƵ͘:ŽƵƌŶĂůŽĨĐŽůůŽŝĚĂŶĚŝŶƚĞƌĨĂĐĞƐĐŝĞŶĐĞ͘ϯϱϭ;ϮϬϭϬͿϭͲϵ͘
Ϯϵ͘ :͘ YŝĂŶ͕ :͘ ŚĞŶ͕ ^͘ ZƵĂŶ͕ ^͘ ^ŚĞŶ͕ Y͘ ,Ğ͕ y͘ :ŝĂŶŐ͕ :͘ ŚƵ͕ ,͘ 'ĂŽ͘ :ŽƵƌŶĂů ŽĨ ĐŽůůŽŝĚ ĂŶĚ
ŝŶƚĞƌĨĂĐĞƐĐŝĞŶĐĞ͘ϰϮϵ;ϮϬϭϰͿϳϳͲϴϮ͘
ϯϬ͘ ^͘ZƵĂŶ͕:͘YŝĂŶ͕:͘ŚƵ͕y͘:ŝĂŶŐ͕Y͘,Ğ͕,͘'ĂŽ͘EĂŶŽƐĐĂůĞ͘;ϮϬϭϰͿ͘
ϯϭ͘ <͘<ŽďĂLJĂƐŚŝ͕,͘zĂŵĂŶĂŬĂ͕&͘zĂŶĂŵŽƚŽ͕D͘KŬƵďŽ͕<͘EŽŐƵĐŚŝ͘'ůŝĂ͘ϲϬ;ϮϬϭϮͿϭϱϮϵͲϭϱϯϵ͘
ϯϮ͘ ͘͘^LJŬĞƐ͕:͘ŚĞŶ͕'͘ŚĞŶŐ͕t͘͘ŚĂŶ͘^ŶĂŶŽ͘;ϮϬϭϰͿ͘
ϯϯ͘ ͘͘ tĂůŬĞLJ͕ :͘͘ KůƐĞŶ͕ ,͘ 'ƵŽ͕ ͘ ŵŝůŝ͕ t͘͘ ŚĂŶ͘ :ŽƵƌŶĂů ŽĨ ƚŚĞ ŵĞƌŝĐĂŶ ŚĞŵŝĐĂů
^ŽĐŝĞƚLJ͘ϭϯϰ;ϮϬϭϮͿϮϭϯϵͲϮϭϰϳ͘
ϯϰ͘ R. Qiao, Q. Jia, S. Hüwel͕ Z͘ yŝĂ͕ d͘ >ŝƵ͕ &͘ 'ĂŽ͕ ,͘Ͳ:͘ 'ĂůůĂ͕ D͘ 'ĂŽ͘ ^ ŶĂŶŽ͘ ϲ ;ϮϬϭϮͿ
ϯϯϬϰͲϯϯϭϬ͘
ϯϱ͘ ,͘'ĂŽ͕͘zĂŶŐ͕^͘ŚĂŶŐ͕^͘ĂŽ͕͘WĂŶŐ͕y͘zĂŶŐ͕y͘:ŝĂŶŐ͘:ŽƵƌŶĂůŽĨŽŶƚƌŽůůĞĚZĞůĞĂƐĞ͘ϭϳϮ
;ϮϬϭϯͿϵϮϭͲϵϮϴ͘
ϯϲ͘ ,͘'ĂŽ͕͘zĂŶŐ͕^͘ŚĂŶŐ͕^͘ĂŽ͕^͘^ŚĞŶ͕͘WĂŶŐ͕y͘:ŝĂŶŐ͘^ĐŝĞŶƚŝĨŝĐƌĞƉŽƌƚƐ͘ϯ;ϮϬϭϯͿ͘
ϯϳ͘ ͘ŚĞŶŐ͕,͘tĂŶŐ͘EĂŶŽŵĞĚŝĐŝŶĞ͘ϴ;ϮϬϭϯͿϭϮϮϱͲϭϮϮϳ͘


15

Fig.1. A: Elucidation of systematic preparation of CDs. B: Elucidation of cellular uptake

procedure of CDs.

Fig.2. Characterization of CDs. A: Dynamic light scattering data; B: TEM image, bar

represented 100 wm; C Absorption spectrum at UV-Vis; D Emission spectrum at different

excitation wavelengths of CDs.

Fig. 3. A: Plasma stability of CDs in different concentrations of plasma for different periods

of time. B: MTT assay of different concentrations of CDs on bEnd.3 cell after 24 h

incubation.

Fig. 4. Hemocompatibility of CDs. A: Time- and concentration-related hemolysis rate of

CDs. B: Images of blood red cells incubated with 1.66 mg/mL CDs for 8 h, bar

represented 50 wm.

Fig. 5. Quantitative cellular uptake using flow cytometer analysis. bEnd.3 cell uptake of

CDs at different concentrations for 2 h (A) and at the equivalent concentration for different

time periods (B).

Fig. 6. bEnd.3 cells uptake of CDs at different concentration for different time, bar

represented wP

Fig. 7. In vivo and ex vivo of normal mice after administrated with CDs. A: Whole body

distribution of CDs at different time point after injection. B: Ex vivo imaging of brain tissue

at 2 h after injection. C-: Distribution of CDs in brain slices, green represented CDs, blue

represented DAPI and bar represented 100 wm.

16

 Highlights
1. A new kind of fluorescent carbonaceous nanospheres (CDs) was prepared using
glutamic acid and glucose as the precursors.

2. The prepared CDs possessed excellent optical properties and good biocompatibility.

3. CDs were used as biological probe for cell imaging in vitro.

4. We report the application of CDs in non-invasive brain imaging.

17

Figure 1
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