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The long-term stability and biocompatibility of

fluorescent nanodiamond

as an in vivo contrast agent

V. Vaijayanthimala

a

,

1

, Po-Yun Cheng

b

,

1

, Shih-Hua Yeh

c

, Kuang-Kai Liu

b

, Cheng-Hsiang Hsiao

d

,

e

,

**

,

Jui-I Chao

b

,

***

, Huan-Cheng Chang

a

,

c

,

*

aInstitute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10672, Taiwan, ROC

bDepartment of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan, ROC cDepartment of Chemistry, National Taiwan University, Taipei 10672, Taiwan, ROC

dDepartment of Pathology, National Taiwan University Hospital, Taipei 10002, Taiwan, ROC

eGeneral Education Center, National Taipei University of Nursing and Health Sciences, Taipei 11219, Taiwan, ROC

a r t i c l e i n f o

Article history:

Received 18 April 2012 Accepted 28 June 2012 Available online 3 August 2012 Keywords: Animal model Biocompatibility Diamond Fluorescence Nanoparticle Sentinel lymph node

a b s t r a c t

Nanocarbon is a promising type of biomaterial for diagnostic and therapeutic applications. Fluorescent nanodiamond (FND) containing nitrogen-vacancy centers as built-influorophores is a new addition to the nanocarbon family. Here, we study the long-term stability and biocompatibility of 100-nm FNDs in rats through intraperitoneal injection over 5 months and develop the potential application of this biomaterial for sentinel lymph node mapping in a mouse model. From both in vivo and ex vivo fluo-rescence imaging as well as transmission electron microscopy, we found that the intradermally administered FND particles can be drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice. Our measurements of water consumption, fodder consumption, body weight, and organ index showed no significant difference between control and FND-treated groups of the rats. Histopathological analysis of various tissues and organs indicated that FNDs are non-toxic even when a large quantity, up to 75 mg/kg body weight, of the particles was administered intraperitoneally to the living animals. With the properties of wide-ranging biocompati-bility and perfect chemical and photophysical stabiocompati-bility, FND is well suited for use as a contrast agent for long-term in vivo imaging.

Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The application of nanotechnology to cancer therapy and drug

delivery is a topic of current interest in nanomedicine

[1

e3]

. It has

opened up many exciting new opportunities for diagnosing, treating,

and tracking the progress of cancer development in molecular and

cellular detail. Nanodiamond (ND) has recently emerged as

a promising biomaterial for such applications

[4

e6]

. The material

can be produced by either detonation,

high-pressure-high-temperature (HPHT), or chemical vapor deposition (CVD) methods,

followed by deagglomeration or milling processes

[7]

. Several

biocompatibility studies of the particles in vitro with various cell

lines have shown that ND is among the least toxic of all

carbon-based nanomaterials tested so far

[8,9]

. Moreover, NDs after

inter-nalization do not cause any obvious cytotoxic or detrimental effects

on the proliferation and differentiation of cells

[10,11]

. The chemical

and biological inertness, augmented by the fact that diamond can be

emit far-red

fluorescence from a variety of optically active defects

[12]

, is a de

finite advantage when using NDs for in vivo applications.

In the past few years, there have been some studies on the

biocompatibility and biodistribution of NDs in vivo. Speci

fically,

Puzyr et al.

[13]

investigated the biocompatibility of detonation

nanodiamonds (DNDs) injected subcutaneously into mice and

found no in

flammatory response at 3 months post-dosing.

However, intravenous administration of DNDs into rabbits could

change the values of some blood biochemical parameters such as

the total bilirubin concentration. Yuan et al.

[14,15]

have recently

studied the in vivo biodistribution and dose-dependent liver and

hematological toxicity of NDs in mice. Intratracheal instillation of

4-nm DNDs and 50-4-nm HPHT-NDs did not show any signi

ficant

* Corresponding author. Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10672, Taiwan, ROC. Tel.:þ886 2 23668260; fax: þ886 2 2362 0200. ** Corresponding author. Department of Pathology, National Taiwan University Hospital, Taipei 10002, Taiwan, ROC. Tel.:þ886 2 23123456xt65457; fax: þ886 2 23934172.

*** Corresponding author. Tel.: þ886 3 5712121; fax: þ886 3 5556219. E-mail addresses: chhsiao7@ntu.edu.tw (C.-H. Hsiao), jichao@ faculty.nctu.edu.tw(J.-I. Chao),hchang@gate.sinica.edu.tw(H.-C. Chang).

1 These two authors contribute equally to this work.

Contents lists available at

SciVerse ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w . e l s e v i e r. co m/ lo ca t e / b i o m a t e ri a l s

0142-9612/$e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.06.084

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pulmonary toxicity and no evidence on lipid peroxidation of the

lung was detected. Marcon et al.

[16]

have further assessed the

in vitro and in vivo toxicity of DNDs with different surface modi

fi-cations (

eOH, eNH

2

, or

eCO

2

H) in HEK 293 cells and Xenopus

laevis. Cell viability assays revealed that NDs were not toxic to HEK

293 cells at concentrations less than 50

m

g/mL, whereas

microin-jection of ND-CO

2

H into early-stage X. laevis embryos caused

embryotoxicity and teratogenicity. Most recently, Rojas et al.

[17]

studied the in vivo biodistribution of DNDs labeled with

18

F in

rats by using positron emission tomography and evaluated the

impact of kinetic particle size on biodistribution. Their results

showed that the radio-labeled DNDs largely accumulated in the

lung, spleen, and liver, and also excreted into the urinary tract.

Removal of larger particles by prior

filtration effectively prevented

the particle accumulation in lung and spleen.

We present herein the result of our study on the long-term

stability and biocompatibility of

fluorescent nanodiamonds

(FNDs) in a rat model through intraperitoneal injection over a time

period of 5 months. It is an extension of our previous studies of

FNDs in Caenorhabditis elegans

[18]

and zebra

fish (Danio rerio)

[19]

,

where we showed that the carbon-based nanomaterial does not

induce any detectable oxidative stress response at the whole

organism level. In this study, after con

firming the non-toxic nature

of the nanomaterial in rats, we apply the FND particles for sentinel

lymph node mapping by both in vivo and ex vivo imaging in mice.

Sentinel lymph node (SLN) mapping is an important step in SLN

biopsy, by which doctors can determine the cancer stage and

accounts for the choice of therapy

[20]

. Although near-infrared

quantum dots (QDs) have been successfully applied for the

purpose

[21]

, the notion of using potentially toxic,

heavy-metal-based QDs for SLN mapping in humans is still under serious

debate

[1,22]

. The non-toxic FNDs clearly offer a favorable

alter-native and, to the best of our knowledge, no such applications have

been reported so far.

The FNDs used in this work were produced by ion irradiation

and subsequent annealing of synthetic type Ib ND powders

[23

e25]

. These particles contain negatively charged

nitrogen-vacancy (NV



) defect centers (with a concentration of up to

10 ppm

[25]

) as built-in

fluorophores. Excitation of the NV



with

green yellow light yields exceptionally stable photoluminescence

with a zero-phonon line at 638 nm, accompanied with a broad

phonon sideband spanning from 600 to 800 nm

[26]

. Nearly 70% of

the

fluorescence lies in the near-infrared window (

Fig. 1

a)

[27]

,

making it suitable for optical bioimaging applications

[28]

. Aside

from this potential usefulness, the

fluorescence lifetime (

s

¼ 11.6 ns

in bulk diamond

[29]

) of the NV



center is substantially longer than

that (typically

s

< 4 ns) of cell and tissue autofluorescence

[30]

,

which allows for the utilization of various time-gating techniques

to enhance the image contrast

[31,32]

. Further improvement of the

contrast is possible by taking advantages of the spin properties of

the NV



center, which is magneto-optical and can be magnetically

manipulated

[33]

.

Three injection methods were employed in this study:

intra-dermal (i.d.), intraperitoneal (i.p.), and subcutaneous (s.c.)

admin-istrations. We

first conducted s.c. injection of FND solution into rat

skin to investigate the long-term stability of the nanomaterial

in vivo for more than 1 month. We then performed i.d. injection of

the FND solution into mouse paws for SLN imaging and short-term

toxicity assessment. With i.p., we were able to inject a relatively

large amount (up to 23 mg per rat) of FNDs into the intraperitoneal

Fig. 1. (a) Comparison of thefluorescence spectrum (red curve) of NVcenters in FNDs with the near-infrared (NIR) window of biological tissues. The black, dark gray, and light gray

curves are the absorption spectra of H2O, oxygen-bound hemoglobin (HbO2), and hemoglobin (Hb), respectively. The absorption spectra were adapted from Ref.[27]. (b) Long-term

stability test of FNDs in a rat after s.c. injection. Images of the same rat were acquired over a time period of more than 37 days. White arrows indicate the site of FND injection. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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cavities of the rats to address its long-term biocompatibility at high

doses. Finally, we carried out detailed histopathological analysis of

rat tissues to reveal whether the injected FND particles would cause

any in

flammation or general toxicity in this animal model.

Impor-tant implications of these results are discussed.

2. Materials and methods 2.1. Production of FNDs

Synthetic type Ib diamond powders (Micronþ MDA 0-0.10, Element Six) were radiation-damaged by using either a 40-keV Heþbeam at a dose ofw1  1014

ions/ cm2or a 3-MeV Hþbeam at a dose ofw1  1016

ions/cm2to create the optimum amount of vacancies in the diamond crystal lattice, as previously described[25]. They were subsequently annealed in vacuum at 800C for 2 h to form FNDs. After

being oxidized in air at 450C for 1 h and washed in concentrated sulfuric and nitric acid (3:1, v/v) solution at 100C for 3 h[11], the NV-containing particles were extensively rinsed in distilled deionized water (DDW) and stored at room temper-ature prior to use. A comprehensive characterization of the FNDs prior to bio-conjugation and injection can be found in[23,24,34]and also inFigures S1 and S2of Supporting Information.

2.2. Preparation of BSA-coated FNDs

Acid-washed FNDs were sonicated in DDW for 30 min and then mixed with bovine serum albumin (BSA) in the weight ratio of 1:2[34]. After vortex mixing for 1 h, excess BSA was removed by DDW wash once, followed byfive washes with phosphate-buffered saline (PBS) using centrifugalfilter devices (Amicon Ultra-15, 100 K or Amicon Ultra-4, 3 K, Millipore). Size distributions of the FND particles before and after BSA coating were determined with a particle size and zeta-potential analyzer (Delsa Nano C, Beckman-Coulter). Thefinal concentration of the BSA-coated FNDs in PBS was 2 mg/mL.

2.3. Animals

BALB/c male nude mice and male Sprague DawleyÒ(SD) rats were purchased from BioLASCO (Taiwan) and acclimated for 2e3 weeks in the animal facility of National Chiao Tung University. Eight-week old mice were used in this experiment and each mouse weighed about 20 g. Similarly, the rats were 8 weeks of age and each weighed approximately of 400e600 g at the beginning of the experiment. Both animals were housed in polycarbonate cages (maximum of 3 rats per cage) with wooden chip bedding and corn stalks. Bedding was changed twice a week. Drinking water and conventional feed were provided ad libitum. The animal facility has a 12-h light/dark cycle with the temperature controlled at 23 1C and a relative humidity

of 39%e43%. All the animals were maintained under specific pathogen-free condi-tions and were treated benevolently to eliminate or reduce suffering during the entire study, approved by the Institutional Animal Care and Use Committee of National Chiao Tung University. The complete study was conducted with compliance of standards established in the Guide for the Care and Use of Laboratory Animals.

2.4. Fodder and water uptake

Water and fodder were added twice a week. The amount of fodder consumed by rats was calculated from the difference between the weight of the ration offered and the amount removed from the feeder. Likewise, the extent of water uptake was calculated from the difference in the volume of water provided and the amount of water that was left in the bottle.

2.5. Weight changes and organ indices

Individual rats of PBS-injected (control) and FND-injected groups were weighed weekly. The mean body weights of the groups were plotted against time to reveal the course of weight gain or loss between the control and test groups. Likewise, organ indices were recorded for control and FND-treated animals. The rats were sacrificed and organs were excised and weighed. Organ indices (in g/g) were calculated from the ratios of the wet weights of the individual organs to the whole body weights (b.w.).

2.6. Subcutaneous administration

SD rats were anesthetized with isoflurane and their dorsal hair was clipped (Animal Clipper Model 900, Thrive) and a depilatory agent was applied to the skin to remove any remaining hair. FNDs were injected subcutaneously into the rightflank of the rat with a dose of 0.5 mg per 500mL PBS.

2.7. Intradermal administration

BALB/c male nude mice were initially anesthetized with isoflurane. BSA-coated FNDs were injected intradermally into the right foot paw at a dose of 40mg in 20mL of PBS. Similarly for the control, 20mL BSA-PBS was injected intradermally to the left paw.

2.8. Intraperitoneal administration

FNDs were injected into SD rats via i.p. administration at a dose of 5 mg/kg b.w. Control groups of the rats were exposed to PBS solution. In order to observe the fate and long-term effect, the rats were injected every week at the same dose (5 mg/kg b.w.) continuously for 12 weeks. They were then sacrificed after 12 weeks of FND injection and their organs such as lung, liver, kidney, spleen, heart and blood were collected for organ index analysis. This group of rats was considered as the non-recovery group. In a parallel experiment, another group of rats were treated for 12 weeks with the same dosage as above and were allowed to recover for 8 weeks. This group was named as the recovery group. After the recovery period, tissue samples were cut apart and weighed for organ index analysis.

2.9. In vivo and ex vivofluorescence imaging

Fluorescence snapshots of mice or rats were acquired after administration of FND particles by either s.c., i.d., or i.p. injection using an in vivo imaging system (Xenogen IVIS Spectrum, Caliper Life Sciences). Background tissue autofluorescence wasfirst measured by photoexcitation of the living animals at the wavelength of 430 nm with a bandwidth of 35 nm. The resultingfluorescence emission was collected at 780 nm with a bandwidth of 20 nm. Samplefluorescence images were then taken by excitation at 605/20 nm and collection of the emission at 780/20 nm. The typical exposure time was 30 s. The acquired images werefinally analyzed with the Living Image 4.1 software.

2.10. Histopathological examination

Dissected lymph nodes and resected tissue specimens werefixed in 10% formalin, embedded individually in paraffin blocks, and sectioned into 5mm thick-ness. The sections were then stained with hematoxylin and eosin (H&E) and examined by using an Olympus BX51 light microscope.

2.11. Transmission electron microscopy

Dissected lymph nodes werefixed in 4% paraformaldehyde and 2.5% glutaral-dehyde in 0.1Mcacodylate buffer (pH 7.4) for 24 h at 48C. After post-fixing in 1% osmium tetroxide for 1 h at room temperature, the samples were dehydrated in a gradient alcohol series, immersed in acetone, infiltrated with the increasing ratio of resin to acetone, and embedded in pure Spurr’s resin (Electron Microscopy Sciences, Hatfield). Ultrathin sectioning was performed on a Leica EM UC7 ultra-microtome. The ultrathin sections (70 nm thickness) were then collected on copper grids, stained with uranyl acetate and lead citrate, andfinally examined under a Hitachi 7000 electron microscope.

3. Results and discussion

3.1. In vivo stability

When a nanoparticle is introduced into the body, it is prone to

enzymatic degradation or phagocytic attack. The durability of the

nanoparticle as a contrast agent in vivo varies, depending on its

composition. Recent studies have shown that porous silicon

nanoparticles are biodegradable

[35]

whereas carbon dots of

similar size are highly biostable

[36]

. In order to develop FND into

a long-term in vivo contrast agent, it is important to study its

stability in small animal models at high doses. The study is also

crucial for its application as an advanced drug delivery device

[37

e42]

. To address this question, we subcutaneously injected bare

FND particles into a SD rat. Compared to in vivo imaging with the

mouse model, the experiment with rats is considerably more

challenging because of their greater skin thickness. The typical

thickness of a rat skin is of the order of 2.0 mm, which is about

5-fold greater than that (0.45 mm) of the mouse skin. Juzenas et al.

[43]

have measured the wavelength dependence of light through

mouse and rat skins of different thickness. They de

fined a

pene-tration depth (

d

) of light into the skin as the depth where the light

intensity decreases to 1/e of the light incident on the skin surface,

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and found that the penetration depth increases from

d

¼ 0.6 mm at

532 nm,

d

¼ 1.7 mm at 605 nm, to

d

¼ 2.6 mm at 780 nm. The

re

flectance of light from the skin surface is w60%.

Fig. 1

b shows in vivo

fluorescence images of a rat after s.c.

injection of bare FNDs (size

w 100 nm

[11]

) into the right

flank at

a dose of 0.5 mg in 500

m

L PBS. The imaging was carried out by

exciting the specimens at 605/20 nm and collecting the

fluores-cence at 780/20 nm. The images were displayed after correction of

tissue auto

fluorescence by subtracting the scaled background filter

images (obtained at 430/35 nm excitation) from the primary

filter

images in order to achieve better image quality

[44]

. As revealed by

the auto

fluorescence-corrected images, the fluorescence intensity

of the injected FNDs stayed essentially the same over 37 days

post-dosing. The weight of the rat, in contrast, is nearly doubled during

the same time period of the study. It indicates that FNDs are robust

in vivo and potentially useful as a long-term imaging agent for

living animals.

3.2. Long-term biocompatibility

With the con

firmation of the robustness of FNDs in vivo, we

conducted additional imaging of the particles administrated by i.p.

injection. The reason to choose this route is because the injection is

simple and reproducible, and frequently generates a depot effect

which allows the nanoparticles to stay in the peritoneum for

several hours. It also eliminates the possibility of particle

accu-mulation in the lung and prohibits the nanoparticles to cross the

blood brain barrier

[45]

.

Fig. 2

a and b shows the results for the i.p.

injection of 2 mg FNDs in 200

m

L PBS for a rat starved for 1 day prior

to imaging to reduce background endogenous

fluorescence

originating from the food in the stomach

[44]

. As seen, the intensity

of the far-red

fluorescence gradually decreases and disappears in

6 min, in stark contrast to that of the s.c. injection. The

disappear-ance of the

fluorescence signal is most likely to be a result that the

injected FNDs are dispersed gradually throughout the peritoneal

cavity rather than being degraded or digested in the cavity. Such

a consideration is indeed supported by an ex vivo imaging study of

the organs and tissues of the rat sacri

ficed on day 8, where most of

the FND particles are found on the surface of mesentery and serosa

of the stomach (

Fig. 2

c). In comparison, the parenchyma of the

abdominal organs is almost free of FND particles.

Next, we performed experiments to study the long-term

biocompatibility of the particles after i.p. injection. Speci

fically,

we studied the sub-acute toxicity of the intraperitoneally

admin-istered FNDs over a time period of 5 months. In this experiment, the

rats were injected weekly at a dose of 5 mg/kg b.w. continuously for

12 weeks. One half of the group (i.e. the non-recovery group) of the

rats was sacri

ficed after 12 weeks of FND injection and another half

group (i.e. the recovery group) was allowed to recover for 8 weeks

without any injection. Measurements of water consumption,

fodder consumption, and body weight of the rats were conducted

every week. The results are displayed in

Fig. 3

and

Figure S3

showing no signi

ficant differences between control (PBS-treated)

and FND-treated recovery/non-recovery groups. In addition to

these physiological parameters, organ indices, which provide

information on the general toxicity, were also measured at the end

of this experiment. The organ index is de

fined as the ratio of the

wet weight of the organ (g) to the whole body weight (g), and

increases or decreases of the indices denote added or reduced

function of the individual organs

[15]

. Shown in

Fig. 4

is a typical set

Fig. 2. In vivo and ex vivo imaging of FNDs in a rat after i.p. injection. (a, b) In vivo images acquired before injection (a) and at 0, 80, 180, and 360 s after injection (b), showing rapid spread of the FND particles in the peritoneal cavity. Blue arrow indicates the site of injection. (c) Ex vivo images of the extracted tissues and organs. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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of data for the measurement. Again, there is no signi

ficant

differ-ence in the organ indices of control and FND-injected rats for both

the recovery and non-recovery groups, indicating that multiple

injections of the bare FNDs do not cause obvious toxicological

effects. Taken together, these results corroborate the suggestion

that the FND administration does not induce any apparent toxicity

in rats during the study period of 5 months. Furthermore, no

abnormal clinical signs or behaviors were detected in either control

or FND-injected animals during the entire course of the study.

To provide more direct information on the existence of FNDs in

the animals, we acquired dissection images of the rats after i.p.

injection of FNDs for 8 weeks. As displayed in

Figure S4

, the FNDs in

the non-recovery group tend to accumulate initially on mesentery

and serous membrane of the stomach, similar to the

finding of

Fig. 2

c. Digestion of the membrane in concentrated nitric acid and

subsequent

fluorescence spectroscopic measurement (procedures

detailed in

Figure S5

along with the

fluorescence spectrum

dis-played in

Figure S6

) con

firmed that the white patches seen on the

serous membrane are FNDs. In comparison, the recovery group of

the FND-injected rats showed much less accumulation of FNDs in

the stomach serous membrane. It suggests that FNDs have been

either absorbed or transported from the site of injection to other

regions of the mouse body after 8 weeks of recovery.

In order to

find out whether the injected FNDs will cause any

damage in vivo, detailed histological examination of all tissue

sections was carried out. Microscopically, no signi

ficant difference

was seen between the FND-treated and control groups (

Fig. 5

a)

except that carbon-laden macrophages (i.e. the dark brown color

spots) cluster on the peritoneal surface of the FND-treated animals

but not in the control group (

Fig. 5

b). Comparing the optical

micrographs of these two groups strongly indicates that the carbon

particles within these macrophages are engulfed FND particles. We

do not observe any in

flammation, necrosis, or tissue reaction

surrounding these carbon-laden macrophages. Moreover, no FNDs

or other speci

fic pathological changes could be identified in the wall

of gastrointestinal tract or parenchyma of visceral organs, such as

liver, spleen, kidney, heart and lung in the treated animals. All the

examinations con

firm the long-term biocompatibility of FNDs. The

excellent biocompatibility of the sp

3

-carbon-based nanomaterial

makes it superior to other nanoparticles, such as nanosilica which

has very recently been reported to cause liver injury after continuous

intraperitoneal injection

[46]

, for in vivo applications.

3.3. Sentinel lymph node imaging

Sentinel lymph node mapping is one of the most important

and routine procedures in cancer treatment. SLNs are the group of

lymph nodes which receive the metastasizing cancer cells from

the primary tumor

[47]

. Thus, the status of SLNs (such as the

axillary lymph nodes, ALNs) is a critical factor in predicting

Body weight (Non-recovery)

Body weight (Recovery)

600

500

400

300

200

100

0

0

2

4

6

8

10

12

Weeks

600

500

400

300

200

100

0

0

2

4

6

8

10

12

14

16

18

20

Weeks

Body weight (g)

Body weight (g)

PBS

FND

PBS

FND

Fig. 3. Comparison of the body weights of PBS-injected (control) and FND-injected SD rats (with a dose of 5 mg/kg b.w. per week) over a time period of 3 and 5 months for the non-recovery and non-recovery groups (n¼ 3 each), respectively. The injection was carried out by i.p. administration.

Fig. 4. Comparison of the organ indices of PBS-injected (control) and FND-injected SD rats (with a dose of 5 mg/kg b.w. per week) after 12 weeks of treatment for the non-recovery and recovery groups (n¼ 3 each), respectively. The injection was carried out by i.p. administration.

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Fig. 5. Histopathological examination of the tissue sections of FND-injected (treated) and PBS-injected (control) SD rats with and without recovery. (a) Images showing no specific pathological changes in both FND-treated and control groups (magnification 200). (b) Images showing clustering of carbon-laden macrophages (appearing as dark brownish spots) on the peritoneal surface of the spleen in the FND-injected rats but not in the BSA-injected rats (magnification 200). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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prognosis of the patients as well as the determination of further

treatment

[48]

. Traditional SLN imaging with radioisotopes and

blue dye is well established in clinical applications, but recent

researches show that nanoparticles with size in the range of

1

e100 nm are also applicable for lymph node mapping

[49]

. To

explore the possibility of using FNDs for this application, nude

mice were injected intradermally with 100-nm FNDs coated

noncovalently with BSA. The coating was made to facilitate the

dispersibility of the particles in PBS as well as in biological

medium

[34]

. The BSA-coated FND particles were then injected

into the right foot paw of a mouse at a dose of 40

m

g in 20

m

L of

PBS.

Fig. 6

a shows an auto

fluorescence-corrected image acquired

on day 8, where the intradermally injected FNDs can be clearly

detected not only at the injection site, but also at the ALN closest

to the right foot paw. To further con

firm that the observed bright

red spot (indicated by a blue arrow) is indeed associated with

SLN, the mouse was sacri

ficed on day 8 and four major lymph

nodes were dissected out for

fluorescence imaging ex vivo

(

Fig. 6

b). In accord with the

finding of in vivo imaging, the FNDs

are predominantly accumulated at the lymph node ALN1 located

at the right axilla of the mouse

[50]

. No FNDs were detected in

the left axillary lymph node (ALN2) and in the brachial lymph

nodes (BLN1 and BLN2) within the sensitivity limit of our

fluo-rescence measurements. The observations are consistent with

a mechanism that most of the intradermally administered FNDs

are accumulated in the dermis and engulfed by the macrophages.

The remaining particles are collected by the dermal lymphatics

and drained into the lymphatic channels of the upper limb. As the

lymphatic channels of the upper extremity converge to the ALN of

the same side, the nanoparticles within the lymphatic channels

are therefore preferentially collected by the lymph node that

first

receives the drainage (i.e. the ALN1).

Fig. 7

presents the result of a histopathological examination for

the skin of a mouse right paw where BSA-coated FNDs were

intradermally injected. Indeed, it is found that the majority of the

FND-containing macrophages (i.e. the dark brown color spots)

aggregate in the dermis of the skin close to the skeletal muscle. No

in

flammation or reaction of any kind to the injected FND particles

was observed. To further examine if the accumulation of the

BSA-coated FNDs in SLNs can lead to any signi

ficant damage or

changes, histological analysis and transmission electron

micros-copy imaging of the resected lymph nodes were performed.

Histologically, clustering of carbon-laden cells is present in the

paracortex of the ALN1 without other noticeable pathological

changes (

Fig. 8

). Ultrastructurally, these carbon particles appear

refractile and electron-dense, which is compatible with the

char-acteristics of FND nanocrystallites (

Fig. 9

). Additionally, these

nanoparticles are located in the cytoplasm of the macrophages,

which are characterized by the presence of abundant lysosomes

and vacuoles in their cytoplasm.

Fig. 6. In vivo and ex vivo lymph node imaging of a nude mouse after i.d. injection of BSA-coated FNDs. (a) In vivo image showing accumulation of the FND particles in the right axillary lymph node (indicated by the blue arrow) on day 8. Note that most of the injected FND particles remain trapped at the injection site. (b) Ex vivofluorescence image of four extracted lymph nodes, where ALN1 and ALN2 are the lymph nodes located at the right and left axilla, respectively, and BLN1 and BLN2 are the lymph nodes located at the right and left brachial regions, respectively. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

Fig. 7. Histological examination of the skin of an FND-treated mouse by H&E staining. The black arrow indicates the epidermis (magnification 100). Macrophages, appearing as dark brownish carbon-laden cells, are found to cluster in the dermis of the right paw, where BSA-coated FNDs were injected. No inflammation, necrosis, or tissue damage occurred in the surrounding cells. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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4. Conclusion

This work demonstrates that diamond nanoparticles containing

built-in NV



fluorophores can be used as an in vivo contrast agent for

small animal models like mice and rats. The high stability of the FND

particles under physiological conditions makes it possible to observe

their

fluorescence emission over 37 days after injection by either

subcutaneous or intraperitoneal administration. Moreover, the

drainage of the 100-nm FND particles from the intradermal injection

site to SLN is readily detectable both ex vivo and in vivo by using

a standard imaging system. Further improvement of the image

contrast is feasible by utilizing the long

fluorescence lifetime and the

magneto-optical property of the NV



centers with more advanced

imaging schemes. The effective SLN mapping with FNDs, as

illus-trated herein, paves the way for the development a

nanodiamond-based real-time optical guidance method, by which surgeons can

precisely identify super

ficial SLNs non-invasively or reveal deep

SLNs by tracing the

fluorescently visualized lymphatics during

surgery. We believe that FND is worthy of further optimization of

performance as well as toxicity evaluations for clinical translation.

Acknowledgments

This work is supported by Academia Sinica and the National

Science Council (NSC) of Taiwan with Grant Nos. NSC

100-2119M-001-028 and NSC 99-2311-B-009-003-MY3. We thank the Taiwan

Mouse Clinic, funded by the National Research Program for

Genomic Medicine (NRPGM) at NSC for technical support in tissue

sectioning. We also thank Wan-Yu Hsieh at the National Taiwan

University and Tzu-Han Hsu at the Core Facility of the Institute of

Cellular and Organismic Biology, Academia Sinica, for assistance in

fluorescence and TEM imaging.

Appendix A. Supplementary material

Supplementary material related to this article can be found

online at

http://dx.doi.org/10.1016/j.biomaterials.2012.06.084

.

References

[1] Service RF. Nanotechnology takes aim at cancer. Science 2005;310:1132e4. [2] Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as

an emerging platform for cancer therapy. Nat Nanotechnol 2007;2:751e60. [3] Sanhai WR, Sakamoto JH, Canady R, Ferrari M. Seven challenges for

nano-medicine. Nat Nanotechnol 2008;3:242e4.

[4] Vaijayanthimala V, Chang HC. Functionalizedfluorescent nanodiamonds for biomedical applications. Nanomedicine 2009;4:47e55.

[5] Xing Y, Dai L. Nanodiamonds for nanomedicine. Nanomedicine 2009;4: 207e18.

[6] Ho D, editor. Nanodiamonds: applications in biology and nanoscale medicine. Morwell: Springer; 2009.

[7] Mochalin VN, Shenderova O, Ho D, Gogotsi Y. The properties and applications of nanodiamonds. Nat Nanotechnol 2012;7:11e23.

[8] Schrand AM, Johnson J, Dai L, Hussain SM, Schlager JJ, Zhu L, et al. Cytotoxicity and genotoxicity of carbon nanomaterials. In: Webster TJ, editor. Safety of Fig. 8. H&E-stained sections of the lymph nodes, (a) ALN1 and (b) ALN2, of a BSA-FND-treated mouse. ALN1 and ALN2 are the axillary lymph nodes dissected from the injected and non-injected sides of the mouse body, respectively. Brownish carbon-laden macrophages are seen to gather in the paracortical zone of the ALN1 lymph node but not in that of ALN2 (magnification 400). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. Electron microscopic examination of carbon-laden cells in the ALN1 lymph node of a BSA-FND-treated mouse after i.d. injection. Refractile and electron-dense FND particles are found in the cytoplasm of the lymph node cells. These cells have many lysosomes and vacuoles in their cytoplasm (L: lysosome, N: nucleus, V: vacuole), which are characteristic of macrophages.

(9)

nanoparticles, nanostructure science and technology. Morwell: Springer; 2009. p. 159e87.

[9] Xing Y, Xiong W, Zhu L, Osawa E, Hussin S, Dai L. DNA damage in embryonic stem cells caused by nanodiamonds. ACS Nano 2011;5:2376e84.

[10] Liu KK, Wang CC, Cheng CL, Chao JI. Endocytic carboxylated nanodiamond for the labelling and tracking of cell division and differentiation in cancer and stem cells. Biomaterials 2009;30:4249e59.

[11] Fang CY, Vaijayanthimala V, Cheng CA, Yeh SH, Chang CF, Li CL, et al. The exocytosis offluorescent nanodiamond and its use as a long-term cell tracker. Small 2011;7:3363e70.

[12] Zaitsev AM. Optical properties of diamond: a data handbook. Morwell: Springer; 2010.

[13] Puzyr AP, Baron AV, Purtov KV, Bortnikov EV, Skobelev NN, Mogilnaya OA, et al. Nanodiamonds with novel properties: a biological study. Diam Relat Mater 2007;16:2124e8.

[14] Yuan Y, Chen Y, Liu JH, Wang H, Liu Y. Biodistribution and fate of nano-diamonds in vivo. Diam Relat Mater 2009;18:95e100.

[15] Yuan Y, Wang X, Jia G, Liu JH, Wang T, Gu Y, et al. Pulmonary toxicity and translocation of nanodiamonds in mice. Diam Relat Mater 2010;19:291e9. [16] Marcon L, Riquet F, Vicogne D, Szunerits S, Bodart JF, Boukherroub R. Cellular

and in vivo toxicity of functionalized nanodiamond in Xenopus embryos. J Mater Chem 2010;20:8064e9.

[17] Rojas S, Gispert JD, Martin R, Abad S, Menchón C, Pareto D, et al. Bio-distribution of amino-functionalized diamond nanoparticles. In vivo studies based on18F radionuclide emission. ACS Nano 2011;5:5552e9.

[18] Mohan N, Chen CS, Hsieh HH, Wu YC, Chang HC. In vivo imaging and toxicity assessments offluorescent nanodiamonds in Caenorhabditis elegans. Nano Lett 2010;10:3692e9.

[19] Mohan N, Zhang B, Chang CC, Yang L, Chen CS, Fang, CY, et al. Fluorescent nanodiamond  a novel nanomaterial for in vivo applications. MRS Proceedings 2011;1362:mrss11-1362-qq06-01.

[20] Bonnema J, van de Velde CJH. Sentinel lymph node biopsy in breast cancer. Ann Oncol 2002;13:1531e7.

[21] Kim S, Lim YT, Soltesz EG, DeGrand AM, Lee J, Nakayama A, et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Bio-technol 2004;22:93e7.

[22] Pons T, Pic E, Lequeux N, Cassette E, Bezdetnaya L, Guillemin F, et al. Cadmium-free CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity. ACS Nano 2010;4:2531e8.

[23] Yu SJ, Kang MW, Chang HC, Chen KM, Yu YC. Bright fluorescent nano-diamonds: no photobleaching and low cytotoxicity. J Am Chem Soc 2005;127: 17604e5.

[24] Fu CC, Lee HY, Chen K, Lim TS, Wu HY, Lin PK, et al. Characterization and application of singlefluorescent nanodiamonds as cellular biomarkers. Proc Natl Acad Sci U S A 2007;104:727e32.

[25] Chang YR, Lee HY, Chen K, Chang CC, Tsai DS, Fu CC, et al. Mass production and dynamic imaging offluorescent nanodiamonds. Nat Nanotechnol 2008;3: 284e8.

[26] Davies G. Vibronic spectra in diamond. J Phys C Solid State Phys 1974;7: 3797e809.

[27] Weissleder R, Ntziachristos V. Shedding light onto live molecular targets. Nat Med 2003;9:123e8.

[28] Hui YY, Cheng CL, Chang HC. Nanodiamonds for optical bioimaging. J Phys D Appl Phys 2010;43:374021.

[29] Collins AT, Thomaz MF, Jorge MIB. Luminescence decay time of the 1.945 eV center in type Ib diamond. J Phys C Solid State 1983;16:2177e81.

[30] Urayama PK, Mycek MA. Fluorescence lifetime imaging microscopy of endog-enous biologicalfluorescence. In: Mycek MA, Pogue BW, editors. Handbook of biomedicalfluorescence. New York: Marcel Dekker; 2003. p. 211e6. [31] Chang CW, Sud D, Mycek MA. Fluorescence lifetime imaging microscopy.

Methods Cell Biol 2007;81:495e524.

[32] Faklaris O, Garrot D, Joshi V, Druon F, Boudou JP, Sauvage T, et al. Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway. Small 2008;4:2236e9.

[33] McGuinness LP, Yan Y, Stacey A, Simpson DA, Hall LT, Maclaurin D, et al. Quantum measurement and orientation tracking of fluorescent nano-diamonds inside living cells. Nat Nanotechnol 2011;6:358e63.

[34] Tzeng YK, Faklaris O, Chang BM, Kuo Y, Hsu JH, Chang HC. Superresolution imaging of albumin-conjugatedfluorescent nanodiamonds in cells by stimu-lated emission depletion. Angew Chem Int Ed 2011;50:2262e5.

[35] Park JH, Gu L, von Maltzahn G, Ruoslahti E, Bhatia SN, Sailor MJ. Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat Mater 2009;8:331e6.

[36] Yang ST, Cao L, Luo PG, Lu F, Wang X, Wang HF, et al. Carbon dots for optical imaging in vivo. J Am Chem Soc 2009;131:11308e9.

[37] Huang H, Pierstorff E, Osawa E, Ho D. Active nanodiamond hydrogels for chemotherapeutic delivery. Nano Lett 2007;7:3305e14.

[38] Liu KK, Chen MF, Chen PY, Lee TJF, Cheng CL, Chang CC, et al. Alpha-bun-garotoxin binding to target cell in a developing visual system by carboxylated nanodiamond. Nanotechnology 2008;19:205102.

[39] Guan B, Zou F, Zhi JF. Nanodiamond as the pH-responsive vehicle for an anticancer drug. Small 2010;6:1514e9.

[40] Liu KK, Zheng WW, Wang CC, Chiu YC, Cheng CL, Lo YS, et al. Covalent linkage of nanodiamond-paclitaxel for drug delivery and cancer therapy. Nanotech-nology 2010;21:315106.

[41] Li XX, Shao JQ, Qin Y, Shao C, Zheng TT, Ye L. TAT-conjugated nanodiamond for the enhanced delivery of doxorubicin. J Mater Chem 2011;21:7966e73. [42] Chow EK, Zhang XQ, Chen M, Lam R, Robinson E, Huang HJ, et al.

Nano-diamond therapeutic delivery agents mediate enhanced chemoresistant tumor treatment. Sci Transl Med 2011;3:73ra21.

[43] Juzenas P, Juzeniene A, Kaalhus O, Iani V, Moan J. Noninvasivefluorescence excitation spectroscopy during application of 5-aminolevulinic acid in vivo. Photochem Photobiol Sci 2002;1:745e8.

[44] Troy T, Jekic-McMullen D, Sambucetti L, Rice B. Quantitative comparison of the sensitivity of detection offluorescent and bioluminescent reporters in animal models. Mol Imag 2004;3:9e23.

[45] Intra J, Salem AK. Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo. J Control Release 2008;130:129e38. [46] Liu T, Li L, Fu C, Liu H, Chen D, Tang F. Pathological mechanisms of liver injury caused by continuous intraperitoneal injection of silica nanoparticles. Biomaterials 2012;33:2399e407.

[47] Jakub JW, Pendas S, Reintgen DS. Current status of sentinel lymph node mapping and biopsy: facts and controversies. Oncologist 2003;8:59e68. [48] Robe A, Pic E, Lassalle HP, Bezdetnaya L, Guillemin F, Marchal F. Quantum dots

in axillary lymph node mapping: biodistribution study in healthy mice. BMC Cancer 2008;8.

[49] Moghimi SM, Hunter AC, Murray JC. Nanomedicine: current status and future prospects. Faseb J 2005;19:311e30.

[50] Jeon YH, Kim YH, Choi K, Piao JY, Quan B, Lee YS, et al. In vivo imaging of sentinel nodes usingfluorescent silica nanoparticles in living mice. Mol Imag Biol 2010;12:155e62.

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