The Effect of Injection Routes on the Biodistribution, Clearance and Tumor Uptake of Carbon Dots.
ABSTRACT The emergence of photoluminescent carbon-based nanomaterials has shown exciting potential in the development of benign nanoprobes. However, the in vivo kinetic behaviors of these particles that are necessary for clinical translation are poorly understood to date. In this study, fluorescent carbon dots (C-dots) were synthesized and the effect of three injection routes on their fate in vivo was explored by using both near-infrared fluorescence (NIRF) and positron emission tomography (PET) imaging techniques. We found that C-dots are efficiently and rapidly excreted from the body after all three injection routes. The clearance rate of C-dots is ranked as: intravenous > intramuscular > subcutaneous. The particles had relatively low retention in the reticuloendothelial system (RES) and showed high tumor-to-background contrast. Furthermore, different injection routes also resulted in different blood clearance patterns and tumor uptakes of C-dots. These results satisfy the need for clinical translation and should promote efforts to further investigate the possibility of using carbon-based nanoprobes in a clinical setting. More broadly, we provide a testing blueprint for in vivo behavior of nanoplatforms under various injection routes, an important step forward towards safety and efficacy analysis of nanoparticles.
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ABSTRACT: During the past ten years significant advances have been achieved in quantum dot (QD) research field. The new synthetic methods and the discovery of new types of QDs have enabled a variety of new applications of QDs for bioimaging and biosensing. This review will focus on the most recent progress of QDs for biomedical applications. Ample examples will be given in this review on newly developed synthetic methods of QDs, non-toxic QDs, QDs for biomolecule detection, cell and animal imaging, and disease therapy.Colloids and surfaces B: Biointerfaces 01/2014; · 4.29 Impact Factor
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ABSTRACT: The therapeutic applications of exogenous nitric oxide are usually limited by its short half-life and its vulnerability to many biological substances, thus straightforward and precise spatiotemporal control of NO delivery may be critical to its therapeutic effects. Herein, the mitochondria-targeted and photoresponsive NO-releasing nanosystem is demonstrated as a new approach for cancer treatment. The nanosystem is fabricated by covalently incorporating a NO photo-donor and a mitochondria targeting ligand onto carbon-dots; accordingly, multi-functionalities (mitochondria-targeting, light-enhanced efficient NO-releasing, and cell imaging) are achieved. The in vitro NO release profiles for the nanosystem show that the duration of NO release from the present C-dot-based nanosystem containing immobilized SNO can be extended up to 8 hours or more. Upon cellular internalization, the nanosystem can target mitochondria and release NO. The action of the nanosystem on three cancer cell lines is evaluated; it is found that the targeted NO-releasing system can cause high cytotoxicity towards the cancer cells by specifically damaging their mitochondria. Additionally, light irradiation can amplify the cell apoptosis by enhancing NO release. These observations demonstrate that incorporating mitochondria-targeting ligand onto a NO-releasing system can enhance its pro-apoptosis action, thereby providing new insights for exploiting NO in cancer therapy.Small 05/2014; · 7.51 Impact Factor
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ABSTRACT: The use of nanoparticles holds promise for medical applications, such as X-ray imaging, photothermal therapy and radiotherapy. However, the in vivo toxicity of inorganic nanoparticles raises some concern regarding undesirable side effects which prevent their further medical application. Ultrasmall sub-5.5 nm particles can pass through the barrier for renal clearance, minimizing their toxicity. In this letter we address some recent interesting work regarding in vivo toxicity and renal clearance, and discuss the possible strategy of utilizing ultrasmall nanomaterials. We propose that small hydrodynamic sized nanoclusters can achieve both nontoxic and therapeutic clinical features.International Journal of Nanomedicine 01/2014; 9:2069-72. · 4.20 Impact Factor
HUANG ET AL.VOL. 7
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June 03, 2013
C2013 American Chemical Society
Effect of Injection Routes on the
Biodistribution, Clearance, and
Tumor Uptake of Carbon Dots
Xinglu Huang,†Fan Zhang,†,‡Lei Zhu,†,‡Ki Young Choi,†Ning Guo,†Jinxia Guo,†,‡Kenneth Tackett,§
ParambathAnilkumar,§Gang Liu,‡QimengQuan,†HakSooChoi,^Gang Niu,†Ya-PingSun,§SeulkiLee,†and
†Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health
(NIH), Bethesda, Maryland 20892, United States,‡Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University,
Xiamen 361005, China,§Department of Chemistry and Laboratory for Emerging Materials and Technology, Clemson University, Clemson, South Carolina 29634,
ing applications in biolabeling and bio-
city and potential environmental hazard of
these inorganic nanomaterials containing
heavy metals such as cadmium limit their
widespread use and in vivo applications in
humans.9?13The emergence of photolumi-
nescent carbon-based nanomaterials pro-
vides an exciting opportunity in the search
for benign (nontoxic) alternative fluorescent
nanomaterials and might offer great potential
for optical imaging and related biomedical
carbon nanodots (C-dots) with ultrasmall sizes
were found to be physicochemically and
photochemically stable.14Combined with
their well-defined, ultrafine dimensions
and a variety of simple, fast, and cheap syn-
thetic routes available,15?23C-dots provide
an encouraging technological platform as
uminescent semiconductor quantum
dots (QDs) have generated much ex-
citement for a wide variety of promis-
an alternative to other carbon-based nano-
and carbon nanotubes and are expected to
have wide applications in preclinical and
potentially clinical studies.24
do not have noticeable signs of toxicity in
treated animals,25demonstrating the feasi-
bility for in vivo applications. The successful
translation of nanoparticle-based biomater-
ials requires nanoparticles (NPs) not only
with well-controlled in vivo behavior26,27
lihood of toxicity. The U.S. Food and Drug
Administration (FDA) has demanded that
the agents injected into the human body,
especially diagnostic agents, should be
cleared completely in a reasonable period
of time,28that such agents should not accu-
mulate in the body, and that their exposure
time should be minimized. To date, the key
determinants of NP biodistribution and
clearance focus on the properties of NPs,
*Address correspondence to
Received for review September 5, 2012
and accepted June 3, 2013.
ABSTRACT The emergence of photoluminescent carbon-based nanomaterials
the in vivo kinetic behaviors of these particles that are necessary for clinical
dots) were synthesized and the effect of three injection routes on their fate in vivo was explored by using both near-infrared fluorescence and positron
emission tomography imaging techniques. We found that C-dots are efficiently and rapidly excreted from the body after all three injection routes. The
clearance rate of C-dots is ranked as intravenous > intramuscular > subcutaneous. The particles had relatively low retention in the reticuloendothelial
system and showed high tumor-to-background contrast. Furthermore, different injection routes also resulted in different blood clearance patterns and
tumor uptakes of C-dots. These results satisfy the need for clinical translation and should promote efforts to further investigate the possibility of using
carbon-based nanoprobes in a clinical setting. More broadly, we provide a testing blueprint for in vivo behavior of nanoplatforms under various injection
routes, an important step forward toward safety and efficacy analysis of nanoparticles.
KEYWORDS: biodistribution.carbon dots.clearance.injection routes.translation.tumor uptake
HUANG ET AL. VOL. 7
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such as the chemical composition, size, shape, and
surface charge.28?31However, very few reports pay
attention to factors applied in the clinic, such as
injection or exposure routes, which are necessary for
clinical translation of nanoformulations. Several stud-
progress has been made in the synthesis strategy of
C-dots, there is no report of C-dots that can target a
disease state and can be efficiently cleared from the
body after different injection routes.
RESULTS AND DISCUSSION
Fabrication and Characterization of near-Infrared C-Dots.
Herein, we prepared near-infrared fluorescent C-dots
(Figure 1A) by coupling the nanoparticles with the
near-infrared dye ZW800, to track their in vivo fates
and the effect of tumor uptake after three injection
routes: intravenous (iv), subcutaneous (sc), and intra-
C-dots were first synthesized according to a previously
reported method.21,22,34Subsequently, surface passi-
vation of C-dots was performed by reacting with
diamine-terminated oligomeric poly(ethylene glycol).
Three major purposes were implemented in the sur-
face passivation process: (1) generation of bright
luminescence;21(2) improvement of hydrophilicity
and stability; (3) surface functionalization of C-dots.
TEM imaging shows ultrasmall surface passivated
C-dots of ∼3 nm (Figure 1B).
The photoluminescence from C-dots is most likely
attributed to the presence of surface energy traps that
become emissive upon stabilization as a result of sur-
face passivation.21Currently, a major reason limiting
in vivo applications of C-dots is their low quantum
yields in the near-infrared (NIR) region (650?900 nm),
an ideal tissue fluorescence imaging window with low
tissue absorption and scattering.35?37To improve the
emission ability of C-dots in the NIR region, ZW800
(excitation ∼770 nm, emission ∼795 nm) was intro-
duced onto C-dots by conjugating the dye NHS ester
with amino groups on the C-dots, to make ZW800-
molecules per particle on average, as estimated by
quantifying the fluorescence intensity and absorbance
values of ZW800 before and after conjugation. C-dot?
ZW800 has absorption peaks at approximately 420 nm
(C-dots) and 770 nm (ZW800) (Figure 2A) and two
emission peaks at approximately 510 and 800 nm after
excitation, respectively (Figure 2B). The imaging ability
more, the low fluorescence background of C-dot?
ZW800 was shown in different biological fluids, such as
fetal bovine serum (FBS), blood, urine, and tissue lysate
(Supporting Information Figure S1). To better study the
in vivo kinetics of C-dots, we also explored the fluores-
fluorescence stability of C-dot?ZW800 was observed in
Figure 1. (A) Schematic structure of C-dot?ZW800 conjugate. (B) TEM image of C-dots.
HUANG ET AL. VOL. 7
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Figure S2, the fluorescence signals derived from C-dots
and ZW800 were virtually unaltered over extended
In our previous study, we demonstrated that the
naked C-dots bear negative charges due to the ex-
istence of ?OH/?COOH groups on the surface, with a
amine, the zeta potential of C-dots was ?14.3 ( 2.8 mV,
and after further modification with ZW800 dye
molecules,39the zeta potential of C-dot?ZW800 be-
notoriously inaccurate technique for the characteriza-
tion of weakly scattering colloids with small sizes, such
als such as gold. To better define the hydrodynamic
diameter (HD) of particles, a gel-filtration chromatog-
raphy (GFC) system was used to characterize small-
sized NPs with various coatings according to previous
reports.29,40First, the protein standards were analyzed
using GFC, including M1 (thyroglobulin; 669 kDa,
18.8 nm HD), M2 (γ-globulin; 158 kDa, 11.9 nm HD),
M3 (ovalbumin; 44 kDa, 6.13 nm HD), M4 (myoglobin;
17 kDa, 3.83 nm HD), and M5 (vitamin B12; 1.35 kDa,
1.48 nm HD) (Figure 2D). Subsequently, a standard
curve of HD vs retention time was established, and the
HD of C-dot?ZW800 was calculated to be 4.1 nm
whether there is potential adsorption of serum pro-
teins, C-dot?ZW800 were incubated in PBS, FBS, or
mouse urine for 1 h at 37 ?C before loading onto the
GFC column. As shown in Figure 2F, slight changes in
Figure 2. Optical properties and hydrodynamic diameters (HD) of C-dot?ZW800. Representative (A) absorbance and (B)
fluorescence emission (λEx= 420, 770 nm) spectra of C-dots, ZW800, and C-dot?ZW800. (C) Fluorescence images of
C-dot?ZW800 based on C-dots and ZW800 by a Maestro imaging system, respectively. (D) Gel-filtration chromatography
(GFC) analysis of protein standards (PBS, pH = 7.4). Molecular weight markers M1 (thyroglobulin; 669 kDa, 18.8 nm HD),
M2 (γ-globulin; 158 kDa, 11.9 nm HD), M3 (ovalbumin; 44 kDa, 6.13 nm HD), M4 (myoglobin; 17 kDa, 3.83 nm HD), and M5
was analyzedby reversed-phase HPLC. (E) GFC standard curve between HD and retention time. (F) Stabilityof C-dot?ZW800
inPBS,FBS,andurineafter1 hincubationat 37?CpriortoloadingontotheGFCcolumn.Theretentiontimeof particlesatan
absorbance of 770 nm was analyzed by reversed-phase HPLC.
HUANG ET AL. VOL. 7
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the peak shape and retention time were found, indi-
cating the possibility of adsorption of plasma protein
onto the surface of C-dot particles.
Blood Circulation of Particles by Three Injection Routes.
Before the NPs can be ready for clinical translation, a
to minimize their potential toxicity. First, we studied
the in vivo kinetics of C-dots by different injection
routes. After injection of C-dots by three routes (iv, sc,
and im), venous blood samples were collected at the
indicated time points and subsequently analyzed by a
blood clearance rates of C-dot?ZW800 are rather differ-
ent after different injection routes, according to the
analysis of fluorescence signals from ZW800 dyes. The
particle concentration in blood within 1 h dramatically
decreased after iv injection (Figure 3A, top), while it was
initially increased after sc (Figure 3A, middle) and im
and 20 min postinjection, respectively. The quantitative
analysis found that the particle fluorescence signal in
than 60 min (n= 4/group, Figure 3B).On the other hand,
both sc and im injection groups showed lower particle
concentration at 1 min postinjection than that at 60 min
by 4.4 ( 1.3-fold and 9.6 ( 2.6-fold, respectively. To
fluorescence images of the collected blood at indicated
time points after iv injection of C-dot?ZW800 were
collected for C-dot and ZW800 (Supporting Information
Figure S3). The same trend of blood clearance was
observed for C-dots and ZW800, implying that the con-
jugated ZW800 dye molecules were not detached from
C-dot?ZW800 in blood after sc and im injection was
significantly higher than that after iv injection, signifying
the impact of injection route on the in vivo behaviors of
rapid blood clearance of C-dot?ZW800 might be due to
serum protein adsorption onto the particle surface, lead-
ing to rapid opsonization followed by removal from the
Biodistribution of C-Dot Nanoparticles by Three Injection
Routes. To minimize the toxicity of NPs, it is necessary
to accountfor and completelyremove all NPs from the
body within a reasonable period of time. After being
introduced into a living subject, NPs are typically
metabolized via two major routes, the liver (into bile)
and the kidneys (into urine).41To fully understand the
clearance route of C-dots, organs were collected at 1
and 24 h after C-dot administration, and the biodis-
tribution of particles was analyzed by quantitative
Information Figure S4A, the majority of C-dot?ZW800
particles were accumulated in the kidneys at 1 h after
injection via all three routes; only a small amount went
to the liver. The signal of C-dot?ZW800 in the kidneys
at the 1 h time point was quantified and ranked based
on the injection routes as im > sc > iv (Supporting
was detected in any organ, suggesting that the parti-
cles were all cleared out of the body regardless of the
injection route (Supporting Information Figure S4C).
To further validate the biodistribution results ob-
tained from optical imaging, we also employed posi-
tron emission tomography (PET) imaging to visualize
and quantify the in vivo kinetics of the C-dots. The
biodistibution of64Cu?C-dot measured by region of
interest (ROI) analysis showed a very similar pattern to
that obtained from optical imaging (Figure 4B). At 24 h
nized and major organs and tissues including blood,
muscle, liver, kidneys, spleen, heart, and lung were
harvested and measured by gamma counting, reveal-
ing less than 1% ID/g radioactivity in any organ mea-
low accumulation of C-dots in the reticuloendothelial
system (RES) and rapid clearance from the body. On
the basis of these results, it was concluded that C-dots
were mainly excreted via the renal route into urine.
Urine Clearance of Particles by Three Injection Routes.
Urine clearance analysis of C-dots after three injection
routes was further performed in order to evaluate the
in vivo behaviors of C-dots. The bladder of each mouse
64Cu?C-dot, the mice were eutha-
Figure 3. Blood circulation of C-dot?ZW800 after three injection routes. (A) After injection of particles, vein blood samples
fluorescence images were acquired. (Top) iv injection, (middle) sc injection, and (bottom) im injection. (B) Fluorescence
time?activity curves derived from signals in (A).
HUANG ET AL. VOL. 7
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after administration of C-dot?ZW800 was exposed
and imaged at the indicated time points. The signal
of C-dot?ZW800 was subsequently quantified to ac-
quire a clearance curve. From Figure 5A and B, a rapid
clearance is observed by iv injection, and the signal in
the bladder plateaued 10 min after administration.
Intravenous injection of C-dots displayed much faster
urine clearance than the sc and im injection route within
10 min postinjection, respectively. To confirm the urine
basedon C-dot emissionover time(Supporting Informa-
tion S5). The urine displayed high background in the
visible region, making it unsuitable for signal quantifica-
tion. However, the signal from C-dot emission demon-
strated the same trend of urine clearance as ZW800
emission, again indicating that the ZW800 fluorescence
signal reflects C-dot?ZW800. The urine activity was also
accordance with the NIR fluorescence imaging results
(Figure 5D). The amount of signals (both quantified from
ZW800 fluorescence and64Cu radiolabel) in the urine
within 1 h postinjection of64Cu?C-dot?ZW800 showed
the pattern of iv > im > sc, in accordance with the
clearance rate from the blood.
Accumulation of Particles at Injection Sites. Besides the
accumulation in the RES, it is possible that the NPs
accumulate at the injection site after sc and im admin-
istration (Figure 6A). For sc and im injection, most
particles initially retained at the injection site have
disappeared by 24 h postinjection. Quantitative anal-
ysis indicates that the signals at sc and im injection
sites at the 24 h time point were 92.3 ( 10.2% and
99.1 ( 9.7% lower than those at 1 min postinjection,
respectively (Figure 6B). These results elucidate that
the NPs do not accumulate at the injection site and
instead rapidly enter the blood circulation.
Tumor Uptake of Particles by Three Injection Routes. For
cancer nanomedicine applications, it is desirable for
NPs to have prominent and prolonged tumor uptake.
C-dot?ZW800 particles were injected via three injec-
tion routes into athymic nude mice bearing subcuta-
neous SCC7 tumors. The images were acquired and
analyzed on a Maestro all-optical imaging system. At
2 h postinjection, tumor waseasilydistinguishablefrom
surrounding normal tissue in all three groups, and the
tumor signal remained high over time (Figure 7A). In
addition to the homogeneous uptake of C-dots in the
injection of particles, the major organs and tissues were harvested from Balb/C mice at the indicated time points, and
middle, sc; bottom, im); right, 24 h postinjection (top, iv; middle, sc; bottom, im). Bright field: 1, liver; 2, spleen; 3, lung; 4,
kidneys; 5, muscle; 6, intestine; 7, heart. (B, C) Quantification of the biodistribution of64Cu-labeled C-dots via three injection
and tissues including blood, muscle, liver, kidneys, spleen, heart, and lung were harvested and measured by gamma counting.
HUANG ET AL. VOL. 7
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excreted through renal filtration (red arrow), as shown
by high tumor-to-background ratio and low level of
fluorescence in other tissues and organs. The iv group
and sc group exhibited higher fluorescence signals in
the tumor area than the im group at 2 h postinjection
(p < 0.05) (Figure 7B). The sc group also showed some-
4, and 24 h postinjection (p > 0.05), presumably due to
the slower blood clearance of C-dots via sc than iv
injection. The imaging results were further confirmed
obtained from mice sacrificed at 2 h postinjection
(Figure 7C). Co-localization of C-dots and ZW800 in the
tissue slices again excluded the possibility of dissocia-
tion of ZW800 from the C-dot?ZW800 conjugate.
In the present study, we found that C-dot?ZW800
urine) and not the liver and spleen (Figure 4). NPs are
often trapped in the RES (mainly liver and spleen) by
macrophages, and PEGylation greatly reduces trap-
ping by the RES compared with the naked NPs. It has
been reported that for PEGylated QDs the hydrody-
namic diameter has to be less than 5.5 nm for efficient
renal excretion,28?30,42with clearance efficiency de-
creasing as hydrodynamic diameter increases. Our
water-soluble C-dots with a core size of 3 nm and HD
Figure 5. Urine accumulation of C-dot?ZW800 after different routes of injection. (A) The mice were kept under isoflurane
anesthesia, the bladder was exposed, and NIR images were acquired at the indicated time points before and after (top) iv
injection, (middle) sc injection, and (bottom) im injection. (B) Quantification of the ZW800 fluorescence signal in (A). (C)
Representative coronal images from 1 h dynamic PET imaging of64Cu?C-dot after three routes of injection: left, iv injection;
middle, sc injection; right, im injection. (D) Urinary bladder ROI analysis of the PET images in (C).
HUANG ET AL. VOL. 7
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of 4.1 nm after PEGylation and ZW800 coupling led to
rapid and efficient elimination of particles from the
body via urinary excretion.
be applied to fit special purposes, such as tumor
targeting, long circulation, or ease of use by the
physician. In this study, we illustrate that efficient
clearance of C-dots is implemented not only after iv
injection but also after sc and im injection with no
(Figures 4 and 6). Owing to the presence of different
biological barriers, the blood clearance and urine
accumulation rate follow the order iv > im > sc
(Figures 3 and 5). Furthermore, tumor uptake of C-dots
by sc and iv injection was higher than that by im
injection. This is probably due to the interplay among
circulation time, clearance rate, and the concentration
routes.28For example, we showed that the blood
clearance of C-dots after iv injection was faster than
the other two injection routes (Figure 5), which in
turn could lead to reduced tumor retention. Thus, the
major organ accumulation, and passive tumor target-
ing, can be easily controlled by the injection route.
Overall, C-dots appear to have great potential as a
24 h after sc injection and im injection: left, bright field images; right, NIR fluorescence images. The injection sites are
indicated by circles. (B) Quantification of the relative fluorescence intensity at the injection sites over time.
Figure 7. Tumor uptake of C-dot?ZW800 after different routes of injection. (A) NIR fluorescence images of SCC-7 tumor-
Ex vivo fluorescence images derived from the emission of C-dots and ZW800 were acquired to confirm tumor uptake of
HUANG ET AL. VOL. 7
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nanoplatform for preclinical biomedical research and
translation into the clinic for optical imaging-guided
It is of note that while 2D fluorescence reflectance
imaging used in this study is useful for qualitative and
comparative analyses, it is unable to provide quantita-
tive information, and it also does not allow for the
assessment of C-dot accumulation in deeper-seated
tissues. 3D fluorescence molecular tomography (FMT),
based on volumetric reconstruction of the concentra-
tion of optical imaging agents, is generally considered
to be able to overcome some of the shortcomings
quantitative and more in-depth analyses.43Combining
conjugate in addition to radiolabeled C-dot?ZW800.
In summary, we explored the effects of different
injection routes on blood circulation, biodistribution,
urine clearance, and passive tumor uptake of C-dots.
The C-dot?ZW800 conjugate was specifically designed
to possess several significant features: NIR fluorescence
emission, high stability in vivo, rapid renal clearance, and
effective passive tumor targeting. Our studies suggest
clearance, the biodistribution of C-dots in major organs
and tissues, and tumor uptake over time. These charac-
teristics make C-dot-based nanoprobes a viable candi-
foundation for testing and designing targeted NPs with
optimal exposure routes to control biodistribution, elim-
ination from the body, and tumor targeting: key criteria
for safety approval and clinical translation.
were synthesized according to previous reports.21,34,44Briefly,
the C-dots were synthesized via laser ablation of a carbon target
in the presence of water vapor with argon. Then, diamine-
terminated oligomeric poly(ethylene glycol), H2NCH2(CH2CH2O)n-
CH2CH2CH2NH2(n = 35, MW ≈ 1500, PEG1500N), was used to
react with the C-dots for surface passivation. In a typical reaction,
PEG1500N(200 mg, 0.13 mmol) was mixed with an acid-treated
particle sample, and the mixture was heated to 120 ?C for 72 h.
After the reaction, the mixture was cooled to room temperature
supernatant containing C-dots was collected for further function-
alization. The resulting particles were observed by transmission
electron microscopy (TEM). The zeta potential of the particles was
between the amino groups of the C-dots and the NHS ester of
ZW800. To remove free PEG before conjugation, the samples were
purified by dialysis membrane (MW ∼3500) in a PBS solution for
5 days by changing to fresh solution every 6?12 h. In a typical
reaction, the C-dots (20 mg) were labeled with ZW800 (1 mg) in
stirred at room temperature for 4 h and then purified by a PD-10
conjugate was completely removed, the product was further
purified by dialysis membrane (MW ∼3500) in a PBS solution for
3 days by changing to fresh solution every 6?12 h. Finally, the
a centrifugal filter (3k cutoff, Millipore). The UV?vis and fluores-
cence spectra of particles were recorded on a Genesys 10s UV?vis
spectrophotometer (Thermo, IL, USA) and an F-7000 fluorescence
spectrophotometer (Hitachi, Japan), respectively.
conjugated with DOTA-NHS ester (Macrocyclics). Briefly, 5 mg
of C-dots was reacted with 0.5 mg of DOTA-NHS ester in 2 mL of
borate buffer (pH = 9.0). The mixture was stirred at room
temperature for 4 h and subsequently purified by a PD-10
desalting column. The product was concentrated into 0.5 mL of
PBS with a centrifugal filter. Then, the C-dot?DOTA conjugate
was labeled with 1 mCi of64Cu for 1 h in NH4Ac buffer (pH = 5.4)
on a solid gold internal target plate of the CS-30 cyclotron
utilizing the nuclear reaction64Ni(p,n)64Cu and separated from
the target material as64CuCl2by anion chromatography.
64Cu labeling, the amino groups of C-dots were first
Gel-Filtration Chromatography. Hydrodynamic diameters of
particles were analyzed by GFC following a previous report.5
Calibration of HD was performed by injecting 100 μL of protein
(M1, 669 kDa, 18.8 nm HD), γ-globulin (M2, 158 kDa, 11.9 nm HD),
ovalbumin (M3, 44 kDa, 6.13 nm HD), myoglobin (M4, 17 kDa,
C-dot?ZW800wereincubated inPBS (pH= 7.4),95% FBS,or95%
urine for 1 h at 37 ?C before loading onto the GFC column.
Tissue Sample Collection and Analysis. Animal procedures were
performed according to a protocol approved by the National
Institutes of Health Clinical Center Animal Care and Use Com-
mittee (NIH CC/ACUC). After the injection of C-dot?ZW800,
Briefly, C-dot?ZW800 in PBS solution (2.5 mg/kg, 50 μL) were
injected by different routes (n = 4/group): intravenous injection
and intramuscular injection (im, muscle of left leg), respectively.
Blood samples (5 μL/withdrawal) were collected from the tail vein
at different time points (1, 2, 5, 10, 15, 20, 25, 30, 45, and 60 min)
postinjection of C-dot?ZW800. The samples were analyzed by a
Maestro all-optical imaging system. For the urine clearance anal-
ysis, the mice were kept under isoflurane anesthesia (2% v/v
isoflurane at 0.2 L min?1O2 flow), and the bladders of the
mice were exposed. Subsequently, the bladders were imaged
before and after three injection routes at indicated time points.
were collected at different time points for ex vivo fluorescence
imaging. NIR fluorescence imaging was acquired by an appro-
priate filter set for ZW800 (excitation at 685?730 nm, emission at
745?800 nm) on a Maestro all-optical imaging system. The
accumulation of particles at injection sites was also studied by
imaged by a Maestro all-optical imaging system.
Tumor Inoculation and Imaging. The murine squamous cell
carcinoma (SCC-7) cells were purchased from the American
Type Culture Collection (ATCC, Rockville, MD, USA). The cells
were maintained in RPMI 1640 medium (Invitrogen) supple-
mented with 10% FBS at 37 ?C with 5% CO2. Flank xenograft
tumors were prepared by subcutaneous injection of 106SCC-7
female athymic nude mouse. When the diameter of the tumors
reached about 8 mm, the mice were divided into three groups
(n = 4/group),and the C-dot?ZW800 particles(2.5 mg/kg,50μL)
were injected by intravenous (iv), subcutaneous (sc), or intra-
muscular (im) route. The tumor uptake of particles was visua-
lized and quantified by a Maestro all-optical imaging system.
HUANG ET AL. VOL. 7
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Dynamic PET scans were performed using an Inveon micro-
of64Cu?C-dot (100 μCi/mouse, 50 μL). The body temperature of
mice was maintained using a thermostat-controlled thermal
heater. PET images were reconstructed by a two-dimensional
ordered-subsets expectation maximum (OSEM) algorithm, and
the frame rates were 10 ? 30, 20 ? 60, 5 ? 120, and 5 ? 300 s.
Histological Analysis. Tissue samples were snap-frozen, and
particles by Olympus fluorescence microscopy. Specifically, the
histology sections were washed twice with PBS and incubated
with Z-fix solution for 10 min. Then, the histology sections were
stained with 40,60-diamidino-2-phenylindole and observed un-
der a fluorescence microscope. The signals derived from C-dots
and ZW800 were observed by appropriate filters and software
of the fluorescence microscope (Olympus IX81).
Statistical Analysis. The level of significance in all statistical
analyses was set at a probability of p < 0.05. Data are presented
as mean ( SD. Analysis of variance and t tests were used to
analyze the data.
Conflict of Interest: The authors declare no competing
Acknowledgment. This work was supported, in part, by the
National Basic Research Program of China (973 Program
2013CB733802), the National Science Foundation of China (NSFC)
(81201086, 81201129, 81101101, and 51273165), the Intramural
Research Program of the NIBIB, NIH, and the Henry M. Jackson
Foundation. S.L. was partially supported by an NIH Pathway to
Independence (K99/R00) Award.
Supporting Information Available: Materials and methods
section and additional figures. This material is available free of
charge via the Internet at http://pubs.acs.org.
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