Post on 26-Jul-2020
Accepted Manuscript
Construction of lanthanide-doped upconversion nanoparticle-Uelx EuropaeusAgglutinin-I bioconjugates with brightness red emission for ultrasensitive in vivoimaging of colorectal tumor
Rongrong Tian, Shuang Zhao, Guifeng Liu, Hongda Chen, Lina Ma, Hongpeng You,Chunming Liu, Zhenxin Wang
PII: S0142-9612(19)30274-1
DOI: https://doi.org/10.1016/j.biomaterials.2019.05.010
Reference: JBMT 19199
To appear in: Biomaterials
Received Date: 18 January 2019
Revised Date: 14 April 2019
Accepted Date: 5 May 2019
Please cite this article as: Tian R, Zhao S, Liu G, Chen H, Ma L, You H, Liu C, Wang Z, Construction oflanthanide-doped upconversion nanoparticle-Uelx Europaeus Agglutinin-I bioconjugates with brightnessred emission for ultrasensitive in vivo imaging of colorectal tumor, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.05.010.
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Construction of Lanthanide-Doped
Upconversion Nanoparticle-Uelx Europaeus
Agglutinin-I Bioconjugates with Brightness
Red Emission for Ultrasensitive In Vivo
Imaging of Colorectal Tumor
Rongrong Tian,a, b Shuang Zhao,a, b Guifeng Liu,c Hongda Chen,a Lina Ma,a
Hongpeng You,a, b Chunming Liu,d, * Zhenxin Wanga, b, *
aState Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China.
bUniversity of Science and Technology of China, Jinzhai Road Baohe District, Hefei,
Anhui, 230026, P. R. China.
cDepartment of Radiology, China-Japan Union Hospital of Jilin University, No. 126,
Xiantai Street, Changchun, 130033, P. R. China.
dCentral Laboratory, Changchun Normal University, Changchun, 130032, P. R.
China.
*E-mail: ccsf777@163.com (CL), wangzx@ciac.ac.cn (ZW)
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ABSTRACT
Lanthanide-doped upconversion nanoparticles (UCNPs)-based active targeting optical
bioimaging has attracted tremendous scientific interest because of its noninvasive
real-time signal feedback, superior tissue penetration depth and high spatial resolution
in early diagnosis of disease. Herein, we synthesize a novel carboxy-terminated silica
coated NaErF4: 10% Yb@NaYF4: 40% Yb@NaNdF4: 10% Yb@NaGdF4: 20% Yb
UCNPs (termed as UCNP@SiO2-COOH) with 808 nm near-infrared (NIR) excitation
and bright 655 nm upconversion luminescence (UCL) emission for realizing deep
tissue imaging. Under 808 nm NIR laser excitation (1.5 W cm-2), the UCL of
UCNP@SiO2-COOH with relative low concentration (2 mg mL-1) can be successfully
visualized under a chicken breast slice with 10 mm thickness. After conjugated with
various molecules including NH2-PEG3400-COOH, peptide D-SP5 and Uelx
Europaeus Agglutinin-I (UEA-I), biodistributions, clearance pathways and
tumor-targeting capacities of the UCNP@SiO2-COOH and corresponding
bioconjugates (termed as UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and
UCNP@SiO2-UEA-I, respectively) were investigated by tracking the UCL intensities
of livers, kidneys and tumors. Both of in vitro and in vivo experimental results reveal
that there is no significant difference for their in vivo biodistributions and clearance
pathways. The UCNP@SiO2-UEA-I exhibits much higher SW480 tumor-targeting
capacity than those of other bioconjugates. In particular, the as-prepared
UCNP@SiO2-UEA-I even to visualize ultrasmall (c.a. 3 mm3 in volume)
subcutaneous SW480 tumor in Balb/c nude mouse through intravenous administration.
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The study implies that the red UCL emitted UCNPs with a minimized heating effect is
suitable for deep tissue biomedical imaging and UCNP@SiO2-UEA-I can serve as an
efficient optical probe for early diagnosis of SW480 tumor.
Keywords: Lanthanide-Doped Upconversion Nanoparticles, Uelx Europaeus
Agglutinin-I, Colorectal Cancer, Molecular Imaging, Active Targeting
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1. Introduction
Optical bioimaging based on lanthanide-doped upconversion nanoparticles
(UCNPs) has recently attracted significant attention for diverse applications in basic
biological research and clinical diagnosis [1-4] due to the outstanding photochemical
properties, including low auto-fluorescence backgrounds, superior photostability, high
penetration depth under near-infrared (NIR) excitation, and weak photodamage of
tissue, etc. [5-9]. Currently, sensitizer Yb3+ ion-doped UCNPs have been extensively
explored in bioimaging applications with 980 nm excitation [10-13]. Unfortunately,
there is a strong water absorption peak at 980 nm, resulting in relatively high
laser-induced thermal damage of tissue and limited tissue penetration depth of 980 nm
laser [14-18]. Meanwhile, green upconversion luminescence (UCL, < 600 nm) of
Yb3+ ion-doped UCNPs are normally much stronger than their red UCL (> 650 nm)
[19, 20]. It is known that short-wavelength light (< 650 nm) shows strong tissue
absorption, which only has shallow penetration depth of tissue. UCNPs with emission
bands in the biologically transparent window (650-950 nm) is a key parameter for
development of in vivo deep tissue optical imaging because the feature can efficiently
minimize light scattering, tissue absorption, and auto-fluorescence backgrounds
[21-24]. In addition, there are plenty of available light sources/detectors for 650-950
nm region. In general, the protocol of employing 650-950 nm excitation/emission
would be a better choice in terms of biological and technological compatibility,
penetration depth (one to several centimeters) and cost performance.
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Recently, Nd3+-sensitized core@shell UCNPs and/or undoped NaErF4@NaYF4
UCNPs have been demonstrated to emit effective single-band red UCL under 800 nm
laser excitation [25-34]. In particular, the Nd3+/Yb3+/activator system can effectively
generate UCL through energy transfer between Nd3+ to Yb3+ to activators (Er3+, Tm3+
and Ho3+) since Nd3+ has intense absorption around 800 nm and the excited Nd3+ can
efficiently transfer energy to Yb3+ [25]. Due to strong energy back-transfer from
activators to Nd3+, a core@shell structure is normally used to separate Nd3+ and
activators, i.e., activators are embedded in the core while Nd3+ is restricted in the
shell. Zhou and coauthors have proposed an interfacial energy transfer (IET) concept
and established a physical model upon an interlayer-mediated nanostructure, which
allows for a fine control of photon upconversion between sensitizer and activator at a
single lanthanide ion level [33]. Very recently, Jang’s group has successfully
synthesized Ce3+ (30 mol%) doped-NaGdF4: Yb, Ho, Ce@NaYF4: Nd, Yb@NaGdF4
core@shell@shell structured UCNPs which emit single-band red (644 nm) under
excitation with 800 nm NIR laser [27].
Tumor-targeting by biomolecular ligands such as peptides, proteins, and folates
have shown great promise for improving tumor accumulation of nanomedicines and
theranostic precision of tumors by biasing recognition at specific receptors
overexpressed in tumor cells [35-37]. For instance, Ren and coauthors have
successfully developed an active-targeting drug-loaded phase-transformation
nanoparticles for low intensity focused ultrasound (LIFU)-assisted tumor ultrasound
molecular imaging and precise therapy through interactions of tumor
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homing-penetrating peptide (sequence CGNKRTR) with overexpressed neuropilin-1
(NRP-1) in human tumors [38]. Compared with the monoclonal antibodies (the
gold-standard of tumor-targeting ligand), peptides offer several advantages including
ease in preparation in large-scale, low immunogenicity and efficient penetration into
target tissue. Accumulating evidences suggest that the presence of specific glycan
epitopes such as truncated mucin-type O-glycans on cell surface are a hallmark
characteristic of various human cancers [39-43]. Lectins, the carbohydrate-binding
proteins, have been immobilized on nanomaterials for tumor-targeting drug delivery
and bioimaging through molecular recognition of glycan epitopes [44, 45]. In the
previous study, we have demonstrated that the lectin Uelx Europaeus Agglutinin-I
(UEA-I) can serve as tumor-targeting molecule to diagnose colorectal tumor [45].
Unlike antibodies, lectins are easy to produce in large quantities because they are
ubiquitous in many plants such as beans and grains.
In this study, we synthesize a novel NaErF4: 10% Yb@NaYF4: 40%
Yb@NaNdF4: 10% Yb@NaGdF4: 20% Yb (termed as Er@Y@Nd@Gd)
core@multishell UCNPs as an efficient 808 nm NIR-to-red (655 nm) luminescence
emission probe for optical bioimaging. After coated with carboxy-terminated silica,
three molecules including NH2-PEG3400-COOH, peptide D-SP5 and UEA-I have been
modified on the UCNPs surface (termed as UCNP@SiO2-PEG, UCNP@SiO2-D-SP5
and UCNP@SiO2-UEA-I, respectively) for studying the effects of ligands on
biodistribution, clearance pathway and tumor targeting capacity of UCNPs. The
results suggest that the surface modification exhibits negligible effect on
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biodistribution and clearance pathway of UCNPs in healthy mice. The
tumor-targeting capacities of UCNPs follow the order, UCNP@SiO2-UEA-I >
UCNP@SiO2-D-SP5 > UCNP@SiO2-PEG > UCNP@SiO2-COOH (i.e.,
carboxy-terminated silica coated Er@Y@Nd@Gd UCNPs). Furthermore, the
UCNP@SiO2-UEA-I exhibits high affinity with SW480 tumor, indicating that it can
serve as an excellent optical probe for precise detection of SW480 tumor at early
stage.
2. Experimental section
2.1 Synthesis of nanocomposites
The hexagonal phase NaErF4: 10% Yb (termed as Er) core, NaErF4: 10%
Yb@NaYF4: 40% Yb (termed as Er@Y) UCNPs, NaErF4: 10% Yb@NaYF4: 40%
Yb@NaNdF4: 10% Yb (termed as Er@Y@Nd) UCNPs, and NaErF4: 10%
Yb@NaYF4: 40% Yb@NaNdF4: 10% Yb@NaGdF4: 20% Yb (termed as
Er@Y@Nd@Gd) core@multishell UCNPs were prepared by previous reported
solvothermal method with slight modifications [32, 33]. The carboxy-terminated
silica coated Er@Y@Nd@Gd UCNPs (termed as UCNP@SiO2-COOH) was
synthesized by our previous report procedure with slight modifications [45]. The
details of synthesis procedures and characterizations of nanocomposites were shown
in the Supporting Information.
2.2 The PEG, D-SP5 and UEA-I functionalized UCNPs (termed as
UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I, respectively)
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The modification procedure of the UCNP@SiO2-COOH was same with our
previous report [45], except that 0.8 mL EDC (0.2 mg mL-1) and 0.8 mL sulfo-NHS
(0.8 mg mL-1) were added to active the surface -COOH groups, and 10 mg
NH2-PEG3400-COOH, 0.1 mg D-SP5 or 0.1 mg UEA-I were added into 3 mL HEPES
(0.1 mM, pH 7.2) containing 0.6 mg nanoparticles, respectively. After the mixtures
were incubated for 3 h at 37 °C with reciprocating oscillation (130 rpm), the
functionlized UCNPs were collected by centrifugation (7000 rpm for 20 min) at 4 °C,
and redispersed in 0.5 mL phosphate-buffered saline (PBS, pH 8.5, 1.5 × 10-3 M
KH2PO4, 8 × 10-3 M Na2HPO4·12H2O, and 137 × 10-3 M NaCl), respectively.
2.3 Cytotoxicity study of the UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I
The colorectal cancer cells (SW480) were cultured in fresh Leibovitz’s L-15
medium supplemented with 10% fetal bovine serum (FBS) and 100 U mL-1
penicillin-streptomycin under a humidified 5% CO2 at 37 °C. To evaluate the
cytotoxicities of the as-prepared UCNP@SiO2-COOH and three bioconjugates
(UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I), the SW480
cells were firstly cultured in 96-well cell-culturing plate (1.5 × 104 cells/well in 100
µL culture medium) for 24 h. After removed the culture medium, 100 µL fresh culture
medium containing the UCNPs with desired concentrations (6.25, 12.5, 25, 50, 100
and 200 µg mL-1) were introduced into the wells and incubated for another 24 h,
respectively. After totally washed by PBS (pH 7.4) for three times, the cell viabilities
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were determined by traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The normal cultured SW480 cells were employed as control
samples.
2.4 UCL imaging of cells
SW480 cells (5 × 104 cells/well in 500 µL culture medium) were seeded in a
48-well plate and incubated for 24 h. After discharged the culture medium and
washed with 0.5 mL PBS (three times), 500 µL fresh culture medium containing
UCNP@SiO2-COOH, UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 or
UCNP@SiO2-UEA-I (100 µg mL-1) were added into the corresponding wells and
incubated for different time (0.5, 1, 2, 4 and 6 h), respectively. After washed with 0.5
mL PBS (three times), the UCNP-stained cells were fixed by 4% (w/v)
paraformaldehyde for 20 min and subjected to UCL imaging by the Nikon Ti-S
fluorescent microscope (Nikon, Tokyo, Japan) under 808 nm NIR laser excitation. For
UCL spectral measurement, UCNP-stained cells were detached by trypsin, counted
with cell counter, collected by centrifugation (1000 rpm, 5 min) and resuspened in
PBS (pH 7.4), respectively. The UCL spectra of 300 µL UCNP-stained cells (1 × 106
cells mL-1) were measured using an external 808 nm laser as excitation source.
2.5 Tissue penetration investigation
For investigating the in vitro tissue penetration of UCNP@SiO2-COOH, chicken
breast slices with different thickness (0, 1, 3, 5, 8 and 10 mm) were covered on the top
of a cuvette which filled with 1 mL UCNP@SiO2-COOH solution (2 mg mL-1). The
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UCNP@SiO2-COOH solution was transversely illuminated by the 808 nm (1.5 W
cm-2) laser. The UCL images of chicken breast slices were recorded. For investigating
the in vivo tissue penetration of UCNP@SiO2-COOH, single dose of
UCNP@SiO2-COOH (0.25 mg in 50 µL 0.9 wt% NaCl solution) was injected
subcutaneously into the upper thigh of a Balb/c mouse. The 808 nm laser (1.5 W
cm-2) entered from opposite position of injection site in thigh and penetrated whole
thigh for exciting UCNP@SiO2-COOH. The UCL image was collected at same
position of 808 nm laser entering the thigh. Besides, single dose of
UCNP@SiO2-COOH (3 mg in 200 µL 0.9 wt% NaCl solution) was injected into the
abdominal cavity of a Balb/c mouse. After 5 min post-injection, the abdominal cavity
of the mouse was illuminated by an 808 nm laser (1.5 W cm-2) and the UCL image
was collected. All of UCL images were recorded by M2590 (GenieTM Nano Cameras)
with a 675 nm short pass filter (SP675, FWHM 150 nm).
2.6 In vivo biodistribution and clearance pathway investigation
200 µL 0.9 wt% NaCl solution containing 1.5 mg mL-1 (Gd3+ content)
UCNP@SiO2-COOH, UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 or
UCNP@SiO2-UEA-I were intravenously injected into healthy Balb/c mice,
respectively. Then, the in vivo UCL images of livers and kidneys were recorded at the
appropriate time points (0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h post-injection)
under the excitation of 808 nm NIR laser (0.6 W cm-2), respectively. In addition, the
mice injected with four UCNPs were sacrificed at 4 and 48 h post-injection,
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respectively, and the organs (heart, liver, spleen, lung, kidneys) were collected for ex
vivo UCL imaging.
2.7 Tumor-targeting capacity investigation
The SW480 tumor-bearing nude mice were injected intravenously with 200 µL
0.9 wt% NaCl solution containing 1.5 mg mL-1 (Gd3+ content) UCNP@SiO2-COOH,
UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 or UCNP@SiO2-UEA-I through tail veins,
respectively. The in vivo UCL images of tumors were recorded at 0, 0.5, 1, 2, 4, 6, 8,
10, 12, 24, 36 and 48 h post-injection under the excitation of 808 nm NIR laser (1 W
cm-2), respectively. Moreover, the tumors and organs of mice were collected for ex
vivo UCL imaging at 8 and 48 h post-injection, respectively.
For the small tumor and ultrasmall tumor detection, in vivo UCL images and ex
vivo UCL images were recorded at 8 h post-injection of the UCNP@SiO2-D-SP5 and
UCNP@SiO2-UEA-I, respectively. The tumor volumes were calculated according to
the following formula: tumor volume (V) = (length × width × hight × π)/6.
2.8 In vivo toxicology investigation
15 healthy Balb/c mice were randomly divided into five groups, which were
received intravenous injections of 200 µL 0.9 wt% NaCl solution only (control
group), 200 µL 0.9 wt% NaCl solution containing 10 mg kg-1 (Gd3+ content)
UCNP@SiO2-COOH, UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 or
UCNP@SiO2-UEA-I through tail veins, respectively. The body weight of each mouse
was monitored every five days. Blood samples and organs (heart, liver, spleen, lung
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and kidneys) were harvested from both the control and the experiment mice at 30 D
post-injection. The organs were fixed in 4% (w/v) paraformaldehyde solution,
embedded in paraffin, sectioned, and finally stained with hematoxylin-eosin (H&E)
for further histological examinations. The blood samples were analyzed by the blood
biochemistry assay.
3. Results and discussion
3.1 Synthesis and characterization of Er@Y@Nd@Gd UCNPs
Recently, UCNPs with bright red emission under 808 nm NIR laser excitation
have sparked a rapidly growing interest because of requirements of deep-tissue
imaging. As shown in Fig. 1, we design and synthesize a novel Er@Y@Nd@Gd
core@multishell UCNPs as an efficient 808 nm NIR-to-red luminescence probe for
bioimaging. As shown in Fig. 2a-h, the as-prepared Er, Er@Y, Er@Y@Nd and
Er@Y@Nd@Gd UCNPs have reasonable monodispersity with mean sizes of 29.5 ±
1.1, 36.5 ± 0.9, 45.3 ± 1.2 and 50.2 ± 1.4 nm in diameters, respectively. The highly
anisotropic structure of Er@Y@Nd is due to that the ionic radius of Nd3+ (0.098 nm)
is much larger than Y3+ (0.089 nm) [46]. The elemental mapping images (Na, F, Er,
Y, Yb, Nd and Gd) in Fig. S1 demonstrate the successful synthesis of the
core@multishell nanoparticles. As shown in Fig. S2, the diffraction peaks of Er,
Er@Y, Er@Y@Nd as well as Er@Y@Nd@Gd UCNPs are all indexed exactly to
pure hexagonal phase of β-NaErF4 (JCPDS NO. 27-0689). The two bands at 1464
cm-1 and 1567 cm-1 (as shown in Fig. S3) can be assigned to the -COOH stretching
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vibration of oleic acid, indicating that the Er@Y@Nd@Gd UCNPs are coated by
oleic acids. The UCL spectra of the nanoparticles are shown in Fig. 2m. Under 808
nm NIR laser excitation, an obvious increase of the red UCL emission intensity can
be observed for Er@Y@Nd@Gd UCNPs, which is likely due to the efficient
suppression of surface-related deactivation and valid promotion of the interaction
between the lanthanide dopants in Shell 3 with that in the Core@Shell 1@Shell
2@Shell 3 configuration [46]. The energy-level diagram and the corresponding
mechanism of the red emission excitation process excited at 808 nm in the
core@multishell nanostructured system are shown in Fig. 1b-c.The energy transfer
process can be described as follows: the 4I9/2 level of Er3+ is populated from 4I15/2 level
by direct absorption of 808 nm (ground state absorption, GSA), and the population of
4S3/2 can be attributed to the efficient cross relaxation (CR) interaction between
up-closed Er3+ ions (24I9/2 → 4S3/2 + 4I13/2). Then, the electrons can decay
nonradiatively from 2H11/2 level to 4S3/2 level of Er3+, resulting in the green emission
bands in the range of 520 nm to 540 nm. Subsequently, the CR process (4S3/2 + 4I9/2 →
24F9/2) leads to an efficient energy transfer (ET) from green emission band to red
emission band (around 650 nm (4F9/2 → 4I15/2)), causing a relatively high red/green
emission ratio. Meanwhile, the Nd3+ ions in Shell 2 serve as the sensitizer to harvest
808 nm photons, resulting in a population of the 4F5/2 state of Nd3+. The photon
energy from the sensitizer Nd3+ can be absorbed by Yb3+ ions in Shell 1 and Shell 3
through interionic cross-relaxation [(4F3/2)Nd, (2F7/2)Yb] → [(4I9/2)Nd, (2F5/2)Yb],
followed by excitation-energy migration over the Yb3+ sublattice. The 2F5/2 level of
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Yb3+ is equal to the 4I11/2 level of Er3+, which is able to serve as a more efficient
energy trapping center through the (4I11/2)Er → (2F5/2)Yb → (4I11/2)Er process, producing
an enhancement in red and green UC emissions. In addition, the Er@Y@Nd@Gd
UCNPs exhibit same characteristic UCL peaks under excitation with 980 nm NIR
laser (as shown in Fig. 2n). Fig. S4 shows the energy-level diagram in this multilayer
system exhibited at 980 nm. Consideration of optically transparent window in the
biological system, 808 nm laser is used for exciting the UCNPs in subsequent in vitro
and in vivo experiments.
3.2 Synthesis and characterization of UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I
In order to generate hydrophilic NIR-to-red UCNPs for biological applications,
the as-prepared hydrophobic Er@Y@Nd@Gd UCNPs were firstly coated with
carboxy-terminated silica shell using our previously reported water-in-oil
microemulsion method with slight modifications [45]. Subsequently, three
biomolecules (NH2-PEG3400-COOH, D-SP5 and UEA-I) were conjugated on
UCNP@SiO2-COOH surface through the reaction between terminal carboxy group of
SiO2 and amine group of these molecules. Among of three biomolecules, PEG
molecules are the U. S. Food and Drug Administration (FDA) approved
pharmaceutical raw materials, which have been extensively used to construct passive
tumor-targeting nanomedicines [47]. D-SP5 has been demonstrated as an effective
tumor-targeting agent, which displays higher binding affinities with tumor
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endothelium and tumor cells [36, 48, 49]. We have demonstrated that UEA-I has high
specificity for SW480 tumor [45]. As shown in Fig. 2i, the HRTEM micrograph of
UCNP@SiO2-COOH shows that a thin and uniform silica shell with thickness of c.a.
3.4 nm is coated on the Er@Y@Nd@Gd UCNPs surface. The morphologies and
monodispersities of UCNPs exhibit a negligible change after silica coating and
biomolecular modifications (as shown in Fig. 2j-l). The UCL intensity of
UCNP@SiO2-COOH is lower (70%) than that of Er@Y@Nd@Gd UCNPs due to the
quenching effect of the SiO2 shell presented around the UCNPs [50]. There is no
significant effect on the UCL intensity of UCNP@SiO2-COOH after biomolecular
modifications. The XPS measurements clearly show the element of Si in
UCNP@SiO2-COOH and the elements of Si and N in UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I (as shown in Fig. S5), respectively.
The hydrodynamic diameters (HDs) of UCNP@SiO2-COOH are 81.5 ± 2.3 nm in
PBS (pH 8.5) and 111.6 ± 1.2 nm in L-15 containing 10% FBS, while the zeta
potentials of UCNP@SiO2-COOH are -37.5 ± 1.3 mV in PBS (pH 8.5) and -16.7 ±
1.2 mV in L-15 containing 10% FBS. The results suggest that UCNP@SiO2-COOH
exhibits good monodispersity and strongly negative surface charge. After
modifications, the HDs and zeta potentials of the UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I are higher than those of
UCNP@SiO2-COOH (as shown in Fig. S6). As shown in Fig. S3, the emergence of
bands at 1560 cm-1 and 1417 cm-1 (symmetric and asymmetric stretching vibration of
-COOH), 1069 cm-1 (stretching vibration of Si-O-Si), 803 cm-1 and 467 cm-1
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(transforming vibration of Si-O) in the FTIR spectrum of UCNP@SiO2-COOH
further confirms the presence of the carboxy-SiO2 on Er@Y@Nd@Gd UCNPs
surface [45]. For the UCNP@SiO2-PEG, the IR characteristic peaks of PEG at 2924
cm-1 (alkyl C-H stretching) and 1092 cm-1 (C-O-C stretching) are clearly observed in
the FTIR spectrum [51], suggesting the successful conjugation of
NH2-PEG3400-COOH. The IR bands at 1633 cm-1 and 1645 cm-1 (the stretching
vibration of C=O in amide bonds) are clearly observed in the FTIR spectra of
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I, suggesting the successful
modifications of D-SP5 and UEA-I on the UCNP@SiO2-COOH surface.
3.3 The interactions of SW480 cells with UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I
The SW480 cells were firstly incubated with UCNP@SiO2-COOH,
UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I over a wide
concentration range (0-200 µg mL-1) for evaluating the cytotoxicities of these UCNPs
by MTT assay, respectively. As shown in Fig. 3a, the viabilities of SW480 cells are
still over 90% after incubation with as high as 200 µg mL-1 UCNPs for 24 h. The
result indicates that UCNP@SiO2-COOH and three bioconjugates do not have
obvious cytotoxicities.
The UCL imaging of SW480 cells was carried out to investigate SW480
cell-targeting capacities of four UCNPs (UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I). In the presence of UCNPs, the UCL
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intensities of SW480 cells are increased by increasing the incubation time (as shown
in Fig. S7). At all time points (0 to 6 h), the UCNP@SiO2-UEA-I stained SW480 cells
show brighter UCL than that of UCNP@SiO2-D-SP5 stained SW480 cells, while both
of UCNP@SiO2-COOH and UCNP@SiO2-PEG stained SW480 cells exhibit weak
UCL. The corresponding UCL spectra of UCNP-stained SW480 cells were also
measured (as shown in Fig. 3b-c). The maximum UCL intensities of UCNP-stained
SW480 cells are linearly increased by increasing incubation time from 0 to 6 h. The
maximum UCL intensity of UCNP@SiO2-UEA-I stained SW480 cells is 1.8 times
that of UCNP@SiO2-D-SP5 stained SW480 cells, 43.4 times that of
UCNP@SiO2-PEG stained SW480 cells, and 42.5 times that of UCNP@SiO2-COOH
stained SW480 cells, respectively. These results demonstrate that the binding affinity
of UCNP@SiO2-UEA-I with SW480 cells is much higher than those of
UCNP@SiO2-D-SP5 and UCNP@SiO2-PEG with SW480 cells.
3.4 Tissue penetration investigation
The in vitro penetration depth of UCNP@SiO2-COOH was examined by using
chicken breast slice to mimic the biological tissue (as shown in Fig. 4a). In this case,
the cuvette was filled with 2 mg mL-1 UCNP@SiO2-COOH solution. The 808 nm
NIR (1.5 W cm-2) laser past through the cuvette from left to right. For examining the
UCL penetration, the top of cuvette was covered by fresh chicken breast slices with
various thickness. As shown in Fig. 4b, although the UCL intensity of
UCNP@SiO2-COOH is decreased by increasing the thickness of chicken breast slice,
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the UCL can be visualized (signal-to-noise ratio (S/N) > 2.6) even as thickness as 10
mm chicken breast slice. The penetration depth can be further enhanced while the
UCNP@SiO2-COOH is excited by higher energy 808 nm laser (as shown in Video
S1). For in vivo bioimaging, single dose of UCNP@SiO2-COOH (0.25 mg in 50 µL
0.9 wt% NaCl solution) and UCNP@SiO2-COOH (3 mg in 200 µL 0.9 wt% NaCl
solution) were injected into the upper thigh region and abdominal cavity of mice,
respectively. As shown in Fig. 4c-f, clearly UCL images can be collected from the
opposite position of thigh (i.e., the 808 nm excited laser and 655 nm UCL pass
through upper thigh of mouse) and the abdomen under excitation with the 808 nm
NIR laser (1.5 W cm-2). The phenomenon confirms the large penetration depth
(penetrating through upper thigh (c.a. 5 mm thickness) and deep abdominal tissue of
living mouse) by using the UCNP@SiO2-COOH. The result suggests that the
combination of 808 nm excitation and 655 nm UCL of the UCNP@SiO2-COOH
results in a large penetration depth for optical bioimaging study of visceral organs.
3.5 In vivo biodistributions and clearance pathways of UCNP@SiO2-COOH,
UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I
The UCL imaging was employed to investigate the biodistributions and
clearance pathways of UCNP@SiO2-COOH and three bioconjugates
(UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I) in healthy
Balb/c mice. The four UCNPs were injected into the mice through tail veins, and the
in vivo UCL images of livers and kidneys were acquired at the desired time-points (0,
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0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h post-injection) since the nanoparticles are
prefer to accumulate in the reticuloendothelial system [51-53]. As shown in Fig. 5a
and S8-S11, the UCL signals of livers/kidneys are increased within 0 to 6 h (livers)
and 0 to 4 h (kidneys) post-injection of UCNPs. After that, the UCL signals of
livers/kidneys are gradually decayed, indicating the excretion of UCNPs from the
body. There is no ligand (PEG, D-SP5 and UEA-I)-dependent difference in UCL
signals of livers/kidneys. The result indicates that UCNP@SiO2-COOH and three
bioconjugates have similar biodistributions and clearance pathways in healthy Balb/c
mice. In addition, the signal changes of the kidneys are smaller than those of livers.
The biodistributions and clearance pathways of UCNPs were further confirmed by ex
vivo UCL imaging of organs and their tissue sections. The organs were harvested
from the mice at 4 and 48 h post-injection, respectively. As shown in Fig. 5b and
S12-S13, liver, spleen, lung and kidneys have strong UCL signals at 4 h
post-injection, while the UCL signals are dominantly detected from the liver, spleen
and lung at 48 h post-injection. The results of in vivo and ex vivo UCL imaging
demonstrate that the UCNPs are eventually accumulated in liver and gradually
excreted from body by liver.
3.6 SW480 tumor-targeting capacity
After confirming their interactions with SW480 cells, we investigated SW480
tumor-targeting capacities of UCNP@SiO2-COOH and three bioconjugates
(UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I). The UCNPs
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were injected into in the SW480 tumor-bearing Balb/c nude mice through the tail
veins, respectively. Under the excitation of 808 nm NIR laser, the UCL signals of
tumor sites were recorded from 0 to 48 h post-injection. As shown in Fig. 6 and
S14-S16, a sustained increase of the UCL signal intensity at tumor site is observed
within 0 to 8 h post-injection, and the maximum UCL signal enhancement is achieved
at 8 h post injection. Notably, the UCL signal intensity in tumor site of
UCNP@SiO2-UEA-I treated mouse is higher than those of UCNP@SiO2-COOH,
UCNP@SiO2-PEG or UCNP@SiO2-D-SP5 treated mice at the same post-injection
time. The maximum UCL signal in tumor site of UCNP@SiO2-UEA-I treated mouse
is 1.9 times that of UCNP@SiO2-COOH treated mouse, 1.8 times that of
UCNP@SiO2-PEG treated mouse, and 1.5 times that of UCNP@SiO2-D-SP5 treated
mouse, respectively. The result indicates that the SW480 tumor-targeting capacities of
UCNPs with active targeting ligands (D-SP5 and UEA-I) are stronger than that of
UCNPs with passive ligand (PEG). The relative strong SW480 tumor-targeting
capacity of UCNP@SiO2-UEA-I may due to its high binding affinity with SW480
cells.
Encouraging by its strong SW480 tumor-targeting capacity, the
UCNP@SiO2-UEA-I was injected into nude mice-bearing small tumour xenografts
(18 mm3 in volume) through the tail veins. Strong UCL signal enhancement of tumor
site is successfully observed at 8 h post-injection (as shown in Fig. 7a-b). In
particular, the UCL signal can clearly be observed at inoculation site (i.e., region of
interest (ROI)) when the Balb/c nude mice were treated by UCNP@SiO2-UEA-I at 15
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D post-subcutaneous inoculation with 5 ×106 SW480 cells (as shown Fig. 7c-d). The
histology analysis verifies that the ultrasmall tumors (c.a. 3 mm3 in volume, as shown
in Fig. S17) are formed under the skin. Under the same experimental conditions, the
UCL signal intensity of ROI in UCNP@SiO2-UEA-I treated SW480 tumor-bearing
Balb/c nude mouse is higher than that of UCNP@SiO2-D-SP5 treated SW480
tumor-bearing Balb/c nude mouse (as shown in Fig. 7 and S18, Table 1). The result
further demonstrates that UCNP@SiO2-UEA-I exhibits high sensitivity for detection
of SW480 tumor.
3.7 In vivo toxicity analysis
The healthy Balb/c mice were intravenously administrated a single dose of
UCNP@SiO2-COOH, UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and
UCNP@SiO2-UEA-I, respectively. The long-term in vivo toxicities of four UCNPs
were assessed by monitoring the bodyweight changes of mice, histology analysis of
major organs, and blood biochemical assays at 30 D post-injection. As shown in Fig.
S19, the bodyweights of mice in all tested groups are increased steadily as the time
prolonged. Comparing with the control group, the main organs (e.g., heart, liver,
spleen, lung, kidneys) of UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 or UCNP@SiO2-UEA-I treated mice show negligible lesions or
abnormalities (as shown in Fig. S20). For blood biochemical assays, there is little
difference between treated groups and control group (as shown in Table S1). The
results further confirm the good biocompatibility of four UCNPs.
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4. Conclusion
In summary, we have designed and synthesized a novel Nd3+-sensitized
Er@Y@Nd@Gd core@multishell UCNPs based on the interfacial energy transfer
(IET) concept. Benefiting from the combined absorption of the Er3+ and Nd3+ ion
from the energy of excitation light and fine controlled lanthanide interactions by the
multi-layer structure, the Er@Y@Nd@Gd UCNPs emit highly bright 655 nm UCL
under 808 nm NIR laser excitation. After coated with carboxy-terminated silica, the
UCNP@SiO2-COOH can serve as an ideal nanoplatform for constructing
passive/active tumor-targeting NIR-to-red probes with excellent tissue penetration and
reasonable biocompatibility. With varying surface functionalized ligands including
NH2-PEG3400-COOH, D-SP5 and UEA-I, we find that there is no ligand-dependent
biodistribution and clearance pathway of different functionalized UCNPs. Both of in
vitro and in vivo experiments demonstrate that the UCNP@SiO2-UEA-I displays high
SW480 tumor-targeting capacity. In vivo UCL imaging with UCNP@SiO2-UEA-I
enables clear visualization of ultrasmall SW480 tumor (c.a. 3 mm3 in volume). The
results suggest that the UCNP@SiO2-COOH bioconjugates hold great promise for
sensitive detection of deep-tissue tumor.
Conflict of Interest
The authors declare no competing financial interest.
Data availability
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The raw/processed data required to reproduce these findings cannot be shared at this
time due to technical or time limitations.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grant
No. 21475126 and 21775145).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.biomaterials.XXXXXX.
References
[1] S. A. Hilderbrand, R. Weissleder, Near-infrared fluorescence: application to in
vivo molecular imaging, Curr. Opin. Chem. Biol. 14 (2010) 71-79.
https://doi.org/10.1016/j.cbpa.2009.09.029.
[2] G. Chen, I. Roy, C. Yang, P. N. Prasad, Nanochemistry and nanomedicine for
nanoparticle-based diagnostics and therapy, Chem. Rev. 116 (2016) 2826-2885.
https://doi.org/10.1021/acs.chemrev.5b00148.
[3] M. Nyk, R. Kumar, T. Y. Ohulchanskyy, E. J. Bergey, P. N. Prasad, High contrast
in vitro and in vivo photoluminescence bioimaging using near infrared to near
infrared up-conversion in Tm3+ and Yb3+ doped fluoride nanophosphors, Nano
Lett. 8 (2008) 3834-3838. https://doi.org/0.1021/nl802223f.
[4] G. Chen, J. Shen, T. Y. Ohulchanskyy, N. J. Patel, A. Kutikov, Z. Li, J. Song, R. K.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
24
Pandey, H. Agren, P. N. Prasad, G. Han, (α-NaYbF4: Tm3+)/CaF2 core/shell
nanoparticles with efficient near-infrared to near-infrared upconversion for
high-contrast deep tissue bioimaging, ACS Nano 6 (2012) 8280-8287.
https://doi.org/10.1021/nn302972r.
[5] H. Dong, S. Du, X. Zheng, G. Lyu, L. Sun, L. Li, P. Zhang, C. Zhang, C. Yan,
Lanthanide nanoparticles: from design toward bioimaging and therapy, Chem. Rev.
115 (2015) 10725-10815. https://doi.org/10.1021/acs.chemrev.5b00091.
[6] W. Zheng, P. Huang, D. Tu, E. Ma, H. Zhu, X Chen, Lanthanide-doped
upconversion nano-bioprobes: electronic structures, optical properties, and
biodetection, Chem. Soc. Rev. 44 (2015) 1379-1415.
https://doi.org/10.1039/C4CS00178H.
[7] Z. Li, S. Lv, Y. Wang, S. Chen, Z. Liu, Construction of LRET-based nanoprobe
using upconversion nanoparticles with confined emitters and bared surface as
luminophore, J. Am. Chem. Soc. 137 (2015) 3421-3427.
https://doi.org/10.1021/jacs.5b01504.
[8] A. Xia, Y. Deng, H. Shi, J. Hu, J. Zhang, S. Wu, Q. Chen, X. Huang, J. Shen,
Polypeptide-functionalized NaYF4: Yb3+, Er3+ nanoparticles: red-emission
biomarkers for high quality bioimaging using a 915 nm laser, ACS Appl. Mater.
Interfaces 6 (2014) 18329-18336. https://doi.org/10.1021/am5057272.
[9] G. Chen, H. Qiu, P. N. Prasad, X. Chen, Upconversion nanoparticles: design,
nanochemistry, and applications in theranostics, Chem. Rev. 114 (2014)
5161-5214. https://doi.org/10.1021/cr400425h.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
25
[10] J. Xu, L. Xu, C. Wang, R. Yang, Q. Zhuang, X. Han, Z. Dong, W. Zhu, R. Peng, Z.
Liu, Near-infrared-triggered photodynamic therapy with multitasking
upconversion nanoparticles in combination with checkpoint blockade for
immunotherapy of colorectal cancer, ACS Nano 11 (2017) 4463-4474.
https://doi.org/10.1021/acsnano.7b00715.
[11] M. Sun, L. Xu, W. Ma, X. Wu, H. Kuang, L. Wang, C. Xu, Hierarchical
plasmonic nanorods and upconversion core-satellite nanoassemblies for
multimodal imaging�guided combination phototherapy, Adv. Mater. 28 (2016)
898-904. https://doi.org/10.1002/adma.201505023.
[12] L. Xia, X. Kong, X. Liu, L. Tu, Y. Zhang, Y. Chang, K. Liu, D. Shen, H. Zhao, H.
Zhang, An upconversion nanoparticle-zinc phthalocyanine based
nanophotosensitizer for photodynamic therapy, Biomaterials 35 (2014) 4146-4156.
https://doi.org/10.1016/j.biomaterials.2014.01.068.
[13] J. Jin, Y. J. Gu, C. W. Y. Man, J. Cheng, Z. Xu, Y. Zhang, H. Wang, V. H. Y. Lee,
S. H. Cheng, W. T. Wong, Polymer-coated NaYF4: Yb3+, Er3+ upconversion
nanoparticles for charge-dependent cellular imaging, ACS Nano 5 (2011)
7838-7847. https://doi.org/10.1021/nn201896m.
[14] R. Weissleder, A clearer vision for in vivo imaging, Nat. Biotechnol. 19 (2001)
316. https://doi.org/10.1038/86684.
[15] Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang, S.
Andersson-Engels, Using 915 nm laser excited Tm3+/Er3+/Ho3+-doped NaYbF4
upconversion nanoparticles for in vitro and deeper in vivo bioimaging without
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
26
overheating irradiation, ACS Nano 5 (2011) 3744-3757.
https://doi.org/10.1021/nn200110j.
[16] S. Han, B. W. Hwang, E. Y. Jeon, D. Jung, G. H. Lee, D. H. Keum, K. S. Kim, S.
H. Yun, H. J. Cha, S. K. Hahn, Upconversion nanoparticles/hyaluronate-Rose
Bengal conjugate complex for noninvasive photochemical tissue bonding, ACS
Nano 11 (2017) 9979-9988. https://doi.org/10.1021/acsnano.7b04153.
[17] X. Xie, N. Gao, R. Deng, Q. Sun, Q. Xu, X. Liu, Mechanistic investigation of
photon upconversion in Nd3+-sensitized core-shell nanoparticles, J. Am. Chem.
Soc. 135 (2013) 12608-12611. https://doi.org/10.1021/ja4075002.
[18] Y. Wang, G. Liu, L. Sun, J. Xiao, J. Zhou, C. Yan, Nd3+-sensitized upconversion
nanophosphors: efficient in vivo bioimaging probes with minimized heating effect,
ACS Nano 7 (2013) 7200-7206. https://doi.org/10.1021/nn402601d.
[19] H. Wen, H. Peng, K. Liu, M. Bian, Y. Xu, L. Dong, X. Yan, W. Xu, W. Tao, J.
Shen, Y. Lu, H. Qian, Sequential growth of NaYF4:Yb/Er@NaGdF4
nanodumbbells for dual-modality fluorescence and magnetic resonance imaging,
ACS Appl. Mater. Interfaces 9 (2017) 9226-9232.
https://doi.org/10.1021/acsami.6b16842.
[20] G. Tian, X. Zheng, X. Zhang, W. Yin, J. Yu, D. Wang, Z. Zhang, X. Yang, Z. Gu,
Y. Zhao, TPGS-stabilized NaYbF4: Er upconversion nanoparticles for dual-modal
fluorescent/CT imaging and anticancer drug delivery to overcome multi-drug
resistance. Biomaterials 40 (2015) 107-116.
https://doi.org/10.1016/j.biomaterials.2014.11.022.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
27
[21] A. M. Smith, M. C. Mancini, S. Nie, Bioimaging: second window for in vivo
imaging, Nat. Nanotechnol. 4 (2009) 710.
https://doi.org/10.1038/nnano.2009.326.
[22] X. Xu, Z. Wang, P. Lei, Y. Yu, S. Yao, S. Song, X. Liu, Y. Su, L. Dong, J. Feng, H.
Zhang, α-NaYb(Mn)F4: Er3+/Tm3+@NaYF4 UCNPs as “band-shape” luminescent
nanothermometers over a wide temperature range, ACS Appl. Mater. Interfaces 7
(2015) 20813-20819. https://doi.org/10.1021/acsami.5b05876.
[23] W. Yin, L. Zhao, L. Zhou, Z. Gu, X. Liu, G. Tian, S. Jin, L. Yan, W. Ren, G. Xing,
Y. Zhao, Enhanced red emission from GdF3: Yb3+, Er3+ upconversion nanocrystals
by Li+ doping and their application for bioimaging, Chem. Eur. J. 18 (2012)
9239-9245. https://doi.org/10.1002/chem.201201053.
[24] G. Tian, Z. Gu, L. Zhou, W. Yin, X. Liu, L. Yan, S. Jin, W. Ren, G. Xing, S. Li, Y.
Zhao, Mn2+ dopant�controlled synthesis of NaYF4: Yb/Er upconversion
nanoparticles for in vivo imaging and drug delivery, Adv. Mate. 24 (2012)
1226-1231. https://doi.org/10.1002/adma.201104741.
[25] Y. Liu, N. Kang, J. Lv, Z. Zhou, Q. Zhao, L. Ma, Z. Chen, L. Ren, L. Nie, Deep
photoacoustic/luminescence/magnetic resonance multimodal imaging in living
subjects using high�efficiency upconversion nanocomposites, Adv. Mater. 28
(2016) 6411-6419. https://doi.org/10.1002/adma.201506460.
[26] D. Chen, L. Liu, P. Huang, M. Ding, J. Zhong, Z. Ji, Nd3+-sensitized Ho3+
single-band red upconversion luminescence in core-shell nanoarchitecture, J. Phys.
Chem. Lett. 6 (2015) 2833-2840. https://doi.org/10.1021/acs.jpclett.5b01180.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
28
[27] A. R. Hong, Y. Kim, T. S. Lee, Y. Kim, K. Lee, G. Kim, H. S. Jang, Intense
red-emitting upconversion nanophosphors (800 nm-driven) with a
core/double-shell structure for dual-modal upconversion luminescence and
magnetic resonance in vivo imaging applications, ACS Appl. Mater. Interfaces 10
(2018) 12331-12340. https://doi.org/10.1021/acsami.7b18078.
[28] Y. Zhong, G. Tian, Z. Gu, Y. Yang, L. Gu, Y. Zhao, Y. Ma, J. Yao, Elimination of
photon quenching by a transition layer to fabricate a quenching�shield sandwich
structure for 800 nm excited upconversion luminescence of Nd3+�sensitized
nanoparticles, Adv. Mater. 26 (2014) 2831-2837.
https://doi.org/10.1002/adma.201304903.
[29] Y. Shang, S. Hao, W. Lv, T. Chen, T. Li, Z. Lei, C. Yang, Confining excitation
energy of Er3+-sensitized upconversion nanoparticles through introducing various
energy trapping centers, J. Mater. Chem. C 6 (2018) 3869-3875.
https://doi.org/10.1039/C7TC05742C.
[30] N. J. J. Johnson, S. He, S. Diao, E. M. Chan, H. Dai, A. Almutairi, Direct
evidence for coupled surface and concentration quenching dynamics in
lanthanide-doped nanocrystals, J. Am. Chem. Soc. 139 (2017) 3275-3282.
https://doi.org/10.1021/jacs.7b00223.
[31] Q. Chen, X. Xie, B. Huang, L. Liang, S. Han, Z. Yi, Y. Wang, Y. Li, D. Fan, L.
Huang, Confining excitation energy in Er3+�sensitized upconversion nanocrystals
through Tm3+�mediated transient energy trapping, Angew. Chem., Int. Ed. 129
(2017) 7713-7717. https://doi.org/10.1002/ange.201703012.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
29
[32] J. Zuo, L. Tu, Q. Li, Y. Feng, I. Que, Y. Zhang, X. Liu, B. Xue, L. J. Cruz, Y.
Chang, H. Zhang, X. Kong, Near infrared light sensitive ultraviolet-blue
nanophotoswitch for imaging-guided “off-on” therapy, ACS Nano 12 (2018)
3217-3225. https://doi.org/10.1021/acsnano.7b07393.
[33] B. Zhou, L. Yan, L. Tao, N. Song, M. Wu, T. Wang, Q. Zhang, Enabling photon
upconversion and precise control of donor-acceptor interaction through interfacial
energy transfer, Adv. Sci. 5 (2018) 1700667.
https://doi.org/10.1002/advs.201700667.
[34] Q. Ju, X. Chen, F. Ai, D. Peng, X. Lin, W. Kong, P. Shi, G. Zhu, F. Wang, An
upconversion nanoprobe operating in the first biological window, J. Mater. Chem.
B 3 (2015) 3548-3555. https://doi.org/10.1039/c5tb00025d.
[35] L. Liang, A. Care, R. Zhang, Y. Lu, N. H. Packer, A. Sunna, Y. Qian, A. V.
Zvyagin, Facile assembly of functional upconversion nanoparticles for targeted
cancer imaging and photodynamic therapy, ACS Appl. Mater. Interfaces 8 (2016)
11945-11953. https://doi.org/10.1021/acsami.6b00713.
[36] Y. Li, Y. Lei, E. Wagner, C. Xie, W. Lu, J. Zhu, J. Shen, J. Wang, M. Liu, Potent
retro-inverso D-peptide for simultaneous targeting of angiogenic blood
vasculature and tumor cells, Bioconjugate Chem. 24 (2013) 133-143.
https://doi.org/10.1021/bc300537z.
[37] W. Zhang, B. Peng, F. Tian, W. Qin, X. Qian, Facile preparation of well-defined
hydrophilic core-shell upconversion nanoparticles for selective cell membrane
glycan labeling and cancer cell imaging, Anal. Chem. 86 (2013) 482-489.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
30
https://doi.org/10.1021/ac402389w.
[38] L. Zhu, H. Zhao, Z. Zhou, Y. Xia, Z. Wang, H. Ran, P. Li, J. Ren,
Peptide-functionalized phase-transformation nanoparticles for low intensity
focused ultrasound-assisted tumor imaging and therapy, Nano Lett. 18 (2018)
1831-1841. https://doi.org/10.1021/acs.nanolett.7b05087.
[39] G. R. Rossi, M. R. Mautino, R. C. Unfer, T. M. Seregina, N. Vahanian, C. J. Link,
Effective treatment of preexisting melanoma with whole cell vaccines expressing
α (1,3)-galactosyl epitopes, Cancer Res. 65 (2005) 10555-10561.
https://doi.org/10.1158/0008-5472.CAN-05-0627.
[40] N. Remmers, J. M. Anderson, E. M. Linde, D. J. DiMaio, A. J. Lazenby, H. H.
Wandall, U. Mandel, H. Clausen, F. Yu, M. A. Hollingsworth, Aberrant expression
of mucin core proteins and O-linked glycans associated with progression of
pancreatic cancer, Clin. Cancer Res. 19 (2013) 1981-1993.
https://doi.org/10.1158/1078-0432.CCR-12-2662.
[41] C. M. Ferrer, V. L. Sodi, M. J. Reginato, O-GlcNAcylation in cancer biology:
linking metabolism and signaling, J. Mol. Biol. 428 (2016) 3282-3294.
https://doi.org/10.1016/j.jmb.2016.05.028.
[42] L. Wen, D. Liu, Y. Zheng, K. Huang, X. Cao, J. Song, P. G. Wang, A one-step
chemoenzymatic labeling strategy for probing sialylated thomsen-friedenreich
antigen, ACS Cent. Sci. 4 (2018) 451-457.
https://doi.org/10.1021/acscentsci.7b00573.
[43] X. Chen, Detecting the sweet biomarker on cancer cells, ACS Cent. Sci. 4 (2018)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
31
428-430. https://doi.org/10.1021/acscentsci.8b00156.
[44] T. Kitamura, S. Sakuma, M. Shimosato, H. Higashino, Y. Masaoka, M. Kataoka,
S. Yamashita, K. Hiwatari, H. Kumagai, N. Morimoto, S. Koike, E. Tobita, R. M.
Hoffman, J. C. Gore, W. Pham, Specificity of lectin�immobilized fluorescent
nanospheres for colorectal tumors in a mouse model which better resembles the
clinical disease, Contrast Media Mol. Imaging 10 (2015) 135-143.
https://doi.org/10.1002/cmmi.1609.
[45] R. Tian, H. Zhang, H. Chen, G. Liu, Z. Wang, Uncovering the binding
specificities of lectins with cells for precision colorectal cancer diagnosis based on
multimodal imaging, Adv. Sci. 2018 1800214.
https://doi.org/10.1002/advs.201800214.
[46] B. Liu, Y. Chen, C. Li, F. He, Z. Hou, S. Huang, H. Zhu, X. Chen, J. Lin, Poly
(acrylic acid) modification of Nd3+�sensitized upconversion nanophosphors for
highly efficient UCL imaging and pH�responsive drug delivery, Adv. Funct.
Mater. 25 (2015) 4717-4729. https://doi.org/10.1002/adfm.201501582.
[47] K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Poly (ethylene glycol) in
drug delivery: pros and cons as well as potential alternatives, Angew. Chem., Int.
Ed. 49 (2010) 6288-6308. https://doi.org/10.1002/anie.200902672.
[48] X. Li, Z. Xie, C. Xie, W. Lu, C. Gao, H. Ren, M. Ying, X. Wei, J. Gao, B. Su, Y.
Ren, M. Liu, D-SP5 peptide-modified highly branched polyethylenimine for gene
therapy of gastric adenocarcinoma, Bioconjugate Chem. 26 (2015) 1494-1503.
https://doi.org/10.1021/acs.bioconjchem.5b00137.
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[49] H. Chen, X. Li, F. Liu, H. Zhang, Z. Wang, Renal clearable peptide functionalized
NaGdF4 danodots for high-efficiency tracking orthotopic colorectal tumor in
mouse, Mol. Pharmaceutics 14 (2017) 3134-3141.
https://doi.org/10.1021/acs.molpharmaceut.7b00361.
[50] G. Jalani, R. Naccache, D. H. Rosenzweig, L. Haglund, F. Vetrone, M. Cerruti,
Photocleavable hydrogel-coated upconverting nanoparticles: a multifunctional
theranostic platform for NIR imaging and on-demand macromolecular delivery, J.
Am. Chem. Soc. 138 (2016) 1078-1083. https://doi.org/10.1021/jacs.5b12357.
[51] F. Liu, X. He, Z. Lei, L. Liu, J. Zhang, H. You, H. Zhang, Z. Wang, Facile
preparation of doxorubicin�loaded upconversion@polydopamine nanoplatforms
for simultaneous in vivo multimodality imaging and chemophotothermal
synergistic therapy, Adv. Healthcare Mater. 4 (2015) 559-568.
https://doi.org/10.1002/adhm.201400676.
[52] L. Xiong, T. Yang, Y. Yang, C. Xu, F. Li, Long-term in vivo biodistribution
imaging and toxicity of polyacrylic acid-coated upconversion nanophosphors,
Biomaterials 31 (2010) 7078-7085.
https://doi.org/10.1016/j.biomaterials.2010.05.065.
[53] L. Cheng, K. Yang, M. Shao, X. Lu, Z. Liu, In vivo pharmacokinetics, long-term
biodistribution and toxicology study of functionalized upconversion nanoparticles
in mice, Nanomedicine 6 (2011) 1327-1340. https://doi.org/10.2217/nnm.11.56.
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Fig. 1. (a) Schematic illustration of the synthesis and modification of core@multishell
NaErF4: 10% Yb@NaYF4: 40% Yb@NaNdF4: 10% Yb@NaGdF4: 20% Yb UCNPs.
From internal NaErF4: 10% Yb core to outer NaGdF4: 20% Yb shell, the
nanoparticles are termed as Er, Er@Y, Er@Y@Nd and Er@Y@Nd@Gd UCNPs,
respectively. (b) Simplified energy level diagrams of Er@Y@Nd@Gd UCNPs: (1)
the NaErF4: 10% Yb, core (It can also be served as a absorption layer.), (2) NaYF4:
40% Yb, the first-transfer layer (shell 1), (3) NaNdF4: 10% Yb, the absorption layer
(shell 2), and (4) NaGdF4: 20% Yb, the second-transfer layer and UCL quenching
reduction layer (shell 3). (c) The proposed energy-transfer mechanism in the
Er@Y@Nd@Gd UCNPs under 808 nm NIR laser excitations. (d) Biodistributions,
clearance pathways and tumor-targeting capacities studies of the as-prepared
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UCNP@SiO2-COOH and three bioconjugates (UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I).
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Fig. 2. TEM micrographs and correspondent size distributions of Er (a and e), Er@Y
(b and f), Er@Y@Nd (c and g), and Er@Y@Nd@Gd (d and h). TEM micrographs of
UCNP@SiO2-COOH (i), UCNP@SiO2-PEG (j), UCNP@SiO2-D-SP5 (k) and
UCNP@SiO2-UEA-I (l). The scale bars of TEM micrographs are 50 nm. Insets of (a)
and (i) are the corresponding HRTEM micrographs of the core Er and
UCNP@SiO2-COOH. The UCL spectra and digital photographs (insets) of
as-prepared nanoparticles under 808 nm (m) or 980 nm (n) NIR laser excitation ((1)
Er, (2) Er@Y, (3) Er@Y@Nd, (4) Er@Y@Nd@Gd, (5) UCNP@SiO2-COOH, (6)
UCNP@SiO2-PEG, (7) UCNP@SiO2-D-SP5 and (8) UCNP@SiO2-UEA-I)).
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Fig. 3. (a) Cell viabilities of SW480 cells after incubated with various concentrations
of UCNP@SiO2-COOH, UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and
UCNP@SiO2-UEA-I for 24 h, respectively. Each cell viability value represents the
mean ± standard deviation of five replicates. (b) The UCL spectra of SW480 cells
incubated with 100 µg mL-1 UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I for various times (0.5, 1, 2, 4 and 6 h),
respectively. These UCL spectra of UCNP-stained cells have the same scale of Y-axis.
(c) The UCL intensities at 655 nm of UCNP-stained cells as a function of incubation
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times.
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Fig. 4. (a) Diagram of experimental setup for investigating the penetration of UCL
emission in chicken breast slice. (b) The intensities and the luminescence images of
UCL passed through chicken breast slices with different thickness (0, 1, 3, 5, 8 and 10
mm, the 0 mm means without chicken breast slice coverage). (c, d) In vivo images of
UCL past through upper thigh regions, and (e, f) in vivo UCL images of abdominal
cavity. c, e were collected under dark-field mode, while d, f were merging images.
The scale bars are 1 cm.
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Fig. 5. (a) The UCL intensities of livers and kidneys of healthy Balb/c mice after
intravenous injection of UCNP@SiO2-COOH, UCNP@SiO2-PEG,
UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I at different time intervals (0, 0.5, 1, 2,
4, 6, 8, 10, 12, 24, 36 and 48 h, the 0 h means pre-injection) of post-injection,
respectively. (b) The brightfield images and UCL images of main organs ((1) heart, (2)
liver, (3) spleen, (4) lung, and (5) kidneys)) of mice at 4 h and 48 h post-injection,
respectively. The scale bars are 1 cm.
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Fig. 6. The UCL intensities of tumor sites as a function of post-injection times. The
UCL intensities were measured after intravenous injection of UCNP@SiO2-COOH,
UCNP@SiO2-PEG, UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I into SW480
tumor-bearing Balb/c nude mice at different time intervals (0, 0.5, 1, 2, 3, 4, 6, 8, 10,
12, 24, 36 and 48 h, the 0 h means pre-injection) of post-injection. The tumor volume
is c.a. 65 mm3.
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Fig. 7. In vivo and ex vivo UCL imaging of small SW480 tumors (c.a. 18 mm3 (a, b)
and 3 mm3 (c, d) in volume) at 8 h post-injection. The SW480 tumor-bearing Balb/c
nude mice were treated with UCNP@SiO2-UEA-I. The scale bars are 1 cm.
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Table 1. The UCL signal intensities of different volume tumors of
UCNP@SiO2-D-SP5 or UCNP@SiO2-UEA-I treated mice.
Region of Interest
(ROI)
UCL Signal Intensity
(UCNP@SiO2-D-SP5)
UCL Signal Intensity
(UCNP@SiO2-UEA-I)
Specific Uptake 1a 93 143
Specific Uptake 2b 84 131
Specific Uptake 3c 66 110
aThe mouse with big tumor (c.a. 65 mm3).
bThe mouse with small tumor (c.a. 18 mm3).
cThe mouse with ultrasmal tumor (c.a. 3 mm3).
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Graphical Abstract