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.

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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