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This article can be cited before page numbers have been issued, to do this please use: X. Ge, L. Dong, L. Sun, Z. Song, R. Wei, L. Shi and H. Chen, Nanoscale, 2015, DOI: 10.1039/C5NR00950B.
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DOI: 10.1039/C5NR00950B
New nanoplatforms based on UCNPs linking with polyhedral oligomeric silsesquioxane (POSS) for multimodal bioimaging
Xiaoqian Gea, Liang Dongc, Lining Sun*a, Zhengmei Songb, Ruoyan Weia, Liyi
a
Research Center of Nano Science and Technology, Shanghai University, Shanghai
200444, P. R. China. E-mail: [email protected] (L. N. Sun); Tel: +86-21-66137153 b
Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444,
China. c
Department of Urology, Renji hospital, School of Medicine, Shanghai Jiao Tong
University, Shanghai 200127, P. R. China. E-mail: [email protected] (H. G. Chen)
Abstract A new and facile method was used to transfer upconversion luminescent nanoparticles from hydrophobic to hydrophilic using polyhedral oligomeric silsesquioxane (POSS) linking on the surface of upconversion nanoparticles. In comparison with the unmodified upconversion nanoparticles, the POSS modified upconversion nanoplatforms [POSS-UCNPs(Er), POSS-UCNPs(Tm)] displayed good monodispersion in water and exhibited good water-solubility, while their particle size did not change substantially. Due to the low cytotoxicity and good biocompatibility as determined by methyl thiazolyl tetrazolium (MTT) assay and histology and hematology analysis, the POSS modified upconversion nanoplatforms were successfully applied to upconversion luminescence imaging of living cells in vitro and nude mouse in vivo (upon excitation with 980 nm). In addition, the doped Gd3+ ion endows the POSS-UCNPs with effective T1 signal enhancement and the POSS-UCNPs were successfully applied to in vivo magnetic resonance imaging (MRI) for Kunming 1
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Shia, Haige Chen*c
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DOI: 10.1039/C5NR00950B mouse, which affords them as potential MRI positive-contrast agent. More importantly,
the corner organic groups of POSS can be easily modified, resulting in kinds of POSS-UCNPs with many potential applications. Therefore, the method and results may provide more exciting opportunities for multimodal bioimaging and
Keywords:
Upconversion
luminescence
nanoplatforms;
POSS;
Hydrophilic;
Bioimaging; Magnetic resonance imaging (MRI).
1. Introduction In recent decades, bioimaging has attracted much attention due to its ability to obtain anatomical and physiological details of bio-systems ranging from cell to ex vivo tissue samples, and to in vivo imaging of living objects.1-3 Bioimaging techniques such as fluorescent imaging, magnetic resonance imaging (MRI) and X-ray tomography (CT) can label targeted object and provide unsurpassed soft tissue details and three dimensional (3D) information of the anatomic structure of tissues, and so on.4-8 Generally, the imaging agents are essential to amplify the signal and the quality of bioimaging deeply depends on the imaging agents used. Rare earth-doped upconversion nanoparticles (UCNPs) based on an anti-Stokes process under continuous excitation with near infrared (NIR) light, are considered as a new promising generation of imaging agents for bioimaging.9-13 Compared with the conventional biological labels, such as organic dyes and quantum dots, UCNPs possess superior physicochemical features, including large anti-Stokes shifts, low auto-fluorescence, low toxicity, high resistance to photobleaching and high penetration depth.14-16 Moreover, multifunctional nanoplatforms based on UCNPs integrated multimodal imaging into a single structure are the next generation of imaging agents for more accurate imaging and diagnosis, which have evoked considerable interest to circumvent the limitation of single imaging platform.10, 17, 18 In general, most of the synthesis processes of UCNPs are performed in oleic acid 2
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multifunctional applications.
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DOI: 10.1039/C5NR00950B (OA) and 1-octadecene (ODE), and their surfaces need to be modified to transfer from
hydrophobic to hydrophilic for successful application of UCNPs in the biological field.19 So far, many surface modification strategies have been developed, including polymer capping.20-22 layer-by-layer assembly.23-25 surface silanization.26-28 etc. More importantly, these modified compounds on UCNPs surface can play the further role to
photodynamic therapy, DNA target recognition and ion sensing.29-34 However, these strategies have some limitations of complicated synthesis processes or post-treatment procedures, fluorescence quenching and aggregation of nanoparticles. For example, the mesoporous silicon capping upconversion nanoparticles offer good water solubility, yet the intensity of fluorescence decreases and usually the toxic surfactants have to be used, also with time-consuming multi-steps.35 For polymer capping, although good water solubility and biocompatibility can be achieved, the worse water molecular access to NaGdF4 nanoparticles, leading to lower relaxivity value than mesoporous silicon capping.36 Therefore, it is necessary to explore new and easier surface modification methods of UCNPs, which make the modified UCNPs possess good water solubility, biocompatibility, and high relaxivity value and can further be applied to other bioapplications with modified compounds. Polyhedral oligomeric silsesquioxane (POSS) is the smallest silica nanoparticle with the general formula of (RSiO1.5)8, which contains a polyhedron silicon−oxygen cube skeleton with intermittent siloxane linkages and tunable organic groups at the silicon atoms.37-39 They are particularly attractive in biological field due to the excellent water solubility, low cytotoxicity and high biocompatibility, well-defined cubic cage structures and ease of functionalization. The eight corner organic groups of POSS can be easily modified to kinds of functional groups, such as amino, thiol, hydroxyl, and halogen, etc.40-42 And some modified POSS have already been used as drug release.43,
44
gene delivery.45,
46
fluorescence imaging.47-49 and other
bioapplications. For example, Liu et al. reported Gd complex was grafted on POSS (POSS–HPG–Gd), which was applied to fluorescence and magnetic resonance dual 3
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loading drug or photosensitizer, grafting DNA and fluorescent dyes, for drug release,
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DOI: 10.1039/C5NR00950B modal imaging due to their excellent water solubility, low cytotoxicity and high
biocompatibility.50 Considering the excellent properties of POSS and UCNPs, the work may open up new perspectives for developing multimodal bioimaging contrast agents and multifunctional bioapplications based on building a system to combine the POSS and UCNPs. To the best of our knowledge, the multifunctional nanoplatforms
In this paper, the NaYF4:Yb,Er and NaYF4:Yb,Tm cores was synthesized, respectively, and NaGdF4 shell was coated to form a core-shell structure [named as UCNPs(Er), UCNPs(Tm)] for potential magnetic resonance imaging (MRI). Then a new and facile method was used to surface modify the UCNPs by using POSS. The POSS modified UCNPs [named as POSS-UCNPs(Er) and POSS-UCNPs(Tm)] have good water solubility and show good biocompatibility and low cytotoxicity in vivo and in vitro. Furthermore, the multifunctional nanomaterials were successfully applied to upconversion luminescence (UCL) imaging of cells in vitro and small animal in vivo (upon excitation with 980 nm), and in vivo MRI for Kunming mouse. 2. Experimental 2.1. Chemicals and materials All the chemicals were used as received without further purification. ErCl3· 6H2O (99.99%), YbCl3· 6H2O (99.99%), YCl3· 6H2O (99.99%), GdCl3· 6H2O (99.99%), TmCl3· 6H2O (99.99%) were purchased from Sigma Aldrich. Sodium hydroxide (NaOH, 96%), ammonium fluoride (NH4F, 98%), methanol (CH4O, 99.5%), and 3-chloroperoxybenzoic acid were obtained from Aladdin. OctaAmmonium Polyhedral oligomeric silsesquioxane (POSS, C24H72N8O12Si8) were purchased from hybrid plastic company. Oleic acid (OA, 90%) and 1-octadecene (ODE, 90%) were obtained from Alfa Aesar. 2.2.
Synthesis
of
NaYF4:Yb,Er,
NaYF4:Yb,Tm,
and
NaYF4:Yb,Er@NaGdF4
[UCNPs(Er)], NaYF4:Yb,Tm@NaGdF4[UCNPs(Tm)] nanocrytals The upconversion nanocrystals of NaYF4:Yb,Er and NaYF4:Yb,Tm were synthesized according to a previously reported method.19, 51, 52 For the synthesis of 4
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based on linking of UCNPs with POSS have not been reported to date.
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NaYF4:Yb,Er@NaGdF4 nanocrystals, the process was similar with DOI: that10.1039/C5NR00950B of NaYF4:Yb,Er. 80 μmol GdCl3 water solution was added to a 100 mL flask, and then heated to 110 °C to evaporate the water. 12 mL oleic acid and 30 mL 1-octadecene were added in when the solution became white powder. The mixture was heated to 150 °C to form a homogeneous transparent solution, and then cooled to room
for another 30 min before heated to 90 °C to remove cyclohexane. Then, 1.25 mL methanol solution of NH4F (0.039 g, 1.05 mmol) and NaOH (0.067 g, 1.68 mmol) was added in and the solution was stirred at 100 °C for a while. After the methanol was evaporated, the solution was heated to 300 °C and kept for 1 h under argon atmosphere and then cooled to room temperature. After centrifugation and washing, the final sample was redispersed in 10 mL cyclohexane. The synthesis of NaYF4:Yb,Tm@NaGdF4 nanocrystals was similar to that of NaYF4:Yb,Er@NaGdF4 except that NaYF4:Yb,Er was replaced by NaYF4:Yb,Tm. 2.3. Synthesis of POSS modified UCNPs nanoplatforms [named as POSS-UCNPs(Er), POSS-UCNPs(Tm)] The epoxidation process of OA molecules on UCNPs was according to the literature.53 2 mL UCNPs(Er) cyclohexane solution (5 mgmL-1), 4 mL CH2Cl2 and 5 mg 3-chloroperoxybenzoic acid were mixed in a 25 mL flask and stirred at 40 °C for 3 h in the dark. After being cooled to room temperature, 10 mg POSS was added and the mixture reacted at 25 °C for 5 h. The obtained product POSS-UCNPs(Er) was separated by centrifugation, washed with water and dispersed in 10 mL water. POSS-UCNPs(Tm)
was
synthesized
according
to
the
procedure
of
POSS-UCNPs(Er) except that UCNPs(Er) was replaced by UCNPs(Tm). 2.4. Characterization The size and morphology of the nanoparticles were characterized on a JEM-2010F low-to-high resolution transmission electron microscope operated at 120 kV. Powder X-ray diffraction (XRD) measurement was performed on a 3 KW D/MAX2200 V PC diffractometer using Cu kα radiation (60 KV, 80 mA) at a step width of 8°min−1. 5
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temperature. 5 mL pre-prepared NaYF4:Yb,Er was added to above mixture and kept
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10.1039/C5NR00950B Fourier transform infrared spectroscopy (FT-IR) spectra were acquired in theDOI: spectral
range from 4000 to 400 cm−1 on an Avatar 370 instrument using the pressed KBr pellet technique. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to detect the concentration of Gd3+ on the surface of nanoparticles. X-ray photoelectron spectroscopy (XPS) were carried out on a RBD upgraded
Al Kα radiation (hν = 1486.6 eV). Upconversion luminescence spectra were recorded on an Edinburgh LFS-920 fluorescence spectrometer with the excitation of an external 0–800 mW 980 nm adjustable CW laser. 2.5. Cell culture and cell viability assay A human cervix cancer cell line (HeLa cells) was provided by Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences. The cells were cultured in RPMI 1640 (Roswell Park Memorial Institute’s medium) supplemented with 10% FBS (fetal bovine serum) at 37 °C and 5% CO2/95% air.54 The cytotoxicity was evaluated by performing methyl thiazolyl tetrazolium (MTT) assays on the HeLa cells. Cells were seeded into 96-well cell culture plates at 5000 per well and cultured for 24 h. Then, culture media containing POSS-UCNPs in different concentrations (0, 12.5, 25, 50, 100, 200, 400 μg/mL) were added to the cells. The cells were subsequently incubated for another 24 h. Then, MTT (10 μl, 3 mg/mL) was added to each wells and the plate was incubated for an additional 5 h at the same condition. Next, 10% sodium dodecyl sulfate (SDS) (100 μL per well) was added to dissolve the formazan crystals. The absorbance was measured with a Bio-Rad model-680 microplate reader at a wavelength of 570 nm. The following formula was applied to calculate the cell viability: cell viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) × 100%.55 2.6. Histology and hematology studies POSS-UCNPs(Er) at a total dose of 15mg/kg were injected into mice through the tail vein and this group of mice were acted as the test group. Mice with saline were acted as the control group. Blood samples were taken from the eye after injected 7 6
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PHI-5000C ESCA system (Perkin-Elmer) with Mg Kα radiation (hν = 1253.6 eV) or
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10.1039/C5NR00950B days. Some important indicators of hepatic and kidney (GLOB = globulin,DOI: ALB =
albumin, ALP = alkaline phosphatase, ALT = alanine aminotransferase, AST= aspartate aminotransferase, T-BIL = total bilirubin, TP = total protein, UA = uric acid, BUN = urea nitrogen and CRE = creatinine) were measured. After the mice were sacrificed, the heart, liver, spleen, lung, and kidney were removed, and immersed in a
sectioned serially and stained by hematoxylin and eosin (H&E). These sections were evaluated by an optical microscope.27 2.7. Laser scanning upconversion luminescence (UCL) microscopy (LSUCLM) imaging in vitro Confocal imaging of cells was performed with a fluorescence microscope and a 60×oil immersion objective lens. The cells were incubated with serum free medium containing 200 μg/mL POSS-UCNPs(Er) for 7 h at 37 °C and washed with phosphate buffered saline (PBS) to remove the excess POSS-UCNPs(Er). The CW laser at 980 nm provided the excitation and the emission was collected at 500–600 nm.56 2.8. Upconversion luminescence imaging in vivo and ex vivo of nude mouse Animal procedures were in agreement with the regulations for the administration of affairs concerning experimental animals. In vivo and ex vivo UCL imaging was performed with a modified Kodak in vivo imaging system using an external 0–5 W adjustable CW infrared laser (980 nm, Shanghai Connet Fiber Optics Co., China) as the excited source and an Andor DU897 EMCCD as the signal collector.57 The POSS-UCNPs(Tm) solution was injected into nude mice via the tail vein. After 15 min, UCL signals were collected at 800 ± 12 nm. Images of luminescent signals were analyzed with Kodak Molecular Imaging Software. 2.9. Longitudinal relaxation time T1 and relaxivity r1 measurement in vitro and magnetic resonance (MR) imaging in vivo for Kunming mouse The longitudinal relaxation time T1 and relaxivity r1 of the POSS-UCNPs was measured with a MR imaging instrument with 3.0 T magnetic fields. Different concentrations of POSS-UCNPs(Er) in water as contrast agent were placed in a series 7
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buffered solution of 4% paraformaldehyde, embedded in paraffin. The specimen was
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10.1039/C5NR00950B of 4 mL tubes for T1 measurement. The resulting T1 values were recorded at DOI: different
concentrations and plotted as 1/T1 (R2) vs. molar concentration of Gd3+ ions; the slope of this line provided the longitudinal relaxivity r1. The T1-weighted MR imaging was conducted on 3.0 T Siemens Magnetom Trio, using a T1-weighted sequence (TR = 800 ms, TE = 12 ms, 256×256 matrix, slice
intravenously injected to Kunming mouse via tail vein and imaged by the MR imaging. 2. Results and discussion 3.1. Synthesis and characterization of POSS-UCNPs Initially, the UCNPs were prepared via the thermal decomposition method using oleic acid (OA) and 1-octadecene (ODE). Then, an epoxidation process was implemented on the carbon-carbon double bond of OA using 3-chloroperoxybenzoic acid. The amino modified POSS was added and linked on the OA via simply addition reaction, in which the POSS modified UCNPs (POSS-UCNPs) can be obtained. The formation of POSS-UCNPs was illustrated in Fig. S1. Considering the excellent water soluble of POSS, there will be more water molecular access to the surface of POSS-UCNPs, which can be applied to examine the MR imaging by Gd3+. More importantly, not only the Si-O-Si structure of POSS can protect UCNPs from kinds of physiological solution, making POSS-UCNPs have better imaging performance in vivo and in vitro, but also the corner organic groups of POSS can be easily modified, resulting in many potential applications of POSS-UCNPs, such as drug release, fluorescence sensing, photodynamic therapy, and target label, etc. The schematic illustration of POSS-UCNPs was displayed in Scheme 1. The morphology and structure of NaYF4:Yb,Er, NaYF4:Yb,Er@NaGdF4 [UCNPs(Er)] and POSS-UCNPs(Er) were characterized by TEM (see Fig. 1). From Fig. 1A, it can be observed that the size of NaYF4:Yb,Er is around 22 nm, and increases to 28 nm after growing with a NaGdF4 shell (Fig. 1B). After surface modification with POSS, no aggregation was observed for POSS-UCNPs(Er), 8
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thickness = 2.0 mm). Then, 200 μL of POSS-UCNPs(Er) (1 mgmL-1) was
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DOI: 10.1039/C5NR00950B showing good monodispersion in water (Fig. 1C). Furthermore, the same method was
successfully applied to surface modification of UCNPs(Tm) nanoparticles, and the TEM images of NaYF4:Yb,Tm, UCNPs(Tm), and POSS-UCNPs(Tm) are displayed in Fig. S2. For POSS-UCNPs(Tm), it shows good dispersity in water and retains the narrow size distribution as the unmodified UCNPs(Tm) nanoparticles. The
scattering (DLS) measurement shows that the POSS-UCNPs(Tm) have an average diameter of 63 nm (Fig. S3). The XRD patterns of the UCNPs(Er), POSS and POSS-UCNPs(Er) are displayed in Fig. 2. Compared with the standard card of hexagonal phase β-NaYF4 (JCPDS:16-0334), the UCNPs(Er) and POSS-UCNPs(Er) both show hexagonal phase structures. In the pattern of POSS-UCNPs(Er), multiple peaks at 10-40 (marked in Fig. 2) can be attributed to the Si-O-Si structure of POSS, which suggested the POSS was successfully assembled on UCNPs. Furthermore, the presence of POSS on UCNPs surface was also confirmed by energy dispersive X-ray (EDX) and X-ray photoelectron spectroscopy (XPS). As shown in Fig. S4, from the EDX spectrum, it can be illustrated the POSS was grafted on the UCNPs by the presence of the elements (Si, Y, Yb, Er, Gd). In addition, from the XPS of POSS-UCNPs (show in Fig. S5), it can be observed the presence of Si element. The results mentioned above indicate the successful modification of POSS-UCNPs using POSS. FT-IR spectra of UCNPs(Er), POSS and POSS-UCNPs(Er) are shown in Fig. 3. In the curve of UCNPs(Er), the peaks at 2923 and 2854 cm-1 are attributed to the asymmetric and symmetric stretching vibration of methylene (CH2) in the long alkyl chain of OA. And the 1551 and 1462 cm-1 bands can be assigned to the asymmetric and symmetric stretching vibration of the carboxylic group (-COOH) in OA. In the spectrum of POSS, the bands located at 1115 and 801 cm-1 were assigned to the Si−O−Si asymmetric and symmetric vibrations, respectively. The peak at 1218 cm -1 is attributed to C-N stretching vibration of POSS. Compared with the POSS, the spectrum of POSS-UCNPs(Er) still exhibit peaks assigned to the Si−O−Si asymmetric 9
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zeta-potential measurement exhibits the value was 3.8 mV, and the dynamic light
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DOI: 10.1039/C5NR00950B and symmetric vibrations and C-N stretching vibration of POSS. In addition, the
peaks at 2923 and 2854 cm-1 assigned to the -CH2 of OA on the UCNPs(Er) surface can be observed in the curve of POSS-UCNPs(Er). Therefore, the FT-IR results further indicate that POSS was linked on the UCNPs and the POSS-UCNPs are successfully formed. upconversion
luminescence
(UCL)
spectra
of
UCNPs(Er)
and
POSS-UCNPs(Er) are shown in Fig. 4. Under CW excitation at 980 nm, the UCNPs(Er) in cyclohexane show bright green light (inset of Fig. 4) and the peaks at 525, 540, and 660 nm are assigned to the transitions of 2H11/2 → 4I15/2, 4S3/2 → 4I15/2, and
4
F9/2 →
4
I15/2 of Er3+, respectively. After modified with POSS, the
POSS-UCNPs(Er) still exhibit bright green color in water under 980 nm laser illumination (inset of Fig. 4), which can be detected by the bare eye. And the emission feature is similar to that of UCNPs except that POSS-UCNPs(Er) present the lower luminescence intensity. To further investigate whether POSS-UCNPs are stable in biological media, the UCL spectra of POSS-UCNPs(Er) in physiology saline were measured over a period of six days. The luminescence intensity of POSS-UCNPs(Er) maintained > 90% of its initial value when measured over a period of six days (Fig. S6). It is illustrated that the POSS-UCNPs possess colloidal stability and photostability, which is responsible for the applications in bioimaging with no dissociation in physiological conditions. In addition, the UCL spectra of UCNPs(Tm) and POSS-UCNPs(Tm) are shown in Fig. S7. They both show the strongest emission around 800 nm due to the 3H4→3H6 transition of Tm3+ and the blue emission peak at 475 nm originated from the 1G4→3H6 transition of Tm3+ion. 3.2. In vitro cytotoxicity studies To investigate the potential biological application of POSS-UCNPs, their biocompatibility is essential to evaluate and the methyl thiazolyl tetrazolium (MTT) assay was employed to study their cytotoxicity. HeLa cells were incubated with POSS-UCNPs at different concentrations ranging from 0 to 400 μgmL-1 for 24 h. As shown in Fig. 5, nearly 100% viability was observed even at the highest concentration 10
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of 400 μgmL−1. These data illustrate that the POSS-UCNPs have low or noDOI: in 10.1039/C5NR00950B vitro cytotoxicity in the dosages range studied. Therefore, the good biocompatibility makes POSS-UCNPs potentially useful for cell imaging in vitro or drug delivery in biomedical applications. 3.3. Histology and hematology results
injected into Kunming mice via the tail vein. And the histochemistry analyses of tissues were performed to determine whether the POSS-UCNPs can cause tissue damage, inflammation or lesion.58 Fig. 6A shows the hematoxylin and eosin (H&E)-stained tissue sections of organs (heart, lung, liver, spleen, and kidney) from mice injected with POSS-UCNPs 7 days post-injection (Test group) and mice receiving injection of normal saline (Control group), respectively, which exhibits no significant difference between Control and Test groups. In all of the tissue sections of Test groups, necrosis was not observed. There were no hydropic degeneration of cardiac myofibersin in heart samples and the hepatocytes in liver samples appeared normal without inflammatory infiltrates.19 Hyperplasia in the periarteriolar lymphoid sheath of white pulp cannot be found in the spleen and no pulmonary fibrosis was observed in the lung samples. In the kidney, glomeruli structures could be easily distinguished. The serum biochemistry assays were used to quantitatively evaluate the potential risks of injury to the liver, spleen and kidney. Fig. 6B and 6C display the serum biochemistry results obtained from mice injected with POSS-UCNPs 7 days post-injection and mice receiving injection of normal saline. As can be seen that seven important hepatic indicators (Fig. 6B) and three indicators of kidney function (Fig. 6C) were measured in the Test and Control groups. All the factors are within normal ranges and at similar levels for the Test groups and the Control groups, suggesting no inflammatory reaction associated with the treatment and no apparent toxicity of POSS-UCNPs in mice at exposure level beyond those commonly used in luminescent in vivo imaging and at exposure time of up to 7 days. Therefore, the results indicate that the POSS-UCNPs have no evident toxic effects in the dosages range studied, which 11
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For the assessment of in vivo toxicity, the POSS-UCNPs nanoplatforms were
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3.4. MR imaging Generally, the materials based on Gd3+ ion have potential as MR imaging contrast agents due to the isotropic electronic state (8S7/2) and half-filled f-orbital (with seven electrons) of Gd3+ ion.59 Thus, the NaGdF4 shell was grown on the core, not only
the POSS-UCNPs can be served as T1-weighted MR contrast agents, the T1 values of them were measured by using a 3.0 T MRI scanner. As shown in Fig. 7a, a positive enhancement of the MR signal was observed as the concentration of Gd3+ increased in color-mapped images based on T1 values. From the liner regression fit of the slop of Gd3+ concentration-dependent relaxation rate 1/T1 (Fig. 7b), the longitudinal relaxivity (r1) is calculated to be 4.98 mM-1· S-1, which is high in comparison with other NaGdF4-based nanomaterials encapsulated with polymer or other shells.60-62 This is possibly because linking of hydrophilic POSS on the surface of UCNPs makes the as-prepared POSS-UCNPs access to more water molecular, which will offer them better MRI property. In addition, the application of POSS-UCNPs for in vivo MR imaging was examined after tail vein injection in Kunming mice at 15 and 30 min. As shown in Fig. 8, the post-injection contrast T1-weighted coronal images and color-mapped images of mouse display enhancement in the liver area (Fig. 8a and b) compared with those of the pre-injection mouse. For transverse cross-sectional images, the higher MR imaging signal appeared significant contrast in the liver after post-injection 15 min compared with pre-injection, whereas the signal at post-injection 30 min decreased (Fig. 8c and d). Moreover, the signal at spleen increased slightly at post-injection 15 and 30 min due to the uptake of nanoparticles in spleen being lower than liver (Fig. 8e and f) . These observations indicate that these nanoparticles could circulate in the liver in a short time. Therefore, it can be deduced that the POSS-UCNPs could be introduced as MRI positive-contrast agents. 3.5. UCL imaging of living cells in vitro 12
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making the luminescence intensity increase but also with MRI ability. To investigate if
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DOI: 10.1039/C5NR00950B Based on the excellent green emission of POSS-UCNPs under NIR light
excitation (Fig. 4), the application in UCL imaging of living cells was investigated. Fig. 9 shows the confocal imaging of HeLa cells incubated with POSS-UCNPs (150 μgmL−1) for 7 h and the typical upconversion green signal (500-600 nm) was detected. The cells exhibited intense upconversion luminescence in intracellular
image (Fig. 9c) also confirms that the upconversion luminescence was localized in the cytosol region rather than merely staining the membrane surface. Importantly, no autofluorescence signal was observed in the UCL image of Hela cells incubated with POSS-UCNPs (Fig. 9b). The absence of background autofluorescence is very important and highly desired feature for UCL imaging in living cells.63 Therefore, the results indicate that the POSS-UCNPs are desirable candidates for upconversion luminescence cell imaging in vitro. 3.6. UCL imaging in vivo and ex vivo Encouraged by the low cytotoxicity in vitro and in vivo (Fig. 5 and Fig. 6) and excellent NIR (980 nm) to NIR (800 nm) upconversion luminescence property (Fig. S4), the UCL imaging capability of POSS-UCNPs(Tm) in vivo was examined on a nude mouse through tail vein injection (100 μL, 1 mgmL−1, under the authorization of the regional ethics committee for animal experiments). The modified Kodak in vivo imaging system was used for in vivo and ex vivo UCL imaging. The in vivo whole-body images of nude mouse are presented in Fig. 10, and the UCL signals were collected at 800 ± 12 nm under CW excitation at 980 nm with a power density of 400 mWcm−2. In Fig. 10a, a significant UCL signal in vivo was observed with high contrast compared with the background after intravenous injection for 15 min. The peritoneum was exenterated and imaged to investigate the distribution of POSS-UCNPs(Tm) in the organs. Fig. 10b and Fig. 10c display the in situ and ex vivo UCL images, respectively, in which the significant UCL emission was observed from the liver, spleen and lung, whereas the other organs (such as heart, kidney) of the mouse showed 13
no
detectable
UCL signals.
This is possibly because
of more
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region of HeLa cells. The overlay of the upconversion image and the bright-field
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DOI:due 10.1039/C5NR00950B POSS-UCNPs(Tm) accumulated in the liver, spleen and lung than other organs to
stronger affinity of the nanoparticles to the liver, spleen and lung tissues. In addition, in vivo UCL imaging after intravenous injection was tested at different time points (15 min, 30 min, and 2 h) (see Fig. S8), which shows that the UCL signal still retained after 2 h post-injection and the POSS-UCNPs was not cleared from blood quickly.64
in vivo upconversion luminescence imaging with high signal-to-noise ratio. 3. Conclusion In summary, we have successfully developed a new and facile strategy to transfer upconversion nanoparticles from hydrophobic to hydrophilic using POSS. After modification, the water-solubility of the POSS-UCNPs has been significantly improved, while the high size monodispersity has been preserved. Furthermore, benefited from low cytotoxicity and excellent biocompatibility, the POSS-UCNPs show good performance in both in vitro cell imaging and small animal in vivo imaging. In addition, the doped Gd3+ ion endows the POSS-UCNPs with effective T1 signal enhancement and the POSS-UCNPs were successfully applied to in vivo MRI for Kunming mouse, which affords them as potential MRI positive-contrast agent as well. Moreover, the method of POSS modification provides an extended and general approach to constructing different water-soluble nanoplatforms based on the easy modification of the corner groups of POSS. Therefore, the approach may open up new perspectives for a unique synthesis of hydrophilic and uniform nanoplatforms for multimodal bioimaging and multifunctional applications. Acknowledgment We are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 21231004, 21201117), Innovation Program of Shanghai Municipal Education Commission (13ZZ073), the Science and Technology Commission
of
Shanghai
Municipality
(13NM1401100,
13NM1401101,
14520722200), Shanghai Rising-Star Program (14QA1401800), and the project from State Key Laboratory of Rare Earth Resource Utilization (RERU2014012). We are also 14
Nanoscale Accepted Manuscript
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The results above show that the POSS-UCNPs can serve as promising candidates for
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10.1039/C5NR00950B grateful to Instrumental Analysis & Research Center of Shanghai University.DOI: We are
also grateful to Prof. Haifang Wang of Institute of Nanochemistry and Nanobiology, Shanghai University for her helpful discussion and suggestion. † Electronic supplementary information (ESI) available: Schematic illustration of the of
POSS-UCNPs.
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NaYF4:Yb,Tm@NaGdF4
TEM
nanoparticles
images in
of
cyclohexane;
NaYF4:Yb,Tm TEM
image
and of
POSS-UCNPs(Tm) in water. DLS of POSS-UCNPs(Tm) in water. Energy dispersive X-ray (EDX) spectrum of POSS-UCNPs(Er). XPS of POSS-UCNPs(Er); XPS of Si element. UCL spectra of POSS-UCNPs(Er) in physiology saline as a function of time. UCL spectra of NaYF4:Yb,Tm@NaGdF4 [UCNPs(Tm)] and POSS-UCNPs(Tm), excited with 980 nm laser (100 mWcm−2). In vivo UCL imaging of Kunming mice after intravenous injection with POSS-UCNPs(Tm) at different time points.
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L. N. Sun, Z. W. Wei, H. Chen, J. L. Liu, J. Guo, M. Cao, T. Wen, andDOI:L.10.1039/C5NR00950B Shi,View Article Online Nanoscale, 2014, 6, 8878-8883. D. Yang, Y. Dai, J. Liu, Y. Zhou, Y. Chen, C. Li, P. a. Ma and J. Lin, Biomaterials, 2014, 35, 2011-2023.
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Scheme 1. Schematic illustration of the POSS-UCNPs nanoplatform.
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Fig. 1. TEM images of NaYF4:Yb,Er (A) and NaYF4:Yb,Er@NaGdF4 (B) nanoparticles in cyclohexane; TEM image of POSS-UCNPs(Er) in water (C).
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Fig. 2. The XRD patterns of UCNPs(Er), POSS, POSS-UCNPs(Er) and the standard card of β-NaYF4 (JCPDS: 16-0334).
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Fig. 3. FT-IR spectra of UCNPs(Er), POSS, and POSS-UCNPs(Er).
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Fig. 4. UCL spectra of UCNPs(Er) and POSS-UCNPs(Er), excited with 980 nm laser (100 mWcm−2). Inset: the bright-field images of UCNPs(Er) and POSS-UCNPs(Er) (a, c); the dark-field images of UCNPs(Er) and POSS-UCNPs(Er) under excitation with 980 nm laser (b, d).
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Fig. 5. In vitro cell viabilities of HeLa cells incubated with POSS-UCNPs(Er) at different concentrations (0, 12.5, 25, 50, 100, 200, and 400 μgmL−1) for 24 h.
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Fig. 6. (A) H&E-stained tissue sections from mice injected with POSS-UCNPs 7 days post-injection (n = 2, dose = 15 mg/kg, Test) and mice receiving injection of normal saline (n = 2, dose = 15 mg/kg, Control). Tissues were harvested from heart, liver, spleen, lung and kidney. (B) Hepatic indicators. (C) Indicators of kidney functions.
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Fig. 7. T1-weighted and color-mapped MR images for various Gd3+ concentrations of POSS-UCNPs(Er) (a); Relaxation rate r1 (1/T1) versus different Gd3+ concentrations of POSS-UCNPs(Er) (b).
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Fig. 8. In vivo T1-weighted MR images of Kunming mouse at pre-injection, post-injection after 15 min and 30 min. The coronal images and color-mapped images (a, b); The transversal cross-sectional images and color-mapped images of liver (c, d); The transversal cross-sectional images and color-mapped images of spleen (e, f)
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Fig. 9. Confocal imaging of HeLa cells incubated with POSS-UCNPs(Er) with a concentration of 150 μgmL−1 for 7 h at 37 °C. Bright-field image (a); UCL image collected at green channel (500–600 nm) (b); merged image of a and b (c).
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Fig. 10. In vivo (a), in situ (b), and ex vivo (c) UCL images of a nude mouse acquired at 15 min. after intravenous injection with 100 μL POSS-UCNPs(Tm) (1 mgmL−1). UCL signals were collected at 800 ± 12 nm. The power density is 400 mWcm−2 on the surface of the nude mouse.
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DOI: 10.1039/C5NR00950B
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: X. Ge, L. Dong, L. Sun, Z. Song, R. Wei, L. Shi and H. Chen, Nanoscale, 2015, DOI: 10.1039/C5NR00950B.
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DOI: 10.1039/C5NR00950B
New nanoplatforms based on UCNPs linking with polyhedral oligomeric silsesquioxane (POSS) for multimodal bioimaging
Xiaoqian Gea, Liang Dongc, Lining Sun*a, Zhengmei Songb, Ruoyan Weia, Liyi
a
Research Center of Nano Science and Technology, Shanghai University, Shanghai
200444, P. R. China. E-mail: [email protected] (L. N. Sun); Tel: +86-21-66137153 b
Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444,
China. c
Department of Urology, Renji hospital, School of Medicine, Shanghai Jiao Tong
University, Shanghai 200127, P. R. China. E-mail: [email protected] (H. G. Chen)
Abstract A new and facile method was used to transfer upconversion luminescent nanoparticles from hydrophobic to hydrophilic using polyhedral oligomeric silsesquioxane (POSS) linking on the surface of upconversion nanoparticles. In comparison with the unmodified upconversion nanoparticles, the POSS modified upconversion nanoplatforms [POSS-UCNPs(Er), POSS-UCNPs(Tm)] displayed good monodispersion in water and exhibited good water-solubility, while their particle size did not change substantially. Due to the low cytotoxicity and good biocompatibility as determined by methyl thiazolyl tetrazolium (MTT) assay and histology and hematology analysis, the POSS modified upconversion nanoplatforms were successfully applied to upconversion luminescence imaging of living cells in vitro and nude mouse in vivo (upon excitation with 980 nm). In addition, the doped Gd3+ ion endows the POSS-UCNPs with effective T1 signal enhancement and the POSS-UCNPs were successfully applied to in vivo magnetic resonance imaging (MRI) for Kunming 1
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Shia, Haige Chen*c
Nanoscale
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DOI: 10.1039/C5NR00950B mouse, which affords them as potential MRI positive-contrast agent. More importantly,
the corner organic groups of POSS can be easily modified, resulting in kinds of POSS-UCNPs with many potential applications. Therefore, the method and results may provide more exciting opportunities for multimodal bioimaging and
Keywords:
Upconversion
luminescence
nanoplatforms;
POSS;
Hydrophilic;
Bioimaging; Magnetic resonance imaging (MRI).
1. Introduction In recent decades, bioimaging has attracted much attention due to its ability to obtain anatomical and physiological details of bio-systems ranging from cell to ex vivo tissue samples, and to in vivo imaging of living objects.1-3 Bioimaging techniques such as fluorescent imaging, magnetic resonance imaging (MRI) and X-ray tomography (CT) can label targeted object and provide unsurpassed soft tissue details and three dimensional (3D) information of the anatomic structure of tissues, and so on.4-8 Generally, the imaging agents are essential to amplify the signal and the quality of bioimaging deeply depends on the imaging agents used. Rare earth-doped upconversion nanoparticles (UCNPs) based on an anti-Stokes process under continuous excitation with near infrared (NIR) light, are considered as a new promising generation of imaging agents for bioimaging.9-13 Compared with the conventional biological labels, such as organic dyes and quantum dots, UCNPs possess superior physicochemical features, including large anti-Stokes shifts, low auto-fluorescence, low toxicity, high resistance to photobleaching and high penetration depth.14-16 Moreover, multifunctional nanoplatforms based on UCNPs integrated multimodal imaging into a single structure are the next generation of imaging agents for more accurate imaging and diagnosis, which have evoked considerable interest to circumvent the limitation of single imaging platform.10, 17, 18 In general, most of the synthesis processes of UCNPs are performed in oleic acid 2
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multifunctional applications.
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DOI: 10.1039/C5NR00950B (OA) and 1-octadecene (ODE), and their surfaces need to be modified to transfer from
hydrophobic to hydrophilic for successful application of UCNPs in the biological field.19 So far, many surface modification strategies have been developed, including polymer capping.20-22 layer-by-layer assembly.23-25 surface silanization.26-28 etc. More importantly, these modified compounds on UCNPs surface can play the further role to
photodynamic therapy, DNA target recognition and ion sensing.29-34 However, these strategies have some limitations of complicated synthesis processes or post-treatment procedures, fluorescence quenching and aggregation of nanoparticles. For example, the mesoporous silicon capping upconversion nanoparticles offer good water solubility, yet the intensity of fluorescence decreases and usually the toxic surfactants have to be used, also with time-consuming multi-steps.35 For polymer capping, although good water solubility and biocompatibility can be achieved, the worse water molecular access to NaGdF4 nanoparticles, leading to lower relaxivity value than mesoporous silicon capping.36 Therefore, it is necessary to explore new and easier surface modification methods of UCNPs, which make the modified UCNPs possess good water solubility, biocompatibility, and high relaxivity value and can further be applied to other bioapplications with modified compounds. Polyhedral oligomeric silsesquioxane (POSS) is the smallest silica nanoparticle with the general formula of (RSiO1.5)8, which contains a polyhedron silicon−oxygen cube skeleton with intermittent siloxane linkages and tunable organic groups at the silicon atoms.37-39 They are particularly attractive in biological field due to the excellent water solubility, low cytotoxicity and high biocompatibility, well-defined cubic cage structures and ease of functionalization. The eight corner organic groups of POSS can be easily modified to kinds of functional groups, such as amino, thiol, hydroxyl, and halogen, etc.40-42 And some modified POSS have already been used as drug release.43,
44
gene delivery.45,
46
fluorescence imaging.47-49 and other
bioapplications. For example, Liu et al. reported Gd complex was grafted on POSS (POSS–HPG–Gd), which was applied to fluorescence and magnetic resonance dual 3
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loading drug or photosensitizer, grafting DNA and fluorescent dyes, for drug release,
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DOI: 10.1039/C5NR00950B modal imaging due to their excellent water solubility, low cytotoxicity and high
biocompatibility.50 Considering the excellent properties of POSS and UCNPs, the work may open up new perspectives for developing multimodal bioimaging contrast agents and multifunctional bioapplications based on building a system to combine the POSS and UCNPs. To the best of our knowledge, the multifunctional nanoplatforms
In this paper, the NaYF4:Yb,Er and NaYF4:Yb,Tm cores was synthesized, respectively, and NaGdF4 shell was coated to form a core-shell structure [named as UCNPs(Er), UCNPs(Tm)] for potential magnetic resonance imaging (MRI). Then a new and facile method was used to surface modify the UCNPs by using POSS. The POSS modified UCNPs [named as POSS-UCNPs(Er) and POSS-UCNPs(Tm)] have good water solubility and show good biocompatibility and low cytotoxicity in vivo and in vitro. Furthermore, the multifunctional nanomaterials were successfully applied to upconversion luminescence (UCL) imaging of cells in vitro and small animal in vivo (upon excitation with 980 nm), and in vivo MRI for Kunming mouse. 2. Experimental 2.1. Chemicals and materials All the chemicals were used as received without further purification. ErCl3· 6H2O (99.99%), YbCl3· 6H2O (99.99%), YCl3· 6H2O (99.99%), GdCl3· 6H2O (99.99%), TmCl3· 6H2O (99.99%) were purchased from Sigma Aldrich. Sodium hydroxide (NaOH, 96%), ammonium fluoride (NH4F, 98%), methanol (CH4O, 99.5%), and 3-chloroperoxybenzoic acid were obtained from Aladdin. OctaAmmonium Polyhedral oligomeric silsesquioxane (POSS, C24H72N8O12Si8) were purchased from hybrid plastic company. Oleic acid (OA, 90%) and 1-octadecene (ODE, 90%) were obtained from Alfa Aesar. 2.2.
Synthesis
of
NaYF4:Yb,Er,
NaYF4:Yb,Tm,
and
NaYF4:Yb,Er@NaGdF4
[UCNPs(Er)], NaYF4:Yb,Tm@NaGdF4[UCNPs(Tm)] nanocrytals The upconversion nanocrystals of NaYF4:Yb,Er and NaYF4:Yb,Tm were synthesized according to a previously reported method.19, 51, 52 For the synthesis of 4
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based on linking of UCNPs with POSS have not been reported to date.
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NaYF4:Yb,Er@NaGdF4 nanocrystals, the process was similar with DOI: that10.1039/C5NR00950B of NaYF4:Yb,Er. 80 μmol GdCl3 water solution was added to a 100 mL flask, and then heated to 110 °C to evaporate the water. 12 mL oleic acid and 30 mL 1-octadecene were added in when the solution became white powder. The mixture was heated to 150 °C to form a homogeneous transparent solution, and then cooled to room
for another 30 min before heated to 90 °C to remove cyclohexane. Then, 1.25 mL methanol solution of NH4F (0.039 g, 1.05 mmol) and NaOH (0.067 g, 1.68 mmol) was added in and the solution was stirred at 100 °C for a while. After the methanol was evaporated, the solution was heated to 300 °C and kept for 1 h under argon atmosphere and then cooled to room temperature. After centrifugation and washing, the final sample was redispersed in 10 mL cyclohexane. The synthesis of NaYF4:Yb,Tm@NaGdF4 nanocrystals was similar to that of NaYF4:Yb,Er@NaGdF4 except that NaYF4:Yb,Er was replaced by NaYF4:Yb,Tm. 2.3. Synthesis of POSS modified UCNPs nanoplatforms [named as POSS-UCNPs(Er), POSS-UCNPs(Tm)] The epoxidation process of OA molecules on UCNPs was according to the literature.53 2 mL UCNPs(Er) cyclohexane solution (5 mgmL-1), 4 mL CH2Cl2 and 5 mg 3-chloroperoxybenzoic acid were mixed in a 25 mL flask and stirred at 40 °C for 3 h in the dark. After being cooled to room temperature, 10 mg POSS was added and the mixture reacted at 25 °C for 5 h. The obtained product POSS-UCNPs(Er) was separated by centrifugation, washed with water and dispersed in 10 mL water. POSS-UCNPs(Tm)
was
synthesized
according
to
the
procedure
of
POSS-UCNPs(Er) except that UCNPs(Er) was replaced by UCNPs(Tm). 2.4. Characterization The size and morphology of the nanoparticles were characterized on a JEM-2010F low-to-high resolution transmission electron microscope operated at 120 kV. Powder X-ray diffraction (XRD) measurement was performed on a 3 KW D/MAX2200 V PC diffractometer using Cu kα radiation (60 KV, 80 mA) at a step width of 8°min−1. 5
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temperature. 5 mL pre-prepared NaYF4:Yb,Er was added to above mixture and kept
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10.1039/C5NR00950B Fourier transform infrared spectroscopy (FT-IR) spectra were acquired in theDOI: spectral
range from 4000 to 400 cm−1 on an Avatar 370 instrument using the pressed KBr pellet technique. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to detect the concentration of Gd3+ on the surface of nanoparticles. X-ray photoelectron spectroscopy (XPS) were carried out on a RBD upgraded
Al Kα radiation (hν = 1486.6 eV). Upconversion luminescence spectra were recorded on an Edinburgh LFS-920 fluorescence spectrometer with the excitation of an external 0–800 mW 980 nm adjustable CW laser. 2.5. Cell culture and cell viability assay A human cervix cancer cell line (HeLa cells) was provided by Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences. The cells were cultured in RPMI 1640 (Roswell Park Memorial Institute’s medium) supplemented with 10% FBS (fetal bovine serum) at 37 °C and 5% CO2/95% air.54 The cytotoxicity was evaluated by performing methyl thiazolyl tetrazolium (MTT) assays on the HeLa cells. Cells were seeded into 96-well cell culture plates at 5000 per well and cultured for 24 h. Then, culture media containing POSS-UCNPs in different concentrations (0, 12.5, 25, 50, 100, 200, 400 μg/mL) were added to the cells. The cells were subsequently incubated for another 24 h. Then, MTT (10 μl, 3 mg/mL) was added to each wells and the plate was incubated for an additional 5 h at the same condition. Next, 10% sodium dodecyl sulfate (SDS) (100 μL per well) was added to dissolve the formazan crystals. The absorbance was measured with a Bio-Rad model-680 microplate reader at a wavelength of 570 nm. The following formula was applied to calculate the cell viability: cell viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) × 100%.55 2.6. Histology and hematology studies POSS-UCNPs(Er) at a total dose of 15mg/kg were injected into mice through the tail vein and this group of mice were acted as the test group. Mice with saline were acted as the control group. Blood samples were taken from the eye after injected 7 6
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PHI-5000C ESCA system (Perkin-Elmer) with Mg Kα radiation (hν = 1253.6 eV) or
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10.1039/C5NR00950B days. Some important indicators of hepatic and kidney (GLOB = globulin,DOI: ALB =
albumin, ALP = alkaline phosphatase, ALT = alanine aminotransferase, AST= aspartate aminotransferase, T-BIL = total bilirubin, TP = total protein, UA = uric acid, BUN = urea nitrogen and CRE = creatinine) were measured. After the mice were sacrificed, the heart, liver, spleen, lung, and kidney were removed, and immersed in a
sectioned serially and stained by hematoxylin and eosin (H&E). These sections were evaluated by an optical microscope.27 2.7. Laser scanning upconversion luminescence (UCL) microscopy (LSUCLM) imaging in vitro Confocal imaging of cells was performed with a fluorescence microscope and a 60×oil immersion objective lens. The cells were incubated with serum free medium containing 200 μg/mL POSS-UCNPs(Er) for 7 h at 37 °C and washed with phosphate buffered saline (PBS) to remove the excess POSS-UCNPs(Er). The CW laser at 980 nm provided the excitation and the emission was collected at 500–600 nm.56 2.8. Upconversion luminescence imaging in vivo and ex vivo of nude mouse Animal procedures were in agreement with the regulations for the administration of affairs concerning experimental animals. In vivo and ex vivo UCL imaging was performed with a modified Kodak in vivo imaging system using an external 0–5 W adjustable CW infrared laser (980 nm, Shanghai Connet Fiber Optics Co., China) as the excited source and an Andor DU897 EMCCD as the signal collector.57 The POSS-UCNPs(Tm) solution was injected into nude mice via the tail vein. After 15 min, UCL signals were collected at 800 ± 12 nm. Images of luminescent signals were analyzed with Kodak Molecular Imaging Software. 2.9. Longitudinal relaxation time T1 and relaxivity r1 measurement in vitro and magnetic resonance (MR) imaging in vivo for Kunming mouse The longitudinal relaxation time T1 and relaxivity r1 of the POSS-UCNPs was measured with a MR imaging instrument with 3.0 T magnetic fields. Different concentrations of POSS-UCNPs(Er) in water as contrast agent were placed in a series 7
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buffered solution of 4% paraformaldehyde, embedded in paraffin. The specimen was
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10.1039/C5NR00950B of 4 mL tubes for T1 measurement. The resulting T1 values were recorded at DOI: different
concentrations and plotted as 1/T1 (R2) vs. molar concentration of Gd3+ ions; the slope of this line provided the longitudinal relaxivity r1. The T1-weighted MR imaging was conducted on 3.0 T Siemens Magnetom Trio, using a T1-weighted sequence (TR = 800 ms, TE = 12 ms, 256×256 matrix, slice
intravenously injected to Kunming mouse via tail vein and imaged by the MR imaging. 2. Results and discussion 3.1. Synthesis and characterization of POSS-UCNPs Initially, the UCNPs were prepared via the thermal decomposition method using oleic acid (OA) and 1-octadecene (ODE). Then, an epoxidation process was implemented on the carbon-carbon double bond of OA using 3-chloroperoxybenzoic acid. The amino modified POSS was added and linked on the OA via simply addition reaction, in which the POSS modified UCNPs (POSS-UCNPs) can be obtained. The formation of POSS-UCNPs was illustrated in Fig. S1. Considering the excellent water soluble of POSS, there will be more water molecular access to the surface of POSS-UCNPs, which can be applied to examine the MR imaging by Gd3+. More importantly, not only the Si-O-Si structure of POSS can protect UCNPs from kinds of physiological solution, making POSS-UCNPs have better imaging performance in vivo and in vitro, but also the corner organic groups of POSS can be easily modified, resulting in many potential applications of POSS-UCNPs, such as drug release, fluorescence sensing, photodynamic therapy, and target label, etc. The schematic illustration of POSS-UCNPs was displayed in Scheme 1. The morphology and structure of NaYF4:Yb,Er, NaYF4:Yb,Er@NaGdF4 [UCNPs(Er)] and POSS-UCNPs(Er) were characterized by TEM (see Fig. 1). From Fig. 1A, it can be observed that the size of NaYF4:Yb,Er is around 22 nm, and increases to 28 nm after growing with a NaGdF4 shell (Fig. 1B). After surface modification with POSS, no aggregation was observed for POSS-UCNPs(Er), 8
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thickness = 2.0 mm). Then, 200 μL of POSS-UCNPs(Er) (1 mgmL-1) was
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DOI: 10.1039/C5NR00950B showing good monodispersion in water (Fig. 1C). Furthermore, the same method was
successfully applied to surface modification of UCNPs(Tm) nanoparticles, and the TEM images of NaYF4:Yb,Tm, UCNPs(Tm), and POSS-UCNPs(Tm) are displayed in Fig. S2. For POSS-UCNPs(Tm), it shows good dispersity in water and retains the narrow size distribution as the unmodified UCNPs(Tm) nanoparticles. The
scattering (DLS) measurement shows that the POSS-UCNPs(Tm) have an average diameter of 63 nm (Fig. S3). The XRD patterns of the UCNPs(Er), POSS and POSS-UCNPs(Er) are displayed in Fig. 2. Compared with the standard card of hexagonal phase β-NaYF4 (JCPDS:16-0334), the UCNPs(Er) and POSS-UCNPs(Er) both show hexagonal phase structures. In the pattern of POSS-UCNPs(Er), multiple peaks at 10-40 (marked in Fig. 2) can be attributed to the Si-O-Si structure of POSS, which suggested the POSS was successfully assembled on UCNPs. Furthermore, the presence of POSS on UCNPs surface was also confirmed by energy dispersive X-ray (EDX) and X-ray photoelectron spectroscopy (XPS). As shown in Fig. S4, from the EDX spectrum, it can be illustrated the POSS was grafted on the UCNPs by the presence of the elements (Si, Y, Yb, Er, Gd). In addition, from the XPS of POSS-UCNPs (show in Fig. S5), it can be observed the presence of Si element. The results mentioned above indicate the successful modification of POSS-UCNPs using POSS. FT-IR spectra of UCNPs(Er), POSS and POSS-UCNPs(Er) are shown in Fig. 3. In the curve of UCNPs(Er), the peaks at 2923 and 2854 cm-1 are attributed to the asymmetric and symmetric stretching vibration of methylene (CH2) in the long alkyl chain of OA. And the 1551 and 1462 cm-1 bands can be assigned to the asymmetric and symmetric stretching vibration of the carboxylic group (-COOH) in OA. In the spectrum of POSS, the bands located at 1115 and 801 cm-1 were assigned to the Si−O−Si asymmetric and symmetric vibrations, respectively. The peak at 1218 cm -1 is attributed to C-N stretching vibration of POSS. Compared with the POSS, the spectrum of POSS-UCNPs(Er) still exhibit peaks assigned to the Si−O−Si asymmetric 9
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zeta-potential measurement exhibits the value was 3.8 mV, and the dynamic light
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DOI: 10.1039/C5NR00950B and symmetric vibrations and C-N stretching vibration of POSS. In addition, the
peaks at 2923 and 2854 cm-1 assigned to the -CH2 of OA on the UCNPs(Er) surface can be observed in the curve of POSS-UCNPs(Er). Therefore, the FT-IR results further indicate that POSS was linked on the UCNPs and the POSS-UCNPs are successfully formed. upconversion
luminescence
(UCL)
spectra
of
UCNPs(Er)
and
POSS-UCNPs(Er) are shown in Fig. 4. Under CW excitation at 980 nm, the UCNPs(Er) in cyclohexane show bright green light (inset of Fig. 4) and the peaks at 525, 540, and 660 nm are assigned to the transitions of 2H11/2 → 4I15/2, 4S3/2 → 4I15/2, and
4
F9/2 →
4
I15/2 of Er3+, respectively. After modified with POSS, the
POSS-UCNPs(Er) still exhibit bright green color in water under 980 nm laser illumination (inset of Fig. 4), which can be detected by the bare eye. And the emission feature is similar to that of UCNPs except that POSS-UCNPs(Er) present the lower luminescence intensity. To further investigate whether POSS-UCNPs are stable in biological media, the UCL spectra of POSS-UCNPs(Er) in physiology saline were measured over a period of six days. The luminescence intensity of POSS-UCNPs(Er) maintained > 90% of its initial value when measured over a period of six days (Fig. S6). It is illustrated that the POSS-UCNPs possess colloidal stability and photostability, which is responsible for the applications in bioimaging with no dissociation in physiological conditions. In addition, the UCL spectra of UCNPs(Tm) and POSS-UCNPs(Tm) are shown in Fig. S7. They both show the strongest emission around 800 nm due to the 3H4→3H6 transition of Tm3+ and the blue emission peak at 475 nm originated from the 1G4→3H6 transition of Tm3+ion. 3.2. In vitro cytotoxicity studies To investigate the potential biological application of POSS-UCNPs, their biocompatibility is essential to evaluate and the methyl thiazolyl tetrazolium (MTT) assay was employed to study their cytotoxicity. HeLa cells were incubated with POSS-UCNPs at different concentrations ranging from 0 to 400 μgmL-1 for 24 h. As shown in Fig. 5, nearly 100% viability was observed even at the highest concentration 10
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The
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of 400 μgmL−1. These data illustrate that the POSS-UCNPs have low or noDOI: in 10.1039/C5NR00950B vitro cytotoxicity in the dosages range studied. Therefore, the good biocompatibility makes POSS-UCNPs potentially useful for cell imaging in vitro or drug delivery in biomedical applications. 3.3. Histology and hematology results
injected into Kunming mice via the tail vein. And the histochemistry analyses of tissues were performed to determine whether the POSS-UCNPs can cause tissue damage, inflammation or lesion.58 Fig. 6A shows the hematoxylin and eosin (H&E)-stained tissue sections of organs (heart, lung, liver, spleen, and kidney) from mice injected with POSS-UCNPs 7 days post-injection (Test group) and mice receiving injection of normal saline (Control group), respectively, which exhibits no significant difference between Control and Test groups. In all of the tissue sections of Test groups, necrosis was not observed. There were no hydropic degeneration of cardiac myofibersin in heart samples and the hepatocytes in liver samples appeared normal without inflammatory infiltrates.19 Hyperplasia in the periarteriolar lymphoid sheath of white pulp cannot be found in the spleen and no pulmonary fibrosis was observed in the lung samples. In the kidney, glomeruli structures could be easily distinguished. The serum biochemistry assays were used to quantitatively evaluate the potential risks of injury to the liver, spleen and kidney. Fig. 6B and 6C display the serum biochemistry results obtained from mice injected with POSS-UCNPs 7 days post-injection and mice receiving injection of normal saline. As can be seen that seven important hepatic indicators (Fig. 6B) and three indicators of kidney function (Fig. 6C) were measured in the Test and Control groups. All the factors are within normal ranges and at similar levels for the Test groups and the Control groups, suggesting no inflammatory reaction associated with the treatment and no apparent toxicity of POSS-UCNPs in mice at exposure level beyond those commonly used in luminescent in vivo imaging and at exposure time of up to 7 days. Therefore, the results indicate that the POSS-UCNPs have no evident toxic effects in the dosages range studied, which 11
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For the assessment of in vivo toxicity, the POSS-UCNPs nanoplatforms were
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DOI: 10.1039/C5NR00950B
3.4. MR imaging Generally, the materials based on Gd3+ ion have potential as MR imaging contrast agents due to the isotropic electronic state (8S7/2) and half-filled f-orbital (with seven electrons) of Gd3+ ion.59 Thus, the NaGdF4 shell was grown on the core, not only
the POSS-UCNPs can be served as T1-weighted MR contrast agents, the T1 values of them were measured by using a 3.0 T MRI scanner. As shown in Fig. 7a, a positive enhancement of the MR signal was observed as the concentration of Gd3+ increased in color-mapped images based on T1 values. From the liner regression fit of the slop of Gd3+ concentration-dependent relaxation rate 1/T1 (Fig. 7b), the longitudinal relaxivity (r1) is calculated to be 4.98 mM-1· S-1, which is high in comparison with other NaGdF4-based nanomaterials encapsulated with polymer or other shells.60-62 This is possibly because linking of hydrophilic POSS on the surface of UCNPs makes the as-prepared POSS-UCNPs access to more water molecular, which will offer them better MRI property. In addition, the application of POSS-UCNPs for in vivo MR imaging was examined after tail vein injection in Kunming mice at 15 and 30 min. As shown in Fig. 8, the post-injection contrast T1-weighted coronal images and color-mapped images of mouse display enhancement in the liver area (Fig. 8a and b) compared with those of the pre-injection mouse. For transverse cross-sectional images, the higher MR imaging signal appeared significant contrast in the liver after post-injection 15 min compared with pre-injection, whereas the signal at post-injection 30 min decreased (Fig. 8c and d). Moreover, the signal at spleen increased slightly at post-injection 15 and 30 min due to the uptake of nanoparticles in spleen being lower than liver (Fig. 8e and f) . These observations indicate that these nanoparticles could circulate in the liver in a short time. Therefore, it can be deduced that the POSS-UCNPs could be introduced as MRI positive-contrast agents. 3.5. UCL imaging of living cells in vitro 12
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making the luminescence intensity increase but also with MRI ability. To investigate if
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DOI: 10.1039/C5NR00950B Based on the excellent green emission of POSS-UCNPs under NIR light
excitation (Fig. 4), the application in UCL imaging of living cells was investigated. Fig. 9 shows the confocal imaging of HeLa cells incubated with POSS-UCNPs (150 μgmL−1) for 7 h and the typical upconversion green signal (500-600 nm) was detected. The cells exhibited intense upconversion luminescence in intracellular
image (Fig. 9c) also confirms that the upconversion luminescence was localized in the cytosol region rather than merely staining the membrane surface. Importantly, no autofluorescence signal was observed in the UCL image of Hela cells incubated with POSS-UCNPs (Fig. 9b). The absence of background autofluorescence is very important and highly desired feature for UCL imaging in living cells.63 Therefore, the results indicate that the POSS-UCNPs are desirable candidates for upconversion luminescence cell imaging in vitro. 3.6. UCL imaging in vivo and ex vivo Encouraged by the low cytotoxicity in vitro and in vivo (Fig. 5 and Fig. 6) and excellent NIR (980 nm) to NIR (800 nm) upconversion luminescence property (Fig. S4), the UCL imaging capability of POSS-UCNPs(Tm) in vivo was examined on a nude mouse through tail vein injection (100 μL, 1 mgmL−1, under the authorization of the regional ethics committee for animal experiments). The modified Kodak in vivo imaging system was used for in vivo and ex vivo UCL imaging. The in vivo whole-body images of nude mouse are presented in Fig. 10, and the UCL signals were collected at 800 ± 12 nm under CW excitation at 980 nm with a power density of 400 mWcm−2. In Fig. 10a, a significant UCL signal in vivo was observed with high contrast compared with the background after intravenous injection for 15 min. The peritoneum was exenterated and imaged to investigate the distribution of POSS-UCNPs(Tm) in the organs. Fig. 10b and Fig. 10c display the in situ and ex vivo UCL images, respectively, in which the significant UCL emission was observed from the liver, spleen and lung, whereas the other organs (such as heart, kidney) of the mouse showed 13
no
detectable
UCL signals.
This is possibly because
of more
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region of HeLa cells. The overlay of the upconversion image and the bright-field
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DOI:due 10.1039/C5NR00950B POSS-UCNPs(Tm) accumulated in the liver, spleen and lung than other organs to
stronger affinity of the nanoparticles to the liver, spleen and lung tissues. In addition, in vivo UCL imaging after intravenous injection was tested at different time points (15 min, 30 min, and 2 h) (see Fig. S8), which shows that the UCL signal still retained after 2 h post-injection and the POSS-UCNPs was not cleared from blood quickly.64
in vivo upconversion luminescence imaging with high signal-to-noise ratio. 3. Conclusion In summary, we have successfully developed a new and facile strategy to transfer upconversion nanoparticles from hydrophobic to hydrophilic using POSS. After modification, the water-solubility of the POSS-UCNPs has been significantly improved, while the high size monodispersity has been preserved. Furthermore, benefited from low cytotoxicity and excellent biocompatibility, the POSS-UCNPs show good performance in both in vitro cell imaging and small animal in vivo imaging. In addition, the doped Gd3+ ion endows the POSS-UCNPs with effective T1 signal enhancement and the POSS-UCNPs were successfully applied to in vivo MRI for Kunming mouse, which affords them as potential MRI positive-contrast agent as well. Moreover, the method of POSS modification provides an extended and general approach to constructing different water-soluble nanoplatforms based on the easy modification of the corner groups of POSS. Therefore, the approach may open up new perspectives for a unique synthesis of hydrophilic and uniform nanoplatforms for multimodal bioimaging and multifunctional applications. Acknowledgment We are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 21231004, 21201117), Innovation Program of Shanghai Municipal Education Commission (13ZZ073), the Science and Technology Commission
of
Shanghai
Municipality
(13NM1401100,
13NM1401101,
14520722200), Shanghai Rising-Star Program (14QA1401800), and the project from State Key Laboratory of Rare Earth Resource Utilization (RERU2014012). We are also 14
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The results above show that the POSS-UCNPs can serve as promising candidates for
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10.1039/C5NR00950B grateful to Instrumental Analysis & Research Center of Shanghai University.DOI: We are
also grateful to Prof. Haifang Wang of Institute of Nanochemistry and Nanobiology, Shanghai University for her helpful discussion and suggestion. † Electronic supplementary information (ESI) available: Schematic illustration of the of
POSS-UCNPs.
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NaYF4:Yb,Tm@NaGdF4
TEM
nanoparticles
images in
of
cyclohexane;
NaYF4:Yb,Tm TEM
image
and of
POSS-UCNPs(Tm) in water. DLS of POSS-UCNPs(Tm) in water. Energy dispersive X-ray (EDX) spectrum of POSS-UCNPs(Er). XPS of POSS-UCNPs(Er); XPS of Si element. UCL spectra of POSS-UCNPs(Er) in physiology saline as a function of time. UCL spectra of NaYF4:Yb,Tm@NaGdF4 [UCNPs(Tm)] and POSS-UCNPs(Tm), excited with 980 nm laser (100 mWcm−2). In vivo UCL imaging of Kunming mice after intravenous injection with POSS-UCNPs(Tm) at different time points.
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Scheme 1. Schematic illustration of the POSS-UCNPs nanoplatform.
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Fig. 1. TEM images of NaYF4:Yb,Er (A) and NaYF4:Yb,Er@NaGdF4 (B) nanoparticles in cyclohexane; TEM image of POSS-UCNPs(Er) in water (C).
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Fig. 2. The XRD patterns of UCNPs(Er), POSS, POSS-UCNPs(Er) and the standard card of β-NaYF4 (JCPDS: 16-0334).
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Fig. 3. FT-IR spectra of UCNPs(Er), POSS, and POSS-UCNPs(Er).
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Fig. 4. UCL spectra of UCNPs(Er) and POSS-UCNPs(Er), excited with 980 nm laser (100 mWcm−2). Inset: the bright-field images of UCNPs(Er) and POSS-UCNPs(Er) (a, c); the dark-field images of UCNPs(Er) and POSS-UCNPs(Er) under excitation with 980 nm laser (b, d).
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Fig. 5. In vitro cell viabilities of HeLa cells incubated with POSS-UCNPs(Er) at different concentrations (0, 12.5, 25, 50, 100, 200, and 400 μgmL−1) for 24 h.
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Fig. 6. (A) H&E-stained tissue sections from mice injected with POSS-UCNPs 7 days post-injection (n = 2, dose = 15 mg/kg, Test) and mice receiving injection of normal saline (n = 2, dose = 15 mg/kg, Control). Tissues were harvested from heart, liver, spleen, lung and kidney. (B) Hepatic indicators. (C) Indicators of kidney functions.
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Fig. 7. T1-weighted and color-mapped MR images for various Gd3+ concentrations of POSS-UCNPs(Er) (a); Relaxation rate r1 (1/T1) versus different Gd3+ concentrations of POSS-UCNPs(Er) (b).
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Fig. 8. In vivo T1-weighted MR images of Kunming mouse at pre-injection, post-injection after 15 min and 30 min. The coronal images and color-mapped images (a, b); The transversal cross-sectional images and color-mapped images of liver (c, d); The transversal cross-sectional images and color-mapped images of spleen (e, f)
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Fig. 9. Confocal imaging of HeLa cells incubated with POSS-UCNPs(Er) with a concentration of 150 μgmL−1 for 7 h at 37 °C. Bright-field image (a); UCL image collected at green channel (500–600 nm) (b); merged image of a and b (c).
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Fig. 10. In vivo (a), in situ (b), and ex vivo (c) UCL images of a nude mouse acquired at 15 min. after intravenous injection with 100 μL POSS-UCNPs(Tm) (1 mgmL−1). UCL signals were collected at 800 ± 12 nm. The power density is 400 mWcm−2 on the surface of the nude mouse.
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