Versatile in situ synthesis of MnO2 nanolayers on upconversion nanoparticles and their application in activatable fluorescence and MRI imaging

We have developed a simple and versatile strategy for in situ growth of MnO2 on the surfaces of oleic acid-capped upconversion nanoparticles by optimizing the component concentrations in the Lemieux–von Rudloff reagent.

S2 obtained from Invitrogen. Dox was purchased from Frontier Scientific. Cell Titer 96 Aqueous One Solution cell proliferation assay (MTS) was purchased from Promega.
All other chemicals were of analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and used without further purification. Ultrapure Milli-Q water (Millipore) was used throughout the experiments.
Normal whole human blood. Normal whole human blood was collected by Xiangya Hospital (Changsha, China) from a volunteer, and prepared using standard protocols. [1] For whole blood experiments, blood was collected in heparin-coated vacutainers (BD, Fisher Scientific) to reduce clotting. Whole blood samples were stored at 4 °C for experiments in one week. All whole blood experiments were performed in compliance with the relevant laws and guidelines from Xiangya Hospital, and the committee of Xiangya Hospital has approved the research experiments. The volunteer has also approved that the blood can be used for research experiments.
Cell lines. Human acute lymphoblastic leukemia CCRF-CEM cell line, human Burkitt's lymphoma Ramos cell line and HeLa cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 0.5 mg/mL penicillin streptomycin at 37 °C under a 5% CO 2 atmosphere. The cell lines were bought from American type culture collection (ATCC), and the cell lines were cultured according to the guidelines of ATCC. Cells were washed before and after incubation with washing buffer. Cells were washed before and after incubation with washing buffer [4.5 g/L glucose and 5 mM MgCl 2 in Dulbecco's PBS with calcium chloride and magnesium chloride (Sigma-Aldrich)]. Binding buffer used for the binding test was prepared by adding yeast tRNA (0.1 mg/mL; Sigma-Aldrich) and BSA (1 mg/mL; Fisher Scientific) to the washing buffer to reduce background binding.

Instruments.
Zeta potential was measured on the Malvern Zetasizer Nano ZS90 (Malvern Instruments, Ltd., Worcestershire, UK). Atomic force microscopy (AFM) images of samples were obtained on a Multimode 8 (Bruker, USA). Transmission electron microscopy (TEM) images and energy-dispersive X-ray analysis (EDX) data were S3 obtained on an H-7000 NAR transmission electron microscope (Hitachi) with a working voltage of 100 kV. Scanning transmission electron microscopy (STEM) imaging was performed by field emission electron microscopy (Titan G2 60-300, FEI company, USA). X-ray photoelectron spectroscopy (XPS) spectra were obtained on an ESCALAB 250Xi XPS system (ThermoFisher-VG Scientific) with a monochromatized Al Kα x-ray source at 12 kV and 6 mA. Powder X-ray diffraction (XRD) spectra were obtained on an XRD-6100 (Shimadzu). FT-IR spectra were obtained on a Nicolet iS10 FT-IR spectrometer (Thermo Scientific). Thermogravimetric analysis (TGA) was performed on an STA449C (Germany) at a heating rate of 10 °C/min under argon.
Fluorescence spectra were recorded on an F4500 spectrometer (Hitachi) in conjuction with a 980 nm diode laser. Fluorescence imaging was recorded on an Olympus FV 500-IX81 confocal microscope equipped with a commercial CW IR laser (980 nm). The MTS assay was obtained on a Multiskan GO UV/Vis microplate spectrophotometer (Thermo Scientific), and the absorbance value at 570 nm was determined by a VersaMax microplate reader (Molecular Devices, Inc.). Synthesis of NaYF4: 24%Gd/20%Yb/2%Er Nanoparticles. In a typical experiment, to a 50-mL flask containing oleic acid (3 mL) and 1-octadecene (7 mL) was added a water solution (2 mL) containing Y(CH3COO)3, Gd(CH3COO)3, Yb(CH3COO)3, and Er(CH3COO)3 with a total lanthanide content of 0.4 mmol. The resulting mixture was heated at 150 °C for 1 h to form lanthanide oleate complexes and then cooled to room temperature. Subsequently, a methanol solution (6 mL) containing NH4F (1.6 mmol) and NaOH (1 mmol) was added and stirred at 50 °C for 30 min. The reaction temperature was then increased to 100 °C to remove the methanol from the reaction mixture. Upon removal of methanol, the solution was heated to 290 °C and maintained at this temperature under an argon flow for 1.5 h, at which time the mixture was cooled to room temperature. The resulting nanoparticles were S4 precipitated out through an addition of ethanol, collected by centrifugation, washed with ethanol, and finally redispersed in 4 mL of cyclohexane.
In situ growth of MnO 2 on the surface of hydrophobic UCNPs (UCNPs@MnO 2 ).

Experimental group:
A mixture of as-prepared UCNPs (0.1 g), cyclohexane (100 mL), tert-butanol (70 mL), water (10 mL) and 5 wt % K 2 CO 3 aqueous solution (5 mL) was stirred at room temperature for about 20 min. Then 20 mL of Lemieux-von Rudloff reagent (17.1 mM KMnO 4 and 0.035 M NaIO 4 aqueous solution) was added dropwise, and the resulting mixture was stirred at about 40 °C for the desired time.
The product was then isolated by centrifugation, washed with deionized water and ethanol, and dried under vacuum.

Response of UCNPs@MnO 2 nanoparticles as MRI contrast agents in normal
human whole blood. Whole blood was collected from a normal human, and was kept in an anticoagulative tube. The T1 relaxation times of UCNPs@MnO 2 (The weight ratio of MnO 2 /UCNPs was 294.64 µg/mg) nanoparticles at different concentrations (0, 10, 25, 50, 100, 200, 400 µg/mL) treated with the fresh whole blood were measured at 1.5 T on a Bruker Minispec analyzer (1.5T).

Preparation of mesoporous silica-coated UCNPs@MnO 2 nanoparticles
(UCNPs@MnO 2 @mSiO 2 ). 0.5 g of CTAC, 0.02 g of TEA and 20 L of water were mixed and stirred at 80 °C until a transparent emulsion was formed. Then 10 mL of the as-prepared UCNPs@MnO 2 (The weight ratio of MnO 2 /UCNPs was 294.64 µg/mg) dispersed in water was added and stirred for 1 h. Next, 0.04 mL TEOS was added dropwise into the solution, followed by stirring for 12 h.
The extraction was repeated three times, and the products were washed with deionized water.

Doxorubicin loading and gelatin capping (Dox-loaded nanosystem). 1 mg of
UCNPs@MnO 2 @mSiO 2 nanosystem was mixed with 0.5 mg of Dox in a 2-mL mixture of DMF and H 2 O (1:1). After shaking for 24 h at RT in the dark, the Doxloaded UCNPs@MnO 2 @mSiO 2 were collected by centrifugation. The residual particles were gently shaken with an aqueous gelatin solution (1 mL, 1%) at 50 °C for 6 h to achieve pore saturation. Then, deionized water (8 mL) at 4 °C was quickly poured into the mixture. After two cycles of centrifugation/water rinsing/redispersion, S7 50 μL of a 1% glutaraldehyde solution was added to crosslink the gelatin at 4 °C. The crosslinking reaction was continued for 8 h. Then, the Dox-loaded nanosystem samples were centrifuged, rinsed with water, and redispersed three times. To evaluate Dox-loading capacity, the supernatant and the washing solution were collected. Dox content in the supernatant solution before and after incubation was measured by a UV-2450 UV-vis spectrophotometer. The drug-loading efficiency was measured to be approximately 0.042 mmol/g. DNA preparation. All DNA synthesis reagents were purchased from Glen Research (Sterling, VA), and all DNA probes (see sequences in Table S1) were synthesized on an ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA) based on solid-state phosphoramidite chemistry at a 1 µmol scale. An amino or phosphate group was coupled on the 5'-ends of primers and templates (see Table S1 for sequences), if applicable. DNA sequences were deprotected according to the The detritylated DNA product was precipitated with NaCl (3 M, 25 µL) and ethanol (600 µL). UV-Vis measurements were performed with a Cary Bio-100 UV/Vis spectrometer (Varian) for DNA quantification.
Electrophoresis was carried out in 1×Tris-borate-EDTA (TBE) buffer (90 mM boric acid, 10 mM EDTA, pH 8.0) at 100 V for 30 min. The sample concentration of each well was 10 µM. The 2.5% agarose gel was stained with ethidium bromide in advance.

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Confocal laser-scanning microscopy imaging. For the internalization test, the sgc8nanosystem (50 µg/mL) were incubated with CEM and Ramos cells (1*10 6 cells/mL) at 4 °C for 0.5 h, followed by washing with washing buffer to remove the unbound samples, then fresh medium with 2% FBS was added to incubate for another 4 h, and then wash the sample with washing buffer. The cells were suspended in washing buffer and analyzed using an FV1000-MPE multiphoton laser scanning confocal microscope (Olympus). The signal was due to the recovered fluorescence of UCNPs. Cytotoxicity study. A sample of 1×10 3 cells in 50 μL of washing buffer was seeded into each test well on a 96-well plate. Then a series of samples at the desired concentration in 50μL of washing buffer were added to the respective test well. The cells were incubated at 37 °C in a 5% CO 2 atmosphere for 2 h. Then, the supernatant was removed from the test well after centrifugation and 100 μL of fresh cell culture medium was added. After another 48h of incubation at 37 °C in a 5% CO 2 atmosphere, a standard MTS assay was performed. The absorbance value at 570 nm was determined by a microplate reader. S11 Table S1. DNA sequences used in this work Scheme S1. a) Each fabrication step of the nanosystem. b) The working priciple for pH-responsive controlled release of the UCNPs@MnO 2 @mSiO 2 -Dox@gel-sgc8 nanosystem (sgc8-nanosystem-Dox). S12 Figure S1 TEM characterization of UCNPs, demonstrating their monodisperse particle size of about 22 nm. The UCNPs were dispersed in cyclohexane. When the content of OA was: wt% OA =10.5%, so theoretically : (2) UCNPs@MnO 2 (12 h) From Figure S6, the ligand content on the surface of UCNPs@MnO 2 was 8.7%, which was OA and AA, so wt OA+AA %=8.7% Hypothesis that the content of OA was x%: wt% OA =10.5% So, W AA = (8.7-x)% According to the equation: So the oxidized ratio of OA (12 h) was: S19 Figure S8. The reaction mixtures of the control experiment in the absence of oleic acid-capped UCNPs.

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To investigate the acid-induced and controlled-release property, the sgc8-nanosystem-Dox was exposed to different buffers with pH values of 4.0, 5.0, 6.0 and 7.4. The precise control of drug release was demonstrated by monitoring the concentration of the released anticancer drug by the measurement of fluorescence intensity at 560 nm (λ ex = 488 nm). Figure S21. Release profiles of Dox from the sgc8-nanosystem-Dox at different pH values. The amount of Dox released from the nanosystem was less than 3.6 % after 5 h at neutral pH, indicating that the gelatin coating on UCNPs@MnO 2 @mSiO 2 exhibits good storage and sealant effects in physiological solution (pH7.4). In contrast, increased acidity resulted in a significant release of Dox within 5 h at pH 6.0, 5.0, and 4.0, respectively, indicating that the release of Dox from the as-prepared nanoplatform is both time-and pH-dependent. As such, these findings revealed that the release rate of the pH-responsive sgc8-nanosystem-Dox increased over the 5 h period in mimicked environments of late endosomes and lysosomes, where the pH values would be in the range of 5.0-6.0.
S33 Figure S22. Colocalization study of sgc8-nanosystem (red) with LysoTracker Green (green) in HeLa cells. LysoTracker Green was used to stain the cell acidic organelles-lysosomes. The red signal was from UCNPs with exciting light of 980 nm laser. Cells were imaged using a 60 oil-immersion objective.
Yellow signal were obtained after green signal merged with red signal, but there were still some red signal in the overlay picture, which, in some extent, confirmed that part of the particles had escaped from lysosomes within 2.5 h.
S34 Figure S23. Cytotoxicity of sgc8-nanosystem on CEM and Ramos cells. The MTT results demonstrate that sgc8-nanosystem without Dox loading has almost no cytotoxicity to CEM and Ramos cells.