A pre-protective strategy for precise tumor targeting and efficient photodynamic therapy with a switchable DNA/upconversion nanocomposite

A pre-protective strategy for precise tumor targeting and efficient photodynamic therapy was developed using a switchable DNA/upconversion nanocomposite.

breast cancer cell line (MCF-7) and normal breast cell line (MCF-10A) was purchased from KeyGEN biotechnology Company (Nanjing, China).

Instruments.
High resolution transmission electron microscopy (HRTEM) was taken on a JEM-2100 electron microscope. Fluorescence spectra were acquired from FLS-980 Edinburgh Fluorescence Spectrometer with a Xenon lamp. Absorption spectra were carried out on a pharmaspec UV-1700 UV-visible spectrophotometer (Shimadzu, Japan). Absorbance was measured in a microplate reader (Synergy 2, Biotek, USA) in the MTT assay. Success of each reaction step was confirmed by monitoring the changes in zeta potential with a Malvern Zeta Sizer Nano (Malvern Instruments). A TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) was used for performing the confocal fluorescence imaging.
To obtain rare earth chlorides, 1 mmol rare earth oxides Y2O3, Yb2O3, and Er2O3 with a stoichiometric ratio of 79.8:20:0.2 were dissolved in hydrochloric acid, and then the solution was stirred and heated to evaporate the water completely. In the typical synthesis procedure, YCl3 (0.798 mmol), YbCl3 (0.20 mmol), and ErCl3 (0.002 mmol) were dispersed in oleic acid (OA, 8 mL) and 1-octadecene (ODE, 18 mL), then the mixture was heated to 160 o C for 30 min. After a homogeneous solution was formed, the mixture was cooled to room temperature. Then, NaOH (0.1 g, 2.5 mmol) and NH4F (0.148 g, 4 mmol) in 10 mL of methanol solution were added dropwise under vigorously stirring for 30 min. The temperature was heated to 100 o C to evaporate methanol, then was raised to 295 o C in an argon atmosphere for 90 min and finally cooled down to room temperature naturally. The resulting NaYF4:Yb,Er nanoparticles were precipitated by adding ethanol and then centrifuged and washed with ethanol and cyclohexane for several times. The precipitates were redispersed in 10 mL hexamethylene solution.
Synthesis of PAA@UCNPs. The PAA@UCNPs synthesis was approved by a literature protocol reported previously. 43 Briefly, 150 mg of PAA (1800 Da) and 15 mL DEG were added to a three-neck flask. The mixture was heated to 110 °C to procedure a cleared solution. Then hexamethylene solution containing 20 mg UCNPs was added slowly and the solution was maintained at 110 °C for 1 h under nitrogen protection. After heating at 240 °C for 1.5 h, the resultant solution was cooled down to room temperature and precipitated with ethanol. The obtained PAA@UCNPs was recovered by centrifugation (14000 rpm for 10 min) and washed three times with ethanol/water (1:1 v/v).

Synthesis of DNA linking of upconversion nanoparticles (UCNPs@PAA-DNA).
The PAA-capped UCNPs were covalently encapsulated with DNA as reported previously. Briefly, 0.5 mg of PAA@UCNPs in 1 mL MES buffer (10 mM, pH 6.0) were activated by EDC and NHS (680 nM) to form the active succinimidyl ester for 30 min. Then the DNA that was dissolves in water was added to the mixture. After 24 h magnetic stirring in dark, the UCNPs@PAA-DNA was purified by centrifugation (14000 rpm for 10 min) and washed with water to remove excess reactant.
After 12 h, the cells was washed with PBS buffer to remove the NPs that were not ingested into the cells. Then, the cells were treated with irradiation of laser powers (1.5 W•cm -2 ) for 5 min. The control group was without any treatment. Then, the cells were further incubated for 24 h. Next, 150 µL MTT solution (0.5 mg/mL) was added to each well to cultivate for 4 h. After removing the MTT solution, 150 µL of DMSO was injected to each well. The absorbance was measured at 490 nm with microplate reader.
MCF-10A cells were first cultured in a confocal dish for 24 h. Then, the UCNPs@PAA-DNA1/2 (100 μg/mL) in pH=6.5 or 7.4 DMEM culture medium were respectively added to the confocal dish. The cells were incubated for 12 h. After that, fresh DMEM culture medium was added to instead of the original solution. The cells were washed with PBS buffer twice to remove the residual nanoparticles. Then they were observed by confocal laser scanning microscopy (CLSM) and confocal images were acquired. (2) The cells were pre-incubated with 50 μM of free FA for 30 min prior to combining FA receptors.

Animal tumor xenograft models.
Nude mice (4-6 week old, female, ~20 g) were fed with normal conditions of 12 h light and dark cycles and given access to food and water ad libitum. In order to establishing the tumor xenograft models, MCF-7 cells were approximately 1×10 6 cells in 150 µL RPMI 1640 and injected subcutaneously into the flanks of the nude mice.
The tumor volume (V) was computed as V=L×W 2 /2 by measuring length (L) and width (W). The relative tumor volumes were calculated for each sample as V/V0 (V0 was the original tumor volume). The treatments were carried out only once when the tumor volume reach to about 100 mm 3 .
After 8 h, laser treatment was performed on the tumor region with 980 nm laser at a power of 1.5 W•cm -2 for 5min. The tumor size and the body weights of the mice were observed every other day for 14 days (day 0, 2, 4, 6, 8, 10, 12 and 14). Table S1. DNA sequences used in this work.   UCNPs@PAA-DNA12 at pH 6.5 when cells were pre-treated with FA or not. Kidney Lung Spleen Liver Heart