Yttrium (III) acetate hydrate (99.9%), ytterbium (III) acetate hydrate (99.9%), erbium (III) acetate hydrate (99.95%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), sodium hydroxide (NaOH, > 98%), ammonium fluoride (NH4F, 99.9%), methanol (99.8%), ethanol (absolute), cyclohexane (99%), tetrahydrofuran (THF, 99.9%), dopamine hydrochloride (99.9%), hydrochloric acid (HCl, 37%), dimethyl sulfoxide (DMSO, 99.9%), hydroxylamine hydrochloride (HH, 99%), superoxide dismutase (SOD), sodium azide (NaN3), and D-mannitol were purchased from Sigma-Aldrich (St Louis, MO, USA). HEPES buffer (1 M, pH 7.2−7.5), N-succinimidyl S-acetylthioacetate (SATA), sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), dihydroethidium (DHE), and SYTOX Green (SG) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Amicon™ Ultra Centrifugal Filter (0.5 mL, 30 K, 5K) was purchased from Millipore (Bedford, MA, USA). 2ʹ,7ʹ-Dichlorofluorescein diacetate (DCFDA) was purchased from Cell Biolabs (San Diego, CA, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Vector Laboratories (Newark, CA, USA). 1,4-Dithiothreitol (DTT) was purchased from Duchefa Biochemie (RV Haarlem, Netherlands).
Methods
Characterization
The morphologies of the UCNPs were characterized by transmission electron microscopy using JEM-2100F (JEOL Ltd., Tokyo, Japan) installed at Hanyang LINC3.0 Analytical Equipment Center (Hanyang University, Seoul, Republic of Korea) at an accelerating voltage of 200 kV. The XRD patterns of the UCNPs were analyzed by grazing incidence X-ray diffraction (GI-XRD) with a fixed incidence angle of 1 ̊, a measuring range from 10 ̊ to 60 ̊ for a 0.04 ̊ step, and a scan speed of 2 ̊ per min. A high-resolution X-ray diffractometer (Smartlab, Rigaku) with a HyPix-3000 detector and Cu-K (= 1.54) radiation operating at 9 kW was used for GI-XRD. DLS and zeta potential measurements of the UCNPs were performed using a Zetasizer Nano ZSP instrument (Malvern Co., Malvern, UK). Fourier transmission infrared (FT-IR) spectra of the UCNPs were obtained using an iS50 FTIR spectrophotometer (Thermo Fisher Scientific). The upconversion PL emission spectra were recorded by the sCMOS camera (Andor, ISTAR-SCMOS-18F-73) attached to Andor’s Kymera 193i spectrometer with external laser excitation at 980 nm (Changchun New Industries Optoelectronics Tech. Co. Ltd. (CNI), Jilin, China). The emission at 610 nm was measured using bandpass filters (ff-01-800/12–25; Edmund Optics, Barrington, NJ, USA) placed in front of the spectrometer. The PL lifetime was measured by exciting the samples with a pulsed 980 nm laser (MDL-III-980; CNI) at a repetition rate of 100 Hz and a pulse width of 100 µs. The emission at 550 nm was selectively measured using a photomultiplier tube detector (H10721-01; Hamamatsu, Shizuoka, Japan) attached to Andor’s Kymera 193i spectrometer, the signal of which was acquired using a digital oscilloscope (RTM3002; Rhode & Schwarz, Munich, Germany). A 550 nm laser (MGL-FN-550; CNI) was used to measure the transmittance of green light to deep tissues.
Preparation of core precursor solution
We prepared the lanthanide precursor solution in a solvent mixture of OA and ODE. In a typical process of preparing the core precursor, Ln(CH3CO2)3 (Ln = Y, Yb, Er total 0.8 mmol) was mixed with 4 mL of OA and 6 mL of ODE. The mixture was heated at 153°C for 1 h with magnetic stirring and then cooled down to room temperature. Subsequently, a 5 mL methanol solution of NaOH (0.25 mmol) and NH4F (0.4 mmol) was added to the oleate-lanthanide solution. The reaction mixture was stirred at 50°C for 1 h. Then, the solution temperature was increased to 110°C, followed by degassing through a vacuum pump at 103°C for 10 min to remove methanol. Finally, the precursor solution was cooled to room temperature and stored in a 50 mL centrifugal tube.
Synthesis of core nanoparticles
In a typical process of core UCNP synthesis, a mixture of 4 mL of OA and 6 mL of ODE was loaded into a 50 mL round bottom flask. Then, the mixture was heated to 103°C for 30 min. Subsequently, the solution was heated to 310°C under argon protection. After the solution temperature reached 310°C, 5 mL of the core precursor was rapidly infected with a one-shot approach, and the solution was stirred at 310°C for 60 min. The resulting nanoparticles were collected by centrifugation, washed with ethanol, and dispersed in cyclohexane.
Synthesis of β-NaYF4:x% Yb3+@NaYF4:2% Er3+ (x = 20, 30, 40, 50 mol%) core-shell nanoparticles
For the synthesis of UCNPs constituted with β-NaYF4:x% Yb3+@NaYF4:2% Er3+(x = 20, 30, 40, 50 mol%), Ln(CH3CO2)3 (Ln = Y, Er, total 0.2 mmol) was mixed with 3 mL of OA and 7 mL of ODE. The mixture was heated at 153°C for 1 h with magnetic stirring and then cooled down to room temperature. Subsequently, the pre-synthesized core nanoparticles were added as seeds with a 3 mL methanol solution of NaOH (0.25 mmol) and NH4F (0.4 mmol). The reaction mixture was stirred at 50°C for 1h and heated at 300°C under an argon flow for 1h before cooling to room temperature. The resulting core-shell nanoparticles were collected by centrifugation, washed with ethanol, and dispersed in cyclohexane.
Surface modification of UCNPs
Dopamine hydrochloride aqueous solution (200 µL, 25 wt %) and UCNPs were dissolved in THF. The solution was added to a flask and heated to 50°C with vigorous stirring. After adding HCl, the resulting UCNP-NH2 was collected through several washing steps. To prepare maleimide-modified UCNPs, 0.5 mg of UCNP-NH2 was resuspended in 10 mM HEPES buffer, and sulfo-SMCC was dissolved in 200 µL of 10 mM HEPES buffer. The UCNP-NH2 and SMCC solutions were mixed and incubated for 5 h. After incubation, the resulting UCNP-SMCC solution was washed. To prepare thiol-modified KR-LP, SATA stock solution was added to KR-LP (50 mg), which was incubated for 30 min. HH stock solution (2 µL at 0.5 M) was added to the reaction solution and incubated for 2h. Finally, the prepared thiol-modified KR-LP and maleimide-modified UCNPs were mixed in HEPES and incubated for 2 h. The thiol group-modified KR-LP was reacted with the maleimide groups on the UCNP surface at pH 7.2. The KR-LP-conjugated UCNP solution was collected through centrifugation. To determine the KR-LP loading amount, the supernatant and residual content were measured using a Nanodrop spectrophotometer at a wavelength of 280 nm.
Expression and refinement of recombinant proteins
For cancer cell targeting, the KR-LP recombinant protein was produced by introducing the LP sequence (Trp–Leu–Glu–Ala–Tyr–Gln–Arg–Phe–Leu) into the pRSETB–His6–KR vector at the C-terminal of KR. This was amplified using an omnidirectional primer (forward primer, 5'–GGG GAT CCC ATG CTG TGC TGT ATG AGA A–3') and three reverse primers (reverse primers, 5'–CCA ATC CTC GTC GCT ACC GAT GGC G–3', 5'–CTG GTA GGC GGC CTC CAG CCA ATC–3', 5'–CCC AAG CTT CTA CAG GAA GCG CTG GTA G–3'). PCR products were cut using restriction enzymes (BamHI and HindIII), separated by agarose gel electrophoresis, and purified using a gel extraction kit. Ligation to the vector was performed at 16°C for 2 h using T4 DNA ligation enzyme. Subsequently, the recombinant vector was transformed into E.coli DH5α strain to extract DNA and transformed into E.coli BL21 (DE3) strain (Novagen, Madison, WI, USA) for protein expression. The transformed E.coli BL21 (DE3) strain was inoculated into 100 mL of Luria broth (LB) culture medium containing ampicillin at a concentration of 100 mg/mL and incubated at 37°C for 24 h for the expression and purification of recombinant proteins. Then, it was inoculated into 500 mL of LB culture solution and cultured at 37°C until the absorbance (optical density, OD) value reached 0.6–0.8. After leaving it at 4°C for 30 min to reduce the temperature of the culture solution, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to reach a final concentration of 1 mM, followed by incubation at 20°C for 20 h to induce protein expression. The cells were centrifuged at 7,800 rpm at 4°C for 20 min and resuspended with 40 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), followed by sonication for 5 min. Then, the cells were centrifuged at 7,800 rpm at 4°C for 20 min, and the supernatant solution was filtered through a 0.45 µm syringe filter. Subsequently, the Ni2+-NTA resin was added, and the solution was incubated for 24 h at 4°C. The protein and bead mixture solution was loaded into a polypropylene column, washed 3 times with 5 mL of washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and purified with 2.5 mL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The size and fluorescence of the purified protein were confirmed by 12% SDS gel electrophoresis. Then, the protein purified with the eluted solution was desalted with PD-10 columns and concentrated using a 50 kDa Amicon Ultra Centrifugation Filter. The protein concentration (mg/mL) was determined by measuring the absorbance at a wavelength of 280 nm using a UV-vis instrument (Cary 60; Agilent Technology), and the sample was stored at − 80°C for further experiments.
Preparation of UCNP-KR-LP
To produce UCNP-maleimide, 0.5 mg of UCNP-NH2 was resuspended in 10 mM HEPES buffer, and sulfo-SMCC was dissolved in 200 µL of 10 mM HEPES buffer. Then, the UCNP-NH2 solution and SMCC solution were mixed and reacted for more than 5 h. The SMCC-UCNP solution was washed with 10 mM HEPES buffer. Next, thiol-KR-LP was prepared by adding a SATA-diluted solution to KR-LP, reacting it for 30 min, and incubating the 0.5 M HH-diluted solution for 2 h. Next, the prepared thiol-KR-LP was mixed with UCNP-maleimide and HEPES buffer and reacted for 2 h. Subsequently, it was washed 3 times by centrifugation to remove unreacted material. Finally, the synthesized UCNP-KR-LP composite was stored at 4°C at a final volume of 200 µL.
Cell culture and MTT assay
MCF-7, MDA-MB-231, U87-MG, SK-BR-3, and MCF-10A cells were cultured in a T-75 cell culture plate using a cell culture medium containing 10% FBS, and 1% penicillin-streptomycin (5% CO2, 37°C). The MCF-7, MDA-MB-231, and U87-MG cancer cell lines were cultured using DMEM, the SK-BR-3 cancer cell line was cultured using RPMI medium, and the MCF-10A cell line (non-malignant) was cultured using mammary epithelial basal medium. At 70% confluency, the cells were washed thrice with DPBS and collected by centrifugation after treatment with 3 mL of trypsin-EDTA. Subsequently, trypan blue was added, and the number of cells was counted using a hematocytometer. Next, the cells were cultured in a 96-well plate at a concentration of 5⋅103 cells per well and incubated for 24 h at 37°C. UCNP, UCNP-KR, and UCNP-KR-LP were added at a final concentration of 100 µg/mL and incubated for 16 h. After removing the culture medium, cells were washed thrice with DPBS and irradiated with a 980 nm NIR laser (1 W/cm2) for 30 min. Next, the cells were washed twice with DPBS, and MTT reagent (0.5 mg/mL) was added and incubated for 4 h at 37°C. After removing the supernatant, 100 µL of DMSO was added to the medium and stored for another 10 min. Subsequently, the absorbance was measured using a microplate reader (Vashokan, Thermo Scientific, USA) at 570 nm with a colorimetric indicator (formazan).
ROS measurement
DHE was used to measure the level of superoxide (O2•–) generated by KR. The final concentration of the UCNP-KR-LP composite prepared by mixing the DHE reaction buffer with PBS was 100 µg/mL, and the final concentration of DHE was 100 µM. NIR irradiation was examined at 10 min intervals using a 980 nm laser for a total of 30 min, and the amount of DHE fluorescence reduction was determined at an excitation wavelength of 370 nm and an emission wavelength of 420 nm using a microplate reader. To confirm the scavenging effect of ROS generation, we used SOD (superoxide anion-specific scavenger), NaN3 (singlet oxygen-specific scavenger), and D-mannitol (C6H14O6, hydroxyl radical specific scavenger). The experiment was conducted by mixing the three scavengers with KR in the ROS reaction buffer. The final concentration was 100 µg/mL KR and 100 µM DHE. Subsequently, the amount of DHE fluorescence reduction before and after light irradiation was measured using a microplate reader. A fluorogenic ROS probe, DCFDA, was used to measure intracellular ROS production. MCF-7 cells were cultured in a 96-well culture plate (5⋅103 cells per well) and incubated for 24 h at 37°C. Then, UCNP, UCNP-KR, and UCNP-KR-LP were added at 100 µg/mL for 16 h. After removing the culture medium, 10 µM DCFDA was added and cultured in the dark. Cells were washed twice with DPBS and irradiated with a 980 nm NIR laser (1 W/cm2) for 30 min. After NIR irradiation, the DCFDA fluorescence signals of cells were detected at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA), microplate reader, and fluorescence imaging system (EVOS M7000 Imaging System, Thermo Fisher Scientific). Flow cytometry was performed by treating the cells with 50 µL of trypsin-EDTA for 1 min and removing the medium from the 96-well culture plate. Then, the plate was washed twice with DPBS, and the cells were filtered with a cell strainer and placed in a cap tube. The cells were kept in an ice bath during analysis, and fluorescence signals were analyzed by detecting DCFDA in the fluorescein (FITC) channel. The experimental results were analyzed through FlowJo (FlowJo™ v10.7; BD Biosciences).
Fluorescence imaging of cells
MCF-7 cells were cultured in a 24-well plate as described above, treated with 200 µg/mL UCNP-KR or UCNP-KR-LP for 16 h, and irradiated with a NIR laser (1 W/cm2) for 30 min. SYTOX Green (a dye that stains the nucleus of dead cells in green) was then added to a final concentration of 261 nM with DAPI (a dye for staining the nucleus of live cell in blue), and fluorescence images were obtained using a confocal fluorescence microscope (Eclipse Ti; Nikon). Experiments on the transmittance of green and NIR light to deep tissues were conducted by placing porcine skin tissues of various thicknesses (0−10 mm) in a 24-well culture plate with MCF-7 cells under a 550 nm green laser and a 980 nm NIR laser for 30 min. To obtain tissues with an accurate thickness, the slice thickness of the tissues was measured using a caliper. The rest of the experimental procedure is similar to the method described above.
Statistical analysis
Statistical analysis was performed using the SPSS software (version 26.0; IBM). Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test as indicated in the figure legends. All data sets were subjected to normality tests (Shapiro-Wilk test) when applicable. Based on the normality of distributions, one-way ANOVA with post-hoc Tukey’s test was performed when the data set was truly normal. N values corresponded to the sample size, and P-values were as follows: *P < 0.05; **P < 0.01; ***P < 0.001.