Reagents
Neodymium (III) acetate hydrate (99.9%), yttrium (III) acetate hydrate (99.9%), ytterbium (III) acetate hydrate (99.9%), thulium (III) acetate hydrate (99.95%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), sodium hydroxide (NaOH, ≥ 98%), ammonium fluoride (NH4F, ≥ 99.9%), ethanol (absolute), methanol (≥ 99.8%), cyclohexane (≥ 99%), tetrahydrofuran (THF, ≥ 99.9%), dopamine hydrochloride (≥ 99.9%), hydrochloric acid (HCl, 37%), dimethyl sulfoxide (DMSO, ≥ 99.9%), and hydroxylamine hydrochloride (HH, 99%) were purchased from Sigma-Aldrich, St Lousis, Mo, USA. HEPES buffer (1 м) was purchased from Gibco. N-succinimidyl S-acetylthioacetate (SATA), sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and CD133 (Prominin-1) Monoclonal antibody were purchased from ThermoFisher Scientific, Waltham, MA, USA. Amicon ultra centrifugal filter (0.5 mL, 30 K, 5K) was purchased from Millipore (Bedford, MA, USA). Anti-EGFR antibody (ab40815), goat anti-rabbit IgG-H&L Alexa Fluor 488 (ab150077) and goat anti rabbit IgG H&L Alexa Fluor 647 (ab150079) were purchased from Abcam, UK. A DAPI mounting kit was purchased from Vector Laboratories, Inc., Burlingame, CA, USA.
Synthesis of nanoparticle
Lanthanide doped nanoparticles were synthesized by thermal decomposition of lanthanide acetate precursors. In a typical process of synthesis, Ln(CH3CO2)3 (Ln = Y, Nd, or Yb total 0.4 mм) were mixed with of Oleic acid (3 mL) and 1-Octadecene (7 mL). The mixture solution was then heated to 150°C and held for 30 min, and subsequently, cooled to 50°C. Continuously, methanolic solution of NaOH (1 mм) and NH4F (1.6 mм, 5 mL) was added to the oleate–lanthanide mixture solution and stirred at 50°C. The obtained solution was then heated to 100°C and degassed through the vacuum pump to remove the residual water and methanol. Thereafter the resultant solution was heated to 300°C, maintained under argon for 1 h, and cooled to room temperature. The precipitated nanoparticle was collected by centrifugation and washed. The final product was re-dispersed in cyclohexane (2 mL).
Surface modification
Nanoparticles (15 mg) were dissolved in THF while dopamine hydrochloride (50 mg) was dissolved in water. The solutions were mixed in a flask (50 mL) and heated to 50°C with a vigorous stirring. Following the HCl addition, the resultant NH2-ligand were collected through several washing steps. To exchange maleimide ligand modified NP, NH2-NP (2 mg) was dispersed in HEPES buffer (10 mм, 200 µL). sulfo-SMCC was dispersed in HEPES buffer (10 mм) and then, these solutions were mixed and incubated for 5 h. To prepare antibody-conjugated NP, SATA stock solution was added to the pristine antibody (50 µg) for a 30 min. continuously, HH solution (1.75 µL, 0.5 м) was added to the above solution and incubated 2 h. Resultant solution was washed using a 30-k centrifuge filter. Subsequently, thiol modified antibody and maleimide modified NP were linked by a click reaction. The concentrations of antibody were determined by the absorption spectrum at 280 nm. Finally, the surface of antibody-NP was covered with a 1% BSA solution as a blocking agent to minimize non-specific binding.
Characterizations of water-heating NIR NP
Sample images were analyzed on JEOL. Ltd. JEN-2100F installed at the Hanyang LINC3.0 Center for Research Facilities (seoul). The XRD patterns of the 1.0NP were characterized by an XRD-7000 diffractometer. A Zetasizer Nano ZSP instrument (Malvern Co., UK) was used to determine the zeta potentials of the water-heating NIR NP. The fourier transform infrared (FT-IR) spectra of the water-heating NIR NP were obtained by using an iS10 Fourier transform infrared spectrophotometer (ThermoFisher Scientific Co., USA). The upconversion PL emission spectra were recorded using a Flame spectrometer (Ocean Optics, Inc., USA) under external excitation at 980 nm provided by an infrared diode laser (Changchun New Industries Optoelectronics Tech. CO., China). A UV-Vis Spectrophotometer Jasco V-700 (Jasco., Japan) was used to measure absorbance spectra. The lifetime and images were obtained by the iStar intensified sCMOS camera (Andor) using a time-gated imaging technology. Their time gate delay was 100 µsec, and the time gate width was 50 µsec. All image was obtained by irradiation of 980 nm pulsed laser (CNI, MDL-III-980-2W) with a power density of 0.5 W m− 2. The NIR emission wavelength was selectively measured using a bandpass filter (Semrock, ff-01-800/12–25, Edmund 980 nm CWL, 12.5 mm Dia., Hard Coated OD 4.0 10 nm) and shortpass filter (Semrock, ff-01-950/sp-25) placed in front of an all detectors.
Photothermal efficiency
Photothermal conversion efficiency of water-heating NIR NP was calculated by the equation described by Roper et al. (Supplementary Fig. S7).
![](data:image/png;base64,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)
Where η is photothermal conversion efficiency, h is a heat transfer coefficient, S is surface area of the container. ΔT represents the temperature difference of the surroundings, I is the power density of the laser, A808 is the absorbance intensity of nanoparticle in water solution at 808 nm, and the QDis represents the heat induced by solvent absorption. hS can be evaluated by the equation below:
![](data:image/png;base64,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)
In our experiments, m is the mass of the solution (0.1 g), and Cp is the heat capacity of solution (4.2 J g− 1), τs is defined by the time data versus -lnθ (85.14 s, Supplementary Fig. S7b). ΔT is 30.8°C (ca. 50.8–20), QDis is expresses heat dissipated from the light absorbed by the quartz sample cell, and it was measured independently to be 25.1 mW using a quartz cuvette cell containing distilled water. I is 2.0 W cm− 2, A808 is 0.15. The photothermal conversion efficiency was found to be 23.3%.
Preparation of orthotopic GBM-bearing mice model
To prepare the orthotopic GBM-bearing mice model, GBM cells (U87MG cell) were intracranially injected into 7-week-old male nude mice. Briefly, male nude mice were anesthetized with isoflurane (3%) and fixed by ear bar in a stereotaxic instrument (Stoelting Co., IL, USA). Once each mouse was anesthetized, the scalp at the surgical position was removed and a small hole positioned at 2 mm right lateral and 2 mm posterior from the bregma was drilled under sterile conditions. PBS (containing 1 × 106 U87MG cells, 8 µL) was loaded into a 26-G Hamilton syringe (Hamilton Company, NV, USA), and then the syringe was placed on the stereotaxic apparatus. After the needle of the syringe was positioned at a 3 mm depth, cells were injected with a 1 µL min− 1 injection rate, followed by 3 min of waiting time to prevent overflow. After injection, the hole was sealed with bone wax and the scalp was closed with suturing. After this procedure, the mice were kept for 3 weeks until the injected cells reached the appropriate size of GBM tissue. 2 weeks of post-surgery, in vivo experiments were undertaken.
Cell Culture and MTT assay
U87MG cells were cultured on a T-75 cell culture plate with DMEM (10 mL) containing 10% FBS and 1% penicillin-streptomycin in a cell incubator under environmental conditions of 5% CO2 and 37°C. When the cells attained more than 70% confluency, they were washed twice with DPBS, treated with Trypsin-EDTA (3 mL), and reacted at 37°C at a constant humidity to separate the cells. After collecting the cells by centrifugation, cells (10 µL) and trypan blue (10 µL) were mixed, the cells were counted using a hematocytometer, and were diluted with a culture medium to the required concentration for each experiment. The cytotoxicity of nanoparticles was evaluated by calculating the cell viability by MTT assay. U87MG cells were seeded in 96-well plates at a density of 1 ⋅ 104 cells per well for 24 h. Cells were treated with UCNP-CD133 at various concentrations of 0, 25, 50, 100, 200, and 400 µg mL− 1, respectively. After reacting for 24 h and 72 h in the dark room without light, MTT reagent (100 µL, 0.5mg mL− 1) was added to each well, and the cells were further cultured for 4 h. Then, the excess MTT solution was removed from each well and the formed formazan was dissolved in DMSO (100 µL). The absorbance of the MTT product was acquired at 570 nm using a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific Inc., USA). Cell viability for each concentration was assessed in triplicate and determined relative to untreated control cells. The following formula was used to calculate the viability of cell growth: Cell viability (%) = (absorbance value of treatment group – blank)/(absorbance value of control group – blank) x 100%
We investigated the concentration- and irradiation dose-dependent PTT effect of Ab-NP on cell viability. U87MG cells were incubated with various concentrations of Ab-NP (0, 50, 100, 200, 400, and 800 µg mL− 1) in 96-well plates, and cells were washed 3 times with PBS to remove excess nanoparticles. Next, the cells were exposed to 808 nm irradiation respectively for 0, 10, 20, and 30 min. Controls were used as water-heating NIR NP with the same loading concentration and duration of NIR exposure. Finally, cell viability was measured by MTT assay after an additional 24 h incubation in the dark.
Flow cytometry analysis
To monitor the effects of cytotoxicity, an analysis was performed using a flow cytometer (FACS Canto, BD Biosciences). U87MG cells were incubated at 37°C for 24 h in 24-well microplates and treated with Ab-NP (200 µg ml− 1). After removing the excess nanoparticles, the cells were subjected to NIR irradiation for 0, 10, and 20 min, and then Sytox-Green was added. Prior to FACS analysis, cells cultured in microplates were detached from the wells using Trypsin-EDTA and centrifuged at 3000 rpm for 3 min. Cells were filtered through 100 µm cell strainer and resuspended in 1× DPBS (200 µL) containing 5% FBS. Between 10,000 and 20,000 events were collected using a forward scatter threshold of 10,000. Data were collected on pulse height, area, and width parameters for each channel. SG collected data using a 488 nm laser and a 530 nm bandpass filter. Flow cytometry data were then analyzed and were plotted in FCS format in a histogram analysis using FlowJo software (ver. 10.8.1., FlowJo, LLC).
Histological study on orthotopic GBM-bearing mice model
Tissues were immobilized in paraformaldehyde (4%) for 2 days and then placed in a Leica TP1020 Semi-enclosed Benchtop Tissue Processor (Wetzlar, Germany) for washing, dehydration, clearing and paraffin infiltration of the tissue samples, followed by embedding in paraffin blocks. The blocks were transverse sectioned at a thickness of 6 µm with a Leica RM2145 Microtome (Wetzlar, Germany). For Nissl staining, the brain slides were stained with a stain solution prepared by dissolving cresyl violet-acetate (0.2 g) in distilled water (150 mL) and a buffer solution containing acetic acid (0.1 м) and sodium acetate (0.1 м). H&E staining were followed the manufacturer’s instruction. For immunofluorescence (IF) staining, GBM tissue slides were stained with anti-EGFR antibody and anti-CD133 antibody and, diluted 1:100 in phosphate buffered saline tween-20 (PBST) and goat serum mixture. Next, goat anti-rabbit IgG-H&L Alexa Fluor 488 and goat anti rabbit IgG H&L Alexa Fluor 647 were used as secondary antibodies, followed by DAPI mounting.
All animals were housed in specific pathogen-free conditions and maintained under the Institutional Animal Care and Use Committee (IACUC: 2020–0081) at Hanyang University.