Poly ethylene glycol methyl ether thiol

Materials. All reagents were of analytical purity and used without further purification. Iron(II) chloride tetrahydrate (FeCl 2 •4H 2 O, 99%), sodium hydroxide (NaOH, 99.99%), diethylene glycol (DEG, 99%), N-methyldiethanolamine (NMDEA, 99%), nitric acid (HNO 3 , 70%), copper(II) nitrate hemi(pentahydrate) (Cu(NO 3 ) 2 •2.5H 2 O, ≥99.99%), polyvinylpyrrolidone (PVP, M w 55 kDa), poly(ethylene glycol) methyl ether thiol (PEG-SH, M w 2 kDa) hydrazine hydrate (55%), ammonium sulfide solution ((NH 4 ) 2 S, 20%), sodium cacodylatetrihydrate (≥98%), glutaraldehyde solution (25% in H 2 O), and formalin solution (10%) were purchased from Sigma-Aldrich (France). Iron(III) chloride hexahydrate (FeCl 3 •6H 2 O, 99%) and ethanol were obtained from VWR (France). Live-Dead Green dead cell and MitoTracker Red CMXRos were purchased from Life Technologies (Thermo Fisher, Belgium).
Iron Oxide Nanoflower Core. IONFs were synthesized using a modified polyol synthesis as previously described. 50 (link) In brief, the iron precursors were solubilized in a DEG and NMDEA mixture (1:1 v/v) and heated to 220 °C for 2.5 h to obtain the alkaline hydrolysis. The obtained magnetic nanoflowers were cleaned with ethanol and ethyl acetate and treated with 10% nitric acid to complete the oxidation. They were then redispersed in water and mixed with 0.3% PVP (55 kDa) prior to the following step.
Copper Sulfide Assembly. The synthesis of the substoichiometric copper sulfide Cu 2-x S assembly has been carried out in the presence or in the absence of an IONF core using a two-step reaction through a template sacrificial synthesis method, modified from ref 33. In the first step, 10 mg of cupric nitrate, Cu(NO 3 ) 2 , was dissolved in 30 mL of Milli-Q H 2 O and mixed with 0.3 g of PVP (55 kDa) and IONF (if present) at [Fe] = 0.3 mM. After 15 min of shaking at room temperature 100 μL of hydrazine 5.5% was added rapidly in the mixture to induce the formation of Cu 2 O NPs. The obtained product was cleaned by centrifugation at 9000g for 45 min and resuspended in 30 mL of Milli-Q H 2 O. The second step of the reaction consisted in the sulfidation of the Cu 2 O shell previously synthesized using 0.1 M sodium sulfide followed by heating at 50 °C for 2 h. After several washings by centrifugation and resuspension in Milli-Q H 2 O, the surface of the nanoassemblies was PEGylated by shaking the sample overnight in the presence of PEG-SH (final concentration 10 mg/ mL) at 4 °C and subsequent washing by centrifugation. These reactions resulted in the production of copper sulfide nanoassemblies (hollow) or of iron oxide nanoflower-like cores surrounded by copper sulfide nanoassemblies (IONF@CuS) when achieved in the presence of IONF (rattle-like).
Morphological and Optical Characterization. TEM images were obtained using a Hitachi HT 7700 TEM operated at 80 kV (Elexience, France), and images were acquired with a charge-coupled device camera (AMT). UV-vis-NIR characterization was performed with a real-time Avaspec-USB2 spectrometer. Cu and Fe concentration was determined by elemental analysis using ICP-AES (iCAP 6500, Thermo Scientific).
Laser-Induced Thermometric Measurements. Heating profiles of aqueous solutions were obtained by placing 10 μL of CuS or IONF@CuS dispersions at concentrations ranging from 0.5 to 40 mM of Cu in a 0.5 mL tube at a 4 cm distance from the laser source. The samples were irradiated with a 1064 nm laser at a power density of 0.3 W cm -2 until equilibrium temperature was reached (typically in 1-2 min, measurements were performed over 5 min to be sure to measure the plateau temperature). The increase in temperature was measured using an FLIR SC7000 infrared thermal camera. The spheroids were analyzed in the same configuration. All values are reported as means of at least three separate experiments.
Cell Culture and Nanoparticle Uptake. Human mesenchymal stem cells were purchased from Lonza and were cultured in hMSCbasal medium at 37 °C, 5% CO 2 , and 95% relative humidity. Human glioblastoma cells U87 were purchased from ATCC and cultured in DMEM medium supplemented with 1% (w/v) streptomycin, 1% (w/ v) penicillin, and 10% (w/v) FBS. At 90% confluence, cells were incubated with the nanoassemblies diluted in serum-free RPMI medium. The copper concentration in the medium ranged from 0.1 to 1 mM. After 4 h of incubation, the medium was removed and the cells were rinsed and incubated further for 2 h in complete hMSC-basal medium to remove any noninternalized copper nanoassemblies. Then, cells were detached by trypsinization, counted, and immediately analyzed or further processed. To assess the intracellular NP content, 2.5 × 10 5 cells were digested in pure nitric acid for 48 h until total dissolution, diluted up to 2% HNO 3 in ultrapure H 2 O, and analyzed by elemental analysis.
Nanotoxicity Study. For the biocompatibility assays, 1.25 × 10 3 or 2.5 × 10 3 cells per well were seeded in 96-multiwell culture plates at 100 μL total volume and incubated overnight. IONF@CuS or CuS nanoassemblies at the concentrations of 0.04, 0.16, 0.32, 1.6, and 3.2 mM of Cu were dispersed in cell culture media and incubated with hMSC cells for 24 h (2.5 × 10 3 cells) or 72 h (1.25 × 10 3 cells). At the end of the incubation, the media was removed and fresh media was provided containing Live-Dead Green dead cell and MitoTracker Red CMXRos (Molecular Probes, Life Technologies Europe, BV, Belgium), after which the cells were further incubated for 30 min in a humidified atmosphere at 37 °C and 5% CO 2 . Next, the cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA), and counterstained with Hoechst 33342 nuclear stain solution (Life Technologies, Belgium). Next, the plates were analyzed using the INCell Analyzer 2000 (GE Healthcare Life Sciences, Belgium), while 2000 cells per condition were acquired in triplicates using a 20× objective lens for the DAPI/DAPI (Hoechst), FITC/FITC (Live-Dead Green), and TexasRed/TexasRed (Mito-Tracker Red CMXRos) channels. The acquired images were processed using the InCell Investigator software (GE Healthcare Life Sciences, Belgium). Cell viability was calculated by segmenting cell nuclei and dead cells (signal crossing the threshold in the green channel overlapping with the nuclei) using the IncCell Developer software (GE Healthcare Life Sciences, Belgium). The number of live cells per each condition was calculated as the total number of nuclei counted minus the number of dead cells. The values were then normalized by the control conditions (= 100%). Finally, for mitochondrial stress, the total area of cellular mitochondria was used, while for mitochondrial ROS, the intensity of the mitochondrial stain was determined. The respective channel was segmented using the Hoechst images as seed, and the total size and intensity of the mitochondrial network were determined for each individual cell. These values were than normalized by the respective control conditions (= 100%). Results represent quantitative data for the analysis of a minimum of 2000 cells per condition. Values are presented as mean + SEM (n = 3).
Stem Cell Spheroid Formation and Characterization. A total of 2.5 × 10 5 stem cells were centrifuged (1200 rpm for 5 min) in 15 mL tubes to form a pellet and cultured in order to induce cell differentiation (chondrogenesis). The cells then spontaneously formed a spheroid, which could be kept in culture for months (here up to 3 weeks of spheroid maturation). The differentiation medium was composed of high-glucose DMEM supplemented with 1% penicillin-streptomycin, 0.1 μM dexamethasone, 1 mM sodium pyruvate, 50 μM L-ascorbic acid 2-phosphate, 0.35 mM L-proline (Sigma), 1% ITS-Premix (Corning), and 10 ng/mL TGF-β3 (Interchim) and was changed twice a week. For histological analysis, spheroids harvested after 21 days of maturation were fixed overnight in 10% formalin, before paraffin inclusion and cutting. Slices that were 4 μm thick were treated with toluidine blue 0.04% for collagen staining and then analyzed by optical microscopy.
At days 1, 3, 9, and 21, the spheroids were fixed with 4% PFA for 2 h at room temperature and transferred in PBS for photothermal analysis, magnetic characterization, and elemental characterization. For the electron microscopy analysis, other spheroids at the same time points were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 h, contrasted with oolong tea extract (OTE) 0.5% in 0.1 M Na cacodylate buffer, postfixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, gradually dehydrated in ethanol (30% to 100%), and gradually embedded in epoxy resins. Ultrathin slices (70 nm) were collected onto 200 mesh copper grids and counterstained with lead citrate prior to being observed by TEM.
Gene Expression Quantification by qPCR. Total RNA was extracted from spheroids at different maturation times using a NucleoSpin RNA II kit (Macheney-Nagel). Reverse transcription into cDNA was achieved using SuperScript II reverse transcriptase (Invitrogen) with random hexamers as primers according to the manufacturer's instructions. qPCR was performed with StepOnePlus (Applied Biosystems) using the SYBR Green reagent (Applied Biosystems). The expression of reference gene RPLP0 was used as a housekeeping transcript gene. Each value is obtained by the average of at least two wells gathering a minimum of three independent repetitions. The sequences of primers used are listed in Table S1.
Magnetometry of the Internalized IONF Core by VSM. Cell magnetization right after IONF@CuS nanoassembly incubation and in samples fixed at different spheroid maturation times was measured by magnetometry using a PPMS device equipped with a vibrating sample magnetometer (VSM) option (Quantum Design). The analysis was performed at 300 K, between 0 and 20 000 Oe, and provides the saturation magnetization of the sample (in emu).
XAS/Synchrotron Measurements. XAS measurements were performed on the spheroids pooled in groups of 4 or 5 for each maturation time to increase the signal-to-noise ratio. Measurements were achieved in the XANES regime at the CRG beamline BM25-SpLine of the European Synchrotron Radiation Facilities (ESRF) in Grenoble (France). The spectra were acquired at the Fe K-edge (7112 eV) and Cu K-edge (8980 eV) at room temperature and atmospheric pressure in transmission and fluorescence modes. Metal foils of Fe and Cu elements were measured as energy calibration references. Iron oxides (as maghemite and ferrihydrite) and several copper-based materials (as copper sulfides, copper oxides, and copper sulfates) were chosen as standards. For each condition (at days 1, 3, 9, and 21 of maturation), a group of 3 or 4 multicellular spheroids was measured to improve the signal-to-noise ratio. XANES spectra of IONF, IONF@CuS, and CuS initial solution samples were also evaluated. Data normalization, energy calibration, and analysis of the XAS data were carried out using the Demeter software package (Athena program). 51 (link) Cancer Cell Spheroid Formation and Photothermal Therapy. In the exact same way as for the stem cells, 2.5 × 10 5 U87 cancer cells were pelleted and kept in complete culture medium for 9 days. At days 1, 3, 6, and 9, the spheroids (5 per day) were collected and transferred to 0.5 mL tubes (one spheroid per tube) in 10 μL of culture medium. The tubes were placed in a thermostatic device so that the sample is maintained at a temperature of 37 °C before exposure to the laser. Heating was achieved with the spheroids placed 4 cm away from the laser source (1064 nm), corresponding to a laser power density of 0.3 W cm -2 .
The samples were irradiated for 10 min. The increase in temperature was measured using an FLIR SC7000 infrared thermal camera. After laser treatment, the spheroids were transferred to a 48multiwell plate, and each single spheroid's metabolic activity was measured 24 h later, by the Alamar blue assay, and renormalized by nontreated control spheroid values (5 control spheroids for each measurement day).
Statistical Analysis. All values are reported as means and standard error of the mean. Significant differences were determined using Tukey's test in one-way analysis of variance (ANOVA). * denotes a p-value < 0.05 (significant result), ** a p-value < 0.01 (very significant), and *** a p-value < 0.001 (highly significant).