Synthesis of nanoparticles
Nanoparticles were synthesized by laser ablation of a hot-pressed boron target (MaTecK Material Technologie & Kristalle GmbH, Juelich, Germany, purity 99.5%, natural isotopic composition) in deionized water (18.2 MΩ at 25 °C) at ambient conditions. A beam coming from a Yb:KGW laser (1025 nm, 480 fs, 8 kHz; Amplitude Systems, France or 1030 nm, 270 fs, Avesta, TETA 10 model, Moscow, Russia) was focused on the surface of the boron target fixed vertically in a quartz cuvette filled with 50 mL of deionized water. The energy was preliminary attenuated to the value of 350 µJ per pulse. The liquid layer thickness between the inner wall of the cuvette and the surface of the target was 3.3 mm. Due to the high energy of the interacting beam, self-focusing effects take place, therefore, the quartz cuvette was moved forward in the direction of the convex lens to focus the beam on the surface of the target and to avoid damaging of the glass wall. To prevent the ablation from the same region, the cuvette with the target was constantly moved by a translation stage. The scan area was set to 7 mm x 7 mm and the displacement speed to 2.5 mm s-1. The laser ablation in water lasted for 7 h.
Boron nanoparticles size, shape and composition were characterized using transmission electron microscopy (TEM, JEOL JEM-3010, 250 kV acceleration voltage, W filament) and scanning electron microscopy (SEM, JEOL JSM-7900F, 0…30 kV) equipped with EDX detector (Brüker). For that, 10 µL of elemental boron particles solution was dropped on the copper-carbon grid (200 mesh, Oxford Instrument) and dried overnight under ambient conditions. Size distribution, fast Fourier transform (FFT) and inverse fast Fourier transform (IFFT) analysis were performed in Fiji ImageJ software. Extinction spectrum was recorded using a UV-Visible spectrophotometer (UV-2600, Shimadzu) and a rectangular quartz cuvette with an optical pathlength of 10 mm.
XRD measurements of dried elemental B NPs powder were performed in a transmission mode. The instrument is equipped with a double reflection mirror (Osmic), an image plate detector (Mar345), and a high brilliancy rotating anode (Rigaku RU-200BH, 50 kV, 50 mA). The radiation is Cu Kα (λ = 1.5418 Å), and the size of the beam is 0.5 x 0.5 mm2. The maximum 2θ value is 65° (0.3° experimental resolution). The powder was obtained by drying a concentrated solution of B NPs in deionized water under ambient conditions. The XRD reflexes were calculated using VESTA (Visualization for Electronic and STructural Analysis) software45 and the data files from Crystallography Open Database (COD ID files: 9011170 for β-Boron and 9014010 for Boric Acid)46,47.
The XPS spectra were recorded using Physical Electronic PHI Versaprobe 5000 equipped with a hemispherical energy analyzer. The hemispherical analyzer was operated in Fixed Analyzer Transmission (FAT) Mode. A monochromic Al Ka X-ray source (1486.60 eV) was operated at 25 W and 15 kV with a beam diameter of 100.0 µm. The energy of the analyzer was operated at a pass energy of 117.5 eV for survey acquisitions and 23.50 eV for high-resolution acquisitions. The energy resolution was 0.020 eV for high resolution spectra, and 1.0 eV for survey spectra. The operating pressure of XPS was around 3.5 x 10-6 Pa. Dual charge neutralization was utilized to reduce the effects of charging on the acquired signal. Survey acquisitions were taken for binding energies from 0 - 1200 eV.
Boron concentration in nanoparticles and supernatant were measured using inductively coupled plasma mass spectrometry (ICP-MS). Nanoparticles were centrifuged at 15 000 g, 30 min, supernatant and particle residue were separated and dissolved in 10% nitric acid under the heating at 80 ⁰C. A mass-spectrometer (NexION 2000, PerkinElmer, USA) was calibrated before the analysis using serial dilutions of sodium tetraborate (Sigma-Aldrich, USA) with known concentrations. 11B peak was used for analysis.
To purify B NPs from the excess of boric acid formed during laser ablation, centrifugation was performed. First, the solution was centrifuged at 5000 g 5 min to separate large particles. Then, the solution was centrifuged twice at 15000 g 15 min and redispersed in deionized water at each step.
To increase boron nanoparticle stability in buffers they were coated with polyethylene glycol (PEG). For this aim 1 mg of particles were dissolved in 1 mL of 95% ethanol and mixed with 100 µL of ethanol containing 100 µg of Silane-PEG (5kDa) and 10 µg of Silane-PEG-carboxyl (5kDa). After that 10 µL of 30% ammonia hydroxide was added to accelerate hydrolysis of silanes. Solution of boron nanoparticles was heated at 60 °C for 2h and then washed 2 times from unreacted chemicals with water by centrifugation (5 000 g 5 min, then 15 000 g, 15 min).
Functionalized nanoparticles characterization
The hydrodynamic diameter and ζ–potential measurements were performed by dynamic and electrophoretic light scattering, respectively, using a Zetasizer Nano ZS (Malvern Instruments, UK) device. Number weighted size distributions were used for analysis. ζ-potential measurements were performed in a 10 mM NaCl solution using the Smoluchowski approximation.
Extinction spectra of aqueous colloids of nanoparticles were recorded by an Infinite M1000 PRO spectrophotometer (Tecan, Austria).
Degradation kinetics measurement
To study boron NP chemical stability in distilled water, PBS (pH = 7.4) or citrate buffer (pH = 4.5), 100 µg of particles were added to 1 ml of the corresponding medium and rigorously mixed. The extinction intensity of NPs at 500 nm was measured to analyze particle concentration at each time point. The obtained intensities were averaged (n=3) and normalized to the initial signal to determine the content of undissolved particles. Before each measurement samples were thoroughly sonicated and stirred.
Cells and Culture Conditions
HeLa cells (ATCC, Manassas, VA) were grown in 100 mm cell culture dishes and maintained in Advanced DMEM media, supplemented with 10% fetal bovine serum, 1% glutamax, 1% Antibiotic Antimycotic Solution at 37°C in a humidified atmosphere containing 5% CO2.
Cytotoxicity of Boron NPs was determined by measuring the viability of cells by the standard MTT assay. Briefly, 2.5 × 103 HeLa cells/well were plated in 96-well plates. After incubation for 24 h, 20 µl of the desired nanoparticle concentration was added to the well containing 180 µl of media. Untreated cells were used as a control. Following a 24 or 72 h incubation period media was removed, and cells were incubated with 100 µl 500 µg/mL methyl thiazolyl tetrazolium (MTT) for 2 h at 37 °C, 5% CO2. The MTT solution was removed, and the formazan reaction products were dissolved in DMSO overnight in the incubator. The optical density of the formazan solution was read on a microplate reader (BioTek Instruments, Inc., Winooski, VT) at 490 nm with a reference wavelength of 630 nm. Cell viability was normalized to untreated cells used as the control.