Materials
Gadolinium chloride hexahydrate (99.999%), ytterbium chloride hexahydrate (99.9%), ammonium fluoride (99.99), sodium hydroxide (97%), oleic acid (90%), 1-octadecene (90%), oleylamine (70%), L-cysteine (97%), diethylenetriaminepentaacetic dianhydride (98%), and H2O2 (30%) were purchased from Sigma-Aldrich. Methanol (ACS reagent grade, ≥99.8%), hexane (ACS reagent grade, ≥98.5%), and chloroform (ACS reagent grade, ≥99.8%) were purchased from Fisher Scientific. Gadolinium and ytterbium standards for ICP is from Inorganic Ventures and high purity nitric acid for quantitative trace metal analysis at the ppb level is from BDH Aristar® Plus. All materials were used as received.
Synthesis
Synthesis of Ultrasmall α-NaGdF4:Yb50%. Ultrasmall nanoparticles were synthesized by modification of a previously reported procedure.[34, 77] To a 100mL three-neck flask containing 0.5 mmol of GdCl3 × 6H2O and 0.5 mmol of YbCl3 × 6H2O was added 9 mL of oleic acid and 15 mL octadecene. The mixture was heated to 160°C and maintained for 1h under argon gas with constant stirring and then cooled to room temperature. Solution of methanol (10 mL) containing 4 mmol NH4F and 2.5 mmol NaOH was added and the mixture was stirred for 30 minutes. Temperature is then increased to 100°C and maintained for 30 minutes to remove methanol. The solution was then heated at 260°C for 10min before cooling to room temperature. The nanoparticles were collected by adding excess amount of ethanol and centrifuged at 7000 rcf for 5 min. The precipitate was washed with ethanol and finally dispersed in 10 mL hexane for further uses.
Synthesis of Ultrasmall β-NaGdF4:Yb50%. Ultrasmall nanoparticles were synthesized following the procedure described for α-NaGdF4:Yb50%, except the solution was stirred for 24 h after the addition of methanol solution (10 mL) containing NH4F (4 mmol) and NaOH (2.5 mmol).
Synthesis of Ultrasmall β-NaGdF4. Ultrasmall nanoparticles were synthesized following the procedure described for α-NaGdF4:Yb50%, except 1.0 mmol of GdCl3 × 6H2O was used.
Synthesis of Ultrasmall α-NaYbF4. Following the procedure described for both α-NaGdF4:Yb50% and β-NaGdF4:Yb50%, except using 1.0 mmol of YbCl3 × 6H2O, resulted to cubic ultrasmall nanoparticles only.
Ligand Exchange Surface Modification. L-Cysteine (60 mg) and diethylenetriaminepentaacetic (DTPA) dianhydride (20 mg) were dissolved in 30 mL H2O at pH 9 in a 100 mL round bottom flask. To this aqueous solution was added 10 mL chloroform solution containing 10 mg of the oleic-capped ultrasmall nanoparticles. The biphasic mixture was stirred vigorously overnight at room temperature to facilitate the transfer of the nanoparticle to the water phase. Excess ligand was removed by twice centrifugation using Vivaspin-20 centrifugal filters (10kDa MWCO) at 3000 rcf for 15 minutes and the collected nanoparticles were redispersed in water and filtered through a 0.2 μm syringe filter.
Folic-acid Functionalized Ultrasmall Nanoparticles (FA-NaGdF4:Yb50%). Five hundred microliters of folic acid dissolved in DMSO (25 mg/mL) in the presence of triethylamine (6.25 μL) was incubated with 6.5 mg of NHS and 6.25 mg of DCC in the dark overnight and then passed through a 0.2 μm filter. The resulting NHS-activated folic acid was then covalently linked to the amino surface of the nanoparticles provided by cysteine ligand by incubating overnight. The resulting NaGdF4:Yb50%-FA was centrifuged at 16000 rcf for 15minutes, washed twice and stored in 1 mL H2O for future use.
Characterization
The size and the morphology of the resulting nanoparticles were characterized by transmission electron microscopy (TEM) using a JEM-2010 microscope at an acceleration voltage of 200 kV. The hydrodynamic size was determined using Malvern Zetasizer NanoZS90. Powder X-ray diffraction (XRD) patterns were recorded by a RigakuUltima IV diffractometer, using Cu Kα radiation (λ = 0.15418 nm). The 2θ angle of the XRD spectra was recorded at a scanning rate of 1°/min. Inductively coupled plasma-optical emission spectrometer (ICP-OES) analysis was performed using a Thermo Scientific iCAP 6000 instrument. CT tests were performed on microCTInveon model scanner (Siemens Medical Solutions USA, Inc.). T1 and T2 rates of the nanoparticles were measured on a 4.7T preclinical MR scanner using increasing concentrations at both 25°C and 37°C with an inversion-recovery, balanced steady-state free precession (IR-bSSFP) sequence, and a multiecho CPMG scan, respectively, as described elsewhere. [78] T1 and T2 relaxivities (mM-1 · s-1) of the nanoparticles were compared to the commercially-available Gd-DTPA contrast agent, Magnevist®.
Elemental analysis using ICP-OES
Acid digestion was performed by dissolving 0.15 mg of the nanoparticles in 0.5 mL concentrated high purity HNO3 acid overnight and diluting with a 2% HNO3 solution to a total volume of 15 mL. The single element standards were prepared with the same acid solution.
Gd3+ ion leaching
The nanoparticles (5 mL, 1 mM Gd) were loaded into a dialysis tubing (Spectrum, 3.5 kD cut-off) and incubated in H2O, or DMEM with 10% fetal bovine serum (FBS), or DMEM with 10% FBS supplemented with 10mM phosphate, at 37ºC under sink conditions, with rocking for 3 days. The amount of released Gd3+ ions in each solution was measured using ICP-OES.
Biodistribution and Clearance
Animal experiments were performed in compliance with guidelines set by the University at Buffalo Institutional Animal Care and Use Committee. Female CD-1 mice were injected intravenously via tail vein with the nanoparticles in 5% dextrose in water at a dose of 2 mg/kg and housed in metabolic cages for 4 days with free access to water and a standard laboratory diet. Urine and feces were collected separately every 4 h and the mice were sacrificed after 96 h through cervical dislocation. Feces and organs including liver, spleen, kidney, brain, heart and lungs were harvested, frozen and weighed prior to digestion. The urine, feces, and isolated organs were individually placed in a screw cap polypropylene sample tube and to each were added 3 mL of concentrated nitric acid and 2 mL peroxide (30% by weight) and pre-digested for 24 h. The tubes were then placed in a sonicated water bath for a total of 8 h until the samples were completely dissolved. After digestion, each sample was diluted to 100 mL with a 2% solution of nitric acid. The samples were then passed through a 0.2 μm filter and the Gd content was quantified with inductively coupled plasma mass spectrometry (ICP-MS) utilizing a Thermo Scientific XSERIES 2 ICPMS Single Quadrupole Mass Spectrometer.
Cytotoxicity Assay
Cell viability was assessed by the PromegaCellTiter 96® AQueous One Solution Cell Proliferation (MTS) Assay. C6 cells were seeded into a 96-well flat-bottom microplate (c.a. 10000 cells/well) at 37°C and 5% CO2 and allowed to attach to the bottom of the microplate overnight. The cells were then treated with different concentrations of NaGdF4:Yb50% nanoparticles for 12, 24, and 48 h. After the treatment, the cellular medium was changed to remove the nanoparticles and cell debris, and the AQueous One Solution reagent (20 µl/well) was added to the cells and incubated for 4 h. Finally, the absorbance was measured at 490 nm using a microplate reader (Opsys MR microplate reader) to determine the percentage of viable cells in the culture relative to the control wells without nanoparticle treatment.
Clonogenic Assay
Clonogenic assay was performed by growing C6 cells in 6-well plates to 90% confluence and were treated with 100 μg/mL concentration of the nanoparticles overnight. Afterwards, cells were irradiated with a 2 Gy X-ray dose using the Faxitron® RX-650 X-ray Irradiator at a dose rate of 0.5 Gy/min delivered using 130 kV energy. Plates were then incubated for 4 h at 37 °C in 5% CO2, and the cells were subsequently harvested and counted. To assess colony formation, cells were then re-plated at 1000 cells/well in 6-well plates and allowed to form colonies consist of 50 cells. Colonies were then gently washed with Hank’s Balanced Salt Solution (Gibco® HBSS) and fixed with ice-cold methanol for 10 minutes, rinsed once again with HBSS and stained with a 0.5% crystal violet solution for another 10 minutes. Plates were then rinsed with H2O to remove excess stain and were left to dry at room temperature. Images of the plates were then acquired and saved in the tagged image file format (Tiff). Colony area for each plate was then measured using the ColonyArea plugin[79] in ImageJ. Surviving colonies were normalized against control wells without nanoparticle treatment.
In vitro BBB Transmigration Assay
We made and validated a cell-based in vitro transwell model of the BBB in our laboratory and used it to examine BBB properties like quantitative permeability and transendothelial migration of nanoparticles. Our 2D in vitro BBB model consists of a two-chamber transwell system in a 12 well culture plate with the upper (luminal) compartment separated from the lower (abluminal) by a semipermeable membrane (polyethylene terephthalate, PET) insert on which the Human brain microvascular endothelial cells (BMVECs) were grown to confluency on the upper side , while a confluent layer of normal human astrocytes (NHAs) was grown on the underside. After tight BBB formation was confirmed by the transendothelial electrical resistance (TEER) measurement, the dispersed nanoparticles (100 µg/mL media) were added to the upper chamber (luminal) and incubated at 37°C in 5% CO2. Media from the lower chamber (abluminal) were collected at 1, 5, 24, 48 and 72 h incubation times and the Gd content was measured using ICP-OES. Percent transmigration was calculated relative to the initial Gd concentration of the media with 100 µg/mL nanoparticles. The TEER was measured again after their crossing of the BBB to make sure that the transmigration was not due to the compromise of BBB.