Aqueous ammonium hydroxide

Europium nitrate hydrate [Eu(NO₃)₂·H₂O], aqueous ammonium hydroxide [aq. NH₄OH, 28–30%], MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), wortmannin, compound C, N^ω-nitro-L-arginine methyl ester hydrochloride (L-NAME), ECM gel and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich, USA and used without further purification. The fluorescent dyes DAF-FM (4-amino-5-methylamino-2′,7′-difluorescein) and DAF-2DA (4,5-diaminofluorescein diacetate) were purchased from Molecular Probes. MnTBAP [Mn(III) tetrakis(4-benzoic acid) porphyrin chloride] and catalase were procured from Calbiochem. Spermine NONOate (SP), 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 8-bromo-guanosine-3′,5′-cyclic monophosphate (8-bromo-cGMP) and 2,3,9,10,11,12-hexahydro-10R-methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl] pyrrolo[3,4-i] [1,6] benzodiazocine-10 carboxylic acid, and methyl ester (KT 5823) were purchased from Santa Cruz Biotech. eNOS (49G3) Rabbit mAb and phospho eNOS Ser1177 antibodies were purchased from Cell Signaling Technology whereas anti-Akt/PKB and anti-phospho-Akt (tyr450) antibodies were obtained from Millipore. Goat anti-rabbit and anti-mouse IgG-ALP and FITC conjugate secondary antibodies were purchased from Genei.

A volume of 1 mL of fractionated PLNPs (2 mg mL⁻¹) was pipetted into a 2 mL microcentrifuge tube. In a different tube, a solution was prepared by adding 221.6 μL of anhydrous ethanol and 246.7 μL of DI water (Millipore Milli-Q), then adding 6.7 μL of tetraethyl orthosilicate (TEOS; 99%; Sigma-Aldrich). The mixture was added to the tube with the PLNPs, and it was placed in a bath sonicator (Fisher Scientific FS30) for 5 min. A volume of 25 μL of aqueous ammonium hydroxide (28–30%; Sigma-Aldrich) was added to the suspension, followed by sonication for another 30 min. The tube with nanophosphors was placed on a room temperature rotator for 7.5 hours. Finally, the particles were washed 3 times by adding 1 mL of anhydrous ethanol and using the centrifuge (Eppendorf Centrifuge 5418), to settle particles and remove the supernatant. The PLNPs were sonicated and vortexed thoroughly during the washings to minimize the formation of aggregates.^{7 (link)}The particle size of the bare and encapsulated nanophosphors was determined by observing the particles dispersed in ethanol under a transmission electron microscope (TEM; JEM-2010F) and the colloidal stability of the silica encapsulated nanophosphors was confirmed by measuring the zeta potential of particles dispersed in ethanol using a Zetasizer (Malvern).