Synthesis and Surface Modification of Mesoporous Silica Nanoparticles

Mesoporous silica nanoparticles, MSNs, were synthesized following a modification of the Stöber method.⁴³ First, cetyltrimethylammonium bromide (CTAB (1 g, 2.74 mmol)), the structure directing agent, was dissolved in H₂O (480 mL) and NaOH (3.5 mL, 2 M) in a 1 L round-bottom flask under moderate magnetic stirring. The mixture was heated at 80 °C, and tetraethylorthosilicate (TEOS (4.5 mL, 20.15 mmol)) was added drop-wise at 0.33 mL/min rate with a pump. For the phosphonate modification, 30 min after the TEOS addition, 3-trihydroxysilylpropyl methylphosphonate (TSPMP (0.5 mL, 1.31 mmol)) was added and heated for a further 1.5 h at 80 °C under magnetic stirring. Then, the solution was centrifuged and washed once with water and twice with ethanol. The product was dried at room temperature under vacuum. The surfactant was removed by ionic exchange using 350 mL of a solution of ammonium nitrate (10 mg/mL) in ethanol (95%) at 80 °C overnight under magnetic stirring. The product was centrifuged, washed 3 times with ethanol, and dried under vacuum.

Rhodamine B-labeled nanoparticles and fluorescein isothiocyanate (FITC)-labeled nanoparticles were synthesized by reacting 1 mg of Rhodamine-B isothiocyanate or fluorescein isothiocyanate with 2.2 μL of (3-aminopropyl)triethoxysilane (APTES) in 100 μL of ethanol for 2 h. Then, this solution was mixed with 4.5 mL of TEOS, and the whole mixture was added to the surfactant solution as previously described. The rest of the procedure was carried out as described above.

For the synthesis of cyanine-7-labeled nanoparticles, 5 mg of cyanine-7 with 4.4 μL of APTES were dissolved in 140 μL of dimethyl sulfoxide and stirred overnight. Then, this solution was mixed with 4.5 mL of TEOS, and the whole mixture was added to the surfactant solution as previously described. The rest of the procedure was carried out as described above.

To perform the PEI coating, 5 mg of phosphonate-modified nanoparticles were dispersed in a solution of 2.5 mg of PEI (5, 8, and 10 kDa) in 1 mL of absolute ethanol. After sonicating for 20 s and stirring for 30 min, the PEI coated nanoparticles (MSNs@PEI) were consequently washed with PBS and ethanol.⁴¹