Supplementary materials and methods
Preparation and characterization of mesoporous silica nanoparticles
Amino-functionalized co-condensed mesoporous silica nanoparticles were synthesized according
to the procedure described by Nakamura et al.1, but with the thiol-silane replaced by 3aminopropyltrimethoxysilane, APS, in accordance with our earlier publications.2 In a typical
synthesis, 1.19 g of tetramethoxysilane (TMOS) was mixed with APS and added to an alkaline
solution containing the structure-directing agent cetyltrimethyl ammonium chloride (CTACl).
For in vitro studies, 250 µl of a solution containing fluorescein isothiocyanate (FITC) in DMF (1
mg/ml) was added to the silane mixture to create inherently fluorescent particles. The resulting
synthesis mixture had a molar ratio of 0.9 TMOS: 0.1 APS: 1.27 CTACl: 0.26 NaOH: 1439
MeOH: 2560 H2O. AlexaFluor®750-labeled particles were prepared similarily, but replacing
FITC with AlexaFluor®750. For smaller particles, a H2O:MeOH ratio of 60:40 was used instead
of 50:50. The mixture was stirred overnight at RT, and thereafter aged for 8 h at static
conditions. The precipitate was either filtered off or separated by centrifugation, washed with
deionized water, methanol and acetone and dried at 318 K in vacuo for 72 h. The structuredirecting agent was subsequently removed by ultrasonic treatment in acidic (HCl) ethanol (0.12
w/w) three times. The mesoscopic ordering of the nanoparticles was confirmed by powder-XRD
using a Kratky compact small-angle system (M. Braun), particle size by scanning electron
microscopy (Jeol JSM-6335F, Jeol Ltd., Japan), redispersability by dynamic light scattering
(Malvern ZetaSizer Nano) and mesoporosity by nitrogen sorption measurements (ASAP 2010
sorptometer, Micromeritics). Surfactant-extracted particles without fluorescent label were
labeled with AlexaFluor® 750 succinimidyl ester in carbonate buffer (pH=8.3) for 1h as a
second approach, and dried in vacuo at RT. Polyethyleneimine, PEI, was grown onto all above
fluorescent mesoporous silica particles by hyperbranching surface polymerization according to
procedures described in our earlier publications.3 After the reaction, the particles were
centrifuged, washed with acetone and vacuum dried for 24 h. For FA-conjugation, 25 mg PEIfunctionalized particles were suspended in MES buffer (pH 4.8). 50 µg of FA was dissolved in
ethanol, to which 20 µl of a 1 µl/ml 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC)solution was added to activate the carboxylic acid groups of FA. This solution was rapidly added
to the particle suspension, after which 25 µl (1 mg/ml in MES) NHS (N-hydroxysuccinimide) solution was also added to the suspension. The suspension was agitated overnight and washed
with deionized water and ethanol, dried in vacuo and stored at 277 K. The amount of conjugated
FA was determined by dissolving a known amount of particles (1mg/ml) in 1M NaOH overnight
and measuring the amount of FA by a UV spectrophotometer reading at λ=280 nm. The
concentrations determined were indeed in the range of 0.2 wt% (corresponding to 4.5 µmol/g)
when the contribution of the fluorophore was subtracted. Impregnation of drug (DAPT) was
performed on vacuum dried, surfactant extracted particles by suspending the particles in
cyclohexane (with the maximum immersion ratio of 0.5 mg/ml). DAPT was separately dissolved
in the same solvent and the desired amount (1 or 5 wt% with regard to the particle mass) was
added to the particle-cyclohexane suspension. The drug-particle-cyclohexane suspension was
subject to ultrasonic treatment, and subsequently agitated overnight protected from light. Drugloaded particles were centrifuged, separated from the solvent and vacuum dried. Successful
loading was corroborated by HPLC measurements, as explained in the Supplementary materials
section.
References
1
T. Nakamura, Y. Yamada, K. Yano, Direct synthesis of monodispersed thiol-functionalized nanoporous silica
spheres and their application to a colloidal crystal embedded with gold nanoparticles. J. Mater. Chem. 17, 2007,
3726-32.
2
J.M. Rosenholm, A. Meinander, E. Peuhu, R. Niemi, J.E. Eriksson, C. Sahlgren and M. Lindén, Targeting of
porous hybrid silica nanoparticles to cancer cells. ACS Nano, 3, 2009, 197-206.
3
a) J.M. Rosenholm, A. Penninkangas, M. Lindén, Amino-functionalization of large-pore mesoscopically ordered
silica by a one-step hyperbranching polymerization of a surface-grown polyethyleneimine. Chem. Commun. (37),
2006, 3909-3911. b) J.M. Rosenholm, M. Lindén, Wet-chemical analysis of surface concentration of accessible
groups on different amino-functionalized mesoporous SBA-15 silicas. Chem. Mater. 19, 2007, 5023-5034. c) J.M.
Rosenholm, A. Duchanoy, M. Lindén, Hyperbranching surface polymerization as a tool for preferential
functionalization of the outer surface of mesoporous silica. Chem. Mater. 20, 2008, 1126-1133.