Support Information. Enzyme encapsulated hollow silica nanospheres for intracellular biocatalysis

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1 Support Information Enzyme encapsulated hollow silica nanospheres for intracellular biocatalysis Feng-Peng Chang, Yann Hung, Jen-Hsuan Chang, Chen-Han Lin, Chung-Yuan Mou* Department of Chemistry, National Taiwan University Taipei 106 Taiwan *To whom correspondence should be addressed 1

2 Materials: Decane, n-hexanol (98%), ammonia (28-30 wt %), tetraethyl orthosilicate (TEOS), 3- aminopropyltrimethoxysilane (APTMS), fluorescein isothiocyanate (FITC), urea and sodium phosphate were from Acrôs [NJ, US]. Polyoxyethylene (5) isooctylphenyl ether (Igepal CA- 520), horseradish peroxidase type VI-A (HRP), 3,3,5,5 -tetramethylbenzidine (TMB), o- phenylenediamine (o-pd), citric acid, trypsin,dimethyl sulfoxide (DMSO), 4',6-diamidino-2- phenylindole (DAPI) and rhodamine B isothiocyanate (RITC) were from Sigma-Aldrich Chemical [MO, US]. N-(3-triethylammoniumpropyl)-4-(4- diethylaminophenylhexatrienyl)pyridinium dibromide (FM4-64) was from Invitrogen [NY, US]. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium [WST-1] was from Clontech [CA, US]. Rhodamine phalloidin was from Millipore [MA, US]. Ultrapure deionized (D.I.) water was generated by a Millipore Milli-Q plus system. Leakage of HRP-RITC from silica nanospheres: Equivalent amount of HRP-RITC (18.6 µg) that was confined in particles (HRP-RITC@HSN (1 mg) and HRP-RITC@SSN (3.4 mg)) or adsorbed onto the particle surface (HRP-RITC incubated with HSN (1mg) and HRP-RITC incubated with SSN (3.4 mg); as control groups) was prepared and suspended in acidic H 2 O (ph: 2, adjusted with 1 N HCl). In acidic water, the electrostatic repulsion between the positively charged HRP (isoelectric point: ph ) and silica nanospheres was strong enough to keep them from nonspecific binding. The fluorescence intensity of each sample was assayed after each washing cycle (centrifugation/resuspension). Excitation: 543 nm; emission: nm. Cell cytotoxicity and proliferation assay: 2

3 cells were seeded in 24-well plates and allowed to attach for 24 h. To determine the particle toxicity, cells were incubated in fresh serum-free medium containing different amounts of BSA-stabilized particles (0-100 µg/ml) for 2.5 h for cytotoxicity assay or further cultured in regular growth medium for 24 h for cell proliferation assay. Particle-treated cells were then washed twice with phosphate-buffered saline (PBS) and incubated with 200 µl WST-1 (10%) in DMEM. Cell viability was determined by the formazan dye generated by the live cells and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, model 680). 3

4 Figure S1. TEM histograms. (a) and (b) Figure S2. TEM image of HSN stained with UA. Scale bar: 50 nm. 4

5 Figure S3. Leakage of HRP-RITC that was entrapped in the particles or adsorbed on the particle surface. Equivalent amount of HRP-RITC that was encapsulated in the SSN and HSN (HRP- and or adsorbed on the SSN and HSN surface (HRP-RITC incubated with SSN and HRP-RITC incubated with HSN, as control groups) were introduced leakage experiment. Each sample was washed (centrifugation/resuspension) with DI water (ph: 2.0) to remove non-entrapped proteins. The fluorescence intensity of particles were assessed and normalized to each sample that was not exposed to washing cycle. 5

6 Figure S4. Calibration curve of fluorescence verses the concentration of HRP-RITC. (a) Concentration-dependent fluorescence spectra of HRP-RITC in 1N NaOH. Excitation: 545 nm. (b) Plot of fluorescence intensity (emission: 575 nm) as a function of the HRP-RITC concentration. Figure S5. HRP activity assay. (a) dose-dependent UV-vis spectrum of o-pd in the presence of HRP@HSN. (b) Lineweaver-Burk plot of HRP@HSN, HRP@SSN, and native HRP. The kinetics of HRP as a function of the concentration of o-pd in the presence of 80 µm H 2 O 2. The Michaelis Menten parameters were obtained from the fitting: 6

7 Figure S6. Confocal images of HeLa cells after treatment with for 24 h. Yellow coloration (Merge channel) indicates co-localization of HRP-RITC (red) and FITC labeled particles (green). Blue, DAPI-stained nucleus. Figure S7. Flow cytometry analysis of cellular uptake of fluorescein-doped HSN. HeLa cells were incubated with F-HSN or at a dose of 30, 60 and 100 µg/ml respectively for 2.5 h. The fluorescence intensity of particle-treated cells was assayed after the extracellular FITC was quenched by trypan blue. The number of positively labeled cells was represented as the percentage of total counting cells in each panel using untreated cells as a reference. 7

8 Figure S8. Mean fluorescence intensity calculated from flow cytometry analysis. Figure S9. The effect of HSN and on cytotoxicity and cell proliferation in HeLa cells. Cells were exposed to HSN or at doses of 0, 30, 60, and 100 µg/ml. Cell viability was examined using WST-1 assay (a) after 2.5 h of particle treatment or (b) after 2.5 h of particle treatment followed by additional 24 h growth. 8

9 Table S1. DLS sizes and zeta values of particles in H 2 O, DMEM culture medium and after stabilized with 2 mg/ml BSA in DMEM. Sample DLS Size (nm) Zeta potential (mv) H 2 O DMEM DMEM+BSA H 2 O DMEM DMEM+BSA HRP@HSN (±5.8) (±13.4) 164.8(±3.4) 20.5(±0.5) -9.9 (±0.5) (±1.4) HSN (±3.6) (±36.1) (±11.3) 21.6 (±0.5) (±0.3) (±1.6) 9