Catalase (EC 1.11.1.6, from bovine liver, lyophilized powder, 2000-5000 units/mg protein; bCAT), horseradish peroxidase (HRP type VI, EC1.11.1.7, lyophilized powder, 275 units/mg solid), tetraethyl orthosilicate (TEOS, reagent grade, 98%), bovine serum albumin (BSA, lyophilized powder), MTT (3-(4-5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich. Polyethylene glycol 6000 (PEG, MW 6000), ammonium hydroxide (28%) and hydrogen peroxide (H2O2, 30%) were obtained from SHOWA. Catalase (EC 1.11.1.6, from Corynebacterium glutamicum, solution, ~500000 U/mL; cgCAT) and indicator fluorescent dye, Ru(II)-tris(4,7-diphenyl-1,10-
phenanthroline) chloride ([Ru(dpp)3]2+, were purchased from Fluka. The
oxygen-insensitive dye, Oregon Green 488-Dextran® (MW 10000) was purchased from Molecular Probe. Ethanol (95%) is obtained from TTL, Taiwan. In addition, water used to prepare solutions was autoclaved double distilled water. DMEM (Dulbecco modified Eagle's minimal essential medium), FBS (fetal bovine serum), penicillin, and streptomycin were purchased from GIBCO Life Technology.
3.2. Preparation of enzyme entrapped sol-gel PEBBLEs
The hydrogen peroxide sensitive PEBBLEs, bCAT-Ru/488-PEBBLE and cgCAT-Ru/488-PEBBLE, that contain fluorescent dyes, [Ru(dpp)3]2+, Oregon Green 488-Dextran® and catalase were prepared as described previously with modification [46].
Briefly, 2.5g poly(ethylene glycol) (PEG, MW 6000 monomethyl ether) was first dissolved in a mixture containing 6 mL ethanol (95%), 25μL Oregon Green 488-dextran (MW 10 000) (stock 0.1 mM in H2O), 200μL [Ru(dpp)3]2+ (stock 0.4 mM in 95% ethanol), and 4.2 mL 28 wt % ammonia water for 30 minutes or until the solution became transparent and viscous. Ammonia is served as a catalyst, while water acts as one of the reactants.
The enzyme stock solution was prepared separately by dissolving enzyme in double distilled water (ddH2O). The specific activity of HRP was determined as described in
Section 3.3 and diluted to 1000 units /mL solution. Various amounts (0.1, 0.08, 0.05, 0.03
and 0.01 g) of catalase from bovine liver (powder, 2000-5000 units/g) were dissolved in 1 mL water to obtained the stock solutions. Whereas, the catalase stock solution from
Corynebacterium glutamicum (~500000 Units/mL buffered solution containing ~30%
glycerol and 10% ethanol) was prepared by diluting 10 μL, 30 μL or 50 μL original enzyme solution in water containing 2.5 mg BSA to give a final volume of 1 mL. The enzyme stock solution (1 mL each) was mixed with 0.5 mL TEOS to make an
enzyme/TEOS sol. The enzyme/TEOS sol was then added into a vigorously stirred PEG
stock solution that was prepared previously by micropipette to initiate the hydrolysis and condensation reaction. About 1/3 of enzyme/TEOS sol was added at a time. The stirring continued for at least 1 h to allow the gelation to proceed thoroughly. To prevent
fluorescent dye from photobleaching, the reaction is performed in the dark.
The whole solution was then transferred to enppendorf tubes and centrifuged at 13,000 rpm for 9 min. The supernatant was discarded to remove the unreacted monomers, such as PEG, ammonia, and dye molecules. The pelleted PEBBLEs were washed by
suspending in the autoclaved PBS buffer and centrifuged at 13,000 rpm for 3 min. Remove and discard supernatant carefully after centrifugation. Repeat washing process at least three times. Finally, PEBBLEs was stored in PBS buffer under 4˚C. The control nanoparticles were also produced by the same process without enzymes and dyes.
3.3. Activity assay of HRP and HRP-entrapped particles
Activity of HRP and HRP-entrapped particles was performed with a conventional peroxidase assay by using ABTS and hydrogen peroxide as subtracts [50]. A standard 1 mL activity assay solution contains ?? units HRP, 0.5 mM ABTS (ammonium salt) (Boehringer Mannheim) and 3 mM H2O2 in 100 mM sodium acetate buffer (pH of 5.5) . ABTS is oxidized by HRP in the presence of H2O2 to form an oxidized form, which exhibits an absorbance at 405 nm (dark green in color). The activity was determined colorimetrically
absorbance with time in the presence of oxidized ABTS. The extinction coefficient of oxidized ABTS (εABTS) at 405 nm is 36.8 mM-1 cm-1 (at 25˚C, pH 5.5) for following calculations. One unit HRP is defined as oxidizing 1 μmole of ABTS within one min. at 25
˚C and pH 5.5.
Particles were sonicated for 10 minutes in PBS buffer to prevent aggregation before activity assay. The activity of HRP-entrapped silica nanoparticle was then measured by using the same assay process as that of free HRP. The background absorption of
HRP-entrapped silica nanoparticle was subtracted from the total absorption. There is no obvious background absorption when the particles used is in the range of 0.01-0.1 mg.
3.4. Activity assay of catalase and catalase-entrapped PEBBLEs
Catalase activity was determined by recording absorbance change at 240 nm of 10 mM hydrogen peroxide in 50 mM potassium phosphate buffer (pH 7.0) on a UV-Vis spectrometer (HITACHI U- 3010). The total volume of reaction mixture is 1 mL. The detecting principle was based on the conversion of hydrogen peroxide and generating H2O and O2. Thus, one unit of catalase is defined as decomposing 1 μmole H2O2 per minute at pH 7.0 and 25°C. The molar absorption coefficient for H2O2 (εH2O2) is 39.4 M-1 cm-1.
The catalase entrapped-PEBBLEs were sonicated for 10 min. in PBS to prevent aggregation prior to activity assay. The activity of catalase entrapped-PEBBLEs was determined as the same assay process as that of free catalase. Catalase
entrapped-PEBBLEs at low concentration give stable absorbance background at 240 nm.
Background absorbance is determined by adding catalase entrapped-PEBBLEs into potassium phosphate buffer as reference before catalase activity assay. However, when catalase entrapped-PEBBLE activity is low, longer incubation is needed to obtain a
significant change in the absorbance of H2O2. Therefore, a calibration curve was conducted by using a catalase/HRP coupled enzyme assay system to avoid the existence of high background due to the addition of large amount of catalase entrapped-PEBBLE particles.
Catalase was diluted by PBS buffer in a concentration of 3.5, 4, 4.5 and 6 units in 10 μL. Each assay solution contained 50 mM potassium phosphate buffer and 10 mM H2O2
with a final volume of 1 mL. The reaction mixture was allowed to stand at room temperature for 30 min. An obvious oxygen bubbles can be observed in tube during reaction. After incubation, the remaining H2O2 in the tube was further detected by a HRP assay. A portion of the above catalase reaction solution (100 μL) was added to HRP assay solution (100 mM sodium acetate buffer, pH 5, containing 0.5 mM ABTS and 10 units of HRP) with final volume of 1mL. The assay mixture was allowed to stand at room
temperature for 5 min. The absorbance of oxidized ABTS was determined on a UV–Vis spectrophotometer (HITACHI U-3010) at 405 nm. Each catalase activity point was repeated for at lease three times. PEBBLEs exhibit no obvious background in following HRP assay at 405 nm.
3.5. Scanning Electron Microscope (SEM) Imaging
PEBBLEs were dispersed in water and sonicated for 20 min. to prevent aggregation.
Place 2 μL of the PEBBLE suspension on the small piece of silica wafer and dried gradually in an anti-humidity cabinet. The sample was then sputter coated with platinum (12V, 90 sec.), and the SEM images were taken on the Thermal Field Emission Scanning Electron Microscope (JEOL, JSM-6500F).
3.6. Fluorescent spectroscopy spectrum of PEBBLEs and fluorescent calibration
PEBBLEs (10 mg) were dispersed in 1 mL water and sonicated for 20 min. to prevent aggregation. In each 10 mg PEBBLEs/H2O suspension solution 1 mL of various
concentrations of hydrogen peroxide was added to make the final concentrations of 0.1, 0.5, 1, 5, 10 and 20 mM. The final volume of reaction mixture is 2 mL. The mixture was then mixed thoroughly in a 3-mL cuvette and incubated at room temperature for 3 minute, followed by wave scanning fluorescence detection on the HITACHI F-4500 with exciting wavelength of 488 nm. The emission scanning range was between 500 and 750 nm. The obtained emission intensity (ransom light units; RLU) of [Ru(dpp)3]2+ at 607 nm was normalized with the emission intensity units of Oregon Green 488-Dextran at 524 nm. The normalized value from control was defined as 100%.
3.7. Cell culture
The human cervical carcinoma cell line (HeLa) was maintained in DMEM (Dulbecco modified Eagle's minimal essential medium) containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (GIBCO Life Technology). Cells were incubated in a
humidified 37˚C incubation chamber containing 5% CO2. Cells were subcultured for every 2-3 days.
3.8. Cell viability test
Cell viability was measured by the MTT (3-(4-5-Dimethylthiazol-2-yl)-2,5
-diphenyltetrazolium bromide) assay. HeLa cells were seeded on 24-well culture plates with 2×104 cells in 500 μL culture medium per well and put in humidified 37˚C incubation chamber for 24 hour prior to assay. Afterward, each well was treated with 100 μg, 200 μg, 300 μg and 400 μg of the sonicated catalase-entrapped PEBBLEs and control (silica-PEG only) particles in PBS for 24 and 48 h. At the end of incubation, cultural medium was removed and cells were washed once with pre-wormed PBS, followed by incubating with 500 μL MTT solution (0.5 mM in culture medium) in a 37 ˚C incubation chamber
containing 5% CO2 for 3 h.
The produced blue-purple formazan in each well was solubilized with 600 μL acidic isopropanol (0.1 N HCl in absolute isopropanol). The absorbance soluble formazan was
viability of the treated group was expressed as the percentage of control group (added no PEBBLEs but PBS buffer only) which was assumed to be 100%.
3.9. Cell images
The location of the PEBBLEs in HeLa cells was monitored using an inverted fluorescence microscope. The HeLa cells (4×105 cells/well) were cultured in a 6-well cultural dish with a sterilized cover glass with in 2 mL culture medium and put in humidified 37˚C incubation chamber for 24 h. Each well is then incubated with the PEBBLEs in PBS. After incubation at 37˚C for 24 h, cells were fixed with 4%
paraformaldehyde in pH 7.4 PBS at room temperature for 20 min, followed by PBS wash for 4 times and monitoring under the inverted fluorescent microscope (Leica DMIL model).
4. Results and discussion