Enhancement of Enzymatic Activity Using Microfabricated Poly(ε-caprolactone)/Silica Hybrid Microspheres with Hierarchically Porous Architecture

In this paper, we present a novel and facile microfluidic method to fabricate hierarchically porous poly­(ε-caprolactone)/silica hybrid microspheres and further investigate in detail their performance as enzyme carriers by three famous proteins and enzymes. Because of the synergy effect between sol–gel process and solvent extraction in microdroplets, hierarchically porous architecture can be formed in situ without the use of porogens and templates. More importantly, the surface porosity or the specific surface area of such microspheres can be precisely tuned via adjusting the hydrolysis/condensation rate by ammonia catalyst and thus the competition between the two above-mentioned processes. Fluorescein isothiocyanate-bovine serum albumin, alcohol dehydrogenase, and superoxide dismutase are immobilized via either physical adsorption or covalent binding to evaluate the performance of hierarchically porous microspheres as enzyme carriers. All the qualitative and quantitative data including fluorescence images, enzymatic activity, immobilization yield, and activity yield prove that enzymes covalently immobilized on hierarchically porous microspheres exhibit the optimal immobilization capacity, enzymatic activity, stability, and reusability, which shows very promising application of such microspheres in enzymatic catalysis

Effect of desferrioxamine on hypoxia-induced Sost expression.

<p>(A) RNA expression level of Sost with DFO as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were cultured for 48 hr under hypoxia (1%O₂), and treated with desferrioxamine (DFO). +:100 uM; ++:200 uM. The RNA level from normoxic condition (20%O₂) group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. A paired <i>t</i>-test was also performed comparing 1% O₂ group and DFO group (+ and ++). **: Two stars indicate statistical significance compared to 1% O₂ group. (B) Western blotting analysis of Sost expression in protein level in osteoblasts under hypoxia. Heat shock protein 90 (HSP90) was used as a loading control.</p

Hypoxia leads to upregulation of Sost gene expression.

<p>(A) Increase of HIF-1α expression in RNA level in osteoblasts under hypoxia. RNA level was normalized to heat shock protein 90 (HSP90). A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. (B) Western blotting analysis of HIF-1α expression in protein level in osteoblasts under hypoxia. Heat shock protein 90 (HSP90) was used as a loading control. (C) Quantification of western blotting of HIF-1α and HSP90 expressions in protein levels. Protein level from normoxic condition (20%O₂) group was normalized to a value of 1. (D) RNA expression level of Sost as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were cultured for 48 hr under hypoxia (1%O₂). RNA was isolated and quantitated by real-time RT-PCR. The RNA level from normoxic condition (20%O₂) group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group.</p

Hypoxia leads to downregulation of Wnt targets.

<p>(A) Increase of HIF-1α expression in RNA level in osteoblasts under hypoxia. RNA levels were normalized to heat shock protein 90 (HSP90). A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. (B) Western blotting analysis of HIF-1α expression in protein level in osteoblasts under hypoxia. Heat shock protein 90 (HSP90) was used as a loading control. (C) RNA expression levels of cyclin D1 and c-Myc as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were cultured for 48 hr under hypoxia (1%O₂). RNA was isolated and quantitated by real-time RT-PCR. The RNA level from normoxic condition (20%O₂) group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. (D) RNA expression levels of cyclin D1 and c-Myc treated with DFO as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were cultured for 48 hr under hypoxia (1%O₂), and treated with desferrioxamine (DFO). +:100 uM; ++:200 uM. The RNA level from normoxic condition (20%O₂) group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing control group (20% O₂) and hypoxia group (1% O₂). *: A star indicates statistical significance compared to control group. A paired <i>t</i>-test was also performed comparing 1% O₂ group and DFO group (+ and ++). **: Two stars indicate statistical significance compared to 1% O₂ group.</p

Hypoxia inhibits osteoblast proliferation.

<p>(A) Osteoblast number counts in the growth medium. MC3T3 osteoblastic cells were cultured in Alpha Minimum Essential Medium, and maintained for different time points as indicated from 4 hr to 72 hr in normoxic (20%O₂) or hypoxia (1%O₂) condition. (B) Inhibition of HIF-1α expression by siRNA resulted in an increase of osteoblast growth. MC3T3 osteoblastic cells were transfected by siRNA, and cultured under hypoxia for 48 hr. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Inhibition of HIF-1α by siRNA results in upregulations of Cyclin D1 and c-Myc expressions in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

HIF-1α inhibits Wnt pathway in vitro.

<p>(A) HIF-1α inhibited Topflash reporter activity in a dose-dependent manner. HEK293 cells were transfected with a Topflash reporter along with 50 ng β-catenin without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ±S.D. (B) HIF-1α cooperated with Osx to inhibit Wnt pathway activity. HEK293 cells were transfected with a Topflash reporter along with 25 ng β-catenin without or with different groups of HIF-1α expression plasmid and Osx plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ±S.D.</p

Binding of the HIF-1 complex to the HRE of Sost promoter under hypoxia.

<p>Nuclear extracts were isolated from MC3T3 cells in either normoxia or hypoxia conditions from 16 h and used as the HIF-1 protein resource. DNA oligonucleotides of <i>Sost</i> were labeled by Biotin. Nuclear extracts and biotin-labeled DNA probe were incubated. Protein-DNA complexes were separated on 4% polyacrylamide gels, and visualized by a Chemiluminescent Nucleic Acid detection Module. Complexes observed in extracts under normoxia (N, lane 1) or hypoxia conditions (Hyp, lane 2) are indicated by the arrows. Two hundred-fold molar excess of unlabeled <i>Sost</i> promoter oligos were used under hypoxia condition (lane 3).</p

Inhibition of HIF-1α by siRNA results in downregulation of Sost expression in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR for HIF-1α and Sost, and HSP90 was used as a negative control. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Effect of HIF-1α on Sost promoter activity.

<p>(A) HIF-1α activates the <i>Sost</i> promoter in a dose-dependent manner. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D. (B) Jab1 does not activate <i>Sost</i> promoter activity. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of a Jab1-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.</p