Microson xl 2000

A pET28b plasmid encoding the Δ3B1-3B2-3B3-3C-His6 sequence from FMDV A₁₀61 with two mutations to enhance enzyme solubility (C95K/C142A) was obtained from Stephen Curry at Imperial College London (50 (link)). Escherichia coli BL21(DE3) pLysS was transformed with this plasmid and grown to an optical density at 600 nm of 0.4 to 0.7 absorbance units. Expression was induced by the addition of 1 mM isopropyl-β-d-thio-galactosidase (IPTG; Sigma) for 4 h at 37°C. Bacteria were harvested by centrifugation and resuspended in a lysis buffer containing 50 mM HEPES, pH 7.1, 200 mM NaCl, 1× HALT protease inhibitor cocktail (Thermo Fisher Scientific), 1% Igepal CA-630 (Sigma), 100 μg/ml lysozyme, 100 μg/ml DNase (Invitrogen) on ice for 1 h. Lysates were subjected to 12, 30-s sonication cycles at an amplitude of 10 μm (Microson XL-2000; Misonix) with 30 s on ice between each sonication. Lysates were clarified by centrifugation and His-tagged 3C^pro purified by nickel ion affinity chromatography using HisTrap columns (GE Healthcare). Elution fractions were analyzed by SDS-PAGE, and fractions with the highest concentration of purified protein were pooled and dialyzed against 50 mM HEPES, pH 7.1, 0.2 M NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, and 5% glycerol. Purified dialyzed proteins were stored at −80°C.

The Sal-Doc SE-NP was prepared by modified single emulsion method (Cui et al., 2014 (link)) with 10 mg copolymer, 1 mg Sal, and 4 mg Doc. Dissolved in 0.5 ml DCM, the mixture was emulsified by sonication with Microson XL2000 (Misonix, USA) in 1.5 ml 3% PVA solution (w/v) with 5 W of power for 60 seconds. The o/w emulsion was then emulsified with 2.5 ml of solution containing 0.5% (w/v) PVA with 2.5% of power by sonication for 10 seconds. The w/o/w emulsion was gently stirred at room temperature and filtered to remove dissociative drugs.

After 28 days of A. actinomycetemcomitans infection, mouse molars and the buccal gingival epithelial layer were harvested and homogenized in PBS containing a complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and 0.05% Triton X-100 by ultrasonic fragmentation using a Microson XL-2000 (Misonix, Farmingdale, NY). Homogenized samples were centrifuged at 13000 g for 10 minutes at 4°C, and the supernatants were collected. MMP-13 in supernatants was measured using an ELISA kit (Cloud-Clone Corp., Houston, TX) according to the manufacturer’s instructions. The minimum detectable concentration was 78 pg/ml.