Cellulose nitrate membrane filter

Pre-treatment of samples and bacterial growth from the filters with heat and ethanol shock, respectively was used to reduce the competing bacteria, to increase the sensitivity of the culture and C. difficile recovery. Water samples (50 ml) were subjected to a heat shock by incubation at 70°C for 20 min. The entire volume was then filtered through 0.2 μm cellulose nitrate membrane filter (Whatman) using Milipore filtering system. Filters were placed on selective agar chromID® C. difficile (bioMerieux) and incubated anaerobically at 37°C for 3 days. After incubation, up to 20 presumptive C. difficile colonies were picked from each filter and subcultured onto blood agar plates (COH, bioMerieux). Remaining bacterial growth was swabbed from the filter, resuspended in 700 μl of absolute ethanol and incubated at room temperature for 30 min. After centrifugation the pellet was inoculated onto chromID® C. difficile plates and incubated anaerobically for 2 days. Up to 10 colonies with suitable C. difficile like morphology were subcultured onto COH plates. Isolates were identified using MALDI-TOF (Biotyper, Bruker).

The release of LID from raw LID, GMS-LID1, GMS-LID2, GMS-LID4, and GMS-LID10 particles was performed in Franz diffusion cells consisting of a donor and a receptor compartment separated by a 0.45 μm cellulose nitrate membrane filter (Whatman, Little Chalfont, UK). Franz cells were kept in an orbital shaker (VWR^® Incubating Mini Shaker, VWR, Chester, PA, USA) at 37 °C under agitation at 100 rpm. The volume of the receptor compartment was 6 mL and the surface available for diffusion was ca. 0.8 cm². The receptor compartment was filled with PBS pH 7.4 medium, and 25 mg of particles were placed in the donor compartment with 0.5 mL of PBS medium. Then, 1 mL-aliquots were taken every hour for the first 8 h and then after 24 h, and the same volume was replaced with fresh PBS solution. Samples were filtered with syringe filters (hydrophilic PTFE, 0.22 μm, Scharlau, Barcelona, Spain) and measured by HPLC (see Section 2.5). The release of LID from SLMPs was compared to the dissolution of 3 mg of pure LID, carried out using the same experimental set-up.
LID release profiles of the drug were fitted to the first-order (Equation (2)) and the first-order with lag time (Equation (3)) release kinetics models: $\%\mathrm{LID}\mathrm{released}=100\times (1-e^{-k_1\times t})$
$\%\mathrm{LID}\mathrm{released}=100\times (1-e^{-k_2\times \left(t-t_{lag}\right)})$
where k₁ and k₂ are the kinetic coefficients, t is the elapsed release time, and t_lag is the lag time.

Bovine serum albumin was dissolved (30% w/v) in a solution containing (mm): 118 NaCl and 2.5 CaCl₂.7H₂O. Visking dialysis tubing (Medicell Membranes Ltd., London, UK) was prepared by boiling for 10 min in a solution containing (mm): 1 EDTA, 119 NaHCO₃, rinsed with ddH₂O and then boiled for another 10 min in 1 mmol l⁻¹ EDTA. The tubing was given a final rinse in ddH₂O before the BSA solution was transferred and left to dialyse for 48 h at 4°C against a 20‐fold larger volume of (mm): 118 NaCl and 2.5 CaCl₂.7H₂O. This removed molecular impurities (molecular mass cut‐off of 12–14 kDa) and saturated the Ca²⁺‐binding sites of BSA (Smith et al. 2010).
All buffers were filtered through a 5.0 μm cellulose nitrate membrane filter (Whatman, Maidstone, UK). Appropriate corrections were made for Na⁺ and Ca²⁺ concentrations in the KH buffer to account for their presence in BSA solution stock (Smith et al. 2010).