Up50h

The biofilms were washed thrice with phosphate-buffered saline at different time points (after 18, 20, 22, 24 h of incubation). The biofilm generated at the bottom of the well was removed by blending in an ultrasonic homogenizer (Hielscher UP50H, Teltow, Germany) for 20 s at 25% amplitude. Serial dilutions of the resultant solution were prepared and seeded in amounts of 100 μL on Sabouraud agar for C. albicans and mitis salivarius-bacitracin agar with sucrose MSBS (containing bacitracin and sucrose) for S. mutans, and then their growth was promoted for the next 48 h under microaerophilic conditions. The colony forming units (CFU/mL) were indicated. The protocol was performed in triplicate.

Polymer nanoparticles with (or without) Paclitaxel were prepared by the emulsification solvent evaporation method. Initially, neat polymeric nanoparticles were prepared by dissolving 50 mg of the TEHA-co-PDLLA block copolymer in 2 mL of dichloromethane (DCM) and homogenized using a probe sonicator model UP50H (Hielscher Ultrasound Technology, Teltow, Germany) at 15 W for 2 min with an aqueous phase containing either a) 10 mL of 0.5% w/v PVA solution, or b) 6 mL of 12 mM sodium cholate hydrate solution. The O/W emulsion formed was gently stirred at room temperature under a fume hood until the evaporation of the organic solvent was completed. Nanoparticles were purified by centrifugation at 9500 rpm for 20 min and reconstituted from the precipitate in fresh water (twice). The resulting suspension was lyophilized (Scanvac, Coolsafe 110-4 Pro, Labogen, Scandinavia) and stored at ambient temperature under vacuum until further study. Nanoparticles with addition of PTX were also prepared based on the above process by adding proper amounts (25 mg in 250 mg of polymer) of PTX in the organic DCM solution. Additionally, for the preparation of core–shell magnetic nanoparticles, 10 mg of superparamagnetic manganese ferrite nanoparticles were added in the PTX-polymer DCM solution and the process was following as described above.

Two mL bioreactor liquid was centrifuged (3200× g, 10 min, 4 °C) and the pellet was solved in 1 mL lysis buffer (8M Urea, 2M Thiourea, 1 mM Phenylmethylsulfonylfluorid). Bacteria were disrupted by bead beating (FastPrep-24, MP Biomedicals, Sanra Ana, CA, USA; 5.5 ms, 1 min, 3 cycles) followed by ultra-sonication (UP50H, Hielscher, Teltow, Germany; cycle 0.5, amplitude 60%) and centrifugation (10,000× g, 10 min) [28 (link)]. The supernatant was used for protein concentration determination using the Pierce^TM 660 nm Protein Assay (Thermo Scientific, Thermo Fischer Scientific, Waltham, MA, USA). Ten micrograms of protein lysate was incubated with 25 mM 1,4-dithiothreitol (in 20 mM ammonium bicarbonate) for 1 h and 100 mM iodoacetamide (in 20 mM ammonium bicarbonate) for 30 min. Protein cleaning, cleavage and peptide cleaning were done with hydrophobic Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (GE Healthcare, Chicago, IL, USA) as described elsewhere [29 (link)]. Proteins were digested with Trypsin (1:50), peptides were eluted with 2% dimethylsulfoxide solved in water without fragmentation. Peptides were solved in 0.1% formic acid for mass spectrometric measurement.

Based on preformulation studies (data unseen) and previous reports, MN loaded LNCs were prepared using lipid matrix of both oleic acid and Labrafac^® oil with ratio 1:1 (Kamel & Basha, 2013; Eissa et al., 2015 (link); Kiani et al., 2017 ). The previously mentioned oil mixture has shown high solubilization power for MN referred to the long chain of oleic acid and the HLB value. Briefly, 20 mg of MN was mixed with lipid matrix (melted in water bath at 80 °C) at different ratios (1:2, 1:3, and 1:4) where soy phosphatidylcholine was used as surfactant (Table 1). A hot aqueous solution was prepared by dissolving soy phosphatidylcholine (5 g) in PG (10 g) in a mass ratio of 1:2. The aqueous solution was added using syringe to the previous lipid phase and the mixture was stirred at 1000 rpm for 15 min at room temperature to obtain the required nanocapsules suspension. The obtained LNCs suspensions were ultrasonicated for 5 min using probe sonicator (UP50H, Hielscher, Teltow, Germany). The composition of prepared polymeric and LNCs formulations is shown in Table 1. The effect of different ratios of MN:lipid matrix (1:2, 1:3, and 1:4) on PS, ZP, and %EE was further studied.

MRC-5 and CCD-1070Sk cells were collected from culture flasks, washed with PBS and the cell lysates were obtained by sonication (30 s × 3 times) on ice with an ultrasonic processor (Hielscher UP50H, Teltow, Germany). The homogenate was centrifuged at 3000× g for 10 min at 4 °C and the supernatants (total proteic extracts) were collected for biochemical assays.

Formulations of EEP-NPs (or Empty-NPs) were prepared using the modified oil-in-water (o/w) single emulsion solvent evaporation method with some modifications [9 (link)]. Briefly, an organic solution consisting of 100 mg/mL EEP and 100 mg PLGA dissolved in DCM was prepared and added dropwise to an aqueous solution containing 0.4% (w/v) PVA and 1% (w/v) CS to obtain the ratio of 1:2 (v/v) of organic and aqueous phases. The resulting solution was stirred, and then sonicated using an ultrasonic processor UP50H (Hielscher Ultrasonics, Hielscher, NJ, USA) at 90% amplitude for 30 min within an ice bath. For complete polymerization, each mixture was stored overnight at room temperature in the dark. Afterwards, the solution was centrifuged at 8800× g for 40 min at 4 °C to obtain NPs. The NPs were further washed once and reconstituted with deionized water before lyophilization. The NP samples were stored at −20 °C until used.

Polar metabolites were extracted from the hearts for the metabolic studies in accordance with Beckonert’s procedure [23 (link)]. In brief, an ice-cold solution of methanol and water (in proportion 8:2.5) was added to the tubes with frozen heart samples. The tissue was homogenized on ice using a Hielscher UP50H ultrasonic processor using 30 s pulses over a period of 5 min. The samples were incubated on ice for 15 min and then centrifuged at 1000 × g for 15 min at 4°C. The supernatant was transferred to a new tube and the solvent was evaporated from the samples using a speed vacuum concentrator. The material was re-suspended in 580 μL of phosphate buffer, pH 7.4 (in D₂O containing Trimethylsilylpropanoic acid (TSP) and NaN₃). The samples were vortexed and centrifuged at 12.000 × g for 5 min and for each sample 550 μL of the supernatant was transferred into the NMR tube (Wilmad Labglass, USA) and kept at 4°C until the NMR analysis.