B 390

A 3² factorial design was tested, in which three different concentrations of GSH and CaCl₂ were examined. The microcapsules were prepared by ionic gelation as described elsewhere [30 (link)]. A 1.5% (w/v) aqueous solution of sodium alginate was prepared and magnetically stirred for 12 h at ca. 25 °C. Then, different amounts of GSH were incorporated: 5%, 10%, or 15% (w/v). These solutions were gelated dropwise through an encapsulator B-390 BÜCHI working at a frequency of 800 Hz, 800 V electrode, 500 mbar air pressure, and nozzle of 200 µm into a CaCl₂ solution at different concentrations: 0.75%, 1.0%, 1.25% (w/v) under stirring for 30 min. The microcapsules were rinsed with distilled water, sieved, and freeze dried at 4 °C. The approximate size of the nanostructures was a function of the microencapsulator used, ranging between 200 and 400 μm.
Table 1 shows the 9 combinations of reactants tested, as well as the treatment codes and replications (n = 3). GSH and CaCl₂ treatments were transformed into coded units (i.e., −1, 0, and +1) to have them in a common scale and to unify their weight during the optimization analysis. The response was expressed as mg of GSH per mg of complex.

Ca-alginate microspheres were prepared using an extrusion method adapted from [15 (link)] and modified. Bacteriophages were encapsulated in alginate (Sigma-Aldrich, Darmstadt, Germany) or alginate with mannitol (Sigma Aldrich, Germany) capsules using encapsulator B-390 (Büchi Labortechnik AG, Flawil, Switzerland). Some 30 mL of T4 lysate was mixed with 90 mL of 2% water solution of sodium alginate. Then, the mixture was extruded through the nozzle with a diameter of 150 µm, 300 µm or 450 µm into the 0.1 M CaCl₂ or 0.1 M CaCl₂ supplemented with 0.3 M mannitol at 23 °C. The following parameters of the encapsulation process were used: air pressure 150 mbar, frequency 800 Hz, and stream height 30 cm. The beads were kept in the solution for 20 min and were allowed to harden. Then, the formed beads were washed with distilled water. The shape and diameter of the capsules were investigated under the light microscope (Zeiss Axio Vert.A1 for wet microspheres and Leica DMI1 for dry microspheres).

Sodium alginate (Pronova UP MVG; FMC Biopolymer AS, Sandvika, Norway) was dissolved in distilled water at a concentration of 1 wt% (w/v) and 6–8 wt% (w/v) HAp (particle size = 50 nm, max., CGBio Inc., Seongnam, Korea) was mixed with a homogenous dissolved alginate solution using a planetary centrifugal mixer and a three-dimensional ultrasonic mixer for 6 min and 15–30 min, respectively. Microsized spherical hybrid structures were fabricated with a mixture of alginate and HAp using an encapsulator (B-390; Buchi, Flawil, Switzerland) in the frequency range of 1500–4000 Hz, electrode range of 1000–2000 V, and pressure range of 150–400 mbar. The fabricated structures were crosslinked in 100 mM CaCl₂ (Sigma, St. Louis, MO, USA) for 30 min under stirring. Crosslinked structures were obtained from the CaCl₂ solution and washed with ethanol. After drying overnight at room temperature, the samples were sintered at 1200 °C. The phase composition of the sintered microbeads was determined via X-ray diffraction (XRD, DMAX-2500; Rigaku, Tokyo, Japan) at an operating voltage of 40 kV. Measurements were taken in the 2θ angle range of 5–55°. Images of the sintered beads were obtained using a tabletop SEM (SNE-4500 M Plus; SEC Co., Ltd., Suwon, Korea).

CTEPH was induced in animals by means of repetitive embolization of distal branches of pulmonary artery with partially biodegradable alginate microspheres (MS) of 160–200 μm in diameter. MSs were produced from ultrapure alginate natrium (Sigma-Aldrich, St. Louis, MO, USA) using 2% barium chloride as a stabilizer with electrostatic encapsulator (B-390, Buchi, Flawil, Switzerland). All MSs were produced in sterile conditions. 50 μL of MSs by volume were suspended in 1 mL of 0.9% NaCl solution and administered via the tail vein 8 times separated by 4-day intervals [26 (link),27 (link)]. Ten rats received injections of 0.9% NaCl solution instead of MSs suspension, thus forming the group of intact animals (INT).