Microcapsules production

Microcapsules were formed from O/W/O double emulsion templates, which were produced by microfluidics³⁸. The three phases of the double emulsion template were: a refined commercial sunflower oil (Liza, Cargill Agricola S.A., Brazil) labeled with an orange food-grade dye as inner phase (μi=55.3mPas); a mixture of 0.5 wt.% low-acyl gellan gum Kelcogel CG-LA (CP Kelco Brasil S/A, Brazil) and 2 wt.% polyoxyethylene sorbitan monolaurate, Tween 20 (Sigma-Aldrich, USA), in ultrapure water with resistivity 18.2 MΩ/cm (Direct-Q3 UV System, Millipore Co., USA) as middle phase; and a sunflower oil dispersion containing 1 wt.% calcium acetate (Sigma-Aldrich, USA) and 5 wt.% polyglycerol-polyricinoleate commercially named Grinstead PGPR super (Danisco Brasil, Brazil) as continuous phase.

Most biodegradable polymers are biocompatible and thus very attractive as shell materials, especially when the application involves interaction with living organisms and natural degradation of microcapsules. Gellan gum is a biocompatible polymer that produces stronger and less permeable gels than the largely used alginate-based ones. It is a linear, anionic, and high molecular weight exopolysaccharide secreted by the bacterium Sphingomonas elodea with some valuable characteristics such as malleability and mucoadhesive ability³⁹. The gelation process of gellan starts with the formation of double helices generated from an initial disordered coil (chain ordering) followed by the connection of the double helices enabled by the presence of cations. The structures formed are thermo⁴⁰ and pH-responsive⁴¹.

To produce monodisperse microcapsules, a three-dimensional coaxial microfluidic device (as sketched in Fig. 10) was attached to a glass slide⁴². It was made of two cylindrical glass-capillaries (World Precision Instruments Inc., USA), with inner and outer diameters of 0.58 mm and 1 mm nested into a square capillary with inner dimension of 1.05 mm (Atlantic International Technology Inc., USA). The tips of the cylindrical glass-capillaries were sanded to the final inner diameters, 50 μm for the injection capillary (left capillary in Fig. 10) and 250 μm for the collection capillary (right capillary in Fig. 10), after being tapered to an inner diameter of approximately 20 μm with a micropipette puller (model P-1000, Sutter Instrument Co., USA). The distance between them was fixed at 75 μm. The collection capillary was treated with a commercial rain repellent Glass Shield (Inove Pack do Brasil, Brazil) for 60 min to render a hydrophobic surface, while the injection capillary was treated with a polyelectrolytes solution composed of 1 wt.% poly(acrylamide-co-diallydimethyl-ammonium chloride) (Sigma-Aldrich, USA) and 2 mol/L NaCl for 60 min to render a hydrophilic surface. Stainless steel dispensing needles of inner and outer diameters 0.66 mm and 0.91 mm (model 304, McMaster-Carr, USA) were fixed at the junctions between capillaries or at their ends to enable fluids injection.

Three-dimensional coaxial microfluidic device used to produce gellan gum microcapsules. (a) Sketch of the microcapillary geometry for generating O/W/O templates and (b) Image of double emulsion production.

The inner phase was pumped through the injection capillary, while the middle and continuous phases were pumped in opposite directions through the interstices between the cylindrical and square capillaries. Then, all fluids were forced through the exit orifice of the collection tube (see Fig. 10). All flow rates were controlled by syringe-pumps (model Pump 11, Harvard Apparatus, USA). The O/W/O template formation was monitored using an inverted microscope (model DMi8, Leica Microsystem, Germany) equipped with a high-speed camera (model Fastcam SA-3, Photron, USA).

The microcapsules were collected in a glass vial with a small volume of hexane. Immediately after, acetate buffer (0.074 mol/L, pH 4.5) was added to the vial, the hexane excess containing the oil from the continuous phase was removed and the residual hexane was evaporated at room temperature for 24 h to ensure an oil-free capsular dispersion. The flow rates of the three phases were varied in order to produce microcapsules with different outer diameter D and shell thickness h³⁷. Microscope images of the microcapsules were captured with the inverted microscope model DMi8 (Leica Microsystems, Germany) and processed using the software Leica Application Suite X (Leica Microsystems, Germany) to determine the outer diameter D and the shell thickness h of the microcapsules. The shear modulus of the shell material was determined using a cantilevered-capillary force apparatus⁴³; the measure value was G=10kPa.