Encapsulation methods

In general microencapsulation procedures are performed in three steps³⁷. The first step is the addition of the biomolecule, or the species which is to be encapsulated, to the encapsulation matrix. This step normally involves the dissolution of the species to be encapsulated into the matrix. The second step includes liquid/liquid or liquid/air mechanical dispersion of encapsulation matrix and the third step is to stabilize microcapsules either by chemical methods such as polymerization or by physical methods such as evaporation, solidification, coalescence or by physicochemical methods such as gelification or coacervation.

Dispersion methods

The common event in all dispersion methods is that the encapsulation matrix containing the target species (which are meant to be encapsulated) must be dispersed in the form of droplets in the stabilizing media to form microcapsules. Prilling, nebulization, emulsification and micro-dispersion are different methods of micro-dispersion, which are explained briefly in the following sections.

Figure 5. Ionotropic affinity between alginate and chitosan.

Prilling methods

Basically, in prilling methods, a laminar flow of the encapsulating matrix and the biomolecule target is created by passing it through a narrow nozzle and this flow is broken up in to small droplets having a relatively small dispersion in size (i.e. with a maximum standard deviation of 10 % of the average particle size). The breaking up of such laminar flow is done by applying an electrostatic potential that reduces the surface tension of the flow (having flow rates of a few milliliters per hour) resulting in formation of small droplets. A schematic operating view of such nozzle is shown in Figure 6a³⁸. It can also be performed using a vibrating nozzle which works well with low viscosity solutions and higher flow rates (a few liters per hour).

Other prilling methods involve cutting off the laminar jet using a rapidly rotating disk (Figure 6b³⁸) or grid, which are effective for higher viscosities (more than 200 mPa·s).

In our study we used the vibrating nozzle prilling method because it gives narrow size dispersion of the beads and also because of its compatibility to operate with the viscosity of sodium alginate solutions.

Figure 6. a) Schematic operation view of a laminar flow breaking up using an electrostatic potential. b) Laminar flow breaking up using a rotating disk. (reproduced from reference 38)

a) b)

Nebulization

Nebulization or spray-drying, is a technique in which a flow of the encapsulation matrix is passed through a liquid/air nozzle or sometimes it is jetted to the surface of a spinning disk at high speed. The droplets formed using this method normally have a relatively wide dispersion, i.e. standard deviation of 30 – 50 % of the average particle size. The source of such a wide dispersion is the use of turbulent flow instead of a laminar flow in comparison with prilling methods. The advantage of this method is the availability of its commercialized equipment in industry for large scale productions.

Emulsification

In this method the encapsulation matrix containing the target species is dispersed in another immiscible liquid and the small droplets are formed with the help of violent agitation using a turbine or stirrer. The droplets may have a wide particle size dispersion (standard deviation of 30-50 %) with the high level of agitation there is a possibility of denaturing the target species; for instance, in case of living cells, they may get disrupted under strong agitation.

For industrial large scale productions (tons per hour), a system of continuous flow has been designed using static mixers.

Microdispersion

The principle of microdispersion is the same as emulsification, which is described in Section 1.4.2.1.3, but the dispersion of two immiscible liquids is done with the help of a surfactant. The average droplet size is relatively small (less that one micrometer) and thermodynamically speaking, such micro-emulsions are generally more stable than normal emulsions.

Stabilization methods

After creating small droplets of the encapsulation matrix in the previous dispersion step, a stabilization method is required so that they are solidified to form microcapsules while trapping the target species inside them. The solidification of hot matrices (previously melted)

can easily be achieved by cooling them to the temperature where they become solid once more.

Sometimes it is possible to evaporate the solvent of the encapsulation matrix using dry hot air and the technique is therefore called evaporation. Many of the polymers used as the matrix of encapsulation can be jellified in a jellification process either by using low temperature or certain chelating ions. For instance, agarose can be jellified by lowering the temperature and alginate can be crosslinked using calcium ions. In the polymerization method, a polymeric network is formed by the polymerization of the monomeric units in the presence of the agent to be encapsulated. For instance, in an emulsion we may incorporate two different monomers, one in each phase. These two monomers can react with each other to create a polymer at the interface of the two phases i.e. around each droplet. In the coacervation method, the precipitation of a polymer is induced by changing the physico-chemical parameters of the media such as acidity, ionic strength, etc.

In our study we used the jellification process of alginate using calcium ions because of the mild conditions for stabilization of microcapsules in the presence of biomolecule targets (GOx in this case) without adding a reactive agent or altering the temperature, which could be devastating to the biomolecule and its functionality.