Alginates are polysaccharide polymers that are found abundantly in the cell walls of brown seaweed (Phaephyceae). Alginic acid is a linear polymer with homopolymeric blocks of (1-4)-linked β-D-mannuronate and its C-5 epimer α-L-guluronate (Figure 3.4), separated by blocks of random or alternating mannuronic and guluronic acids24.
Figure 3.4 Chemical Structure of a typical alginate monomer. M and G denote the mannuronate
and guluronate blocks, respectively.
The proportion of such blocks as well as the molecular weight of the alginate differs per alginate source and preparation, resulting in different physical properties25. Importantly,
alginic acid can be hydrated forming viscous hydrogels that are heat-stable at room temperature25, where the water molecules are entrapped within the alginate matrix due
to capillary forces, but are still able to move within the polymer26.
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Figure 3.5 The ionotropic cross-linking of alginate using calcium ions. The calcium ions
specifically bind the guluronic acid blocks, and co-ordinated alginate chains overlay adjacent
chains forming an “egg box” structure. Diagram adapted from referenced publication27.
Alginate can exist in a soluble salt form, such as when bound to sodium (NaC6H7O6).
Monovalent metal ions form soluble alginate salts, whilst multivalent cations (such as Ca2+ but excluding Mg2+) cross-link guluronic acid blocks within the alginate to form
ionotropic alginate hydrogels via what is commonly known as the “egg-box” model 28
(Figure 3.5). Covalently bonded alginate hydrogels can also occur when alginate chains are cross-linked using poly(ethylene glycol)-diamines 25. Alginate polymers can
be degraded using enzymatic processes29 or microwaves exposure30. Ionotropic
alginate hydrogels can be easily dissolved using a calcium ion chelation agent such as EDTA 31.
3.2.2.1 Alginate properties and applications
The physical properties of alginate hydrogels are dependent on the ratio of guluronic to mannuronate blocks (G/M ratio), with alginates rich in guluronic presenting higher gel strength, swelling and viscoelasticity due to the higher affinity of guluronic acid residues to divalent ions. Alginates rich in mannuronic acid are believed to offer better long-term stability32. Alginates with a high molecular weight provide gels with more robust
mechanical properties33.
Alginate hydrogels respond to mechanical stress differently depending on whether they are ionically or covalently cross-linked. Ionotropic alginate hydrogels give rise to plastic
123 deformation under mechanical stress, which causes loss of water content and a stochastic reformation of ionic bonds. Covalently linked alginate hydrogels do not experience the dissociation of bonds, and therefore give rise to elastic deformation34.
Alginate and its related salt forms are generally regarded as biocompatible 35-39,
although impurities may remain as it is obtained from natural sources40. Thus, they are
extensively employed within the biomedical, pharmaceutical and cosmetic industries due to their thickening, gel-forming and stabilizing properties41. The ability to
controllably gel alginate into capsules or beads makes it suitable for encapsulation processes for drug delivery25, 32, 39, 42, 43, cell encapsulation38, 44-50 or the formation of
biomimetic, soft matter constructs51, 52. Alginate hydrogels present numerous physical
characteristics that are suitable for these encapsulation purposes, and make them ideal for the encapsulation of DIB networks, as it has already been shown that DIBs can form between aqueous droplets and hydrogels2 and between alginate hydrogel
shapes53. A summary of the physical characteristics of alginate hydrogels is given in
Appendix 4.
Additionally, practical considerations allow alginate to be used to generate monodisperse capsules within a microfluidic device, using gelation methods that can be classified as being either internal or external42. Internal gelation requires an
insoluble calcium salt to be present in the same solution containing alginate, such as calcium carbonate, which can solubilise upon acidification. Conversely, external gelation occurs when an alginate solution is brought into contact with a solution containing divalent cations, where this solution can be miscible with the alginate or not. Gelation method is reported to significantly change certain hydrogel properties such as pore size and matrix density54, and particular purposes will benefit from an
appropriately selected method. For example, for drug delivery, internally gelled alginate hydrogels offer a lower encapsulation efficiency and faster release than externally
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gelled alginate hydrogels42. Internally gelled alginate hydrogels are reported to be more
homogeneous55, with externally gelled alginates beads often reported to contain a
liquid core of the un-gelled alginate solution51, 52, 56, 57.
In the context of the production of droplet interface bilayer networks encapsulated within a hydrogel shell, an internal mode of gelation is more desirable because of the improved homogeneity and permeability of such hydrogels42, 55, which are required in
order to provide with robustness and the ability to interact with an aqueous environment, respectively.
3.2.2.2 Alginate gelation & microfluidics
Alginate solutions are shear-thinning fluids (and therefore Non-Newtonian) 58 of which
role in the rheology of droplet formation is not yet fully understood 59, but is known to be
problematic with regards to droplet monodispersity60, 61. In comparison to Newtonian
fluids, the extensional viscosity of shear-thinning fluids can cause an alginate solution stream to resist droplet pinch-off and give rise to fluid jets which break off into heterogeneous droplets60. Effective capillary numbers can be calculated that account
for shear-thinning effects and used to generate monodisperse droplets via flow rate modulation 59.
Furthermore, it is important that the rate of gelation is controlled in order to avoid early and undesired formation of hydrogel which may obstruct continuous fluid flow, especially at the point of droplet formation. For this reason, an internal gelation method is preferred using low solubility calcium salts such as calcium sulphate and calcium carbonate, as this offers a higher degree of control than external gelation methods, which have been reported to give rise to quick, but poorly controlled gelation62. Using
internal gelation methods, the rate of acidification, and hence gelation, can be extensively controlled63 via the choice and concentration of acid, initial pH of the
125 alginate solution, the use of a buffer solution, and the concentration and particle size of calcium carbonate.
Alginate hydrogels in the form of capsules and beads have effectively been produced using microfluidic methods 47, 48, 64-69 which present with significant advantages in terms
of control, homogeneity and uniformity in comparison to previous methods, including conventional emulsification70 and electrostatic droplet extrusion71. Droplets of oil have
been encapsulated within alginate shells57, 67 using sequential coaxial droplet
generating flows and an external gelation method relying on the partition of un-gelled O/W/O emulsion droplets from an oil phase into an aqueous phase containing calcium chloride67.