IGEM:IMPERIAL/2009/Encapsulation/Phase2/Alginate

Specifications of encapsulation
These include: 1) protection against low pH 2) attachment in intestines 3) efficient release of the bacteria within the gastrointestine 4) use of materials that are inexpensive, stable, and of food grade 5) Inducibility and possibly 6) protection against oxygen, heat, and other environmental stresses during drying, formulation, and storage

Microencapsulation with alginate
These capsules are primarily composed of alginate, a naturally occurring polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues.

Calcium ions are used to cross-link G-rich regions of the alginate chains, and the resulting calcium alginate (CaAlg) hydrogel beads are coated with crosslinkers to strengthen the bead surface and control permeability. A final coating with alginate is applied in order to hide the PLL from the host(4) and make the capsules biocompatible.

Microencapsulation has been applied to enhance the viability of probiotic bacteria during processing and also for targeted delivery to the gastrointestinal tract.

Benefits:
 * mild gelation conditions
 * biocompatibility
 * biodegradability
 * nontoxicity
 * pH dependency

Method
Bifidobacterial cells were centrifuged and added to alginate solution. These were extruded to 0.1M calcium chloride through the end of a blunt needle using compressed air The cross-linking material were added. The beads were gently stirred and hardened for an hour.

Spheres of diameter 1.5mm formed - corresponded to previous study that gel diameters of 1-3mm needed to protect bifidobacteria

Various materials can be used for coating: polydextrose, soy fibre, skim milk, yeast extract, kappa- carageenen, chitosan and whey protein

Acid resistance
Exposed to simulated gastric juice pH 1.5

in one paper, skimmed milk exhibited highest resistance

testing different materials

Proposed reason:

The increase of the carboxy charges of polymeric matrices serves to - neutralise acid and enhancing the buffering effects This improved gastric stability of entrapped bacteria

moderate protection (22–26 %) afforded by native alginate beads seems related to the availability of D-mannuronate carboxylate groups to intercept proton access

properties of alginate beads

Attachment
The mechanism of sustained drug release is attributable to the fact that alginate is a mucoadhesive polymer; the enhanced gastrointestinal residence time is likely to be responsible for the improvement in drug bioavailability

The ability of chitosan to modulate the intestinal tight junctions is an added virtue, which helps the encapsulated drugs in crossing the permeability barriers

Release
usage as anti-tuberculosis drug carriers

nominal release (less than 7% of the encapsulated drug) in the SGF throughout the 72 h study period.

In the SIF, the release of rifampicin was less (16%) compared with isoniazid (20.6%) or pyrazinamide (22.1%) in the initial 6 h. Subsequently, there was a slow but sustained release of each drug, limited to less than 3% of the encapsulated drug

Drugs encapsulated in alginate–chitosan microspheres attained Cmax at 24 h as against 1 h in the case of orally administered parent drugs. In case of free drugs, the Cmax was achieved instantaneously. The sustained release allows a reduction in dose/dosage frequency

Control: polycationic macromolecules such as chitosan not only stabilize the alginate microspheres but also control the porosity of alginate to enhance the sustained release effect

Proposed reasons:

A decrease in the pH leads to shrinkage in the alginate gel and a reduced permeability of the alginate–chitosan microspheres. In a neutral/alkaline medium, the interpolymeric complex swells and disintegrates to release the drugs, assisted by the sequestration of calcium ions by the phosphate present in the SIF

When pH is lowered below the pKa values of d-mannuronic and l-guluronic acid (3.6 and 3.7, respectively) alginate is converted to alginic acid with release of calcium ions

Membrane Preparation for Alginate coating using a Protein Sequence with high Ca2+ binding affinity
Inspiration from |this article

Protein sequence with a high calcium binding affinity has been characterised. GGXGXDXUX (X, any amino acid; U, large hydrophobic amino acid). In this way we could express this sequence on the surface membrane of our cell (on the fimbria/ membrane proteins etc), and then this would give a calcium coating to the cell. alginate could then be added to the solution, and this should form a capsule around the cell.