IGEM:IMPERIAL/2009/Encapsulation/Phase2

Alginate-chitosan-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

Sporulation

An endospore is a dormant, tough, and non-reproductive structure produced by Gram-positive bacteria. They form through the production of an encapsulating spore coat within the spore-forming cell. Examples include Bacillus and Clostridium.

Sporulation and E.coli

E.coli is non-sporulating. Therefore the idea to clone the genes for sporulation from Bacillus subtilis into E.coli was investigated. A paper (below) used the amino-acid sequence deduced from the nucleotide sequence of the spolIAC gene of Bacillus subtilis which has been shown to be homologous to that of the sigma subunit of the Escherichia coli RNA polymerase. Results show that this gene can be cloned in E. coli only under conditions in which it is not expressed.

E.coli and sporulation.

The sigma-like products of the sporulation gene spolIA C of Bacillus subtilis is toxic to Escherichia coli.
The main chassis we would therefore use is B.subtilis as it is the easiest to manipulate and well characterised.

It is now agreed that spore formation in bacteria is a form of differentiation in which there is an ordered, temporal sequence of events and a degree of commitment.

Bacteria will produce a single endospore internally. The spore is sometimes surrounded by a thin covering known as the exosporium, which overlies the spore coat. The spore coat, which acts like a [sieve] that excludes large toxic molecules like lysozyme, is resistant to many toxic molecules and may also contain enzymes that are involved in germination. The cortex lies beneath the spore coat and consists of peptidoglycan. The core wall lies beneath the cortex and surrounds the protoplast or core of the endospore. The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs, that protect the spore DNA from UV radiation and heat. The core also contains normal cell structures, such as ribosomes and other enzymes, but is not metabolically active.

The Sporulation Process & Genes Involved At Each Stage

Sporulation in Bacillus subtilis is a fertile system for studying development because of the detailed genetic understanding of this process. A summary from the paper is shown, identifying the key steps in the sporulation process.

1. Entry into sporulation

• Spo0A, which in its phosphorylated form stimulates expression of early sporulation genes and inhibits the expression of genes whose function is to prevent sporulation.
• SinR is required for the development of competence and motility, but inhibitory for sporulation and the production of exoproteases.
• SinI binds tightly to SinR, thus preventing SinR from binding to promoter DNA.

2. Final commitment

• SpoIIAB can bind to sigmaF and prevent sigmaF from combining with core RNA polymerase and transcribing from sigmaF directed promoters.

3. Spore coat synthesis and assembly

• GerE is the latest acting of four regulatory factors (sigmaE-SpoIIID-sigmaK-GerE) which ensure the correct temporal appearance of gene products in the mother cell.
• GerE is a 74 residue DNA-binding protein that acts both as a repressor and as an activator of expression of a number of coat proteins. Its function is to control the synthesis and assembly of the spore at the level of transcription.
• SpoIVA induced by sigma E and controls the assembly of a ring of CotE proteins.
• Coat protein genes (eg CotG) induced under the control of sigma K and the DNA-binding protein GerE.

Control, Genes Involved and Potential Solution?

There are a number of transcription factors that regulate each stage of the sporulation process. Each has a number of operons that it influences.

At the sporulation initiation stage, Spo0A controls 62 operons (found using DBTBS). The exact function of each gene is not known. However, there have been various studies done on isolating and mutating a specific gene to find the effect it has on the coat formation produced.
Therefore, the mechanism known is more of a 'linked' pathway and the role of all individual genes is not known.

Therefore, instead of finding the minimum number of genes required for each stage, an idea we came up with was to keep as many genes as possible and add in an inducible promoter for production of the initial stage of sporulation. Therefore, we can control Spo0A without subjecting it to nutrient starvation processes. This can be done in a plasmid in vitro and then inserting this into the cell.

Since the forespore will be the part producing the spore (as the mother cell lyses), it is important for our protein produced and contained within the forespore. As a result we would try to target the sigma factors that are only expressed in the forespore to produce the protein we require to transport. An example is sigma G. For this a promoter needs to be found that acts on sigma G, and the protein gene placed under the control of this promoter. The next steps in the sporulation process will be the same.

However, the process produces a number of extracellular and intracellular proteases that may degrade the protein we are trying to deliver. For this purpose, the genes involved in the production of protein degrading enzymes has been investigated (see paper below).

• So if we want to control the initial step we would need to modify the chromosome, which in B.subtilis is more feasible than in other chassis such as E.coli. Tom and Chris have recently ordered a B.subtilis sporulation deficient strain, might be interesting to read how this was done.
• Id be interested to hear more of a comparison about the physical properites of the spore vs the other encapsulation methods you have looked at. Tianyi has outlined some key features that need to be met, e.g. resistance to pH as low as 2, stable at body temperature

The spore offers unique resistance properties and can survive under extreme conditions, such as excessive temperature, desiccation, and exposure to solvents and other noxious chemicals.
These features would make the spore an ideal vehicle for delivery of heterologous antigens to extreme environments such as the gastrointestinal tract.

Oral administration of a Bacillus subtilis spore-based vaccine

Useful Links

Link to a patent describing the surface display of enzymes on spore proteins

Structural studies of Key Regulators of Bacterial Sporulation