BACKGROUND a. Field of the Invention

The present invention relates to products for delivering viable bacteria or other microorganisms to the human or animal digestive tract, and to methods of making such products. b. Related Art

Fecal microbiota transplant (FMT) is a known therapeutic method for delivering viable and beneficial bacteria to the digestive tract, and in particular to the intestinal tract, of an animal, including humans. During such therapy the stool of a healthy individual, containing fecal bacteria, is transplanted into a patient. FMT has been demonstrated as a therapy or treatment for patients showing Clostridium difficile infection (CDI). FMT is described in: S. Han, S. Shannahan, and R. Pellish,“Fecal microbiota transplant: Treatment options for Clostridium difficile infection in the intensive care unit,” J. Intensive Care Med., vol. 31, no. 9, pp. 577-586, 2016. CDI is reported to cause several diseases, such as diarrhea, pseudomembranous sepsis, and toxic megacolon. In the worst case CDI can be fatal.

FMT has also been reported as a therapy or treatment for gastrointestinal diseases, for example inflammatory bowel disease (P. Moayyedi, J. K. Marshall, Y. Yuan, and R. Hunt, “ Canadian Association of Gastroenterology position statement: Fecal microbiota transplant therapy,” Can. J. Gastroenterol. Hepatol., vol. 28, no. 2, pp. 66-68, 2014).

Currently, FMT is administered by nasojejunal tubes, colonoscopy or orally by an encapsulated product (S. D. Goldenberg et al.,“ Comparison of Different Strategies for Providing Fecal Microbiota Transplantation to Treat Patients with Recurrent Clostridium difficile Infection in Two English Hospitals: A Review,” Infect. Dis. Then, vol. 7, no. 1, pp. 71-86, 2018). These ways to administer the FMT are commonly seen as unappealing.

It has been demonstrated that viable microbial cells could be encapsulated within hollow poly(organosiloxane) microcapsules by templating water-in-oil Pickering emulsion droplets via the interfacial reaction of alkylchlorosilanes ( J . van Wijk, T. Heunis, E. Harmzen, L. M. T. Dicks, J. Meuldijk, and B. Klumperman, “Compartmentalization of bacteria in microcapsules,” Chem. Commun., vol. 50, no. 97, pp. 15427-15430, 2014). Viable cells have also previously been encapsulated in gels, including hydrogels, calcium alginate and sol-gel products. However, encapsulated bacteria may not be suitable for forming viable colonies in the gastrointestinal tract because the cells are retained within the capsule wall.

A particle stabilized emulsion or Pickering emulsion is an emulsion that is stabilized by solid particles such as silica. In principle, a broad variety of monomers or crosslinkers can be used for template immobilization via a Pickering emulsion polymerization. Polymerisation of Pickering emulsions stabilized by silica nanoparticles was performed using methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) as functional monomer and crosslinking agent ( Shen , X., & Ye, L. (2011). Interfacial molecular imprinting in nanoparticle-stabilized emulsions. Macromolecules, 44(14), 5631-5637). In another approach, polymerization of 2- (dimethylamino)ethyl methacrylate (DMAEMA) and EGDMA in Pickering emulsions stabilized by silica particles and Triton X - 100 was performed {Zhou, T., Kamra, T., & Ye, L. (2018). Preparation of diclofenac-imprinted polymer beads for selective molecular separation in water. Journal of Molecular Recognition, 31(3), 1-7). It has also been revealed that functional monomers 4-vinylpiridine, 2-(dimethylamino)ethyl methacrylate (DMA) and acrylamide, as well as the crosslinkers EGDMA and divinylbenzene (DVB) can be used for the polymerization of Pickering emulsions stabilized by silica nanoparticles and Triton X - 100 ( Kujawska , M., Zhou, T., Trochimczuk, A. W., & Ye, L. (2018). Synthesis of naproxen - imprinted polymer using Pickering emulsion polymerization. Journal of Molecular Recognition, 31, 1- 12) . The use of Pickering emulsion polymerization to synthesize polymer beads with microbial recognition sites has been reported (X. Shen et al.,“Bacterial imprinting at pickering emulsion interfaces,” Angew. Chemie - Int. Ed., vol. 53, no. 40, pp. 10687-10690, 2014). Bacteria were first treated with a vinyl-containing pre-polymer, N-acryl chitosan (NAC) to provide a bacterial template, and then used to stabilize an oil-in-water emulsion composed of cross-linking monomers that were dispersed in an aqueous buffer. Polymerization of the oil phase, followed by removal of the bacterial template, resulted in well-defined polymer beads bearing bacterial imprints.

The strategy of using the chitosan bacteria network to produce microspheres with bacteria immobilized onto their surface was also used for adsorption and biodegradation of organic compounds ( Cai , Y., Yang, S., Chen, D., Li, N., Xu, Q., Li, H., ... Lu, J. (2017). A novel strategy to immobilize bacteria on polymer particles for efficient adsorption and biodegradation of soluble organics. Nanoscale, 9(32), 11530-11536). They immobilized Pseudomonas putina and Paracoccus dentrificans on microspheres and reported that the immobilized bacteria were able to biodegrade the organic compounds phenol and DMF. Their aim was to produce material feasible for removing organic compounds from wastewater. They showed that it is possible to immobilize different kinds of bacteria. They also claimed: “Moreover, it is worth noting that this method could also be used to immobilize bacterial cells on many other functional polymer matrixes for the removal of contaminants in wastewater.” However, they did not present any results which would support their statement in this paper.

It has further been demonstrated that“intact, washed bacteria alone could stabilize oil-water emulsions by attaching to the interface, analogous to silica particles with contact angles intermediate between water-wet and oil-wet.” ( Dorobantu , L. S., Yeung, A. K. C., Foght, J. M., & Gray, M. R. (2004). Stabilization of Oil-Water Emulsions by Hydrophobic Bacteria. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70(10), 6333-6336). Their experiments were based on four different bacteria strains ( P . fluorescens, R. suberifaciens, R. erythropolis, A. venetianus), which were tested to stabilize oil in water emulsions of hexadecane/water. Their results revealed that the ability of bacteria to stabilize such a Pickering emulsion depends on the contact angle it has at the oil/water interface which in turn depends on the hydrophilicity/hydrophobicity of the bacteria surfaces. Their work clearly demonstrates that in principle only bacteria are needed to stabilize a Pickering emulsion, although not every bacteria strain is feasible to stabilize any oil phase.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a method of making polymer beads with viable gastrointestinal microorgansims attached to the surface of the beads, for delivering viable gastrointestinal microorganisms, the method comprising:

a) preparing an emulsion stabilised by live gastrointestinal microorganisms; and b) converting the emulsion to polymer beads with viable gastrointestinal microorganisms attached to surfaces of the beads.

In some embodiments step a) preferably comprises:

combining an organic monomer phase and an aqueous phase containing the live gastrointestinal microorganisms to produce a two-phase mixture;

emulsifying the two-phase mixture to produce an emulsion comprising droplets of monomer phase stabilised by live gastrointestinal microorganisms; and step b) preferably comprises:

polymerising the monomer droplets to produce polymer beads with viable gastrointestinal microorganisms attached to surfaces of the beads.

The monomer phase preferably comprises a mixture of styrene and a cross-linking agent, preferably 1 ,4- divinylbenzene. The monomer phase may further include a polymerization initiator, preferably 2,2’-azo(bis)(2-methylpropionitrile) (AIBN). The polymerization is preferably carried out at a temperature in the range 35-39°C, more preferably about 37°C.

In other embodiments step a) preferably comprises:

combining a solution of a polymer in an organic solvent and an aqueous phase containing the live gastrointestinal microorganisms to produce a two-phase mixture;

emulsifying the two-phase mixture to produce an emulsion comprising droplets of solvent phase stabilised by live gastrointestinal microorganisms;

and wherein step b) comprises:

evaporating at least some of the solvent phase to produce polymer beads with viable gastrointestinal microorganisms attached to surfaces of the beads.

The polymer may be poly-(L,L-lactide).

The method may further comprise the steps of removing the aqueous phase and washing the beads with water.

The aqueous phase preferably comprises the gastrointestinal microorganism in a lysogeny broth (LB) medium. The aqueous phase is preferably prepared by adding the gastrointestinal microorganism to the LB medium without pre-treatment of the gastrointestinal microorganism with a pre-polymer.

The gastrointestinal microorganism may be a non-spore-forming bacterium, preferably E. coli.

A second aspect of the present invention provides a polymer particle with viable gastrointestinal microorganisms attached to the surface of the particle, obtainable by the method according to the first aspect of the invention.

A third aspect of the present invention provides a polymer particle according to the second aspect of the invention for use in fecal microbiota transplant (FMT) therapy.

A fourth aspect of the present invention provides a polymer particle according to the second aspect of the invention for use in transporting probiotics into the human or animal gastrointestinal tract.

A fifth aspect of the present invention provides a polymer particle according to the second aspect of the invention for use as a medicament.

A sixth aspect of the present invention provides a polymer particle according to the second aspect of the invention for use in the treatment of Clostridium difficile infection.

A seventh aspect of the present invention provides a polymer particle according to the second aspect of the invention for use in the treatment of gastrointestinal diseases.

An eighth aspect of the present invention provides a polymer particle according to the second aspect of the invention for use in the treatment of inflammatory bowel disease.

It has surprisingly been found that it is possible to synthesize polymer (micro)beads with viable gastrointestinal bacteria on their surfaces. The bacteria are firmly bound to the surfaces but can nonetheless form bacteria colonies when provided with a suitable growth medium. The invention is particularly useful for providing microbeads with viable bacteria inhabiting the human gastrointestinal tract (eg E. coli).

Polymer microbeads with viable fecal bacteria attached may be used in FMT therapy and are envisaged to provide improved patient acceptance and standardization, because they can be produced in“clean” industrial environments.

In addition to use in FMT therapy, other applications are envisaged for providing beneficial substances to the human or animal digestive tract, for example for transporting probiotics to the human gastrointestinal tract.

The term “viable” is used herein to mean that the microorganism is capable of reproducing to produce a microorganism colony if provided with a suitable nutrient medium; for example a lysogeny broth (LB) medium for bacterial cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 shows chemicals used in a polymerization reaction in an embodiment of the present invention;

Figure 2 is a schematic diagram of a process in accordance with an embodiment of the invention;

Figure 3 shows a viability test of polymer beads in accordance with an embodiment of the invention, on LB-agar plates;

Figure 4 is a scanning electron microscopy image of a polymer bead with E. coli attached to its surface, in accordance with an embodiment of the invention; and

Figure 5 is a scanning electron microscopy image of E. coli attached to the bead surface of Figure 4.

DETAILED DESCRIPTION

Figure 1 illustrates the chemicals used for the polymerization. Generally, 0.5mL Styrene (Merck KGaA, 99% purity) and 0.5mL divinylbenzene (Merck KGaA, 60% - 65% isomer ratio) were mixed together and 18mg of AIBN (Sigma Aldrich, 98% purity), which works as an initiator of the polymerization, was diluted in the monomers. The stabilizer in styrene and divinylbenzene was removed before synthesis.

Figure 2 illustrates the procedure of the Pickering emulsion polymerization which was used to synthesize polymer beads with viable E. coli attached to its surface. The procedure was performed under sterile conditions. For this, the monomer solution was transferred into a sterile falcon tube. A washed pallet of cultivated E. coli W (ATCC 9637) was suspended in 4ml_ autoclaved LB- medium (1 Og/L Proteose Peptone, 5g/L NaCI, 5g/L Yeast Extract and 1 g/L a-D-Glucose monohydrate). 1.2mL of the E. coli suspension was transferred to the falcon tube with the monomers. The mixture in the falcon tube contained two separate phases which could be emulsified by shaking for 30 seconds. This was a surprising result, as X. Shen et al had found that no stable Pickering emulsion could be obtained when the bacteria or NAC were used on their own. Afterwards, the polymerization was performed for 16.5h at 37°C. The resulting polymer beads contained viable E. coli attached to their surface.

Figure 3 explains the viability test, which was performed to ensure, that E. coli has survived the polymerization. The viability test was performed under sterile conditions. For this, the liquid phase was removed after the polymerization and the beads were washed two times with autoclaved water. Afterwards, the beads were dried in a sterile fume hood and spread onto LB- agar plates (15g/L Agar, 10g/L Proteose Peptone, 5g/L NaCI, 5g/L Yeast Extract and 1 g/L a-D-Glucose monohydrate). The plates were incubated for 48 hours at 37°C. After the incubation E. coli colonies were observable around the bead, which confirmed the viability of the bacteria on the polymer beads.

Figure 4 shows a scanning electron microscopy image captured from a polymer bead which was synthesized as described above. It is clearly observable that E. coli is attached all over the surface of the beads, which ensures the loading of a large number of bacteria per bead. A closer look on the arrangement of E. coli on the surface of the beads is illustrated in Figure 5.

Although the invention has been illustrated with respect to the use of styrene monomer, it will be understood that the monomer type is not limited providing that the resulting product is physiologically acceptable to a human or animal subject. In particular, it is considered that biodegradable polymer matrices may be employed as the bead-forming material.

The invention has been illustrated with reference to thermal-initiated polymerization. However, it will be understood that the polymerization could alternatively be photo- initiated for suitable monomers, providing that the wavelength and intensity of light used does not render the bacteria or other microorganism non-viable.

However, other techniques than emulsion/suspension polymerization can be used to fabricate biodegradable polymer microspheres: One opportunity to synthesize such polymers is the solvent evaporation method. An example of biodegradable polymer spheres synthesized by this method is reported by Ito and Kawakani, who fabricated poly(lactide-co-glycolide) (PLGA) nanospheres in an oil in water emulsion via the solvent evaporation method {Ito, F., & Kawakami, H. (2015). Colloids and Surfaces A : Physicochemical and Engineering Aspects Facile technique for the preparation of monodispersed biodegradable polymer nanospheres using a solvent evaporation method. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 482, 734-739 ). The work of Demina et al. demonstrates that the solvent evaporation method can be combined with Pickering emulsion stabilization. They synthesized poly-(L,L-lactide) microparticles via solvent evaporation in an emulsion stabilized by chitin nanocrystals ( Demina , T. S., Sotnikova, Y. S., Istomin, A. V, Grandfils, C., Akopova, T. A., & Zelenetskii, A. N. (2018). Preparation of Poly ( L , L-Lactide) Microparticles via Pickering Emulsions Using Chitin Nanocrystals. Advances in Materials Science and Engineering, 2018, 1-8). Without wishing to be bound by theory, we believe that immobilization of bacteria on polymer microspheres, notably biodegradable polymer microspheres, can alternatively be achieved via the combination of Pickering emulsion stabilization followed by subsequent solvent removal by evaporation.

Other modifications and variations not explicitly disclosed above may also be contemplated without departing from the scope of the invention as defined in the appended claims.