WO2021022134A1

WO2021022134A1 - Bioactive compositions and applications thereof

Info

Publication number: WO2021022134A1
Application number: PCT/US2020/044453
Authority: WO
Inventors: Aaron ANSELMO; Kunyu QIU; Isabella YOUNG; Blaide WOODBURN
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 2019-08-01
Filing date: 2020-07-31
Publication date: 2021-02-04

Abstract

In one aspect, bioactive compositions are described herein comprising a composite film including one or more bacterial species disposed in a polymeric matrix, the polymeric matrix comprising at least one biocompatible polymer. In some embodiments, the one or more bacterial species are embedded in the polymeric matrix. The polymeric matrix may also comprise one or more components of a bacterial culture medium, such as MRS.

Description

BIOACTIVE COMPOSITIONS AND APPLICATIONS THEREOF

RELATED APPLICATION DATA

The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to United States Provisional Patent Application Serial Number 62/881,745 filed August 1, 2019, which is incorporated herein by reference in its entirety.

FIELD

The present invention relates to bioactive compositions and, in particular, to bioactive compositions comprising composite films including one or more bacterial species.

BACKGROUND

Recent interest in the microbiome is partly due to its role in regulating human health and disease. Significant efforts have described connections between the microbiome and diseases such as cancer, metabolic disease, depression, and diabetes. Resulting from these initial findings and correlations, efforts to modulate the microbiome composition to provide therapeutic benefit represent a highly active area of research. To date, the most established approaches to modulate the microbiome are the use of antibiotics, changes to diet, and the administration of microbes that were harvested from a host source. To a lesser extent, efforts utilizing prebiotics and probiotics have also been described; although, microbiome modulation resulting from prebiotic and probiotic use remains a debated topic. In any case, the administration of living bacteria in the form of probiotics or microbe-based therapeutics has been investigated as an approach to modulate the microbiome. The advantages of therapeutic bacteria over small molecule drugs and biologies are their abilities to modulate multiplex host physiology, re-establish dysbiotic microbiomes and sustainably produce therapeutics in situ. Examples of this include fecal transplants, engineered probiotics, or spore consortia. Many of these microbe-based

therapeutics have been investigated and shown to be efficacious in clinical trials for the treatment of IBD or Cdiff However, the delivery of living microbes to the GI tract represents a significant challenge due to the susceptibility of microbes to their surrounding environment. As such, there is growing need to design formulations for bacteria that can improve their storage and delivery.

Currently, the commonly used methods for manufacturing orally delivered bacteria in clinical settings include lyophilization followed by capsule encapsulation and direct freezing of bacteria slurry at extremely low temperature. Due to the harsh conditions during lyophilization, viability of bacteria will almost inevitably decrease even though the protectants such as saccharides are added. Freezing bacteria slurry at -80°C or even lower temperature can well preserve viability for a very long time, however, it demands specific equipment and thus not applicable to daily storage for patients in reality.

Additionally, spatiotemporal distribution of bacteria along the GI tract has been shown to play an important role in the interaction with the host as well as the existing microbiome. Thus, it calls for sophisticated design of delivery systems for bacteria to maximize therapeutic effects while minimizing safety issues. However, lyophilized bacteria powder and frozen slurry are challenging to further tune their delivery.

SUMMARY

In view of the foregoing disadvantages, new compositions are needed for the storage and delivery of living bacterial species to the GI tract. In one aspect, a bioactive composition described herein comprises a composite film including one or more bacterial species disposed in a polymeric matrix, the polymeric matrix comprising at least one biocompatible polymer. In some embodiments, the one or more bacterial species are embedded in the polymeric matrix.

The polymeric matrix may also comprise one or more components of a bacterial culture medium, such as MRS. The polymeric matrix, in some embodiments, comprises a mixture of

biocompatible polymers. Two or more biocompatible polymers of the mixture can exhibit different degradation rates for controlling bacterial release profile of the composite film, in some embodiments. Moreover, loading of the bacterial species in the composite film is at least 10⁸ CFU, in some embodiments.

In another aspect, methods of making bioactive compositions are described herein. In some embodiments, a method of making a bioactive composition comprises providing a mixture including one or more bacterial species disposed in a polymeric solution, the polymeric solution comprising at least one biocompatible polymer, and depositing the mixture on a substrate. The mixture is subsequently dried to form a composite film comprising the one or more bacterial species embedded in a polymeric matrix, the polymeric matrix comprising the at least one biocompatible polymer.

These and other embodiments are further described in the following detailed description BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and IB illustrate various aspects of delivering bacterial species to a tissue environment via composite films described herein, according to some embodiments.

FIG. 2 is a schematic of a synthesis process for L. casei composite films, according to some embodiments.

FIG. 3 is a schematic of a synthesis process for L. casei composite films, according to some embodiments.

FIG. 4 is a schematic of a synthesis process for L. casei composite films, according to some embodiments.

FIG. 5A is a scanning electron microscopy (SEM) image of L. casei ATCC393 employed in composite films, according to some embodiments.

FIG. 5B is a top down SEM of a composite film comprising L. casei ATCC393, according to some embodiments.

FIG. 5C is a cross-section SEM of a composite film comprising L. casei ATCC393, according to some embodiments.

FIG. 5D is a camera image of the composite film of FIGS. 5B and 5C.

FIG. 6A provides spectra of . casei ATCC393, film without L. casei ATCC393, and films with 10⁸ or 10⁹ L. casei ATCC393 loading, according to some embodiments.

FIG. 6B provides the Zeta potential for plain L. casei ATCC393 and L. casei ATCC393 after exposure to 3% PVA, 5.2% MRS, and 1% glycerol. Error bars represent standard deviation (n = 3).

FIG. 7A illustrates the viability of L. casei ATCC393 with different combinations of excipients at 4°C.

FIG. 7B illustrates the viability of L. casei ATCC393 with different combinations of excipients at 37°C.

FIG. 8 illustrates weight loss percentage from films containing 10⁸ L. casei ATCC393, according to several embodiments.

FIG. 9 illustrates storage of L. casei ATCC393 at room temperature in composite films with MRS and glycerol using different polymers. Each error bar represents standard deviation (n=3). FIG. 10A illustrates microbe distribution on the intestinal surface under fast and slow release conditions, according to some embodiments.

FIG. 10B provides intestinal surface coverage of microbes under fast and slow release conditions, according to some embodiments.

FIG. IOC depicts storage of . casei ATCC393 in 3%PVA+2%NaCMC composite films at 4°C.

FIG. 10D illustrates microbe release kinetics from 3%PVA films with 0%, 1%, or 2% NaCMC added, according to some embodiments.

FIG. 10E illustrates microbe release kinetics from 3%PVA+2%NaCMC at various film thicknesses, according to some embodiments.

FIG. 11 A illustrate apparatus for measuring mucoadhesion of composite films described herein, according to some embodiments.

FIG. 1 IB illustrates results of mucoadhesion testing according to some embodiments.

FIG. 12A illustrates a rhodamine-dyed composite film, according to some embodiments. FIG. 12B illustrates folding of the composite film of FIG. 12 A.

FIGS. 12C and 12D illustrate insertion and capping of the composite films of FIG. 12A into a 00-sized oral capsule.

FIG. 12E illustrates ten rhodamine-dyed composite films and an empty 00-sized capsule. FIGS. 12F and 12G illustrate insertion and capping of the ten composite films of FIG.

12E into a 00-sized oral capsule.

FIG. 12H characterizes storage of composite films (3%PVA+MRS+glycerol with 10^X /.. casei ATCC393) in 00-sized capsules, according to some embodiments.

FIG. 121 characterizes capsule dissolution time with or without film inclusion, according to some embodiments.

FIG. 12J provides statistical analysis conducted using one-way ANOVA followed by post hoc Dunnett’s test (statistical significance defined at p < 0.05). *: significantly different from initial loading.

DETAILED DESCRIPTION

Embodiments described herein can be understood more readily by reference to the following detailed description and examples and their previous and following descriptions. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

I. Bioactive Compositions

As described herein, a bioactive composition comprises a composite film including one or more bacterial species disposed in a polymeric matrix, the polymeric matrix comprising at least one biocompatible polymer. In some embodiments, the one or more bacterial species are embedded or encapsulated in the polymeric matrix.

Turning now to specific components, the polymeric matrix can comprise any

biocompatible polymer or mixture of biocompatible polymers not inconsistent with the technical objectives described herein. In some embodiments, biocompatible polymer of the matrix comprises polyvinyl alcohol (PVA). One or more biocompatible polymers of the matrix can also be selected from Table I.

Table I - Matrix forming biocompatible polymers

Biocompatible polymers of the matrix can exhibit any desired number average molecular weight or weight average molecular weight. Specific identity of biocompatible polymer(s) employed in matrix formation can be selected according to several considerations including, but not limited to, identity of the bacterial species carried by the matrix, desired degradation and release characteristics of the matrix, and/or desired tissue adhesion characteristics of the matrix. In some embodiments, for example, the bacterial release profile from the matrix can be tuned by employing two or more polymers of differing degradation rates.

As described herein, one or more bacterial species are carried by the polymeric matrix.

In some embodiments, the bacterial species are embedded within or encapsulated by the polymeric matrix. Any bacterial species not inconsistent with the technical objectives detailed herein can be used. In some embodiments, the one or more bacteria are probiotics. Bacteria carried by the polymeric matrix can be aerobic or anaerobic. Bacteria carried by the polymeric matrix, in some embodiments, can be selected from the Lactobacilli genus, Bifidobacteria genus, and other lactic acid bacteria, including Lactococci and Streptococci. Additional probiotic strains comprise bacterial genera Bacillus , Escherichia , and Propionibacterium. Table II provides a non-limiting listing of various bacterial strains for use with polymeric matrices of composite films described herein.

Table II - Bacterial Strain of Composite Films

In some embodiments, the one or more bacterial species are dispersed throughout the polymeric matrix. Alternatively, the bacterial species may be heterogeneously distributed in the polymeric matrix. Any desired loading of bacterial species can be present in the polymeric matrix. A composite film, for example, can exhibit an initial bacterial loading of at least 10⁸ CFU. In some embodiments, the initial bacterial loading in the polymeric matrix is 10⁸ to 10¹² CFU. Moreover, the bacteria of the composite film can exhibit viability for extended periods of time after incorporation in to the polymeric matrix. In some embodiments, the bacteria can remain viable for at least 6 months or at least one 1-year subsequent to polymeric incorporation when the composite film is stored at a temperature of 4°C. Additionally, the bacteria can exhibit viability for at least one week subsequent to polymeric incorporation when the composite film is stored at room temperature. In some embodiments, the pre-film formulation can exhibit viability after storage of the composite film mixture (prior to film formation) at temperatures as low as - 80°C for at least one week, and subsequently maintain the properties needed to form films.

Embedding or encapsulating the bacteria in the polymeric matrix can also eliminate or reduce oxygen exposure, thereby mitigating oxygen-mediated viability loss for anaerobic bacterial species. In some embodiments, bacterial loading of the composite film decreases less than an order of magnitude over the storage time periods described herein.

Composite films described herein can further comprise one or more components of bacterial culture media, in some embodiments. The composite film, for example, can comprise one or more components of MRS. Alternatively, the composite film can comprise the complete culture medium composition, such as a complete formulation of MRS. The culture medium, in some embodiment, can comprise milk, such as skim milk. A culture medium can be present in the polymeric matrix of the composite film in any desired amount. In some embodiments, one or more culture media are present in an amount of 0.1 to 20 weight percent or 1 to 15 weight percent of the polymeric matrix.

One or more additive may also be present in the polymeric matrix. Additive can be employed to vary rheological properties of the composite film. In some embodiments, the additive comprises a plasticizer, such as glycerol.

The polymeric matrix of the composite film can have any desired thickness. In some embodiments, thickness of the polymeric film ranges from 100 pm to 3 mm. Thickness of the polymeric film can be controlled in the fabrication methods described herein.

FIGS. 1 A and IB illustrate various aspects of delivering bacterial species to a tissue environment via composite films described herein, according to some embodiments. As illustrated in FIG. 1 A, composite films (MBT-film) are formed followed by encapsulation in oral capsules with enteric coatings. After oral administration, the capsules pass through the stomach, dissolve and release the composite film in the intestines. In FIG. IB, the polymeric matrix of the composite films can be engineered for mucoadhesion and tunable release for modifying the spatiotemporal distribution of the bacterial species along the intestines.

II. Methods of Making Bioactive Compositions

The mixture comprising the one or more bacterial species disposed in polymeric solution can be fabricated in various manners. FIG. 2 is a schematic of the synthesis process for /.. casei ATCC393 composite films, according to some embodiments. Poly(vinyl alcohol) (PVA), MRS broth, and glycerol were first dissolved in water and then L. casei ATCC393 was added to the aqueous mixture. The mixture was then cast into 24-well plates followed by a 2-day dehydration process. FIG. 3 is a schematic for the synthesis process for L. casei ATCC393 composite films. The steps in FIG. 3 are similar to those in FIG. 2. FIG. 4 is a schematic of an alternative synthesis process relative to FIGS. 2-3 for L. casei ATCC393 composite films, according to some embodiments.

The composite films can be dried under various conditions. In some embodiments, the composite films are dried at a temperature of 1-10°C or 2-5°C. In other embodiments, the composite films can be dried in a desiccator or anaerobic chamber at room temperature. Drying conditions can be selected according to several considerations including, but not limited to, specific composition of the polymeric matrix and specific identity of the microbes embedded in the polymer matrix.

Composite films formed according to methods described herein can have any

compositions and/or properties set forth in Section I above.

These and other embodiments are further illustrated in the following non-limiting examples. EXAMPLE 1 - Composite Films

Synthesis and characterization ofL. casei ATCC393 in Composite b4BT) films

Polymer solution casting is a facile and scalable approach for fabricating polymeric films that contain a variety of excipients and payloads. Here, this approach was used to synthesize MBT-films of different polymer compositions with a number of excipients of distinct physicochemical properties for /.. casei ATCC393 encapsulation. Briefly, aqueous mixtures of polymer, excipients, and bacteria were mixed and cast onto polystyrene well-plates where the water was then allowed to evaporate at 4°C, leaving behind the solids in the form of a polymeric MBT-film, as illustrated in FIG. 2.

Poly(vinyl alcohol) (PVA) was chosen as the main polymer encapsulant for the present examples because of its widespread use in FDA-approved oral formulations and minimal cytotoxicity, established use as a film-former, and compatibility with additive blending approaches to make multifunctional composite films. Scanning electronic microscopy (SEM) was used to determine if L. casei ATCC393 (FIG. 5 A) was encapsulated in PVA. SEM imaging of an intact MBT-film with L. casei ATCC393 from a top-down view demonstrated that L. casei ATCC393 were not visible on the surface or exterior of MBT-films (FIG. 5B). However, SEM imaging of MBT-film cross-sections revealed the presence of L. casei ATCC393 inside the MBT-films, confirming encapsulation (FIG. 5C). Potential surface interactions between PVA and L. casei ATCC393 were then studied with both FTIR and zeta potential measurements. Minor differences of -OH related peak shifts in FTIR were observed when comparing solid formulations with and without L. casei ATCC393 (FIG. 6A), which indicate potential interactions between PVA and /.. casei ATCC393. Zeta potential measurements were performed to compare L. casei ATCC393: (i) without treatment, and (ii) exposed to MRS, glycerol and 3% PVA, followed by extensive washing. Following exposure to film constituents, zeta potential of L. casei ATCC393 slightly increased (FIG. 6B). This slight change indicates that possible interactions between neutrally charged PVA and the bacterial surface may occur at these specific conditions. Storage ofL. casei ATCC393 in MBT-films

Film formation involves dehydration which can cause loss of microbe viability as shown with other MBT dehydration approaches such as lyophilization. As such, it was sought to develop films for long-term storage of L. casei ATCC393 by including storage excipients within the films. DeMan-Rogosa-Sharpe (MRS) broth, a lactobacillus-specific culture formulation, was added as an excipient component of the film because it was hypothesized that the constituents of MRS broth (e.g. sugars, amino acids) could provide storage benefits as they have been shown to protect other sensitive cargos like biologies. Furthermore, it was confirmed that MRS broth exhibited minimal toxicity against intestinal epithelial cells. Glycerol was also investigated as an excipient since it is the most commonly used protectant for microbe storage. Additionally, glycerol is a known plasticizer that grants flexibility to the films, a feature that could allow the films to be manipulated into different geometries. These excipients were investigated

individually and in combination, both with film-encapsulated L. casei ATCC393 and non- encapsulated (plain) L. casei ATCC393. MBT-films that contained all excipients maintained structural integrity after dehydration and removal from well-plate templates (FIG. 5D), and exhibited high consistency in loading.

Storage of L. casei ATCC393 was first investigated at 4°C (FIG. 7 A), to evaluate the role that each of the above excipients play at long-times. Plain L. casei ATCC393 (no excipients or polymers) suffered a nearly 4 log reduction in CFU after 1 week and were undetectable thereafter. The main conclusion was that MRS-containing formulations provided significantly higher maintenance of L. casei ATCC393 as compared to non-MRS formulations at each timepoint. MRS alone, without PVA and glycerol, provided the largest benefit to L. casei ATCC393 storage at 1 year at 4°C. Interestingly, a possible trend at increasing PVA

concentration was observed, despite all PVA-containing formulations demonstrating

significantly lower CFU maintenance as compared to MRS alone. To investigate the possible trend with PVA further, a separate storage study was performed at an increased temperature of 37°C (FIG. 7B), which mimics developing- world conditions, and at PVA amount ranging from 1 to 20%. Complete loss of L. casei ATCC393 CFU viability was lost after three days of storage in all groups, except those with 10% and 20% PVA concentration (FIG. 7B), indicating that PVA, and higher PVA percentages provide advantages at storage under higher temperature conditions. Altogether, a complex relationship between excipients, PVA concentration, and storage conditions was observed.

Towards defining a mechanism for MRS-based storage enhancement, various film and pre-film characteristics were examined. The pH of various pre-dried film solutions was evaluated and of the final film formulation to determine if pH differences could be responsible for viability loss, but no significant differences in pH were observed, as evidenced in Table 3.

Table 3 - pH of various film solutions and final film formation

Loss on drying (FIG. 8) was then evaluated as an indicator of moisture content but no correlation between percent moisture content with storage viability was observed. Finally, it was investigated whether glucose, the most abundant protectant contained in MRS, was responsible for conferring the storage benefits in the MRS formulations. MRS was replaced by glucose at the same concentration of glucose in the MRS broth. Within 8 weeks, a continuous viability loss was observed at 4°C, indicating that while glucose is likely an important storage component in our MRS formulation, glucose alone cannot recapitulate the complete storage benefits provided by MRS. Given that MRS improved bacteria viability regardless of the presence of PVA (FIG. 7 A), it was postulated that the MRS-based storage strategy is compatible with other polymers. As such, the compatibility of the present film system with other commonly used polymers for oral delivery such as sodium alginate, PVP K90, HPMC and gelatin type B was investigated. Storage studies at room temperature with L. casei ATCC393 demonstrated that high viability was maintained for 1 week in all these biomaterials, with statistically significant reduction in CFUs for alginate and PVP (FIG. 9).

Tunable Release ofL. casei ATCC393 from MBT-films

To evaluate whether different release profiles could affect spatial distribution of MBTs along the intestines, a custom 3D-printed holder was used for mounting sections of intestines. At one end, a hole was created for infusion of suspensions that recapitulate different release rates of MBTs on the intestinal surface so as to mimic transit time in the intestines. A bioluminescent non-pathogenic E. coli strain was chosen for the study for ease of quantification and to visualize microbe distribution along the intestine via IVIS. To mimic burst release, as is provided by current clinical MBT capsules, 10⁷ CFU were introduced as a bolus at the inlet, followed by immediate infusion of water at 1 mL min ¹ for 6 minutes at room temperature to mimic physiological flow rates in the intestines. To mimic slow release, an equivalent amount of bacteria were introduced as an infusion using identical flow conditions and for the same amount of time. At the end of the infusion, the signals of total bioluminescent flux for the two release groups were comparable, indicating similar total dosing. Effectively, this allowed comparison of how release rate of MBTs affects their intestinal distribution at short timepoints.

The results demonstrate that the slow release groups provided 1.5 fold more intestinal surface area coverage of MBT as compared to the fast release group (FIGS. 10A and 10B).

These differences in MBT distribution motivated the development of film formulations that enable distinct release profiles towards controlling intestinal distribution. However, when attempted to modulate the release of 3% PVA films, it observed that PVA exhibited rapid release (< 15 min), independent of film thickness, indicating that PVA on its own provides a burst-like release profile. To modify the present film system to achieve sustained release for our base PVA films, a high-molecular-weight (700 kDa) sodium carboxymethylcellulose (NaCMC) was blended in, since NaCMC has been shown to provide sustained release properties to various formulations. A blend of NaCMC and PVA was chosen because NaCMC films without PVA exhibited poor pliability and flexibility, thus rendering them difficult to for post-fabrication handling and eventual dosing. It was first confirmed that addition of NaCMC into the PVA films did not compromise the storage of L. casei ATCC393 (FIG. IOC), and then systematically investigated how addition of NaCMC affected release profiles. It was demonstrated that as NaCMC amount increases in relation to PVA, release of MBTs is slowed (FIG. 10D). It was then investigated how film thickness can be used to modulate release. Although release was not affected by thickness for PVA-only films, increasing thickness of PVA-NaCMC films substantially prolonged bacteria release profiles (FIG. 10E), providing 3 distinct release profiles that achieve 99% total CFU release in 15 minutes, 2 hours, or 8 hours, depending on film thickness. Finally, it was demonstrated that increasing surface area of MBT films containing NaCMC, while maintaining identical volume and CFUs, could also be used to provide sustained release profiles.

Mucoadhesion ofMBT-films

Mucoadhesive properties facilitate strong interactions between macroscopic delivery systems and the GI tract. These properties enable prolonged GI residence time or even enhanced retention to specific GI areas. NaCMC, used above to provide sustained release properties to MBT-films, is also a mucoadhesive polymer, and as such PVA-NaCMC films were examined for mucoadhesion. The mucoadhesive force, measured by a pulley system, was defined as the minimal force to detach MBT-films from the lumen side of ex vivo porcine intestine (FIG. 11 A). A mucoadhesive force of over 40 mN was recorded for the NaCMC containing MBT-films, over 100 times higher than the MBT-film weight (FIG. 1 IB).

Compatibility ofMBT-films with current clinical capsules

It was then sought to demonstrate that the MBT-film platform is compatible with existing oral delivery modalities that are used in the clinic for MBT delivery. To better visualize the compatibility ofMBT-films and capsules, rhodamine B was used to dye the PVA MBT-film.

The flexibility of PVA MBT-films, granted by plasticizing effects of glycerol, enabled folding and subsequent insertion into a standard, 00-sized oral capsule (FIGS. 12A-D). Slow-releasing PVA-NaCMC-MBT films of the highest NaCMC concentration tested could also be inserted into a standard 00-sized oral capsule. Furthermore, a single 00-sized capsule could be used to deliver at least ten MBT-films without affecting the integrity of the capsule system (FIGS. 12E-G). Storage compatibility with capsules was also evaluated with ten PVA MBT-films stored at 4°C inside capsules for five days, demonstrating statistically insignificant viability loss (FIGS. 12H). Furthermore, capsule dissolution time comparing empty capsules and capsules with ten films inside demonstrated no significant differences in release (FIG. 121). Through increasing encapsulation of L. casei ATCC393 up to 10⁹ CFU per MBT-film whilst maintaining long-term storage benefits (FIG. 12J), it was demonstrated that the MBT-film system described herein could deliver 10¹⁰ CFU of L. casei ATCC393 via a single capsule, a dose that would meet the requirements of many clinically used MBTs.

Here, a polymer-based encapsulation system is reported that simultaneously enables the long-term storage of L. casei ATCC393 and facilitates new delivery modalities for MBTs such as mucoadhesion and tunable release of MBTs. The MBT-film system employed here exhibits additive properties as different excipients and polymers are included at the synthesis stage; thus, our MBT-films have the potential to form the basis of a modular platform for MBT delivery (FIG. 2). Through the addition NaCMC, the MBT-film system was modified for sustained release and mucoadhesive functions. Furthermore, compatibility of the MBT-film system with other polymers such as alginate and gelatin was shown which are commonly used in many MBT formulations. ^[31] It was believed that due to the tunable and additive nature of this system, our MBT-films can be engineered for storage and delivery of a variety of clinically-relevant MBTs, beyond the /.. casei ATCC393 example highlighted here. Future development of these systems may lead to approaches to modulate GI residence time and the spatial distribution of microbes in oral delivery.

It was first confirmed that /.. casei ATCC393 was completely encapsulated in the MBT- films following dehydration by visualizing the surface and cross-sections of MBT-films. SEM imaging of the films highlighted how . casei ATCC393 was only observed in the films interior following cross-sectioning (FIG. 5). It was then demonstrated that MRS, both alone and when formulated with PVA, preserves L. casei ATCC393 at 4°C (FIG. 7A) and 37°C (FIG. 7B). Compared with 4°C, storage at high temperatures resulted in faster loss of viability, which is consistent with the trend reported in other studies. ^[32] However, conflicting results were observed at 4°C (FIG. 7A) and 37°C (FIG. 7B). MRS alone provided enhanced storage at 52 weeks compared to PVA+MRS formulations, but the opposite trend was observed at 37°C. Towards describing this, the components of MRS that likely contribute to enhanced storage (e.g. glucose, amino acids) were closely analyzed. Both sugar and amino acid excipients have been demonstrated to require hydrophobic, ion-dipole, and hydrogen-bonding interactions to stabilize surface proteins to grant storage benefits. Since MBTs are protein-rich, the benefits that glucose and amino acids provide in stabilizing proteins may underlie the storage benefits that the MRS components contribute to MBTs. Because of this, it was hypothesized that as PVA is introduced into the formulation, the physical interactions between storage excipients and the MBTs that occur at the excipient-microbe interface are changed, since excipient mass remained constant for each group. Altering the interactions between excipients and MBTs may be responsible for the demonstrated differences in storage. This hypothesis is supported by our characterization work that suggests PVA and excipients may interact with the MBT surface (FIG. 6). Interestingly, at both temperatures a trend was observed that at higher PVA concentrations, storage was improved.

Since oxidation-mediated death of dehydrated microbes has been shown for many other strains as they are exposed to storage conditions in oxygen-rich environments and the fact that PVA is widely-known and used as an oxygen barrier, it was postulated that increasing PVA amount can potentially ameliorate oxidation-mediated viability loss via PVA limiting oxygen diffusion. Considering these results and our hypotheses, it is clear a complex relationship between PVA, excipients, and storage conditions (e.g. temperature, humidity) exists and that further study is needed to define optimal formulations for specific storage conditions. The compatibility of MRS with polymers other than PVA (FIG. 9) was then demonstrated, which potentiates the application of MRS in the context of a wide range of biomaterials, further supporting the MBT-film system and MRS as a potential platform for MBT storage and delivery. However, inclusion of MRS and potentially other components that are widely used with bacteria for a variety of reasons (i.e. as growth or storage media) should be considered for their toxicity against mammalian cells. Since the specific combination of MRS ingredients, such as peptone, yeast extracts and a variety of salts could pose toxicity risks against mammalian host cells, cytotoxicity on an intestinal epithelial cell line was assessed. No statistical significance of cell viability was identified among the MRS treated groups and control, implicating MRS has minimal, if any, impact on Caco-2 cells in vitro.

A 3D-printed holder was the used to mount sections of intestines for investigation of how different MBT release profiles alters their distribution along the intestinal surface. The aim was to recreate and subsequently compare two extreme MBT release profiles, bolus and sustained release. This experiment was conducted at room temperature to minimize bacteria growth, which could potentially confound the observations. In the bolus case, which mimics current delivery of MBTs via capsule, 10⁷ CFU were exposed directly to the surface of the intestines followed by water flow to mimic intestinal transport. In the sustained release case, which could potentially be mimicked by our tunable MBT-film system, an identical number of microbes were exposed to the same area of the intestines, but over a longer period of time. Effectively, it was compared how microbes exposed to the intestines over a short period of time at high concentration differs in intestinal distribution as compared to a longer period of time at lower concentration. Both cases were exposed to identical physiologically relevant flow rates during the duration of the experiment. Quantitative imaging analysis showed that slower release rate correlated with higher surface area coverage as compared to the bolus release (FIG. 10A). While it has not been shown in any previous known work, it is likely that altered distributions of MBTs along the GI tract play a role in the ability of the MBT to colonize. This is likely the case because the first step in MBT colonization is adhesion, and adhering to a larger surface area may increase the number of individual MBT-intestinal interactions, potentially increasing the likelihood of colonization. Considering another main therapeutic function of MBTs, secretion of drugs, increasing surface area coverage would lead to higher absorption of drugs across the epithelium. As such, modifying the spatial distribution of MBTs through formulation design has the potential to alter how MBTs interact with target tissues. However, this experimental system relied on a static depot of MBTs as the source of MBTs, a situation that may be more difficult to achieve in a dynamic in vivo situation. Taken together, it was desirable to create a sustained release and mucoadhesive delivery system towards tailoring microbe distribution profiles in the GI tract.

As such, the development of MBT-films with distinct release profiles was a focus. The initial result showed that NaCMC alone exhibited poor flexibility and pliability as compared to PVA+NaCMC films which would render NaCMC films incompatible with post-fabrication handling for incorporation into standard oral capsules. Flexibility and pliability maintains structural integrity of the films throughout the fabrication and capsule-loading process, and potentially under the dynamic gastrointestinal conditions in vivo, which is important as geometry (FIG. 1 IE) and surface area are shown to be key structural parameters in ensuring reproducible and predictable release profiles. As a result, the strategy of polymer blending with PVA and slow-releasing NaCMC to slow film dissolution was chosen. Through the additive blending of NaCMC, our MBT-films provided sustained release of MBTs over a period of up to 10 hours or over a period of as short as 15 minutes (FIG. I I). It was further demonstrated that tuning the NaCMC content (FIG. 1 ID), thickness (FIG. 1 IE), and surface area are all viable strategies to grant sustained release properties. Many other potential strategies exist to slow dissolution of PVA films, such as inducing crystallization of PVA by treating the film with freeze-thaw cycles and chemical crosslinking of PVA chains. However, these methods introduce additional physical or chemical challenges to the encapsulated microbes which may reduce viability during fabrication.

Mucoadhesive properties allow formulations to reside in the dynamic environment of GI tract for extended periods of time. It was shown that the addition of NaCMC into PVA films enables mucoadhesion on intestines ex vivo, where the magnitude of the adhesive force is comparable to that of other intestinal devices reported in the literature. It was envisioned that in the context of drug-secreting MBTs, prolonged residence of MBTs enabled by mucoadhesion could extend the time for which MBTs secrete drugs at sites of absorption (e.g. the small intestine), which would potentially lead to higher therapeutic doses or longer duration of therapy. Also, mucoadhesion interfaces MBTs close to host tissues and thus could alter or accelerate interactions between MBTs and the mucus, since colonization requires adherence to mucus. Translation of these advantages towards in vivo performance of the films to manipulate GI residence time requires further evaluation. Generally, microbiome manipulation with MBTs in treating recurrent Clostridium difficile infections and inflammatory bowel diseases is assumed to require sustainable colonization for boosting therapeutic efficacy. However, modulating colonization of MBTs through controlling their interactions at sites of desirable colonization represents an under-explored area. By providing a mucoadhesive anchor of MBT-formulations upstream of colonization sites and subsequently providing tunable release kinetics, MBTs may interact with colonization sites in varied and controllable spatiotemporal profiles (FIG. 2), which may influence whether and how MBTs colonize. As such, mucoadhesive and tunable release properties of PVA-NaCMC films may serve as an enabling tool to systematically study the impacts of spatiotemporal factors of MBT delivery on the colonization and therapeutic outcome in vivo.

Finally, it was demonstrated compatibility of our MBT-films with standard capsules (FIG. 12) to highlight that our MBT-films can additionally use the existing advantages of capsules. For example, enteric coatings can be used to bypass the stomach in an effort to limit interactions between MBTs and the acid environment. Moreover, multiple MBT-films, possibly of different MBT composition or even release rates, can be included in a single capsule with up to 10¹⁰ CFU capsule ¹ (FIG. 12J), which satisfies the dosing requirements of clinical studies that require the highest dose of MBTs. Given that each film can include unique bacteria, it was speculated that the film-capsule combinatorial system can also be used for delivering microbial consortia. Overall, the present application reports a multifunctional polymeric film system for storage and delivery of L. casei ATCC393. It was found MRS enhanced storage of L. casei ATCC393 at two different temperatures. Mucoadhesion and tunable release properties of the film system may serve as a powerful tool to control parameters of oral microbe delivery including residence time and spatial distribution. The additive approach was used to endow additional functions such as improved storage, tunable release, or mucoadhesion highlight the potential of using this system as a future platform for MBT delivery.

Various embodiments of the invention have been described in fulfillment of the various objects of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A bioactive composition comprising:

a composite film including one or more bacterial species disposed in a polymeric matrix, the polymeric matrix comprising at least one biocompatible polymer.

2. The bioactive composition of claim 1, wherein the one or more bacterial species are embedded in the polymeric matrix.

3. The bioactive composition of claim 1, wherein the polymeric matrix comprises one or more components of a bacterial culture medium.

4. The bioactive composition of claim 3, wherein the bacterial culture medium comprises MRS.

5. The bioactive composition of claim 3, wherein the bacterial species exhibits full viability in the polymeric matrix for at least 8 weeks at a storage temperature of 4°C.

6. The bioactive composition of claim 3, wherein the bacterial species exhibits full viability in the polymeric matrix for at least 1 week at a storage temperature of 23°C.

7. The bioactive composition of claim 1, wherein the polymeric matrix comprises a mixture of biocompatible polymers.

8. The bioactive composition of claim 7, wherein two or more biocompatible polymers of the mixture exhibit different degradation rates for controlling bacterial release profile of the composite film.

9. The bioactive composition of claim 1, wherein loading of the bacterial species in the composite film is at least 10⁸ CFU.

10. The bioactive composition of claim 1, wherein loading of the bacterial species in the composite film is at least 10⁹ CFU.

11. The bioactive composition of claim 1, wherein the biocompatible polymer is selected from the group consisting of polyvinyl alcohol (PVA), hydroxypropyl methycellulose (HPMC), polyvinylpyrrolidone (PVP), gelatin, polylactic acid (PLA), polyglycolic acid (PGA), poly lactic- co-glycolic acid (PLGA), and mixtures thereof.

12. The bioactive composition of claim 1, wherein the one or more bacterial species are probiotic.

13. The bioactive composition of claim 12, wherein the bacterial species is Lactobacillus cae i.

14. The bioactive composition of claim 1, wherein the composite film exhibits a bioahesive force to intestinal surfaces of at least 40 mN.

15. The bioactive composition of claim 1, wherein the composite film is positioned in a capsule.

16. The bioactive composition of claim 1, wherein the polymeric matrix further comprises plasticizing agent.

17. A method of making a bioactive composition comprising:

providing a mixture including one or more bacterial species disposed in a polymeric solution comprising at least one biocompatible polymer;

depositing the mixture on a substrate; and

drying the mixture to form a composite film comprising the one or more bacterial species embedded in a polymeric matrix, the polymeric matrix comprising the at least one biocompatible polymer.

18. The method of claim 17, wherein the mixture further comprises one or more components of a bacterial culture medium.

19. The method of claim 18, wherein the bacterial culture medium comprises MRS.

20. The method of claim 17, wherein loading of the bacterial species in the composite film is at least 10⁸ CFU.

21. The method of claim 17, wherein loading of the bacterial species in the composite film is at least 10⁹ CFU.

22. The method of claim 17, wherein the biocompatible polymer is selected from the group consisting of polyvinyl alcohol (PVA), hydroxypropyl methycellulose (HPMC),

polyvinylpyrrolidone (PVP), gelatin, polylactic acid (PLA), polyglycolic acid (PGA), poly lactic- co-glycolic acid (PLGA), and mixtures thereof.

23. The method of claim 17, wherein the polymeric solution comprises biocompatible polymers of differing degradation rates to modulate bacterial release rates from the composite film.

24. The method of claim 17 further comprising varying thickness of the composite film to modulate bacterial release rates from the composite film.

25. The method of claim 17, wherein the bacterial species in the polymeric matrix of the composite film exhibit full viability for at least 8 weeks at a storage temperature of 4°C.

26. The method of claim 17, wherein the bacterial species in the polymeric matrix of the composite film exhibit full viability for at least 1 week at a storage temperature of 23°C.

27. The method of claim 17 further comprising loading the composite film into a capsule.