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Biomaterials

Biomaterials - Poster Session D

Poster P5 - “Bugs For Drugs”: Encapsulation of a Cooperative Bacterial Consortia as a Therapeutic to Resolve Recurrent C. difficile Infection

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Clostridioides difficile infection (CDI) poses a significant healthcare challenge, characterized by high recurrence rates and limited treatment options. CDI is the primary cause of antibiotic-associated and infectious diarrhea in hospitals, with an estimated prevalence of 400,000 cases annually, and growing incidence and mortality rates. High-risk conditions for this infection include prolonged hospital stays, older age, weakened immune system, and, notably, recent antimicrobial therapy. In most cases, healthy gut bacteria prevent C. diff from colonizing, but when broad-spectrum antibiotics are used to treat other health conditions, the normal flora is disrupted, increasing susceptibility to CDI. CDI poses a unique problem due to its cyclical nature; it has high recurrence rates. Around 1 in 6 CDI patients will contract it again in the 2-8 weeks following recovery. Typical treatments for CDI involve narrow-spectrum antibiotics, which continue to disrupt healthy gut bacteria, making recovery more difficult and contributing to high relapse rates. Due to these factors, there is a large need for a more accessible and affordable treatment for recurrent CDI that can restore healthy microbiota by re-establishing natural colonization resistance. This study aims to address this issue by proposing the development of an encapsulated biologic of cooperative bacteria as a novel therapeutic approach for CDI. Using computational modeling, a consortium of bacteria comprising Bifidobacterium longum ATCC 55813, Escherichia coli K12, Roseburia intestinalis DSM 14610, and Streptococcus thermophilus LMD-99 was previously identified as effective in combating CDI while preserving the gut microbiota.

Materials and

Methods:  To achieve delivery of the cooperative consortia of bacteria to the intestine, we chose to design an encapsulation and delivery strategy that utilizes hydrogel microparticles to carry and protect bacteria through the gastrointestinal tract. Norbornene-functionalized carboxymethyl cellulose (cCMC) was chosen as the hydrogel microparticle material for this encapsulation, as it is a versatile and robust polymer that can be used to create a hydrogel with tunable stiffness and degradation behavior. Our proposed mechanism of delivery involved encapsulating each species of bacteria in the consortia directly within hydrogel microparticles. These microparticles are then encapsulated in a different, water-soluble material that “bundles” the microparticles together into a hydrogel matrix in a form that resembles a thin “film” that can be taken orally by a patient. Upon arrival in the intestine, conditions present and specific to the intestine trigger degradation of the microparticles, releasing the bacteria to begin their therapeutic activity in the gut microbiome. cCMC was synthesized with bacteria present within the hydrogel, and formed into microparticles using syringe extrusion. To confirm the stability of the hydrogel microparticles in physiologically relevant conditions, stability studies were performed at various temperatures, pH levels, and using pepsin enzymes. After conducting the stability studies, the encapsulated bacteria were re-cultured in order to confirm viability after experimentation and confirmed using colony forming unit quantification.

Results, Conclusions, and Discussions: The encapsulation and microparticle formation methods were successful, allowing for GFP tagged E. coli to be localized within carboxymethyl cellulose particles, with particle sizes ranging from 30-100 um. After four hours of incubation of the cCMC microparticles in water at room temperature and internal body temperature, no significant difference was observed between the percent mass change of the hydrogel at the differing temperature conditions. Similarly, the particles were tested in water and pepsin solution, at a concentration of 0.75 mg/mL, mimicking that of the internal stomach environment. Again, no significant difference was observed. Physiologically relevant pH level tests were conducted with the cCMC microparticles, at pH levels of 1.5, 4.5, 7, and 8 over four hours. Mass loss occurred in the 1.5 pH level, with a percent change of -54.57%, indicating degradation at this pH. Following the stability studies, viability students were conducted to confirm the viability of the bacteria within the microparticles. After degrading the cellulose and performing serial dilutions of the bacteria for CFU quantification, growth of the bacteria was seen for all temperature and pH levels of 1.5, 4.5, 7, and 8. Growth was observed at all dilutions (10-1, 10-2, 10-3, 10-4) for every experimental group aside for 1.5 pH, in which growth only was visible at the 10-1 and 10-2 dilutions. While this is likely due to degradation, the mass loss occurred only after the first hour, and still allowed viable bacteria to be recovered.
A method for the encapsulation of these bacteria in a biocompatible hydrogel matrix of norbornene-functionalized carboxymethylcellulose (cCMC) was developed and optimized to ensure stability and efficacy under physiologically relevant conditions. Stability studies, including exposure to varying pH levels, temperature, and enzymatic degradation, demonstrated the viability of encapsulated bacteria over time. Notably, the encapsulation method exhibited minimal degradation in acidic conditions mimicking the gastrointestinal tract. Overall, the development of an encapsulated biologic offers a promising advancement in CDI therapeutics, with the potential to introduce new, improved treatment outcomes and alleviate the burden of this persistent healthcare challenge.

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