M.Sc. Lara Luana Teruel Enrico

In ophthalmology, living biomaterials have appeared as promising drug delivery and biosensor devices to tackle dynamic sensing and delivery of compounds. Their living character complicates their assessment with the also dynamic ocular tissues. The use of animal experiments increases complexity, and most animal ocular models are anatomically different from humans. Thus, in vitro ocular systems applied specifically to living biomaterials are required to assess their safety, compatibility and efficacy. Here, we report on an in vitro cornea model for co-cultures with Corynebacterium glutamicum-polyvinyl alcohol living biomaterials, which are reported as suitable living contact lenses, to study their cytocompatibility to the eye. We co-cultured this living biomaterial with human primary corneal cells (epithelial and fibroblasts) for 7 days, mimicking contact lens extended wear. We studied bacterial proliferation, biocontainment and biosafety. We investigated potential cytotoxicity and pro-inflammatory responses of living biomaterials to corneal cells. Our results revealed that the living biomaterial does not trigger cytotoxicity or pro-inflammatory phenotypes on corneal cells during the 7-day co-culture. We placed the living biomaterial on top of the corneal epithelium, observing no cytotoxic effects. Overall, these findings highlight the potential of in vitro investigations for living biomaterials and the applicability of these devices for ophthalmology purposes.

Living therapeutic materials (LTMs) are an emerging class of biomaterials that integrate living cells within engineered polymer matrices to provide dynamic and responsive functionalities. In this study, we engineered the robust, nonpathogenic, and GRAS-certified microorganism Corynebacterium glutamicum into a multifunctional biofactory for LTM applications. Using synthetic biology, we designed and constructed C. glutamicum strains capable of sensing, reporting, and producing the extremolyte ectoine. Ectoine is a clinically used compatible solute with cytoprotective and anti-inflammatory properties that is widely applied in dermatological formulations, nasal sprays, and ophthalmic preparations for the treatment of inflammatory and stress-related conditions. The engineered strains were further encapsulated in polymer-based living materials, including membrane-in-gel patches and core–shell hydrogel systems, to create skin-compatible and ocular-applicable therapeutic platforms. We developed genetic biosensors that detect diaminobutyric acid (DABA), a key intermediate in the ectoine biosynthesis pathway, to enable the time-resolved monitoring of cellular function. These biosensors, which are integrated with fluorescence and enzymatic reporter systems, allowed the noninvasive visualization of metabolic activity. Encapsulation strategies were optimized to ensure high metabolic activity, structural stability, and biocontainment, along with the controlled release of ectoine for potential applications in drug delivery and protective therapies. This work highlights the potential of C. glutamicum as a versatile platform for next-generation LTMs, offering precise monitoring and targeted therapeutic capabilities toward multifunctional living materials for precision medicine and environmental biosensing applications.

The increasing prevalence of dry eye syndrome in aging and digital societies compromises long-term contact lens (CL) wear and forces users to regular eye drop instillation to alleviate discomfort. Here a novel approach with the potential to improve and extend the lubrication properties of CLs is presented. This is achieved by embedding lubricant-secreting biofactories within the CL material. The self-replenishable reservoirs autonomously produce and release hyaluronic acid (HA), a natural lubrication and wetting agent, long term. The hydrogel matrix regulates the growth of the biofactories and the HA production, and allows the diffusion of nutrients and HA for at least 3 weeks. The continuous release of HA sustainably reduces the friction coefficient of the CL surface. A self-lubricating CL prototype is presented, where the functional biofactories are contained in a functional ring at the lens periphery, outside of the vision area. The device is cytocompatible and fulfils physicochemical requirements of commercial CLs. The fabrication process is compatible with current manufacturing processes of CLs for vision correction. It is envisioned that the durable-by-design approach in living CL could enable long-term wear comfort for CL users and minimize the need for lubricating eye drops.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, antibacterial, anticancer and other beneficial effects but are only produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.