M.Sc. Ketaki Deshpande

Engineered living materials (ELMs) made of bacteria in hydrogels have shown considerable promise for therapeutic applications through controlled and sustained release of complex biopharmaceuticals at low costs and with reduced wastage. While most therapeutic ELMs use E. coli due to its large genetic toolbox, most live biotherapeutic bacteria in development are lactic acid bacteria due to native health benefits they offer. Among these, lactobacilli form the largest family of probiotics with therapeutic potential in almost all sites of the body with a microbiome. A major factor limiting the use of lactobacilli in ELMs is their limited genetic toolbox. This study expands on recent work to expand the genetic programmability of probiotic Lactiplantibacillus plantarum WCFS1 for protein secretion and encapsulate it in a simple, cost-effective, and biocompatible core–shell alginate bead to develop an ELM. The controlled release of recombinant proteins is demonstrated, even up to 14 days from this ELM, thereby terming it PEARL – Protein Eluting Alginate with Recombinant Lactobacilli. Notably, lactobacillus encapsulation offered benefits like bacterial containment, protein release profile stabilization, and metabolite-induced cytotoxicity prevention. These findings demonstrate the mutual benefits of combining recombinant lactobacilli with alginate for the controlled and sustained release of proteins.

In this study novel polymeric materials based on chitosan (CS) were synthesized by chemically modifying CS with two bioactive moieties: eugenol and a compound containing a thiazolium group. These modifications aimed to impart antioxidant and antimicrobial properties to the matrix. Additionally, the scaffolds were reinforced with chitin nanowhiskers (Nw) to improve their mechanical strength and stability. Porous three-dimensional scaffolds were fabricated via the freeze-drying process, resulting in highly interconnected pore networks suitable for cell infiltration and nutrient transport. Biological characterization revealed that the incorporation of the two bioactive groups significantly enhanced the antioxidant activity and antimicrobial efficacy against both Gram-positive and Gram-negative bacteria to the scaffolds. Mechanical testing demonstrated that the Nw reinforcement increased scaffold stiffness and resilience without compromising porosity. In vitro biological assays using fibroblasts showed favorable cytocompatibility and promoted sustained cell proliferation over three weeks. Fluorescence microscopy confirmed fibroblast adhesion and morphological adaptation within the scaffold architecture. Additionally, the scaffolds were evaluated for their immunomodulatory effects using macrophage cultures, revealing a balanced immune response with reduced proinflammatory signaling, which is critical for successful integration and reduced fibrosis in vivo. These results indicate that those are promising candidates for tissue engineering and regenerative medicine applications.

Advances in the past decades in materials science, biofabrication methods, and synthetic biology have given rise to new fields like living materials. A living material is a class of biohybrid composite with living elements, including bacteria, yeasts, fungi, and mammalian cells, integrated with nonliving components. (1−6) These materials combine the advantages of both living and nonliving components to generate novel functions such as responses to environmental parameters and syntheses of complex biomolecules. (7) The nonliving aspect combines diverse chemistries and manufacturing techniques to support or enhance the functions of the living part. (6) Living materials as therapeutics (Living Therapeutic Materials, LTMs) bring revolutionary options to diagnostic and therapeutic practice, offering a solution to life-concerning issues by life itself (Figure 1). Living Therapeutic Materials are revolutionizing classical drug delivery devices, as they can produce therapeutics long-term, in situ, and on demand. This represents a more sustainable way for treatment. To realize Living Therapeutic Materials in the clinic, more preclinical studies need to be carried out so the concerns in terms of safety are well understood and their capacity as a more efficient delivery system is assessed. In the past decade, there has been a rise in the number of proof-of-concept LTMs and yet, the preclinical investigation of these materials is just starting.

Living therapeutics are attractive candidates to tackle the limitations of classically delivered therapeutic peptides, which are often poorly stable and require cost-intensive modifications. Their functional assessment is limited to animal experiments, which increase the complexity to evaluate the dynamic nature of these systems. Therefore, we developed an in vitro model of endotoxemia using macrophages to assess early-stage anti-inflammatory Living therapeutics. We refined the model based on three anti-inflammatory peptides (KCF-18, I6P7, and α-MSH) and identified suitable therapeutic concentrations and treatment durations. We applied the model to Lactiplantibacillus plantarum TF103, a probiotic engineered to secrete these peptides. The model revealed that Living therapeutics enhanced the effects of the peptides, requiring lower amounts of anti-inflammatory effects. This points to potential synergistic effects between peptides and bacteria. The model presented here allows the investigation of dynamic regimes, which could be useful in the development of complex systems such as the ones encountered in Living therapeutics.

The most common characteristic observed in numerous diseases like rheumatoid arthritis or psoriasis is chronic inflammation. Endotoxemia is an important factor in these conditions as it is triggered by prolonged exposure to lipopolysaccharide (LPS), leading to inflammation and immune dysregulation. Therapeutic peptides are promising options to treat these chronic diseases with inflammatory characteristics. However, the applicability of therapeutic peptides is limited due to their poor stability in the body, which is typically overcome by cost-intensive modifications. Living therapeutics are emerging as a more cost-effective strategy to tackle this limitation by engineering microbes to produce and deliver the peptides right where they are needed. We developed an in-vitro endotoxemia (and psoriatic) model to test living therapeutics secreting anti-inflammatory peptides: KCF-18, I6P7, α-MSH (secreted from a genetically modified lactic acid-free strain of Lactiplantibacillus plantarum (TF103)) on murine macrophages, characterized the dose-response effects of these peptides and performed multi-array cytokine analysis. The model revealed that this living therapeutic approach enhanced the effects of the peptides, requiring lower amounts to achieve anti-inflammatory effects. Notably, α-MSH secreted by TF103 L. plantarum achieved significant pathway suppression, comparable to or exceeding that of synthetic controls, without inducing cytotoxicity. This points to potential synergistic effects between the peptides and the intrinsic anti-inflammatory properties of lactic acid bacteria. We will expand the applicability potential of these anti-inflammatory living therapeutic materials in an in vitro model of psoriasis.

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.