bims-enlima Biomed News
on Engineered living materials
Issue of 2025–07–20
38 papers selected by
Rahul Kumar, Tallinna Tehnikaülikool



  1. bioRxiv. 2025 Jun 08. pii: 2025.06.08.658465. [Epub ahead of print]
      Microfluidic systems incorporating or contained within hydrogels are important in creating microphysiological systems (MPSs). Often naturally derived hydrogels are used, as their inherent bioactivity supports dynamic cellular behaviors. Hydrogel biomaterials that are partly or fully synthetic are desirable in engineering systems with specific, designed properties, though they typically lack bioactive features of natural materials without additional molecular design. In particular, permissive biomaterials enable physiologically relevant dynamic cellular behaviors. Granular hydrogels offer inherent permissiveness, owning to porosity between particles and dynamic behaviors in the absence of interparticle crosslinking. However, applying these in MPS to model tissues requires stable channels to perfuse fluid in these dynamic systems. Here, we establish channels within granular hydrogels to enable perfusion through spatially controlled interparticle crosslinking. Selective crosslinking allowed for the formation of stable channels while allowing the microparticles of a granular hydrogel between two channels to remained uncrosslinked. This allowed spatiotemporal control of signals within an environment established from microparticles without interparticle crosslinking. Fluorescently tagged molecules allowed for the visualization of controlled soluble gradients between two channels within the device. Additionally, embedded 3D printing processes can be used to specify material composition within the system, demonstrating integrated technology for engineering well-defined hydrogel systems. Integrated microfluidic-based control over soluble signals in a system that is compatible with 3D printing processes will establish a basis for building MPSs for broad applications, and the ability to maintain granular systems in culture without interparticle crosslinking will enable design of synthetic hydrogels that access unique dynamic properties within these systems.
    DOI:  https://doi.org/10.1101/2025.06.08.658465
  2. Can J Microbiol. 2025 Jul 17.
      M13 bacteriophages form self-assembled nanorods with the ability to self-assemble into complex materials with higher-order structures. These features make them useful templates for material fabrication. Their use in soft materials, bio-nano systems, and biomedical applications is well established. For these bio-interfacial applications, it is crucial that phages remain biocompatible and their production sustainable. Here, we review the bioprocessing of M13 phages and genetic engineering strategies that retain their natural assets in nanomaterials or bulk materials. Specifically, we highlight the extensively studied fermentation process of M13 phages with Escherichia coli (E. coli) and common downstream processing methods suitable for materials manufacturing. The ease of phage production contributes to its wide use for phage display, enabling the creation of large libraries of functional mutants. For materials purposes, genetic engineering often targets the pIII and pVIII proteins, enabling different geometries and fragment sizes. We also review common peptides displayed on phages, including RGD, used for SPR probes, targeted medicine, cell regeneration, or tissue scaffolding. We study glutamate-modified phages for metal binding, biomineralization, and electronics in bulk materials. By considering self-assembly, bioprocessing, and genetic engineering, material engineers can fully harness M13 phages for diverse functional and sustainable devices.
    DOI:  https://doi.org/10.1139/cjm-2025-0019
  3. bioRxiv. 2025 Jul 09. pii: 2025.07.09.663982. [Epub ahead of print]
      Electroactive microbes can be used as components in electrical devices to leverage their unique behavior for biotechnology, but they remain challenging to engineer because the bioelectrochemical systems (BES) used for characterization are low-throughput. To overcome this challenge, we describe the development of the Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP), which allows for samples to be arrayed and characterized in individually addressable microwells. This device reliably reports on the current generated by electroactive bacteria on the minute time scale, decreasing the time for data acquisition by several orders of magnitude compared to conventional BES. Also, this device increased the throughput of screening engineered biological components in cells, quickly identifying mutants of the membrane protein wire MtrA in Shewanella oneidensis that retain the ability to support extracellular electron transfer (EET). BiCASP is expected to enable the design of new components for bioelectronics by supporting directed evolution of electroactive proteins.
    The bigger picture: Devices that interface microbes and materials, known as bioelectronics, can be used to sense environmental chemicals in real time, generate energy from sugars, and synthesize chemicals. While these devices leverage the unique capabilities of living systems as components in devices, such as their ability to convert chemical information in the environment into electrical information at the cell surface, it remains challenging to engineer these cellular components and their biomolecules for new applications, largely because commercially available bioelectrochemical systems for monitoring current generated by electroactive microbes are costly and require large culture volumes, needs continuous monitoring for days to obtain stable signals, and multichannel potentiostats to monitor multiple microbes in parallel.To overcome these challenges, we created the Bioelectrochemical Crossbar Architecture Screening Platform or BiCASP that is easy to fabricate, enables parallel analysis of microbial samples in flexible arrayed formats, and yields a stable signal on the minute time scale. This device is expected to enable the application of combinatorial protein engineering methods, such as directed evolution, to proteins that control microbial current production, by allowing for fast screening of cells expressing protein mutant libraries. As a proof-of-concept, we demonstrate that this device can screen for cells that express mutants of decaheme cytochromes that retain the ability to electrically connect cells to electrodes. This device will simplify the engineering of cells and proteins that function as electrical switches as well as the diversification of bioelectronic devices for real-time sensing of chemicals in the environment.Furthermore, BiCASP is promising as a high-throughput screening (HTS) platform, enabling rapid, parallel analysis of cellular and molecular interactions of diverse biological systems through label-free electrochemical methods. Such capabilities could transform drug discovery, personalized medicine, and functional genomics, supporting systematic genetic and chemical screens even at single-cell resolution.
    Highlights: A high-throughput screening platform with individual addressabilityA device with a flexible crossbar architecture that simplifies current analysisReproducible detection of real-time cellular current on the minute time scaleThe device can be used to screen a library for cells with functional protein wires.
    DOI:  https://doi.org/10.1101/2025.07.09.663982
  4. bioRxiv. 2025 Jun 09. pii: 2025.06.06.658326. [Epub ahead of print]
      Polymeric hydrogels are valuable platforms for determining how specific mechanical properties of native tissue extracellular matrix (ECM) regulate cell function. Recent research has focused on incorporating viscous and elastic properties into hydrogels to investigate cellular responses to time-dependent mechanical properties of the ECM. However, a critical aspect often overlooked is that cells continuously remodel their microenvironment in hydrogels, such as by the deposition of newly secreted (nascent) ECM. While this nascent ECM has been demonstrated to play a vital role in transmitting mechanical signals across various biological contexts, the mechanisms by which it regulates cellular function in response to time-dependent mechanical properties remain poorly understood. In this study, we developed an interpenetrating polymer network that enables independent control of viscous and elastic hydrogel properties. We show that cells cultured on high-viscosity hydrogels deposit increased nascent ECM which also correlates with enhanced hydrogel remodeling. Interestingly, higher nascent ECM deposition on high-viscosity hydrogels was decoupled from intracellular contractility. These results establish a relationship between hydrogel viscosity and nascent ECM deposition that may extend to diverse cell types and offer new insights into cell-hydrogel interactions.
    DOI:  https://doi.org/10.1101/2025.06.06.658326
  5. Cell. 2025 Jul 09. pii: S0092-8674(25)00691-9. [Epub ahead of print]
      Multicellular coordination enhances biological complexity, yet the widely used yeast Saccharomyces cerevisiae possesses limited multicellular capabilities. Here, we expand the possibilities for engineering multicellular behaviors in yeast by developing modular toolkits for two key mechanisms in multicellularity, contact-dependent signaling and specific cell-cell adhesion. MARS (mating-peptide anchored response system) enables contact-dependent signaling via surface-displayed peptides and G protein-coupled receptors, mimicking juxtacrine communication, while Saccharomyces SATURN (adhesion toolkit for multicellular patterning) uses adhesion-protein pairs for the creation of programmable cell aggregation patterns. Combining these allows the construction of multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on local population. We further created JUPITER (juxtacrine sensor for protein-protein interaction), a genetic sensor based on MARS and SATURN, for assaying protein-protein interactions and selecting high-affinity nanobody binders. Collectively, these toolkits present versatile building blocks for constructing complex, user-defined multicellular yeast systems and expand the scope of its biotechnological applications.
    Keywords:  GPCR signaling; cell-cell adhesion; cell-cell communications; juxtacrine signaling; protein-protein interactions; synthetic biology; synthetic biology toolkit; synthetic multicellularity; yeast engineering
    DOI:  https://doi.org/10.1016/j.cell.2025.06.025
  6. Nat Rev Bioeng. 2025 Feb;3(2): 108-125
      Additive manufacturing is an engineering tool that enables the creation of complex structures for biomedical use, such as for 3D scaffolds for tissue engineering and regenerative medicine and in vitro disease models for drug testing. Lithography-based techniques (e.g., digital light processing DLP, volumetric additive manufacturing VAM) have particularly advanced in recent years for the 3D processing of photoreactive resins into structured hydrogels. The aim of this review is to introduce the various light-based lithographic 3D printing methods that are being used to process hydrogels, provide a guide to lithography-based printing from bioresin selection to the optimization of print parameters, highlight examples of in vitro and/or in vivo biomedical applications of hydrogels where lithography-based approaches have been leveraged, and discuss recent advanced efforts to process hydrogels into heterogenous structures with multi-scale organization. Finally, a perspective on the challenges and opportunities ahead in this field is provided.
    DOI:  https://doi.org/10.1038/s44222-024-00251-9
  7. bioRxiv. 2025 Jun 11. pii: 2025.06.07.658428. [Epub ahead of print]
      Towards achieving biomimetic complexity in biofabricated systems, an all-granular bioprinting system might use particle-based hydrogel inks to establish structures within a particle-based support matrix. In such a system, the granular support matrix can be designed to persist in the final construct and include cells incorporated prior to printing. To biofabricate complexity, bioprinting can introduce high-resolution heterogeneous structures that guide cell behaviors. The designs of the granular ink and support hydrogels are crucial to achieving complexity. High resolution structures and channels depend on small particles that flow and can be stabilized, and that can be printed and then removed, respectively. Herein, an all-granular system is described that used a granular formulation of an established, tunable hyaluronic acid-based hydrogel as the basis for a support matrix and a small particle gelatin hydrogel as an ink. Towards facilitating stabilization of the printed structure and flow during printing, the support and ink materials included soluble, interstitial components, and all exhibited yield stress behaviors characteristic of granular hydrogel systems. The support matrix's viscoelastic properties were dependent on intraparticle hydrogel network design, and it could be stabilized against flow by photoinitiated crosslinking. The gelatin ink could form fine filaments, as small as 100 µm in testing here, and melted to leave channels within crosslinked support matrices. Channels could support flows introduced by hydrostatic pressure and could be used to rapidly transport soluble factors into the construct, which could be used to establish soluble gradients by diffusion and support cell viability. The all-granular system supported printing of complex, multimaterial structures, with feature resolution on the order of 100 µm and spatial positioning on the order of 10s µm. The process and materials exhibited biocompatibility with respect to cells included within the support matrix during printing or introduced into channels to begin establishing endothelialized bioprinted vessels.
    DOI:  https://doi.org/10.1101/2025.06.07.658428
  8. bioRxiv. 2025 Jun 24. pii: 2025.06.18.660409. [Epub ahead of print]
      Human induced pluripotent stem cells (hiPSCs) offer patient-specific and immune-evasive sources for generating diverse cell types; yet lack of vascularization in hiPSC-derived tissues remains a major limitation for both therapeutic applications and disease modeling. Elucidating the mechanisms underlying vascular network formation in hiPSC-derived cells is therefore imperative. We and others have previously demonstrated that hiPSC-derived endothelial progenitor cells (hiPSC-EPs) self-assemble into lumenized microvascular networks when cultured in 3D norbornene-functionalized hyaluronic acid-based hydrogels. Herein we investigated the early period of culturing to characterize contractility of hiPSC-EPs. We hypothesized that multi-cell cooperativity would increase over time and would be dependent on the original hydrogel storage modulus. To quantify cellular contractility either 4 or 7 days after en-capsulation, 3D kinematic analysis was performed on single and small multi-cell clusters of hiPSC-EPs embedded in NorHA-based hydrogels. Contractile responses were significantly and non-linearly influenced by multicellularity, culture duration, and hydrogel stiffness. Novel to this study was the observation that NorHA hydrogels exhibited compressible behaviors, with greater compressibility occurring in NorHA hydrogels with lower stiffness. Hence, the kine-matic analysis was modified to incorporate separate deviatoric and volumetric strain indices. At day 7, multicellularity synergistically increased both strain components. These findings indicated that hiPSC-EP contractility and mechanical interactions with the hydrogel are governed by culture duration, multicellularity, and hydrogel stiffness; providing mechanical insight on hiPSC-EP self-assembly into microvasculature networks, a critical step towards development of functional vascular tissues for regenerative medicine and disease models.
    DOI:  https://doi.org/10.1101/2025.06.18.660409
  9. Commun Mater. 2025 ;6(1): 148
      Damping technologies aim to control the loads and deformations generated by ambient or forced vibrations in structures and machineries used in transport applications and construction. Traditionally, the materials used in damping devices are of fossil origin, but viscoelastic biobased resources are an alternative source of damping materials. Here, we develop an alginate-based hydrogel system with diverse porosity topologies by including poloxamer 407 as a sacrificial porogen at varying concentrations. Vibration transmissibility tests and dynamic mechanical analysis reveal these gels exhibit loss factors between 16% and 28% in the 100-300 Hz frequency range and that the dynamic modulus increases over an order of magnitude compared to the static modulus, reaching approximately 3 MPa. The visco- and poroelastic and pneumatic-like effects from the tunable porous structures contribute significantly to this damping effect. Furthermore, these hydrogels are biosourced and biodegradable, providing a sustainable alternative to conventional fossil-based damping materials.
    Keywords:  Gels and hydrogels; Mechanical engineering
    DOI:  https://doi.org/10.1038/s43246-025-00857-5
  10. Sci Adv. 2025 Jul 18. 11(29): eadv2681
      Silicone elastomers are widely used in biomedical devices and soft machines because of their compliance, inertness, and biocompatibility. Their sol-gel transition during curing enables mold casting and layer-by-layer manufacturing, allowing the fabrication of fully elastomeric and hybrid soft-rigid devices. However, controlling adhesion at material interfaces remains elusive, especially under diverse temperature conditions. This study introduces a framework that relates adhesion strength to a dimensionless reaction coordinate coupling time and temperature. This reaction coordinate can be used to predict the transition from bulk fracture to adhesive failure, which is crucial to create robust devices with strong interfaces. Using this framework, we fabricated elastomeric robotic actuators and demonstrated 3D printing with direct ink writing. The actuators achieved 50% higher curvature with the same design, and the 3D-printed parts exhibited over 200% improvement in interlayer adhesion. This work serves as a tool for optimizing interfacial adhesion for soft materials across different fabrication approaches.
    DOI:  https://doi.org/10.1126/sciadv.adv2681
  11. Nature. 2025 Jul 16.
      Living biological systems rely on the continuous operation of chemical reaction networks. These networks sustain out-of-equilibrium regimes in which chemical energy is continually converted into controlled mechanical work and motion1-3. Out-of-equilibrium reaction networks have also enabled the design and successful development of artificial autonomously operating molecular machines4,5, in which networks comprising pairs of formally-but non-microscopically-reverse reaction pathways drive controlled motion at the molecular level. In biological systems, the concurrent operation of several reaction pathways is enabled by the chemoselectivity of enzymes and their cofactors, and nature's dissipative reaction networks involve several classes of reactions. By contrast, the reactivity that has been harnessed to develop chemical reaction networks in pursuit of artificial molecular machines is limited to a single reaction type. Only a small number of synthetic systems exhibit chemically fuelled continuous controlled molecular-level motion6-8 and all exploit the same class of acylation-hydrolysis reaction. Here we show that a redox reaction network, comprising concurrent oxidation and reduction pathways, can drive chemically fuelled continuous autonomous unidirectional motion about a C-C bond in a structurally simple synthetic molecular motor based on an achiral biphenyl. The combined use of an oxidant and reductant as fuels and the directionality of the motor are both enabled by exploiting the enantioselectivity and functional separation of reactivity inherent to enzyme catalysis.
    DOI:  https://doi.org/10.1038/s41586-025-09291-6
  12. Nat Biotechnol. 2025 Jul;43(7): 1027
      
    DOI:  https://doi.org/10.1038/s41587-025-02746-1
  13. Biomacromolecules. 2025 Jul 14. 26(7): 3901-3902
      Over the past three decades, the field of peptide-based materials has been rapidly expanding and evolving, becoming a multidisciplinary area, with new developments and applications being consistently discovered. The purpose of this Peptide Materials Special Issue is to highlight research presented at the first Gordon Research Conference on Peptide Materials in January, 2023. Consequently, we invited eminent scientists with primary research interests in Peptide Materials to contribute original research articles or short reviews in this area. This thematic issue is focused on the materials aspects of peptides and their derivatives and mimics, including both fundamental research in peptide design, synthesis, assembly, micellization, gelation, and coacervation, as well as disparate technological applications, including functional materials for energy storage, catalysis, drug delivery, regenerative medicine, adhesion, protein purification, and nanotechnology. As peptides are composed of amino acids─the fundamental building blocks of proteins─they serve as a natural bridge between small-molecule supramolecular assemblies and large biomacromolecular constructs. Their ability to adopt well-defined secondary and tertiary structures, undergo hierarchical self-assembly, and exhibit tunable biochemical properties and distinct structural features highlights their importance and relevance within the broader landscape of biomacromolecular research. The peptide materials field has become a well-established, interdisciplinary area that attracts chemists, chemical engineers, material scientists, physicists, and biomedical engineers. The papers collected in this special issue demonstrate the growing recognition of peptides, polypeptides, proteins, and their derivatives and mimics as a versatile and critical class of biomacromolecules, poised to drive continued growth and innovation across diverse scientific and technological disciplines.
    DOI:  https://doi.org/10.1021/acs.biomac.5c00733
  14. Small. 2025 Jul 17. e02262
      Microgels offer unique advantages over bulk hydrogels due to their improved diffusion limits for oxygen and nutrients. Particularly, stimuli-responsive microgels with inherently bioactive and self-supporting properties emerge as highly promising biomaterials. This study unveils the development of interparticle-crosslinked, self-supporting, ion-responsive microgels tailored for 3D and 4D (bio)printing applications. A novel strategy is proposed to develop microgels that enabled interparticle crosslinking, eliminating the need for filler hydrogels and preserving essential microscale void spaces to support cell migration and vascularization. Additionally, these microgels possessed unique, ion-responsive shrinking behavior primarily by the Hofmeister effect, reversible upon the removal of the stimulus. Fabricated microgel-based constructs supported angiogenesis with tunable vessel size based-on interstitial void spaces while demonstrating excellent shear-thinning, self-healing properties and high print fidelity. Various bioprinting techniques are employed and validated using these microgels, including extrusion-based, embedded, intraembedded, and aspiration-assisted bioprinting, facilitating the biofabrication of scalable constructs. Multi-material 4D printing is achieved by combining ion-responsive microgels with non-responsive microgels, enabling programmable shape transformations upon exposure to ionic solutions. Utilizing 4D printing, complex, dynamic structures are generated such as coiling filaments, grippers, and folding sheets, providing a foundation for the development of advanced tissue models and devices for regenerative medicine and soft-robotics, respectively.
    Keywords:  3D bioprinting; 4D printing; Microgels; stimuli‐responsive; vascularization
    DOI:  https://doi.org/10.1002/smll.202502262
  15. Small Sci. 2025 Jul;5(7): 2500104
      Sustainable materials that effectively combine sophisticated functionality with eco-friendly materials are critical for next-generation technologies. Herein, a novel, fully bioderived, 4D printable shape memory polymer with linear tunability and remotely controlled actuation capabilities is presented. Using a linearly tunable matrix based on plant-derived acrylates with biosourced carbon content ranging from 75% to 87%, such as acrylated rapeseed oil, isobornyl acrylate, and isobornyl methacrylate, precise linear control over glass transition temperatures and mechanical properties is achieved. Furthermore, incorporating up to 0.2 wt% carbon nanotubes enhances electrical and thermal conductivity, enabling Joule heating and light-driven activation of 4D-printed actuators. These materials demonstrate remarkable shape fixity and recovery ratios above 90%, validated through thermomechanical analysis. Complex geometries, including auxetic and spiral structures, are successfully fabricated using vat photopolymerization 4D printing, highlighting exceptional resolution and defect-free printing. Dual-stage actuation and modular recovery capabilities are demonstrated for multifunctional applications. The materials reported here outperform conventional petroleum-based acrylates, requiring significantly lower activation voltages while maintaining rapid and efficient recovery. Developed biobased systems open pathways for sustainable applications in soft robotics, aerospace, adaptive medical devices, and smart textiles, paving the way for greener technologies.
    Keywords:  3D printing; actuators; additive manufacturing; plant‐based acrylates; sustainability
    DOI:  https://doi.org/10.1002/smsc.202500104
  16. bioRxiv. 2025 Jun 20. pii: 2025.06.17.660155. [Epub ahead of print]
      Cell barcodes are essential for a wide array of experimental applications, including lineage tracing, genetic screening, and single-cell analysis. An optimal barcode library would provide high diversity, live-cell compatible identification, and simple readout. In this work, we introduce single chain tandem fluorescent protein (sctFP) barcodes, constructed by linking different fluorescent proteins (FPs) into a single polypeptide chain with varied copy numbers. We found that the fluorescence signal intensity ratio at different wavelengths can reliably differentiate sctFPs generated using cnidarian FPs, but not prokaryotic FPs that require exogenous cofactors. The sctFPs enable the multiplexing of genetically encoded fluorescent biosensors, enhancing current biosensor multiplexing methods through a simplified imaging and analysis pipeline that support high-throughput applications. Their robust spectral profiles are compatible with a broad range of biosensor types. Using sctFPs, we demonstrate simultaneous tracking of various signaling activities with biosensors of different spectral properties. Together, this strategy provides a robust and scalable method for barcoding cells across diverse experimental contexts.
    DOI:  https://doi.org/10.1101/2025.06.17.660155
  17. Angew Chem Int Ed Engl. 2025 Jul 13. e202513147
      The development of high-performance elastomers for additive manufacturing requires overcoming complex property trade-offs that challenge conventional material discovery pipelines. Here, a human-in-the-loop reinforcement learning (RL) approach is used to discover polyurethane elastomers that overcome pervasive stress-strain property tradeoffs. Starting with a diverse training set of 92 formulations, a coupled multi-component reward system was identified that guides RL agents toward materials with both high strength and extensibility. Through three rounds of iterative optimization combining RL predictions with human chemical intuition, we identified elastomers with more than double the average toughness compared to the initial training set. The final exploitation round, aided by solubility prescreening, predicted twelve materials exhibiting both high strength (>10 MPa) and high strain at break (>200%). Analysis of the high performing materials revealed structure-property insights, including the benefits of high molar mass urethane oligomers, a high density of urethane functional groups, and incorporation of rigid low molecular weight diols and unsymmetric diisocyanates. These findings demonstrate that machine-guided, human-augmented design is a powerful strategy for accelerating polymer discovery in applications where data is scarce and expensive to acquire, with broad applicability to multi-objective materials optimization.
    Keywords:  3D printing; Machine learning; Polymers; multi-objective optimization; reinforcement learning
    DOI:  https://doi.org/10.1002/anie.202513147
  18. Angew Chem Int Ed Engl. 2025 Jul 13. e202510449
      Whole-plant biomass from non-agricultural sources and waste biomass from processing agricultural products are both promising feedstocks for biopolymer production because they are abundant and do not compete with food production. However, their processing steps are notoriously tedious with the final materials often displaying inferior performance and limited scope in their properties. Here, we report a strategy to integrate whole-cell spirulina, a green-blue algae, into robust hybrid algae-polyimine networks by leveraging a mechanochemical ball milling method. This strategy provides a greener synthetic approach to conventional solvent casting methods for polyimine synthesis; it simultaneously overcomes persistent constraints encountered in biomass processing and derivatization. The hybrid algae-based materials retain adaptability and recyclability imparted by their underlying dynamic covalent polymer matrix and display enhanced mechanical properties compared to their all-synthetic equivalents. These advantageous properties are attributed to the unique morphology of the ball milled hybrid materials which is facilitated by integration of the spirulina into the polymer matrix. Substituting spirulina with alternative biomass sources such as waste agricultural products also yields robust hybrid materials, thus highlighting the broad utility of this straightforward mechanochemical synthesis.
    Keywords:  Biomass; Green Chemistry; Mechanochemistry; polymer; sustainability
    DOI:  https://doi.org/10.1002/anie.202510449
  19. Small Sci. 2025 Apr;5(4): 2400320
      Artificial cells serve as promising micro-robotic platforms that replicate cellular features. One ubiquitous characteristic of living cells is compartmentalization of content in distinct and well-defined locations. Herein, a microfluidic strategy to mimic compartmentalization is developed through the production of micron-scale two and three compartment biomimetic microgels, where hydrogel compartment number, composition, size, and shape can be controlled. Our lab-on-chip system enables the incorporation of various synthetic organelles into spatially separated compartments within the microgels. This design concept allows for the introduction of a variety of individually triggered bioinspired behaviors, including protein capture, enzyme-mediated content release, and stimuli-triggered motility, each isolated in a distinct compartment enabling the use of the microgels as compartmentalized artificial cells. With this approach, the division of content and function seen in biological cells can be mirrored, which will underpin the generation of increasingly sophisticated and functional soft matter microdevices using bottom-up synthetic biology principles.
    Keywords:  artificial cells; biomimicry; compartmentalization; hydrogels; microfluidics
    DOI:  https://doi.org/10.1002/smsc.202400320
  20. Rheol Bull. 2024 Jul;93(1): 20-27
      Rheology is the science of how materials deform and flow and is a critical aspect of understanding the biomechanical functions of cell and tissue. Historically, scientists have designed simple and cost-effective instruments for assessing the mechanical properties of biological materials to inform their functionality. Cells and tissue are heterogeneous and possess complex mechanical properties. Yet, simple instruments such as falling ball viscometers and torsion pendulums, can often accurately capture and measure different aspects of how biological materials deform that are relevant to physiological conditions. Here we review the application of simple, home-built instruments suitable for probing the viscoelastic properties of biological materials, underscoring the importance of creativity and innovation of experimental tool design in the field of biomechanics.
  21. ACS Appl Mater Interfaces. 2025 Jul 18.
      Piezoelectric materials offer wireless bond cleavage in remote areas using ultrasound or other mechanical pressure. Although this approach has enormous therapeutic application potential, the appropriate design of such materials is limited and yet to be explored. In this study, we demonstrate the use of BaTiO3 nanoparticles for piezocatalytic bond cleavage and the controlled release of organic boronates within live cells, facilitated by medical-grade ultrasound. We demonstrated that piezocatalytically generated hydrogen peroxide (H2O2) can facilitate bond cleavage without inducing cytotoxicity. However, this process requires optimization of nanoparticle shape, with nanospheres proving to be more suitable compared to nanorods or nanowires. We have demonstrated piezocatalytic uncaging of various biorelevant organic boronates via deboronative hydroxylation outside/inside living cells, and extended this approach to 3D tumor spheroid models. The proposed approach offers a promising pathway for the ultrasonic activation of boronate-based drugs in remote areas of the body, enabling enhanced therapeutic performance.
    Keywords:  biorthogonal chemistry; hydrogen peroxide; medical ultrasound; nanoparticle; piezocatalysis; reactive oxygen species; responsive drug delivery
    DOI:  https://doi.org/10.1021/acsami.5c10556
  22. Biomater Adv. 2025 Jul 09. pii: S2772-9508(25)00235-3. [Epub ahead of print]177 214408
      3D in vitro model systems, such as hydrogels, have garnered popularity due to their ability to more accurately recapitulate in vivo environments compared to 2D cell culture systems. However, methods which involve casting hydrogels by hand may be time consuming, have poor reproducibility, and reduced capacity to generate complex structures. Hence, 3D bioprinting has emerged as a useful tool for the high throughput production of in vitro tissue models such as hydrogels and complex constructs. Here, we demonstrate the mechanical properties, printability, and ability to support single cells and spheroids in culture for two highly characterised composite bioinks: Alginate/Gelatin (AlgGel), which is ionically crosslinked, and Alginate/Gelatin Methacrylate (GelMA) (AlgGelMA), whereby the GelMA is crosslinked by illumination with UV light. In this study, we engineered gels that exhibit a wide range of stiffnesses, which vary due to the concentration of crosslinking polymer present. AlgGel hydrogels were softer (1.5-4.5 kPa), and stiffness decreased with time in culture, however, AlgGelMA hydrogels were stiffer (6-40 kPa), and the stiffness increased with time. Microarchitectural studies using Scanning Electron Microscopy and Microcomputed Tomography (μCT) revealed that hydrogels produced using both bioinks bore a highly porous structure, further simulating in vivo conditions. To assess the ability of both bioink families to support cell culture, the Acute Myeloid Leukaemia cell line THP-1 and human Mesenchymal Stem Cells (hMSCs) as single cells and spheroids were bioprinted in each bioink. Interestingly, THP-1 cells formed larger clusters when cultured within AlgGel bioinks compared to AlgGelMA. Additionally, hMSCs appeared to be unable to migrate through the AlgGel matrix, as single hMSCs displayed rounded morphologies and hMSC spheroid shape was not disrupted after seven days. Contrastingly, hMSCs and spheroids cultured within AlgGelMA hydrogels were able to invade the gel matrix and migrate. Together, these data demonstrate that both AlgGel and AlgGelMA bioinks show promise for use as the basis of 3D bioprinted in vitro tissue models.
    Keywords:  Alginate; Bioprinting; Endothelial cells; Gelma; Stem cells
    DOI:  https://doi.org/10.1016/j.bioadv.2025.214408
  23. Acta Biomater. 2025 Jul 15. pii: S1742-7061(25)00531-8. [Epub ahead of print]
      The use of bacteria to fabricate materials has garnered significant attention in both fundamental and applied research. In this study, we introduce a method for producing bacteria-based materials utilizing the bacterial capacity to colonize 2D nanomaterials, which in turn provides additional robustness to the resulting bacterial film. Specifically, we demonstrate the fabrication of self-standing films based on a bacterium Bacillus pumilus reinforced with 2D nanosheets derived from Layered Double Hydroxides. The Live/Dead staining, followed by Confocal Laser Scanning Microscopy (CLSM), revealed an anisotropic distribution of live and dead bacteria within the mature biofilms leading to a stratified biofilm architecture. Scanning Electron Microscopy (SEM) confirmed the presence of endospores in the reinforced biofilm structure. Biological assays further supported this observation. Notably, simulated gastrointestinal passage experiments showed that the presented bacterial films provide significant protection to probiotic bacteria under harsh gastrointestinal conditions, suggesting their potential applicability in advanced delivery systems. The present study offers a useful approach for future innovations in bacteria-based material fabrication. STATEMENT OF SIGNIFICANCE: In this manuscript we present a sustainable approach for producing bacteria-based materials by harnessing the natural ability of bacteria to colonize 2D nanomaterials. Applying 2D LDH nanosheets contributed to endospores formation and structural robustness leading to the reinforced self-standing bacterial films. Moreover, the reinforced biofilms effectively protect probiotic bacteria under harsh gastrointestinal conditions. Our findings can contribute to the development of innovative bacteria-based living materials with potential applications in advanced delivery systems and regenerative medicine.
    Keywords:  2D Nanomaterials; Biofilm engineering; Layered Double Hydroxides; Living materials; Probiotic Delivery
    DOI:  https://doi.org/10.1016/j.actbio.2025.07.034
  24. Sci Adv. 2025 Jul 18. 11(29): eadu6897
      Biological lifeforms can heal, grow, adapt, and reproduce, which are abilities essential for sustained survival and development. In contrast, robots today are primarily monolithic machines with limited ability to self-repair, physically develop, or incorporate material from their environments. While robot minds rapidly evolve new behaviors through artificial intelligence, their bodies remain closed systems, unable to systematically integrate material to grow or heal. We argue that open-ended physical adaptation is only possible when robots are designed using a small repertoire of simple modules. This allows machines to mechanically adapt by consuming parts from other machines or their surroundings and shed broken components. We demonstrate this principle on a truss modular robot platform. We show how robots can grow bigger, faster, and more capable by consuming materials from their environment and other robots. We suggest that machine metabolic processes like those demonstrated here will be an essential part of any sustained future robot ecology.
    DOI:  https://doi.org/10.1126/sciadv.adu6897
  25. bioRxiv. 2025 Jun 17. pii: 2025.06.17.660209. [Epub ahead of print]
      Pathogenic Bordetella bacteria infect the ciliated respiratory epithelia of mammalian and avian hosts. Several bacterial proteins mediate host cell adhesion, but filamentous hemagglutinin (FhaB) is a principal adhesin because mutants lacking this protein exhibit profound colonization defects. Here, we show that FhaB carries a C-terminal microtubule-binding domain (FhaB-CT), which is translocated into the host-cell cytoplasm to promote bacterial colonization. Cryogenic electron microscopy of microtubule-bound FhaB-CT shows that the domain binds primarily to α-tubulin through a network of polar interactions. Live-cell microscopy of infected tracheal explants reveals that FhaB-CT delivery is required for Bordetella to occupy a niche at the base of cilia on airway epithelia. Finally, we demonstrate that the microtubule-binding domain is required for long-term colonization of the mouse nasal cavity by B. pertussis . These observations suggest that the FhaB-CT domain is delivered into motile cilia, where it interacts with axonemal microtubules. We propose that Bordetella initially adhere to the tips of cilia, then deploy multiple FhaB adhesin molecules to migrate to the base of the cilial forest. This mechanism enables Bordetella to resist removal by the mucociliary 'escalator' that clears the respiratory tract of microbes and debris.
    DOI:  https://doi.org/10.1101/2025.06.17.660209
  26. bioRxiv. 2025 Jun 14. pii: 2025.06.12.659214. [Epub ahead of print]
      Precise spatial regulation of site-specific DNA recombination (SSR) in vivo remains a challenge due to limited tunability of current platforms. Here, we present an optogenetic approach that overcome these limitations by employing engineered light-regulated recombinase E-LightR-Cre and tunable wireless implantable optoelectronic devices. E-LightR-Cre meets the key criteria for spatial regulation of SSR in vivo , showing no detectable activity in the dark, while demonstrating robust activation upon blue-light illumination. To achieve local E-LightR-Cre activation in murine lungs, we developed wireless, fully-implantable optoelectronic devices enabling focal illumination with no discernible organ damage. By modulating illumination intensity and duration, we can control the size of the activated area. Local expression of oncogenic KRas-G12D in a photoactivated subpopulation of cells in vitro revealed rapid reprogramming of the mutant expressing cells and their non-activated neighbors. Light-guided activation of E-LightR-Cre in mouse lungs resulted in focal expression of a reporter gene and allowed us to induce local formation of oncogenic lesions in vivo .
    DOI:  https://doi.org/10.1101/2025.06.12.659214
  27. Trends Biotechnol. 2025 Jul 12. pii: S0167-7799(25)00252-5. [Epub ahead of print]
      Synthetic metabolism has the potential to transform carbon capture, bioremediation, or bioproduction strategies. To transfer metabolic designs from an in vitro context to living model systems such as the bacterium Escherichia coli, metabolic engineers incentivize the maintenance and use of the introduced metabolic module by making cell survival dependent on it (growth-coupled selection). However, creating and characterizing appropriately rewired selection strains is nontrivial and requires labor-intensive growth phenotyping in various conditions. To enhance the community use of extant selection strains, we compiled designs covering the central, amino acid, and energy metabolism of E. coli for this review, and we revisit the key concepts of growth-coupled selection.
    Keywords:  E. coli engineering; auxotrophs; growth-coupled selection; selection strains; sustainability; synthetic metabolism
    DOI:  https://doi.org/10.1016/j.tibtech.2025.06.015
  28. bioRxiv. 2025 May 10. pii: 2025.05.10.653182. [Epub ahead of print]
      Gene expression is constantly regulated by gene regulatory networks that consist of multiple regulatory components to mediate cellular functions. An ideal tool for analyzing gene regulation processes would provide simultaneous measurements of the dynamics of many components in the gene regulatory network, but existing methodologies fall short of simultaneously tracking the dynamics of components over long periods of time. Here, we present CytoTape-a genetically encoded, modular, and scalable analog recorder for continuous, multiplexed in situ recording of gene regulation dynamics over multiple days and weeks at single-cell resolution. CytoTape consists of a flexible, thread-like, elongating intracellular protein self-assembly engineered via AI-guided rational design. Gene regulation dynamics, together with timestamps for reconstruction of the continuous time axis, are directly encoded via distinct molecular tags distributed along single CytoTape assemblies in live cells, to be readout at scale after fixation via standard immunofluorescence imaging. CytoTape recorders are modularly designed to record gene expression driven by a variety of activity-dependent promoters. We demonstrated the utility of CytoTape in mammalian embryonic kidney cells, cancer cells, glial cells, and neurons, achieving simultaneous recording of five cell plasticity-associated transcription factor activities and immediate early gene expression levels, namely CREB, c-fos, Arc, Egr1, and Npas4 activities, within single cells in a spatiotemporally scalable manner. CytoTape revealed complex waveforms and nonlinear temporal couplings among these cellular activities, enabling investigations of how gene regulation histories and intrinsic signaling states shape transcriptional logics. We envision CytoTape to have broad applications in both basic and disease-related cell biology research.
    DOI:  https://doi.org/10.1101/2025.05.10.653182
  29. Adv Healthc Mater. 2025 Jul 16. e2501616
      Three-dimensional bioprinting has emerged as a promising strategy in tissue engineering, enabling the fabrication of biomimetic tissue constructs for regenerative medicine, disease modeling, and drug screening. A key challenge in this field is the development of organ-specific bioinks capable of recapitulating native microenvironments to support cell viability, proliferation, and tissue-specific maturation. In this study, a novel photocrosslinkable bioink derived from methacrylated decellularized porcine kidney extracellular matrix (KdMA) is reported. The decellularization process effectively removed cellular components while preserving key extracellular matrix constituents. The resulting KdMA bioink exhibited favorable rheological properties, including tunable stiffness and rapid photocuring kinetics, making it compatible with both digital light processing-based stereolithography and extrusion-based bioprinting platforms. Encapsulated human embryonic kidney cells maintained high viability and formed multicellular spheroids, demonstrating the bioink's cytocompatibility and structural support. Additionally, the KdMA bioink enabled stable multilayer bioprinting with preserved structural integrity and tunable mechanical properties. These results underscore the utility of KdMA as a kidney-specific bioink and its promise as a versatile platform for advancing renal tissue engineering and organoid maturation.
    Keywords:  3D bioprinting; decellularized extracellular matrix; kidney tissue engineering; photocrosslinkable bioink
    DOI:  https://doi.org/10.1002/adhm.202501616
  30. bioRxiv. 2025 Jul 09. pii: 2025.07.09.663984. [Epub ahead of print]
      Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency.
    DOI:  https://doi.org/10.1101/2025.07.09.663984
  31. bioRxiv. 2025 Jun 29. pii: 2025.06.27.661910. [Epub ahead of print]
      Unlike many physical nonequilibrium systems, in biological systems, the coupling to external energy sources is not a fixed parameter but adaptively controlled by the system itself. We do not have theoretical frameworks that allow for such adaptability. As a result, we cannot understand emergent behavior in living systems where structure formation and non-equilibrium drive coevolve. Here, using ecosystems as a model of adaptive systems, we develop a framework of living circuits whose architecture changes adaptively with the energy dissipated in each circuit edge. We find that unlike traditional nonequilibrium systems, living circuits exhibit a phase transition from equilibrium death to a nonequilibrium dissipative state beyond a critical driving potential. This transition emerges through a feedback mechanism that saves the weakest edges by routing dissipation through them, even though the adaptive rule locally rewards the strongest dissipating edges. Despite lacking any global optimization principle, living circuits achieve near-maximal dissipation, with higher drive promoting more complex circuits. Our work establishes ecosystems as paradigmatic examples of living circuits whose structure and dissipation are tuned through local adaptive rules.
    DOI:  https://doi.org/10.1101/2025.06.27.661910
  32. bioRxiv. 2025 May 25. pii: 2025.05.20.654730. [Epub ahead of print]
      Natural tissue comprises fibrous proteins with complex fiber alignment patterns. Here, we develop a reproducible method to fabricate biomimetic scaffolds with patterned fiber alignment along multiple orientations. While extrusion-based approaches are commonly used to align fibrous polymers in a single orientation parallel to the direction of flow, we hypothesized that extrusion-based 3D printing could be utilized to achieve more complex patterns of fiber alignment. Specifically, we show control of lateral spreading of a printed filament can induce fiber alignment that is either parallel or perpendicular to the flow direction. Theoretical prediction of the printing parameters that control fiber orientation was experimentally validated using a collagen biomaterial ink. The velocity ratio of the printhead movement relative to the ink extrusion rate was found to dictate collagen fiber alignment, allowing for the informed fabrication of collagen scaffolds with prescribed patterns of fiber alignment. For example, controlled variation of the ink extrusion rate during a single print resulted in scaffolds with specified regions of both parallel and perpendicular collagen fiber alignment. Human corneal mesenchymal stromal cells seeded onto the printed scaffolds adopted a spread morphology that aligned with the underlying collagen fiber patterns. This technique worked well for filaments either printed into air or extruded within a support bath using embedded 3D printing, enabling the fabrication of 3D structures with aligned collagen fibers. Taken together, this work demonstrates a theoretical and experimental framework to achieve the reproducible fabrication of 3D printed structures with controlled collagen fiber patterns that guide cellular alignment.
    DOI:  https://doi.org/10.1101/2025.05.20.654730
  33. RSC Chem Biol. 2025 Jul 14.
      Mucin proteins are essential for life but are challenging to study due to their complex glycosylation patterns. Synthetic mimics have become vital tools for understanding and modulating the roles of mucins in human health and disease. These materials also have diverse biomedical applications as lubricants and anti-infectives, in vaccine formulations, and more. We developed a chemoenzymatic approach to prepare polypeptide-based synthetic mucins displaying a variety of glycans with native linkages and orientations. By combining the polymerization of glycosylated amino acid N-carboxyanhydrides with enzymatic sialylation and fucosylation, we produced a tunable panel of synthetic mucins. These polymers were recognized by natural glycan-binding and glycan-degrading enzymes, providing insights into the structural preferences of these proteins. Glycan- and linkage-dependent effects on proteolysis were observed. Further, investigation of the influence of glycans on peptide backbone secondary structure revealed that both sialylation and linkage at Ser vs. Thr have profound effects on hierarchical conformation. Overall, our methodology offers versatile tools for exploring the diverse glycobiology of mucins.
    DOI:  https://doi.org/10.1039/d5cb00111k
  34. Biofabrication. 2025 Jul 14.
      Melt electrowriting (MEW) is capable of generating highly defined microarchitectures suitable for tissue engineering applications. The main biodegradable polymer typically utilized for MEW processing, poly(ε-caprolactone), is prone to creep under dynamic loads and plasticization due to water absorption, making its use problematic for situations demanding dynamic loading in aqueous media. Photocrosslinking during processing can eliminate these problems while also allowing for manipulation of mechanical properties. However, photocrosslinking strategies utilized to date have either limited processing time or require prolonged UV irradiation. Herein we demonstrate the potential of a cyclic trimethylene carbonate monomer bearing a pendant coumarin moiety (MUM) for creating MEW processable copolymers that are thermally stable and photocrosslinkable. The MUM was copolymerized with caprolactone to form copolymers that were MEW processed into both linear and crimped fibre structures followed by long-wave UV photocrosslinking yielding high modulus scaffolds with very low sol content. The photocrosslinked scaffolds were also cytocompatible. The ability to copolymerize MUM with other cyclic lactone monomers allows for the generation of a variety of MEW processable polymers with tunable properties. Collectively, the findings demonstrate the potential of MUM containing copolymers for MEW generation of scaffolds for a range of tissue engineering applications.&#xD.
    Keywords:  additive manufacturing; coumarin; melt electrowriting; photocrosslinking; tissue engineering
    DOI:  https://doi.org/10.1088/1758-5090/adef81
  35. Adv Mater. 2025 Jul 14. e2507859
      Piezoelectric materials that are simultaneously healable, stretchable, and degradable have remained an unmet challenge, limiting advancements in wearable and implantable electronics, where devices face multidimensional mechanical deformation, causing a risk of damage. To address this critical gap, a biocompatible piezoelectric material is developed for ultrahigh piezoelectric effects with DL-alanine amino acid crystals, which is stretchable, healable, and degradable. The in situ grown DL-alanine piezoelectric crystals within an ionically cross-linked gelatin hydrogel matrix strengthen the piezoelectric properties with an ultrahigh voltage coefficient of 1.6 Vm N-1. The combination of the piezo-ionic property and crystal alignment results in a record-breaking energy harvesting figure-of-merit value at 57.6 pm2 N-1 to deliver outstanding mili-watt level power outputs in proof-of-concept devices which can power up even several electric light bulbs. An elastically stretchable, damage resistant strain sensor is further optimized for real-time healthcare monitoring and biomechanical motion tracking. By integrating machine learning algorithms, the sensing system intelligently classifies biomechanical activities with high accuracy, enabling advanced applications in healthcare, rehabilitation, and sports monitoring.
    Keywords:  biodegradable; energy harvesting; piezoelectric; self‐healing; stretchable
    DOI:  https://doi.org/10.1002/adma.202507859
  36. Proc Natl Acad Sci U S A. 2025 Jul 22. 122(29): e2502921122
      Optical switches and bifurcation rely on the nonlinear response of materials. Here, we demonstrate linear temporal bifurcation responses in a passive multimode microresonator, with strongly coupled chaotic and whispering gallery modes (WGMs). In microdisks, the chaotic modes exhibit broadband transfer within the deformed cavities, but their transient response is less explored and yields a random output of the analog signal distributed uniformly from "0" to "1." Here, we build chaotic states by perturbing the multimode microring resonators with densely packed silicon nanocrystals on the waveguide surface. In vivo measurements reveal random and "digitized" output that ONLY populates around 0 and 1 intensity levels. The bus waveguide mode couples first to chaotic modes, then either dissipates or tunnels into stable WGMs. This binary pathway generates high-contrast, digitized outputs. The fully passive device enables real-time conversion of periodic clock signals into binary outputs with contrasts exceeding 12.3 dB, data rates of up to 107· bits per second, and 20 dB dynamic range.
    Keywords:  TEM; chaotic states; random number generator; silicon nanocrystals; silicon photonics
    DOI:  https://doi.org/10.1073/pnas.2502921122
  37. bioRxiv. 2025 May 02. pii: 2025.04.30.651527. [Epub ahead of print]
      Single-molecule force spectroscopy enables the detailed probing of molecular interactions, providing new insights into molecular mechanisms-yet studying biology "one molecule at a time" can lead to throughput challenges that limit applications. While multiplexed single-molecule assays can address these issues, suitable functionalization of surfaces is required, which remains a technical challenge-many commonly used approaches are constrained by random and sparse biomolecules arrangements, limiting programmability and throughput. An ideal anchoring method would enable (i) high surface densities to maximize throughput, (ii) precise control of spatial position and molecular identity for maximum control, (iii) covalent linking for high force application, and (iv) efficient patterning without the need for expensive facilities to maximize accessibility. To achieve these aims, we have developed a light-guided surface patterning method that can covalently organize oligonucleotides (oligos) without the need for lithographic equipment. Oligos with 3-Cyanovinylcarbazole (CNVK) nucleoside are crosslinked by UV patterns reflected through a digital micromirror device (DMD), with beads arranged accordingly. To demonstrate compatibility with established single-molecule methods, we performed single-molecule force spectroscopy experiments on patterned coverslips, using both magnetic tweezers and hydrodynamic-based systems. Our light-guided approach provides a scalable and accessible solution for biomolecular patterning that allows precise control over molecular identity and spatial positioning, enabling high-throughput measurements in single-molecule research.
    DOI:  https://doi.org/10.1101/2025.04.30.651527
  38. ACS Pharmacol Transl Sci. 2025 Jul 11. 8(7): 2180-2191
      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 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.
    Keywords:  Lactiplantibacillus plantarum; cytokines; immune response; probiotics; therapeutic peptides
    DOI:  https://doi.org/10.1021/acsptsci.5c00216