Biology subjects

Bhusari, S.

Publications and source records attributed to Bhusari, S..

5 recordsLinked to original sources

bioRxiv2023

Rheological behavior of Pluronic/Pluronic diacrylate hydrogels used for bacteria encapsulation in living materials

Pluronic (Plu) hydrogels containing Pluronic diacrylate (PluDA) have become popular matrices to encapsulate bacteria in engineered living materials. For this purpose, 30 wt% Plu/PluDA hydrogels with variable fraction of covalently crosslinkable PluDA in the hydrogel composition are used. The degree of covalent crosslinking and the consequent different mechanical properties of the hydrogels have been shown to affect bacteria growth, but a systematic investigation of the mechanical response of the hydrogels is still missing. Here we study the rheological response of 30 wt.% Plu/PluDA hydrogels with increasing PluDA fraction between 0 and 1. We quantify the range of viscoelastic properties that can be covered in this system by varying in the PluDA fraction. We present stress relaxation and creep-recovery experiments, and analyze the variation of the critical yield strain/stress, relaxation and recovery parameters of Plu/PluDA hydrogels as function of the covalent crosslinking degree using the Burgers and Weilbull models. We expect this study to help users of Plu/PluDA hydrogels to estimate the mechanical properties of their systems, and eventually to correlate them with the behaviour of bacteria in future Plu/PluDA devices of similar composition.

bioengineering↗

bioRxiv2022

Plasmonic Stimulation of Gold Nanorods for the Photothermal Control of Engineered Living Materials

Engineered living materials (ELMs) use encapsulated microorganisms within polymeric matrices for biosensing, drug delivery, capturing viruses, and bioremediation. It is often desirable to control their function remotely and in real time. Suitable, genetically engineered microorganisms respond to changes of their environment. Here, we combine this local sensitivity with a nanostructured encapsulation material to sensitize the ELM for infrared light. Previously, blue light has been used to stimulate microorganisms that contain optogenetic modules responsive to those wavelengths without the need for exogenous cofactors. Here, we use plasmonic gold nanorods (AuNR) that have a strong absorption maximum at 808 nm, a wavelength where human tissue is relatively transparent. Biocompatible composites of a Pluronic-based hydrogel and AuNR are prepared without agglomeration; they react to illumination by local heating. We measure a photothermal conversion efficiency of 47 % in transient temperature measurements. Steady-state temperature profiles from local photothermal heating are quantified using infrared photothermal imaging, correlated with measurements inside the gel, and applied to stimulate thermoresponsive bacteria. Using a bilayer ELM construct with the thermoresponsive bacteria and the thermoplasmonic composite gel in two separate but connected hydrogel layers, it is shown that the bacteria can be stimulated to produce a fluorescent protein using infrared light in a spatially controlled manner.

bioengineering↗

bioRxiv2022

Encapsulation of bacteria in bilayer Pluronic thin film hydrogels: a safe format for engineered living materials

In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.

bioengineering↗

bioRxiv2022

In vitro evaluation of immune responses to bacterial hydrogels for the development of living therapeutic materials

In living therapeutic materials, organisms genetically programmed to produce and deliver drugs are encapsulated in porous matrices or hydrogels acting as physical barriers between the therapeutic organisms and the host cells. The therapeutic potential of such constructs has been highlighted in in vitro studies, but the translation to in vivo scenarios requires evaluation of the immune response to the presence of the encapsulated, living organisms. In this study, we investigate the responses of human peripheral blood mononuclear cells (PBMCs) exposed to a living therapeutic material consisting of engineered E. coli encapsulated in Pluronic F127-based hydrogels. The release of inflammation-related cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN{gamma}) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) in response to the bacterial hydrogels, as well as the subsets of natural killer cells and T cells after exposure to the bacterial hydrogel for up to three days were examined. In direct contact with PBMCs, both E. coli and its endotoxin-free variant, ClearColi, induce apoptosis of the immune cells and trigger IL-6 release from the surviving cells. However, we found that encapsulation of the bacteria in Pluronic F127 diacrylate hydrogels considerably lowers their immunogenicity and practically abolishes apoptosis triggered by ClearColi. In comparison with E. coli, free and hydrogel-encapsulated ClearColi induced significantly lower levels of NK cell differentiation into the more cytolytic CD16dim subset. Our results demonstrate that ClearColi-encapsulated hydrogels generate low immunogenic response and are suitable candidates for the development of living therapeutic materials for in vivo testing to assess a potential clinical use. Nevertheless, we also observed a stronger immune response in pro-inflammatory PBMCs, possibly from donors with underlying infections. This suggests that including anti-inflammatory measures in living therapeutic material designs could be beneficial for such recipients.

immunology↗

bioRxiv2022

Regulating bacterial behavior within hydrogels of tunable viscoelasticity

Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in materials composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELMs function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells.

bioengineering↗