OBJECTIVE: Staphylococcus aureus is an opportunistic pathogen whose treatment is further complicated by its ability to form biofilms. In this study, we examine the impact of growing S. aureus biofilms on different polymerizing surfaces, specifically agar and agarose, on the pathogen\'s tolerance to fluoroquinolones.
RESULTS: Biofilms of two methicillin-resistant strains of S. aureus were grown on agar or agarose in the presence of the same added nutrients, and their antibiotic susceptibility to two fluoroquinolones, moxifloxacin (MXF) and delafloxacin (DLX), were measured. We also compared the metabolism and extracellular polymeric substances (EPS) production of biofilms that were grown on agar and agarose.
CONCLUSIONS: Biofilms that were grown on agarose were consistently more susceptible to antibiotics than those grown on agar. We found that in biofilms that were grown on agar, extracellular protein composition was higher, and adding EPS to agarose-grown biofilms increased their tolerance to DLX to levels that were comparable to agar-grown biofilms.

Exploiting high-performance magnetic beads for specific enrichment of ribonucleic acid (RNA) has important significance in the biomedical research field. Herein, a simple strategy was proposed for fabricating boronate-decorated polyethyleneimine-grafted magnetic agarose beads (BPMAB), which can selectively isolate cis-diol-containing substances through boronate affinity. The size of the basic magnetic agarose beads was controlled through the emulsification of the water-in-oil emulsion with a high-speed shear machine, which enhanced the specific surface area of BPMAB. Subsequently, to modify more boronic acid ligands, branched PEI with excellent hydrophilicity and numerous reaction sites was grafted. 2,4-Difluoro-3-formylphenyl boronic acid (2,4-DFPBA) was covalently immobilized for selectively capturing cis-diol-containing substances under physiological condition (pH 7.4). The BPMAB with a diameter range from 1.86 μm to 11.60 μm possessed clearly spherical structure, and excellent magnetic responsiveness and suspension ability in aqueous solution. β-Nicotinamide adenine dinucleotide (β-NAD), a short-chain cis-diol carrying agent, was selected as a target molecule for evaluating the adsorption property of BPMAB and the maximum adsorption capacity of BPMAB for β-NAD could reach 205.11 mg g-1. In addition, the BPMAB as adsorbent was used to selectively enrich RNA from mammalian cells. The maximum adsorption capacity of BPMAB for RNA was 140.50 mg g-1. Under optimized conditions, the BPMAB-based MSPE successfully enriched the high-quality total RNA with 28S to 18S ribosomal RNA ratios ranging from 2.06 to 2.16. According to the PCR analysis of GADPH gene, the extracted total RNA was successfully reverse transcribed into cDNA. Therefore, we believe that the BPMAB-based MSPE could be applicable for the specific enrichment of RNA from complex biological systems.

The realization of soft robotic devices with life-like properties requires the engineering of smart, active materials that can respond to environmental cues in similar ways as living cells or organisms. Cell-free expression systems provide an approach for embedding dynamic molecular control into such materials that avoids many of the complexities associated with genuinely living systems. Here, we present a strategy to integrate cell-free protein synthesis within agarose-based hydrogels that can be spatially organized and supplied by a synthetic vasculature. We first utilize an indirect printing approach with a commercial bioprinter and Pluronic F-127 as a fugitive ink to define fluidic channel structures within the hydrogels. We then investigate the impact of the gel matrix on the expression of proteins in E. coli cell-extract, which is found to depend on the gel density and the dilution of the expression system. When supplying the vascularized hydrogels with reactants, larger components such as DNA plasmids are confined to the channels or immobilized in the gels while nanoscale reaction components can diffusively spread within the gel. Using a single supply channel, we demonstrate different spatial protein concentration profiles emerging from different cell-free gene circuits comprising production, gene activation, and negative feedback. Variation of the channel design allows the creation of specific concentration profiles such as a long-term stable gradient or the homogeneous supply of a hydrogel with proteins.

Bacteria are often found in environments where space is limited, and they attach themselves to surfaces. One common form of movement on these surfaces is bacterial twitching motility, which is powered by the extension and retraction of type IV pili. Although twitching motility in unrestricted conditions has been extensively studied, the effects of spatial confinement on this behaviour are not well understood. In this study, we explored the diffusive properties of individual twitching Pseudomonas aeruginosa cells in spatially confined conditions. We achieved this by placing the bacteria between layers of agarose and glass, and then tracking the long-term twitching motility of individual cells. Interestingly, we found that while confinement reduced the immediate speed of twitching, it paradoxically increased diffusion. Through a combination of mechanical and geometrical analysis, as well as numerical simulations, we showed that this increase in diffusion could be attributed to mechanical factors. The constraint imposed by the agarose altered the diffusion pattern of the bacteria from normal to superdiffusion. These findings provide valuable insights into the motile behaviour of bacteria in confined environments.

As an emerging biomedical material, wound dressings play an important therapeutic function in the process of wound healing. It can provide an ideal healing environment while protecting the wound from a complex external environment. A hydrogel wound dressing composed of tilapia skin gelatin (Tsg) and fucoidan (Fuc) was designed in this article to enhance the microenvironment of wound treatment and stimulate wound healing. By mixing horseradish peroxidase (HRP), hydrogen peroxide (H2O2), tilapia skin gelatin-tyramine (Tsg-Tyr), and carboxylated fucoidan-tyramine in agarose (Aga), using the catalytic cross-linking of HRP/H2O2 and the sol-gel transformation of Aga, a novel gelatin-fucoidan (TF) double network hydrogel wound dressing was constructed. The TF hydrogels have a fast and adjustable gelation time, and the addition of Aga further enhances the stability of the hydrogels. Moreover, Tsg and Fuc are coordinated with each other in terms of biological efficacy, and the TF hydrogel demonstrated excellent antioxidant properties and biocompatibility in vitro. Also, in vivo wound healing experiments showed that the TF hydrogel could effectively accelerate wound healing, reduce wound microbial colonization, alleviate inflammation, and promote collagen deposition and angiogenesis. In conclusion, TF hydrogel wound dressings have the potential to replace traditional dressings in wound healing.

Biotherapeutics is the fastest growing class of drugs administered by subcutaneous injection. In vitro release testing mimicking physiological conditions at the injection site may guide formulation development and improve biopredictive capabilities. Here, anin vitrorelease cartridge (IVR cartridge) comprising a porous agarose matrix emulating subcutaneous tissue was explored. The objective was to assess effects of medium composition and incorporation of human serum albumin into the matrix. Drug disappearance was assessed for solution, suspension and in situ precipitating insulin products (Actrapid, Levemir, Tresiba, Mixtard 30, Insulatard, Lantus) using the flow-based cartridge. UV-Vis imaging and light microscopy visualized dissolution, precipitation and albumin binding phenomena at the injection site. Divalent cations present in the release medium resulted in slower insulin disappearance for suspension-based and in situ precipitating insulins. Albumin-binding acylated insulin analogs exhibited rapid disappearance from the cartridge; however, sustained retention was achieved by coupling albumin to the matrix. An in vitro-in vivorelation was established for the non-albumin-binding insulins.The IVR cartridge is flexible with potential in formulation development as shown by the ability to accommodate solutions, suspensions, and in situ forming formulations while tailoring of the system to probe in vivo relevant medium effects and tissue constituent interactions.

Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE.

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived fromin vivostudies in mice, asin vitrostudies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survivalin vitrofor up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCsin vitro. Our innovative model system is suitable for in-depthin vitrostudies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Cancer poses a significant threat to human health, and monotherapy frequently fails to achieve optimal therapeutic outcomes. Based on this premise, porphyran (PHP), a marine polysaccharide with immunomodulatory function, was used as a framework to coat gold nanorods and construct a novel nanomedicine (PHP-MPBA-GNRs) combining photothermal therapy and immunotherapy. In this design, PHP not only maintained the dispersion stability and photothermal stability of gold nanorods but also could be released under weakly acidic conditions to activate anti-tumor immunity. In vivo studies have shown that PHP-MPBA-GNRs can effectively inhibit tumor cell proliferation and reduce metastasis under near-infrared (NIR) light irradiation. Preliminary mechanistic investigations revealed that PHP-MPBA-GNRs could increase reactive oxygen species (ROS) and induce apoptosis in cancer cells. The PHP in PHP-MPBA-GNRs can also activate dendritic cells and up-regulate the expression of co-stimulatory molecules and antigen-presenting complexes. All biological experiments, including in vivo tests, demonstrated that PHP-MPBA-GNRs achieved a combination of photothermal therapy and immunotherapy for tumors.

The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.