Immunomodulatory effect of mycobacterial outer membrane vesicles coated nanoparticles.

Tuberculosis (TB) is one of the most widely prevalent infectious diseases that cause significant mortality. Bacillus Calmette-Guérin (BCG), the current TB vaccine used in clinics, shows variable efficacy and has safety concerns for immunocompromised patients. There is a need to develop new and more effective TB vaccines. Outer membrane vesicles (OMVs) are vesicles released by Mycobacteria that contain several lipids and membrane proteins and act as a good source of antigens to prime immune response. However, the use of OMVs as vaccines has been hampered by their heterogeneous size and low stability. Here we report that mycobacterial OMVs can be stabilized by coating over uniform-sized 50 nm gold nanoparticles. The OMV-coated gold nanoparticles (OMV-AuNP) show enhanced uptake and activation of macrophages and dendritic cells. Proteinase K and TLR inhibitor studies demonstrated that the enhanced activation was attributed to proteins present on OMVs and was mediated primarily by TLR2 and TLR4. Mass spectrometry analysis revealed several potential membrane proteins that were common in both free OMVs and OMV-AuNP. Such strategies may open up new avenues and the utilization of novel antigens for developing TB vaccines.

High ligand density drives extensive spreading and motility on soft GelMA gels.

In comparison to synthetic hydrogels where ligand density and stiffness can be independently tuned, cell responses are expected to deviate on native biopolymer networks where ligand density and stiffness are coupled. Here we probe the tensional homeostasis of fibroblasts on methacrylated gelatin (GelMA) gels, which are widely used in tissue engineering applications. On 5%-15% GelMA gels which are very soft (10-100's of Pa's in stiffness), fibroblasts were found to spread extensively and assemble prominent stress fibers and focal adhesions. Probing of contractile mechanics using trypsin-induced detachment revealed adhesive drag, but not contractility, was sensitive to GelMA concentration. Contractility-altering drugs blebbistatin and nocodazole, which exhibited opposite effects on focal adhesion size, both led to reduction in adhesive drag and cell rounding. However, cell motility was impacted only in nocodazole-treated cells. Collectively, our experiments suggest that on soft GelMA gels, contractility-independent adhesion clustering mediated by high ligand density can drive cell spreading and motility.

Silver-Nanoparticle-Entrapped Soft GelMA Gels as Prospective Scaffolds for Wound Healing.

Gelatin-based hydrogels have received particular attention for tissue-engineering applications given their biocompatibility, ease of tuning their physical properties through chemical modifications, and incorporation of antibacterial activity. While several studies have focused on the detailed quantification of biomechanical properties of these gels, considerably less attention has been paid to understanding how adhesivity of these gels impacts single as well as collective cell migration, which directly determines the efficacy of wound healing. In this study, we address this question by quantifying fibroblast motility and antibacterial activity of silver nanoparticle (AgNP)-entrapped methacrylated gelatin (GelMA) hydrogels. Using 5 and 15% GelMA soft gels cross-linked with 1 min UV exposure, we first show that cells spread more and migrate faster on 15% GelMA gels. Next, we show that ∼10 nm AgNPs entrapped in 15% GelMA gels get released over a time-scale greater than 72 h and exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria at concentrations nontoxic to cells. Finally, using a polydimethylsiloxane (PDMS) device for simulating wound healing, we show that closure of ∼800 µm gaps on GelMA gels is significantly faster compared with other conditions. Together, our findings illustrate the potential of AgNP-entrapped soft GelMA gels as scaffolds for achieving accelerated wound healing of deep dermal wounds by enabling fast fibroblast migration and minimization of microbial infections.

Engineering interfacial migration by collective tuning of adhesion anisotropy and stiffness.

Interfacial migration is central to multiple processes including morphogenesis and wound healing. However, the sensitivity of interfacial migration to properties of the interfacial microenvironment has not been adequately explored. Here, we address this question by tracking motility of 3T3 fibroblasts at the interface of two hydrogels. By sandwiching cells between two adhesive gels (composed of methacrylated gelatin) or between an adhesive and a non-adhesive gel (composed of gellan), we show that cells are more motile in case of the latter. By tuning the bulk stiffness of the gellan gel, we then show that motility is tuned in a stiffness-dependent manner. Fastest motility observed in case of the stiffest gel was associated with increased cell height, suggestive of stiffness-mediated cytoskeletal assembly. Inhibition of cell motility by contractile agonists and actin depolymerizing drugs is indicative of a mode of migration wherein cells combine contractile tractions exerted at their base and actin-based pushing forces on the top surface to propel themselves forward. Together, our results suggest that dorso-ventral adhesion anisotropy and stiffness can be collectively tuned to engineer interfacial migration. STATEMENT OF SIGNIFICANCE: It is increasingly understood that cells migrate in vivo through confining spaces which typically occur as pores in the matrix and through naturally occurring interfaces that exist between neighbouring ECM fibers, or between the stroma and the vasculature. Such interfaces are also created when treating wounds on the skin surface by covering the wounds with adhesives. How multiple cues impact interfacial migration has not been adequately addressed. By studying cell migratory behaviour at the interface of two hydrogel substrates, we identify adhesivity and stiffness as two critical factors that can be tuned to maximize cell migration. We foresee a potential use of this knowledge in the design of tissue adhesives for wound healing applications.

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif.

Amyloids are highly ordered, cross-ß-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.