Antibacterial 3D-Printed Silver Nanoparticle/Poly Lactic-Co-Glycolic Acid (PLGA) Scaffolds for Bone Tissue Engineering.

Infectious bone defects present a major challenge in the clinical setting currently. In order to address this issue, it is imperative to explore the development of bone tissue engineering scaffolds that are equipped with both antibacterial and bone regenerative capabilities. In this study, we fabricated antibacterial scaffolds using a silver nanoparticle/poly lactic-co-glycolic acid (AgNP/PLGA) material via a direct ink writing (DIW) 3D printing technique. The scaffolds' microstructure, mechanical properties, and biological attributes were rigorously assessed to determine their fitness for repairing bone defects. The surface pores of the AgNPs/PLGA scaffolds were uniform, and the AgNPs were evenly distributed within the scaffolds, as confirmed via scanning electron microscopy (SEM). Tensile testing confirmed that the addition of AgNPs enhanced the mechanical strength of the scaffolds. The release curves of the silver ions confirmed that the AgNPs/PLGA scaffolds released them continuously after an initial burst. The growth of hydroxyapatite (HAP) was characterized via SEM and X-ray diffraction (XRD). The results showed that HAP was deposited on the scaffolds, and also confirmed that the scaffolds had mixed with the AgNPs. All scaffolds containing AgNPs exhibited antibacterial properties against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). A cytotoxicity assay using mouse embryo osteoblast precursor cells (MC3T3-E1) showed that the scaffolds had excellent biocompatibility and could be used for repairing bone tissue. The study shows that the AgNPs/PLGA scaffolds have exceptional mechanical properties and biocompatibility, effectively inhibiting the growth of S. aureus and E. coli. These results demonstrate the potential application of 3D-printed AgNPs/PLGA scaffolds in bone tissue engineering.

Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

Influenza A virus poses a constant challenge to human health. The highly conserved influenza matrix-2 (M2) protein is an attractive target for the development of a universal antibody-based drug. However, screening using antigens with subphysiological conformation in a nonmembrane environment significantly reduces the generation of efficient antibodies. Here, M2(1-46) was incorporated into nanodiscs (M2-nanodiscs) with M2 in a membrane-embedded tetrameric conformation, closely resembling its natural physiological state in the influenza viral envelope. M2-nanodisc generation, an antigen, was followed by Chiloscyllium plagiosum immunization. The functional vNARs were selected by phage display panning strategy from the shark immune library. One of the isolated vNARs, AM2H10, could specifically bind to tetrameric M2 instead of monomeric M2e (the ectodomain of M2 protein). Furthermore, AM2H10 blocked ion influx through amantadine-sensitive and resistant M2 channels. Our findings indicated the possibility of developing functional shark nanobodies against various influenza viruses by targeting the M2 protein.

Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer.

Smoothened (SMO) protein is a member of the G protein-coupled receptor (GPCR) family that is involved in the Hedgehog (Hh) signaling pathway. It is a putative target for treating various cancers, including medulloblastoma and basal cell carcinoma (BCC). Characterizing membrane proteins such as SMO in their native state is highly beneficial for the development of effective pharmaceutical drugs, as their structures and functions are retained to the highest extent in this state. Therefore, although SMO protein is conventionally solubilized in detergent micelles, incorporating the protein in a lipid-based membrane mimic is still required. In this study, we used styrene maleic acid (SMA) copolymer that directly extracted membrane protein and surrounding lipids as well as formed the so-called polymer nanodiscs, to solubilize and purify the SMO transmembrane domain encapsulated by SMA-nanodiscs. The obtained SMA-nanodiscs showed high homogeneity and maintained the physiological activity of SMO protein, thereby enabling the measurement of the dissociation constant (Kd) for SMO ligands SMO-ligands Shh Signaling Antagonist V (SANT-1) and Smoothened Agonist (SAG) using ligand-based solution nuclear magnetic resonance spectroscopy. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment.

Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain.

OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.