Drug-resistant bacterial infections and their lipopolysaccharide-related inflammatory complications continue to pose significant challenges in traditional treatments. Inspired by the rapid initiation of resident macrophages to form aggregates for efficient antibacterial action, this study proposes a multifunctional and enhanced antibacterial strategy through the construction of novel biomimetic cell membrane polypeptide nanonets (R-DPB-TA-Ce). The design involves the fusion of end-terminal lipidated polypeptides containing side-chain cationic boronic acid groups (DNPLBA) with cell membrane intercalation engineering (R-DPB), followed by coordination with the tannic acid-cerium complex (TA-Ce) to assemble into a biomimetic nanonet through boronic acid-polyphenol-metal ion interactions. In addition to the ability of RAW 264.7 macrophages cell membrane components\' (R) ability to neutralize lipopolysaccharide (LPS), R-DPB-TA-Ce demonstrated enhanced capture of bacteria and its LPS, leveraging nanoconfinement-enhanced multiple interactions based on the boronic acid-polyphenol nanonets skeleton combined with polysaccharide. Utilizing these advantages, indocyanine green (ICG) is further employed as a model drug for delivery, showcasing the exceptional treatment effect of R-DPB-TA-Ce as a new biomimetic assembled drug delivery system in antibacterial, anti-inflammatory, and wound healing promotion. Thus, this strategy of mimicking macrophage aggregates is anticipated to be further applicable to various types of cell membrane engineering for enhanced antibacterial treatment.

Type I interferons (IFN-Is) are critical antiviral cytokine in innate immunity but with limited direct defense ability against bacterial infections in mammals. In bony fish, despite all the IFN-Is (IFN1-4) act in antiviral immunity, studies demonstrate that IFN1 can remarkably contribute to host defense against bacterial infections. In this study, we found that IFN1 from grass carp (Ctenopharyngodon idella) contains an unusual cationic and amphipathic α-helical region (named as gcIFN-20, sequence: SYEKKINRHFKILKKNLKKK). The synthesized peptide gcIFN-20 could form α-helical structure in a membrane environment and exerts potent antimicrobial activity against multiple species of Gram-negative (G-) and Gram-positive (G+) bacteria with negligible toxicity. Mechanism studies showed gcIFN-20 kills G+ bacteria through membrane disruption and cytoplasm outflow while G- bacteria through membrane permeation and protein synthesis inhibition. In two mouse bacterial infection models, gcIFN-20 therapy could significantly reduce tissue bacterial loads and mortalities. In addition to the direct antibacterial activity, we also found that gcIFN-20 could significantly suppress the lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro and in vivo, obviously alleviated lung lesions in a mouse endotoxemia model. The mechanism is that gcIFN-20 interacts with LPS, causes LPS aggregation and neutralization. The antimicrobial and anti-inflammatory activities in vivo of gcIFN-20 in mammalian models suggested a promising agent for developing peptide-based antibacterial therapy. IMPORTANCE Type I interferons play crucial role in antiviral immunity in both vertebrates and invertebrates. The powerful antimicrobial activity is recently reported in nonmammalian vertebrates. The present study identified a novel antimicrobial peptide (gcIFN-20) derived from grass carp interferon 1, found gcIFN-20 exhibits forceful bactericidal and anti-inflammatory activity in mammals, and efficient therapeutic effect against two clinical severe extraintestinal pathogenic Escherichia coli and a mouse endotoxemia models. The antimicrobial mechanisms are membrane disruption and cytoplasm overflow for Gram-positive bacteria, while membrane permeation and protein synthesis inhibition for Gram-negative bacteria. The anti-inflammatory mechanisms can be aggregating and neutralizing lipopolysaccharide to attenuate the binding with receptors and facilitate phagocytosis. The results indicate that gcIFN-20 can be a promising novel therapeutic agent for bacterial diseases and inflammatory disorders, especially as a potential weapon for multidrug resistant strain infections.

Lipopolysaccharide (LPS) has been implicated as a major cause of inflammation and an uncontrolled LPS response increases the risk of localized inflammation and sepsis. While some native peptides are helpful in the treatment of LPS-induced inflammation, the use of these peptides is limited due to their potential cytotoxicity and poor anti-inflammatory activity. Hybridization is an effective approach for overcoming this problem. In this study, a novel hybrid anti-inflammatory peptide that combines the active center of Cathelicidin 2 (CATH2) with thymopentin (TP5) was designed [CTP, CATH2 (1-13)-TP5]. CTP was found to have higher anti-inflammatory effects than its parental peptides through directly LPS neutralization. However, CTP scarcely inhibited the attachment of LPS to cell membranes or suppressed an established LPS-induced inflammation due to poor cellular uptake. The C-terminal amine modification of CTP (CTP-NH2) was then designed based on the hypothesis that C-terminal amidation can enhance the cell uptake by increasing the hydrophobicity of the peptide. Compared with CTP, CTP-NH2 showed enhanced anti-inflammatory activity and lower cytotoxicity. CTP-NH2 not only has strong LPS neutralizing activity, but also can significantly inhibit the LPS attachment and the intracellular inflammatory response. The intracellular anti-inflammatory effect of CTP-NH2 was associated with blocking of LPS binding to the Toll-like receptor 4-myeloid differentiation factor 2 complex and inhibiting the nuclear factor-kappa B pathway. In addition, the anti-inflammatory effect of CTP-NH2 was confirmed using a murine LPS-induced sepsis model. Collectively, these findings suggest that CTP-NH2 could be developed into a novel anti-inflammatory drug. This successful modification provides a design strategy to improve the cellular uptake and anti-inflammatory activity of peptide agents.

Intestinal inflammatory disorders, such as inflammatory bowel disease (IBD), are associated with increased pro-inflammatory cytokine secretion in the intestines. Furthermore, intestinal inflammation increases the risk of enteric cancer, which is a common malignancy globally. Native anti-inflammatory peptides are a class of anti-inflammatory agents that could be used in the treatment of several intestinal inflammation conditions. However, potential cytotoxicity, and poor anti-inflammatory activity have prevented their development as anti-inflammatory agents. Therefore, in this study, we designed and developed a novel hybrid peptide for the treatment of intestinal inflammation. Eight hybrid peptides were designed by combining the active centers of antimicrobial peptides, including LL-37 (13-36), YW12D, innate defense regulator 1, and cathelicidin 2 (1-13) with thymopentin or the active center of thymosin alpha 1 (Tα1) (17-24). The hybrid peptide, LL-37-Tα1 (LTA), had improved anti-inflammatory activity with minimal cytotoxicity. LTA was screened by molecule docking and in vitro experiments. Likewise, its anti-inflammatory effects and mechanisms were also evaluated using a lipopolysaccharide (LPS)-induced intestinal inflammation murine model. The results showed that LTA prevented LPS-induced impairment in the jejunum epithelium tissues and infiltration of leukocytes, which are both histological markers of inflammation. Additionally, LTA decreased the levels of tumor necrosis factor-alpha, interferon-gamma, interleukin-6, and interleukin-1β. LTA increased the expression of zonula occludens-1 and occludin, and reduced permeability and apoptosis in the jejunum of LPS-treated mice. Additionally, its anti-inflammatory effect is associated with neutralizing LPS, binding to the Toll-like receptor 4-myeloid differentiation factor 2 (TLR4/MD-2) complex, and modulating the nuclear factor-kappa B signal transduction pathway. The findings of this study suggest that LTA may be an effective therapeutic agent in the treatment of intestinal inflammation.

Intestinal inflammation is an inflammatory disease resulting from immune dysregulation in the gut. It can increase the risk of enteric cancer, which is a common malignancy globally. As a new class of anti-inflammatory agents, native peptides have potential for use in the treatment of several intestinal inflammation conditions; however, their potential cytotoxicity and poor anti-inflammatory activity and stability have prevented their development. Hybridization has been proposed to overcome this problem. Thus, in this study, we designed a hybrid peptide (LL-37-TP5, LTP) by combing the active centre of LL-37 (13-36) with TP5. The half-life and cytotoxicity were tested in vitro, and the hybrid peptide showed a longer half-life and lower cytotoxicity than its parental peptides. We also detected the anti-inflammatory effects and mechanisms of LTP on Lipopolysaccharide (LPS)-induced intestinal inflammation in murine model. The results showed that LTP effectively prevented LPS-induced weight loss, impairment of intestinal tissues, leukocyte infiltration, and histological evidence of inflammation. Additionally, LTP decreased the levels of tumour necrosis factor-alpha, interferon-gamma, and interleukin-6; increased the expression of zonula occludens-1 and occludin; and reduced permeability in the jejunum of LPS-treated mice. Notably, LTP appeared to be more potent than the parental peptides LL-37 and TP5. The anti-inflammatory effects of LTP may be associated with the neutralization of LPS, inhibition of oxidative stress, and inhibition of the NF-κB signalling pathway. The findings of this study suggest that LTP might be an effective therapeutic agent for treating intestinal inflammation.