To Örebro University

Bacterial cellulose (BC) is a clinically established nanofibrillar wound dressing material that promotes healing by maintaining a moist and protected wound microenvironment. BC dressings can remain on wounds for extended periods, improving patient outcomes and reducing healthcare costs. However, BC lacks intrinsic antimicrobial properties, and infections in contaminated wounds remain a clinical concern, particularly in vulnerable patient populations. In this study, we present a benign and scalable self-assembly strategy to functionalize clinically used BC dressings with presynthesized colloidal silver nanoparticles (AgNPs) and antimicrobial peptides (C5), resulting in dual-action antimicrobial activity while preserving beneficial BC material properties. Colloidal AgNPs were efficiently adsorbed into the BC matrix by tailoring the interaction potential between the BC nanofibrils and the nanoparticles. Subsequent functionalization with C5 provided complementary antimicrobial mechanisms. The resulting dressings exhibited potent antimicrobial activity against Staphylococcus aureus, while maintaining high cytocompatibility with human primary keratinocytes and fibroblasts. By enabling tunable silver content, improved antimicrobial performance, and low cytotoxicity, the platform offers a promising route toward infection control in hard-to-heal wounds using clinically approved advanced BC dressings.

Mesoporous silica materials are promising carriers for antimicrobial peptides (AMPs), offering a versatile platform for combating bacterial infections. However, achieving high loading efficiency and controlled AMP release under physiological conditions remains a challenge. This study introduces a protein-capped mesoporous silica-based delivery system for treating topical bacterial infections. The system leverages elevated protease activity at infection sites to trigger the release of the sequence-optimized antimicrobial lipopeptide L-6-C5 (SOAP), facilitating efficient bacterial killing. SOAP was loaded into aminopropyl-functionalized SBA-15 mesoporous silica (amino-SBA-15) and capped with bovine serum albumin (BSA) or casein, forming amino-SBA-15-SOAP@protein. Protein adsorption prevented premature SOAP release while enabling protease-triggered delivery. BSA capping achieved 92.6 ± 0.2 % loading efficiency and enhanced peptide retention by 4.5-fold compared to non-capped particles, while casein yielded only a 1.25-fold increase. In the absence of proteases, SOAP release followed first-order kinetics, resulting in sustained release over 6 days. When exposed to trypsin, the release mechanism changed from diffusion-based to anomalous non-Fickian transport with zero-order kinetics, enabling rapid and efficient SOAP release. Proteolytic degradation of the protein cap also accelerated particle degradation and aggregation, offering insights into release dynamics under physiological conditions. The BSA-capped systems (amino-SBA-15-SOAP@BSA) showed effective bacteriostatic activity against Staphylococcus aureus (S. aureus), low hemolytic activity, and high cytocompatibility toward human dermal fibroblasts, outperforming free SOAP. Additionally, BSA capping reduced nonspecific protein binding in serum-rich media. By integrating sustained SOAP delivery with protease-triggered release, the amino-SBA-15-SOAP@BSA system addresses key limitations in AMP delivery, providing a promising strategy for controlled and localized AMP delivery in the treatment of topical bacterial infections.

Flaviviruses, including West Nile virus, Zika virus, and Dengue virus, pose global health challenges due to their distribution, pathogenicity, and lack of effective treatments or vaccines. This study investigated the antiviral activity of novel truncated peptides derived from the two-peptide plantaricins PLNC8 αβ, PlnEF, PlnJK, and PlnA. The antiviral potential was predicted using machine learning tools, followed by in vitro evaluation against the Kunjin virus using plaque reduction assays in Vero cells. Molecular docking assessed peptide interactions with KUNV and ZIKV. Full-length and truncated peptides from PlnA, PlnE, PlnF, PlnJ, and PlnK demonstrated limited antiviral efficacy against KUNV in vitro, despite in silico predictions suggesting antiviral potential for PlnA, PlnE, and PlnJ. Large discrepancies were observed between the predicted and experimentally determined activities. However, complementary two-peptide plantaricins PlnEF and PlnJK exhibited significant synergistic effects. Furthermore, the truncated peptides PLNC8 α1-15 and PLNC8 β1-20 reduced KUNV viral load by over 90%, outperforming their full-length counterparts. Molecular docking revealed interactions of PLNC8 α and PLNC8 β, and their truncated variants, with KUNV and ZIKV, suggesting a mechanism involving viral envelope disruption. These findings highlight the potential of plantaricin-derived peptides as promising antiviral candidates against flaviviruses, warranting further investigation into their mechanisms and applications.

Wounds are highly prone to infection, which can delay healing and lead to severe complications such as gangrene and sepsis. Non-healing wounds significantly impact patients' physical and mental well-being and place a substantial financial burden on healthcare systems. Timely and effective treatment of wound infections is critical, but the rise of antibiotic-resistant pathogens complicates this process. In this study, we investigate a potent protease resistant antimicrobial peptide (AMP), PLNC8 αβ, for the treatment of wound infections and present a strategy for localized AMP delivery using functionalized advanced nanocellulose (NC) wound dressings. Two types of NC dressings were explored: bacterial cellulose (BC) and TEMPO-oxidized nanocellulose derived from wood powder (TC). In a porcine wound infection model, PLNC8 αβ exhibited high antimicrobial activity, successfully eradicating the infection while promoting wound re-epithelialization. To achieve controlled release of PLNC8 αβ from the NC dressings, the peptides were either physisorbed directly onto the nanofibrils or encapsulated within mesoporous silica nanoparticles (MSNs) that were incorporated into the dressings. The PLNC8 αβ functionalized dressings demonstrated low cytotoxicity toward human primary fibroblasts and keratinocytes. Both BC and TC dressings showed efficient contact inhibition of bacteria but were less effective in inhibiting bacteria in suspension. In contrast, MSN-functionalized dressings, displayed significantly enhanced peptide-loading and sustained release capacities, resulting in improved antimicrobial efficacy. These findings highlight the potential of PLNC8 αβ and PLNC8 αβ-functionalized nanocellulose wound dressings for the treatment of infected wounds, offering an effective alternative to conventional antibiotic therapies.

Wound infections result in delayed healing, morbidity, and increased risks of sepsis. Early detection of wound infections can facilitate treatment and reduce the need for the excessive use of antibiotics. Proteases are normally active during the healing process but are overexpressed during infection as part of the inflammatory response. Proteases are also produced by the bacteria infecting the wounds, making proteases a highly relevant biomarker for infection monitoring. Here, we show a fluorescence turn-on sensor for real-time monitoring of protease activity in advanced nanocellulose wound dressings for rapid detection of wound pathogens. Colloidal gold nanoparticles (AuNPs) were adsorbed on bacterial cellulose (BC) nanofibrils by using a carefully optimized self-assembly process. The AuNPs could either be homogeneously incorporated in BC dressings or 3D printed in wood-derived cellulose nanofiber (CNF) dressings using a BC-AuNP ink. The BC-adsorbed AuNPs were subsequently functionalized with fluorophore-labeled protease substrates. Cleavage of the substrates by proteases produced by the wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa resulted in a significant increase in fluorescence that correlated with the growth phase of the bacteria. Wound dressing with integrated sensors for the detection of proteolytic activity can enable the sensitive and rapid detection of infections, allowing for optimization of treatment and reducing the risks of complications.

PLNC8 alpha beta is a cationic antimicrobial peptide that previously has been reported to express both antibacterial and antiviral properties. This study aimed to further elucidate the antiviral effects of PLNC8 alpha beta and its impact on virus-induced cytotoxicity and inflammatory signaling in human alveolar epithelial cells (A549) infected with the flavivirus Kunjin. Complementary in silico analyses using molecular dynamics (MD) simulation were conducted to investigate the mechanism of action of PLNC8 alpha beta by studying the interaction of PLNC8 alpha and beta with models of a flavivirus membrane and a eukaryotic plasma membrane, respectively. Our findings demonstrated that PLNC8 alpha beta significantly reduces both extracellular and intracellular viral loads, as confirmed by plaque reduction assays and RT-PCR. The peptide also mitigated virus-induced cytotoxicity and inflammation. Notably, PLNC8 alpha beta modulated the virus-induced dysregulation of key signaling and inflammatory genes, such as TLR9, TLR3, NOD2, FOS, JUN, IL6, and CXCL8. MD simulation revealed that PLNC8 alpha beta exhibits higher binding affinity for a flavivirus membrane model compared to a model of the plasma membrane, likely due to stronger electrostatic interactions with anionic phospholipids. This selective interaction possibly accounts for a potent antiviral activity of PLNC8 alpha beta combined with a minimal cytotoxicity toward human cells. Overall, PLNC8 alpha beta shows significant promise as an antiviral agent against flavivirus infections and warrants further exploration for peptide-based antiviral therapies.

Bacterial resistance towards antibiotics is a major global health issue. Very few novel antimicrobial agents and therapies have been made available for clinical use during the past decades, despite an increasing need. Antimicrobial peptides have been intensely studied, many of which have shown great promise in vitro. We have previously demonstrated that the bacteriocin Plantaricin NC8 αβ (PLNC8 αβ) from Lactobacillus plantarum effectively inhibits Staphylococcus spp., and shows little to no cytotoxicity towards human keratinocytes. However, due to its limitations in inhibiting gram-negative species, the aim of the present study was to identify novel antimicrobial peptidomimetic compounds with an enhanced spectrum of activity, derived from the β peptide of PLNC8 αβ. We have rationally designed and synthesized a small library of lipopeptides with significantly improved antimicrobial activity towards both gram-positive and gram-negative bacteria, including the ESKAPE pathogens. The lipopeptides consist of 16 amino acids with a terminal fatty acid chain and assemble into micelles that effectively inhibit and kill bacteria by permeabilizing their cell membranes. They demonstrate low hemolytic activity and liposome model systems further confirm selectivity for bacterial lipid membranes. The combination of lipopeptides with different antibiotics enhanced the effects in a synergistic or additive manner. Our data suggest that the novel lipopeptides are promising as future antimicrobial agents, however additional experiments using relevant animal models are necessary to further validate their in vivo efficacy.

The skin is the largest organ of the human body. Wounds disrupt the functions of the skin and can have catastrophic consequences for an individual resulting in significant morbidity and mortality. Wound infections are common and can substantially delay healing and can result in non-healing wounds and sepsis. Early diagnosis and treatment of infection reduce risk of complications and support wound healing. Methods for monitoring of wound pH can facilitate early detection of infection. Here we show a novel strategy for integrating pH sensing capabilities in state-of-the-art hydrogel-based wound dressings fabricated from bacterial nanocellulose (BC). A high surface area material was developed by self-assembly of mesoporous silica nanoparticles (MSNs) in BC. By encapsulating a pH-responsive dye in the MSNs, wound dressings for continuous pH sensing with spatiotemporal resolution were developed. The pH responsive BC-based nanocomposites demonstrated excellent wound dressing properties, with respect to conformability, mechanical properties, and water vapor transmission rate. In addition to facilitating rapid colorimetric assessment of wound pH, this strategy for generating functional BC-MSN nanocomposites can be further be adapted for encapsulation and release of bioactive compounds for treatment of hard-to-heal wounds, enabling development of novel wound care materials.

Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αβ, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αβ is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αβ against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αβ that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (μM), which is surprisingly efficient considering the high content of cholesterol (8–35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αβ, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αβ can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines.

Multidrug resistance bacteria constitue an increasing global health problem and the development of novel therapeutic strategies to face this challenge is urgent. Antimicrobial peptides have been proven as potent agents against pathogenic bacteria shown by promising in vitro results. The aim of this study was to characterize the antimicrobial effects of PLNC8 αβ on cell signaling pathways and inflammatory responses of human keratinocytes infected with S. aureus. PLNC8 αβ did not affect the viability of human keratinocytes but upregulated several cytokines (IL-1β, IL-6, CXCL8), MMPs (MMP1, MMP2, MMP9, MMP10) and growth factors (VEGF and PDGF-AA), which are essential in cell regeneration. S. aureus induced the expression of several inflammatory mediators at the gene and protein level and PLNC8 αβ was able to significantly suppress these effects. Intracellular signaling events involved primarily c-Jun via JNK, c-Fos and NFκB, suggesting their essential role in the initiation of inflammatory responses in human keratinocytes. PLNC8 αβ was shown to modulate early keratinocyte responses, without affecting their viability. The peptides have high selectivity towards S. aureus and were efficient at eliminating the bacteria and counteracting their inflammatory and cytotoxic effects, alone and in combination with low concentrations of gentamicin. We propose that PLNC8 αβ may be developed to combat infections caused by Staphylococcus spp.