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.

Modern medicine relies on the access to effective antibiotics. They are not only necessary to treat infections but enable the invasive therapies and surgeries to which we are accustomed today. Hence, the significant rise of bacterial resistance towards antibiotics threatens to topple a large part of global health care. This thesis investigates the potential of two antimicrobial peptides (AMPs), namely the bacteriocin Plantaricin NC8 ∝β (PLNC8 ∝β), and a novel synthetic lipopeptide derived from PLNC8 β termed 6-C5-N, for the topical treatment of infected wounds. Through a series of studies, the effectiveness and broad-spectrum activity of both these AMPs in vitro is demonstrated, and their influence on human cells in regard to toxicity, inflammation and survival is evaluated. Both AMPs exhibit low cytotoxicity in vitro and modulate important cytokines and growth factors in relation to infection and wound healing. Furthermore, utilizing ex vivo and in vivo models, it is demonstrated that 6-C5-N is an interesting candidate for the topical treatment of infected wounds. Additionally, a possible explanation of the complex problem with bacterial resistance to AMPs is presented, by demonstrating how extracellular divalent cations can be utilized by gram negative bacteria as protection against positively charged antibacterial peptides. In conclusion, PLNC8 ∝β and its derivative lipopeptide 6-C5-N are promising candidates for topical treatment of infected tissues and could play a role in the struggle against the development of antimicrobial resistance.

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.

Introduction/Objectives: Wound infections are a major clinical challenge, often leading to severe complications and impeding the normal healing process, thereby resulting in chronic, non-healing wounds. These wounds impose a profound physical and psychological burden on patients while significantly increasing healthcare costs. Effective and timely treatment of wound infections is imperative; however, the escalating prevalence of antibiotic-resistant pathogens complicates infection management. This study aims to evaluate the antimicrobial efficacy of the potent two-peptide bacteriocin PLNC8 αβ for treating wound infections and to develop a localized delivery system using advanced functionalized nanocellulose (NC) wound dressings.

Methods: The in vivo efficacy of PLNC8 αβ was evaluated using an infected porcine wound model. To develop a system for controlled release of PLNC8 αβ, two types of nanocellulose-based wound dressings were investigated: bacterial cellulose (BC) and TEMPO-oxidized nanocellulose derived from hardwood (TC). To endow the dressings with antimicrobial properties, they were functionalized with PLNC8 αβ. Functionalization with PLNC8 αβ was achieved by either direct physisorption onto the nanocellulose fibrils or by encapsulation within mesoporous silica nanoparticles (MSNs), which were integrated into the BC dressings.

Results: In a porcine wound infection model, PLNC8 αβ effectively eradicated bacterial infections while promoting re-epithelialization. The PLNC8 αβ functionalized dressings demonstrated low cytotoxicity toward human primary fibroblasts and keratinocytes. Both BC and TC dressings demonstrated robust contact-based antibacterial activity but exhibited limited efficacy against bacteria in suspension. MSN-functionalized BC dressings significantly improved peptide-loading capacity and provided sustained release of PLNC8 αβ, leading to superior antimicrobial performance.

Conclusions: This study underscores the potential of PLNC8 αβ for treatment of wound infections and demonstrates PLNC8 αβ functionalized nanocellulose wound dressings as an innovative therapeutic approach for managing infected wounds. By combining potent antimicrobial activity with advanced delivery mechanisms, this platform offers a promising alternative to conventional antibiotic treatments, addressing the growing challenge of antibiotic resistance.

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.

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.

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.