Bacterial biofilm-related infections are a leading cause of delayed healing in chronic wounds. Within biofilms, bacteria are embedded in self-produced matrices of extracellular polymeric substances (EPS). EPS enable cell-to-cell communication (quorum-sensing) (Hausner et al., 1999), provide bacterial cells with a constant supply of nutrients, and maintain their ability to respond to environmental variations (Bridier et al., 2011). Furthermore, EPS hinder biofilm eradication, acting as a shield against host immune responses and antibiotics to protect encapsulated bacteria (Flemming et al., 2016). Consequently, biofilm-producing bacteria can be up to 1000-fold more resistant to antibiotics than planktonic cells of the same strain (Cascioferro et al., 2021). Therefore, effective biofilm eradication strategies are crucial for addressing antibiotic resistance in the treatment of bacterial biofilm-related infections (Bowler et al., 2020).

In recent years, bioactive compounds (Pompilio et al., 2023) and gas therapies (Yuan et al., 2021) have been explored for their potential applications in combating bacterial biofilms. On one hand, α-amylase (Am), one of the most popular bioactive anti-biofilm agents, not only hydrolyzes the polysaccharides matrix of EPS but also triggers pro-inflammatory responses, which results in the prevention of bacterial replication and reduction of the biofilm (Lahiri et al., 2021). For instance, commercially available Am has been reported to reduce biofilms in methicillin-resistant Staphylococcus aureus (MRSA) strains (Craigen et al., 2011). In contrast, nitric oxide (NO) acts as a quorum-sensing molecule, influencing biofilm dispersal and the conversion to the planktonic mode of growth, leading to the shift from a sessile biofilm phenotype to a free-swimming dispersal phenotype (Heckler et al., 2019). In addition to its anti-biofilm function, NO has been reported to trigger angiogenesis, a crucial process for tissue remodeling in infected wounds (Cooke et al., 2002). However, biofilm-dispersing agents alone, such as Am and NO, proved inadequate for complete biofilm eradication (Craigen et al., 2011, Choi et al., 2020). We hypothesize that due to their distinct mechanisms of action, the concurrent use of Am and NO would yield greater efficacy in eradicating biofilms. Biofilm dispersants can expose bacteria encapsulated within biofilms, rendering them more susceptible to antibiotics. Therefore, a combination of antibiotics and biofilm dispersants represents an effective approach for combating mature biofilms (Hawas et al., 2022). Furthermore, the addition of antibiotics to these two biofilm-dispersing agents may lead to a promising clinical strategy for bacterial biofilm control.

However, as a free radical gas with a short half-life, NO is notoriously difficult to deliver and achieve controlled release (Rong et al., 2019). The most versatile option for NO delivery is to use NO donors that can liberate NO either spontaneously or under specific conditions, such as those controlled by light, heat, transition metals, and enzymes (Scatena et al., 2005, Yang et al., 2021). Among these molecules, stable NO donors are activated by photothermal stimulation or reactive oxygen species. For example, NO release from L-arginine can be induced by endogenous H2O2; however, the release profile is uncontrollable (Chen et al., 2023). Photothermally sensitive NO donors can easily cause additional thermal damage, as photothermal therapy lacks effective temperature control (Fu et al., 2023, Lv et al., 2023). Other NO donors are less stable, resulting in a short half-life ranging from several minutes to a few hours (Cai & Webb, 2020). Novel delivery systems can prolong the release of NO. Topological supramolecular nanocarriers prolong the half-life of N-diazeniumdiolates (typical NO donors) from several seconds to 5 h (Li et al., 2023). Approximately 60% of the NO was released from the donors within 24 h of immersion when the donors were loaded onto chitosan films (Choi et al., 2020). However, the application of these reported delivery systems is limited due to the short NO release periods (less than 1 d) and the constraints on co-delivering hydrophobic and hydrophilic drugs or biologics simultaneously, making it difficult to implement a combined therapy of biofilm eradication and antibiotics within a single delivery system.

Herein, we fabricated a novel nanocomposite hydrogel (NC gel) which achieved long-term controlled release of NO and co-delivery of hydrophilic drugs and biologics. In our previous study, the surface vicinal diols of poly(lactic acid)-hyperbranched polyglycerol (PLA-HPG) based nanoparticles could be oxidated into aldehydes, rendering tissue adhesion properties for the nanoparticles as the aldehydes interact protein amines via Schiff-base linkages (Deng et al., 2015). Thus the oxidized nanoparticles were known as bioadhesive nanoparticles (BNPs). In this study, a spontaneous NO donor molecule, S-Nitrosotetradecanethiol (SNO), was loaded into BNPs, which prolonged the release of NO from a few hours to 3 days. A novel NC gel was prepared from BNPs and the hydrophilic carboxymethyl chitosan (CS) via Schiff-base linkages, which is suitable for the co-delivery of SNO, Am and an antibiotic cefepime (Cef). As shown in Fig. 1, a reversible crosslink is formed between the aldehyde groups on the BNPs and the amine groups on the CS via Schiff base reaction, allowing the NC gel to switch between liquid and solid states. The hydrophobic NO donor, SNO, is encapsulated in BNPs, which spontaneously release NO in a sustained and controlled manner, while Am and Cef are loaded into the porous structure of the NC gel (SNO/BNP/CS@Am-Cef). Following delivery to MRSA biofilm-infected sites, SNO/BNPs are released from SNO/BNP/CS@Am-Cef and adhere to the wound area. While Am hydrolyzes EPS to degrade the biofilm, and SNO/BNPs generate NO to promote the release of biofilm bacteria via quorum sensing. The dispersion of the biofilm by SNO and Am causes the bacteria to transition from the sessile to planktonic phase, enhancing the interaction between the bacteria and the antibiotic Cef that is released from the NC gel. Such combined therapy can be used to eradicate MRSA biofilms and trigger angiogenesis to facilitate tissue healing. SNO/BNP/CS@Am-Cef has shown improved therapeutic efficacy in MRSA biofilm-infected mice, which highlights the advantages of the BNP/CS NC gel for co-delivery of hydrophobic drugs, hydrophilic compounds, and biologics, and emphasizes combination therapy to efficiently combat bacterial biofilm-related infections.