Introduction

With the acceleration of aging in the global population, chronic wounds have become a significant public health threat and healthcare burden. A positive correlation was observed between the age of patients and the healing period of chronic wounds.1 Bacterial infection is the most common complication of chronic wounds, and most bacteria in chronic wounds occur as biofilm, which is a leading cause of delayed wound healing.2 Significant types of chronic wounds include diabetic ulcers, traumatic ulcers, pressure ulcers, and venous ulcers. Chronic non-healing wounds often deteriorate into multidrug-resistant (MDR) bacterial infections, producing a large amount of purulent discharge with malodor, presenting an urgent problem that requires clinical treatment.

The traditional treatment for bacterial biofilm removal in chronic wounds involves antibiotic therapies. Currently, studies have shown that the overuse of antibiotics significantly promotes the emergence of “multidrug-resistant bacteria”, which not only increases treatment costs but also can raise case fatality rates. The distribution of drug-resistant pathogens was identified as a significant global public health problem. Nanoparticles have been reported to exhibit high antimicrobial activity against “multidrug-resistant bacteria”, and due to multiple mechanisms acting simultaneously, it is difficult for new drug-resistant strains to emerge.3 Recent microbiological research has begun to focus on antibacterial nanoparticles, whose effectiveness in killing various pathogens in wounds, such as bacteria, fungi, and viruses, has been demonstrated in many laboratory studies.4

Recent studies have elucidated the mechanisms and advantages of nanoparticles in inhibiting bacterial growth and biofilm formation by comparing various types of nanomaterials—such as metal and polymer nanoparticles—regarding their antimicrobial properties.3 In addition, researchers have categorized these nanoparticles into four main types, each employing different mechanisms against biofilms. These mechanisms include the direct killing of bacteria within the biofilm, disruption of the biofilm’s structure, release of antimicrobial agents, and enhancement of immune function to combat infections5 However, many existing studies primarily focus on a single mechanism of action for nanoparticles against bacterial biofilms and lack a systematic analysis of the dynamic process of bacterial biofilm formation in chronic wounds.

In this paper, we explore the complex relationship between bacterial biofilms, non-healing chronic wounds, and recurrent infections. The formation of bacterial biofilms is a complex, multi-stage process. To effectively target these biofilms in chronic wounds, we have divided the mechanisms by which nanoparticles inhibit biofilm formation into four stages: (1) preventing the initial settlement and attachment of planktonic bacteria, (2) interfering with bacterial reproduction and colony formation, (3) disrupting mature bacterial biofilms within the chronic wound, and (4) reducing the regeneration and spread of bacteria on the surfaces of mature biofilms. It is important to note that these four stages are continuous and highly coordinated processes. Furthermore, we aim to summarize the factors influencing the efficacy of nanoparticles in inhibiting bacterial biofilm formation in chronic wounds, to guide rational design and promote the broader application of nanoparticles in treating chronic wound infections.

The Truth About Chronic Wound Infection

A chronic wound is defined as one that fails to achieve anatomical and functional integrity through a normal, orderly, and timely repair process. Key reasons contributing to delayed wound healing include infection, malnutrition, and poor coagulation function. The two typical clinical characteristics of chronic wounds are: (i) healing is delayed in infected wounds, with no healing tendency observed after more than one month, and (ii) repeated wound infections.6 Biofilms are inherently resistant to antimicrobial treatment, and the distinct bacterial state in the biofilm environment can promote a unique reinfection mechanism.

Wound healing is a refined tissue regeneration process that includes four distinct yet overlapping phases: hemostasis, inflammation, cell proliferation, and remodeling. A review indicates that due to the complexity of wounds, there is currently a lack of sufficient wound treatment materials that can systematically regulate the unique wound microenvironment. Hydrogels present significant advantages in wound therapy by allowing for spatiotemporal control of the healing process and tissue remodeling7 (Figure 1). For instance,Qi developed an “all-in-one” in situ injectable hydrogel (QOP) made from a polysaccharide matrix and polydopamine nanoparticles, which facilitates the sequential treatment of diabetic wounds. This hydrogel enhances diabetic wound repair by establishing a barrier that neutralizes inflammatory cytokines, inhibits bacterial growth, and eliminates reactive oxygen species, thereby accelerating the transition from the inflammatory to the proliferative phase. It also provides a three-dimensional network structure that supports cellular repair and tissue remodeling, ultimately being fully absorbed by the body.8 Therefore, chronic wound infections require smarter and more sophisticated designs to offer comprehensive and precise treatment.

Figure 1 Schematic illustration of the influence of peptide-based self-assembling hydrogels on the wound healing process.

Notes: Reproduced from Guan T, Li J, Chen C et al Self-Assembling Peptide Based Hydrogels for Wound Tissue Repair. Adv Sci (Weinh). 2022,9(10):e2104165. Creative Commons.7

Chronic Wound Infection: How Bacteria Become the Main Enemy? Dynamic Synergistic Effect of the Abundance of Bacteria in Biofilms

Clinically, the abundant bacteria in the wound is crucial to clinical efficacy. Some studies have demonstrated that debridement can disrupt the diversity of wound bacterial communities due to the obstruction of anaerobic bacterial growth after epithelial tissue repair.9 Therefore, the relatively high abundance of bacteria in the biofilm can promote the chronicity of wound infections, especially with anaerobic ammonium oxidation bacteria.

The composition of bacterial diversity in biofilms undergoes a dynamic pattern of change over time. During the initial stage of wound formation, the normal skin flora dominates, consisting primarily of gram-positive aerobic bacteria. At this point, the biofilm is not pathogenic, and the activated host immune system can facilitate wound healing within weeks. With nutrient depletion, Corynebacterium and Staphylococcus begin colonizing the wound, resulting in a considerably different microbiome from the adjacent skin. Then, facultative anaerobic gram-negative bacteria begin to invade the wound, benefiting from aerobic bacteria that consume oxygen and create an anaerobic environment conducive to survival. The balance between microorganisms and the host’s innate immune system is threatened at this stage. Eventually, the wound becomes further infected, with specific bacteria causing stronger immune and inflammatory responses that affect deep tissues, such as muscle and bone. Simultaneously, blocked blood flow leads to local tissue ischemia, promoting the rapid growth of anaerobic microorganisms in a hypoxic environment, further delaying the healing of chronically infected wounds. Therefore, bacterial diversity and dynamic synergy are strongly associated with wound chronicity.10

Extracellular Polymeric Substance (EPS) Matrix Forms a Stable Three-Dimensional Protective Barrier

The EPS matrix surrounds the bacteria and displays a fibrous grid structure with high electron density, consisting of extracellular polysaccharides, proteins, lipids, and extracellular DNA (eDNA). This EPS matrix can prevent and delay the penetration of antibiotics into the biofilm, forming a protective barrier giving mature bacterial cells located deep in the matrix more time to develop resistance, resulting in the persistence of a chronic wound. Most studies have concluded that excessive EPS matrix harms the wound environment and can lead to non-healing chronic wounds. For example, membrane-attached lipoproteins can interact with eDNA in the matrix and promote biofilm formation associated with chronic wound infections.11

It is a matter of concern that the structure of EPS can change to adapt to the surrounding environment. Studies have shown that EPS matrix components are loosely adhered to the polystyrene surface in the in vitro pore plate model. As a result, enzymes quickly destroy biofilms and induce high levels of diffusion. However, in both microscopic and in vivo models, EPS exhibits tighter attachment and greater resistance to dispersion, possibly due to the activity of binding proteins. In addition, the expression of genes related to polysaccharide production in EPS is altered in different models.12 It has been fully confirmed that the biofilm EPS matrix plays a significant role in the occurrence and development of chronic wound infections. Therefore, changes in EPS stability and barrier function in different chronic wound infections are closely related to wound healing.

Lateral Transfer of Resistance Genes

The lateral transfer of resistance genes is an evolved survival strategy for bacteria. Resistance genes spread and exchange with different pathogenic bacterial strains through this mechanism. Increasing evidence demonstrates that acquiring exogenous DNA through lateral gene transfer (LGT) is a common phenomenon in bacteria, including transformation and conjugation. When resistance genes transfer horizontally, bacteria acquire intrinsic resistance to antibiotics, leading to chronic wound infections that are difficult to heal.

First, drug-resistant genes are widespread in bacterial biofilm communities of chronic wounds. In 2004, the first vancomycin-resistant Staphylococcus aureus was isolated by Cosgrove from the chronic wounds of patients receiving antibiotics.13 Similarly, a study on wound bacterial biofilm specimens to analyze the characteristics of antibiotic resistance genes found that more than 50% contained resistance genes for aminoglycosides (clindamycin), macrolides (erythromycin), and tetracyclines (minocycline), indicating that the presence of a wide range of resistance genes can be related to horizontal gene transfer.14 Further research confirmed that extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli colonizing burn wounds can transfer beta-lactamase genes to non-ESBL-producing Escherichia coli, which can rapidly dissolve β-lactam antibiotics. These plasmids carry various antibiotic resistance genes that play a predominant role in gene transfer.15 In addition, Kalan conducted a time-series shotgun metagenomics analysis of the microbial community (at the strain level) in chronic wound specimens from patients.16 The results indicated that S. aureus genomes associated with poor wound healing contained multiple antibiotic-resistance genes. These superantigens exacerbate the inflammatory response by non-specific stimulation of T cells, resulting in delayed wound healing.

Second, bacteria in chronic wound biofilms are in close contact with each other, facilitating lateral transfer between bacteria.17 The success rate of plasmid binding and transfer of antibiotic resistance genes is high, demonstrating a wide host range and expanding the antibiotic resistance spectrum of recipient bacteria.18 Some studies have shown that improper treatment of wounds infected with multiple bacteria can also cause the rapid transfer of drug-resistant genes to other pathogenic bacteria, which increases the treatment difficulty.19 These processes are interconnected and complementary. Therefore, the infection process of chronic wounds is related to the lateral transfer of resistance genes in bacterial biofilms.

Quorum Sensing (QS): Defense Against Host Immune Function and Epithelium Barrier Remodeling

Quorum sensing (QS) refers to the ability of bacteria to spontaneously produce and release specific signaling molecules, perceive their concentration changes, and regulate the group behavior of microorganisms in biofilms. QS is critical in electrical signaling, information propagation, and intercellular communication in chronic wound bacterial biofilms. A biofilm matrix structure that allows a high density of bacteria to inhabit a narrow space can enhance the signal transmission efficiency of QS. QS was identified as a critical factor in wound chronicity in recent years.

The QS system of bacterial biofilms can influence host mitochondria’s network morphology and energy characteristics, further regulating epithelial barrier properties, stimulating surrounding skin and mucous membranes, and delaying wound healing. Josephson et al found that QS molecules, such as 3-oxo-dodecyl-L-methoserine lactone (3O-C-HSL), can induce mitochondrial fragmentation, disrupt the inner mitochondrial membrane and cristae structure, alter mitochondrial respiratory enzymes and bioenergetics, and decrease mitochondrial membrane potential.20 This is mechanically accompanied by co-differential expression of the mitochondrial proteome and epithelial cell type-specific components, involving their structural organization, electron transport chain complexes, and stress responses.

The QS system of bacterial biofilms can also promote the chronicity of wounds by attenuating the host’s innate immune responses. The response of host immune cells to bacterial biofilms depends on the substantial state change in the QS system. A previous study showed that the QS system of Streptococcus can inhibit macrophage viability and decrease the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-β, and nuclear factor (NF)-κB, which can lead to the occurrence of necrotizing skin disease.21

The QS system of bacterial biofilms also enables bacteria in chronic wounds to sense the presence of other bacteria by secreting and receiving chemical signals. Once the biofilm reaches a certain level of cell density or saturation, it will turn off the gene expressing EPS and reactivate the flagellum motility gene, allowing the bacteria to spread Planctomycetes to the surrounding area to find a new suitable environment, further expanding the chronic wound area.

Interestingly, He found that QS-defective mutants of bacterial biofilms, known as “cheaters”, are usually seen in chronic infectious wounds. Biofilms composed of QS-dysfunctional bacteria have an enhanced ability to kill human phagocytes, helping bacteria evade the immune system and making infection control more difficult.22 Similarly, earlier studies verified that the degree of infection of wild-type bacterial biofilms was lower than that of mutant bacterial biofilms in a mouse chronic burn wound model. QS receptors can utilize the signal molecules produced by others to increase the release of virulence factors, making bacteria more adaptive.23

Relation Between Bacterial Biofilm and Repetitive Wound Infection Bacteria Pool

A biofilm continuously releases Planctomycetes as a bacterial repository. Regular antibiotic doses can eliminate Planctomycetes but cannot completely eradicate the bacteria in the biofilm. When the body’s defense system cannot control the bacterial biofilm, releasing bacteria from the biofilm to other parts of the body can cause reinfection, subsequently forming persistent foci and recurrent infections. Garcia analyzed the wounds of patients infected with Mucus using whole-genome sequencing techniques. The results showed that the outbreak of invasive wound Mucormycosis was primarily caused by biofilms with various Mucus strains in the environment rather than direct contact transmission between patients.24

“Dormant State” of Central Bacteria

Switching between a metabolically active and dormant state protects from environmental stresses and enables bacteria to overgrow under favorable conditions. Although a rapid increase in metabolically active bacteria can lead to complications, many dormant bacteria can also be problematic, as they tend to be more virulent and resistant to antibiotics. During the final phase of the biofilm’s life cycle, the structure of the biofilm is destroyed, and the dormant bacteria within the biofilm resuscitate and multiply swiftly, potentially causing recurrent infections in wounds. There are two main reasons for this: first, the dormant bacteria at the biofilm’s center exhibit strong drug resistance. Cellular dormancy triggered by toxin/antitoxin pairs (p)ppGpp, SOS response, and ATP levels are known to be the mechanistic basis of persistence (Figure 2).25 Conlon showed that the Planctomycetes isolated from the biofilm maintain a dormant phenotype, which can develop severe antibiotic resistance. Although the dormant bacteria in a biofilm are in a single free-floating state, their biological state remains unaltered.26 In addition, dormant cells in the biofilm can be activated under appropriate conditions and re-enter an active state, thus triggering the recurrence of the infection. This heterogeneity in the maturation process of biofilms, where cells differentiate to form two subpopulations of active and dormant cells, is the key to the high degree of drug resistance in biofilms.27 Second, the dormant bacteria in the center of the biofilm are characterized by low levels of inflammation. Thurlow’s study demonstrated that staphylococcal biofilms reduce inflammation in mouse macrophages by promoting the differentiation of these host immune cells compared to planktonic cells.28 Therefore, the immune evasion of bacterial biofilms is likely due primarily to the resulting lower inflammatory effects rather than the suppression of host immune defense mechanisms.

Figure 2 Formation of bacterial persistence via cell dormancy. Various pathways could trigger bacterial cells into dormant state upon antibiotic stress. A considerable number of TA modules could free toxins upon stresses, which result in persister formation by inhibiting DNA replication, or transcription, or translation processes, or downregulating proton motive force (PMF) that is under the control of SOS response. Dormancy could be also triggered by accumulated alarmone molecules and decreased intracellular ATP levels. The gray layers represent the outer membrane and inner membrane, respectively, and the red square linkage represents the peptidoglycan and periplasmic space. Reproduced with permission from Zou J, Peng B, Qu J et al Are Bacterial Persisters Dormant Cells Only? Front Microbiol. 2021;12:708,580. Creative Commons.25

Interestingly, Bacterial persisters are not only dormant cells; studies have shown that metabolically active bacteria can maintain persistence by lowering intracellular antibiotic concentrations through efflux pumps. Cell wall-deficient bacteria (CWDB), including L-type and globular bodies produced by β-lactam antibiotics, are associated with antibiotic persistence.25

Antimicrobial Mechanism of Nanoparticles on Bacterial Biofilms in Chronic Wounds

Nanoparticles are considered safe and effective antibiotic alternatives due to their antibacterial activity, low toxicity, and difficulty producing drug-resistant bacteria. They have promising applications in the field of biomedicine. Skin cuts and abrasions are prone to infection, and it is crucial to swiftly eliminate microbes from festering wounds to accelerate healing and reduce morbidity. Numerous studies have applied nanoparticles to treat skin wounds, and the results indicate that nanoparticles are highly effective in treating chronic wounds.29 Nanotechnology offers a novel approach to treating chronic open wound infections. As a result, the antimicrobial mechanisms of nanoparticles have become a research focus. Scholars’ attention is mainly directed toward physical damage to bacterial cell membranes, the oxidative stress effect of reactive oxygen species (ROS), and the regulation of bacterial gene expression. However, these mechanisms do not account for bacterial biofilms’ dynamic formation and structure in chronic wounds, and their complex interactions remain unclear.

Biofilms shield bacteria in chronic wounds, leading to and exacerbating the inflammatory response. Inhibitors tend to be unable to penetrate the biofilm, causing repeated infections of surrounding soft tissues and resulting in non-healing wounds. The life cycle of chronic wound biofilms consists of four stages: (1) the initial settlement and attachment of planktonic bacteria; (2) the reproduction and formation of early bacterial colonies; (3) the maturation of bacterial biofilms; and (4) the dissemination of bacteria on the biofilm surface. The types and states of bacteria vary significantly across these stages. Studies investigating how nanoparticles suppress biofilms in chronic wounds are still in the preliminary stages. Further exploration of the dynamic mechanisms between nanoparticles and chronic wound biofilms holds significant theoretical value. Therefore, this section primarily summarizes the role of nanoparticles in the different stages of chronic wound biofilms. Nanoparticles typically inhibit bacterial biofilm formation through multiple antimicrobial mechanisms. In most cases, the antimicrobial effect is not limited to a specific stage of the biofilm cycle but coincides in multiple phases (Figure 3).

Figure 3 Antibacterial mechanism of nanoparticles on chronic wound biofilm.

Preventing the Initial Settlement and Attachment of Planktonic Bacteria

Bacterial biofilms begin with planktonic bacteria near the wound surface, which neutralizes the electrostatic repulsion between the bacterial and wound surfaces, allowing initial settlement and attachment. Depending on the environment to which the wound is exposed, different types of biofilms are formed, and the initial attachment of bacteria is a dynamic selective process critical in shaping the composition of early colonies. A clinical study proved that while the removal of diseased tissue through surgical debridement successfully cleared biofilms from chronic wounds, the biofilms were reestablished within 48 hours after the initial debridement, and a substantial population of bacteria was detected in mature biofilms 72 hours post-debridement.30 This indicates a window of opportunity following debridement, during which the planktonic bacteria adhering to the wound surface are susceptible to therapeutic interventions. Therefore, effectively preventing initial bacterial attachment can hinder biofilm reformation.

Bacterial attachment to the wound involves the specific binding of bacterial surface adhesion molecules to particular protein receptors on the wound surface. Thus, bacterial adhesion can be inhibited by targeting bacterial adhesion molecules (protein structures31 and glycolipid components32) or by inhibiting the expression of adhesion-related genes. Disrupting the attraction between bacterial cells, closely associated with the bacteria’s surface charge, can prevent adhesion. The findings indicate that using nanoparticles to prevent initial bacterial attachment is crucial for exploring treatment strategies to regulate and control bacterial biofilm formation in chronic wounds.

Electrostatic Interaction

Due to the typically negative charge on bacterial surfaces within the milieu of chronic wounds, nanoparticles can influence bacterial adherence directly or indirectly, subsequently impacting the development of bacterial biofilms in such wounds.

Positively charged nanoparticles, for instance, have the potential to be drawn electrostatically towards the negatively charged bacterial cells present in the exudate of chronic wounds, reducing bacterial adhesion to the wound surface. Mei observed abundant adherence of positively charged nanomaterials to bacterial biofilms via electrostatic forces, a process that successfully eradicated bacteria and accelerated the healing of infected wounds in diabetic rats without inducing antibiotic resistance. The study suggests that the possible mechanism involves nanomaterials making close contact with the biofilm through electrostatic interaction, destroying the bacterial membrane, and inhibiting intracellular enzyme activity (Figure 4).33 Several other studies indicate that nano-carriers carrying a positive charge employ an identical mode of action involving the electrostatic attraction between the positively charged lipids in non-lamellar lyotropic liquid crystalline nanoparticles and the anionic bacterial biofilm. This interaction leads to the fusion of the bacterial membrane and subsequent disintegration of the biofilm’s architecture. Wang formulated mesoporous silica (MS) nanoparticles impregnated with tannic acid (TA), which were capable of creating a blood-clotting seal on injuries while also inhibiting bacterial adhesion via chemical bonds and electrostatic forces. These loaded MS nanoparticles demonstrated good antibacterial activity and can promote wound healing.34

Figure 4 (A) Functional Silver Nanoparticle as a Benign Antimicrobial Agent That Eradicates Antibiotic-Resistant Bacteria and Promotes Wound Healing. The scanning electron microscopy (SEM) image shows that EPL-g-butyl@AgNPs was achieved by anchoring on the bacterial cell surface, damaging the cell walls as well as membranes, inhibiting intracellular biological functions, and finally leading to the cell death. Reproduced with permission. Copyright 2016, American Chemical Society. (B) TEM images of a thin section of (B−D) S. aureus and (E−G) P. aeruginosa (B and E) before and (C, D, F, and (G) after incubation with AgNPs@PDMAEMA-C4 for 30 min. (C) Optimization of synthesis conditions of AgNPs@PDMAEMA-C4. (H) The effect of a different alkyl chain on MIC value; (I and J) the effect of a different alkyl chain on the cytotoxicity effect of the nanoparticles with different concentrations (I and J are 1.0 and 2.0 μg/mL, respectively) against NIH3T3 cells; the effects of molar ratio of PDMAEMA-C4 and silver on (K) UV−vis spectrum, (L) size, Zeta potential, and (M) MIC value; the effects of volume of reducing agent on (N) UV−vis spectrum, (O) size, Zeta potential, and (P) MIC value; the effects of molecular weight of PDMAEMA-C4 on (Q) UV−vis spectrum and (S) MIC value; and (R) the GPC traces of PDMAEMA with different molecular weight. Reproduced with permission from Mei L, Lu Z, Zhang X, et al. Polymer-Ag nanocomposites with enhanced antimicrobial activity against bacterial infection. ACS Appl Mater Interfaces. 2014;6(18):15813–15821. Copyright 2014, American Chemical Society.33

In contrast, negatively charged nanoparticles can enhance electrostatic repulsion within the wound, disrupting bacterial cell attachment and serving an antimicrobial function. For example, silver nanoparticles (Ag-NPs) with reducing amino acids, which have a net negative surface charge, maximize electrostatic repulsion in the alkaline exudate environment of the wound and prevent the adhesion of drug-resistant bacteria.35 In addition, nanoparticles can be stabilized to prevent agglomeration by maintaining a negatively charged surface. For example, alginate silver nanoparticles exhibit potent germ-fighting capabilities, believed to result from the electrostatic repulsion of scattered nanoparticles coupled with an increase in surface area, enhancing interaction with bacterial cells in the biofilm. These nanoparticles also reduce the secretion of proinflammatory cytokines by macrophages, leading to a synergistic antibacterial effect.36

Prevent Bacterial Attachment

After bacterial adhesions (such as flagella, proteins, and others) attach to corresponding receptors on the exterior of host cells (such as extracellular matrix, glycoproteins, and others), bacteria can firmly adhere to the wound surface, causing wound infection.37,38

Initially, nanoparticles inhibit the production of adhesions, preventing bacteria from adhering to the wound matrix. For example, the adhesion of S. epidermidis primarily relies on the synthesis of polysaccharide intercellular adhesion (PIA) encoded by the ICAADBC locus, which can be suppressed by silver nanoparticles.39

Secondly, nanoparticles competitively bind to bacteria by mimicking bacterial adhesion receptors, effectively reducing bacterial binding to receptors in chronic wound biofilms. Halbus et al reported that MS silica nanoparticles with rough surfaces can form a reversible covalent bond with the diol group on the bacterial membrane, independent of electrostatic adhesion. This approach aimed to prevent bacteria from binding to receptors and maximize bacteriostatic efficacy (Figure 5).40

Figure 5 (A) The mechanism of self-grafting/covalent binding of the sugar groups expressed on the bacterial cell wall and boronic acid-functionalized surface-rough SiO2NPs. (B) The viability of the bacterial cells after treatment with surface-functionalized rough SiO2NPs pro-duced using two different catalysts, NH4OH (or NaOH), in the Stöber process, respectively. (C) SEM images show that the strong covalent interaction of the HPBA-functionalized CuONPs/SiO2 with the cell walls is probably the main contributor toward bacterial walls disruption and damage. Reproduced with permission from Halbus AF, Horozov TS, Paunov VN. “Ghost” Silica Nanoparticles of “Host”-Inherited Antibacterial Action. ACS Appl Mater Interfaces. 2019;11(42):38519–38530.. Copyright 2019, American Chemical Society.40

Ultimately, nanoparticles can modulate the expression of genes associated with bacterial adhesion. For example, with increasing selenium nanoparticle (SENP) content, the expression of protein genes (CSGA and CSGG) related to E. coli-mediated adhesion decreased gradually.41 In addition, membrane-related genes (OMPA and OMPF) were downregulated, while genes associated with producing destructive ROS (AHPF) were up-regulated. Similar correlations have been observed in multiple studies. Liu demonstrated that a Ti surface modified by AgNPs can regulate the gene expression linked to biofilm adhesion (ICAA and ICAR of S. epidermidis; FNBA and FNBB of S. aureus), preventing bacterial adhesion and biofilm formation.42 Shariati also confirmed that curcumin nanoparticles downregulated the transcription level of Pseudomonas aeruginosa adhesion genes.43

Interferes with Bacterial Reproduction and Colony Formation

Biofilm formation in chronic wounds differs from that observed in other infections. Mature large biofilms are less likely to be present in the chronic wound environment, whereas microcolony biofilm formation is more common. Certain species of bacteria, such as Pseudomonas aeruginosa, have been shown to exhibit already the effect of elevated levels of auto-inducing molecules (AIs) during the colony formation phase, and AIs can directly influence host cell immune function, so the presence of these colonies can promote wound chronicity.44 In addition, phagocytes cannot engulf larger colonies, and failed phagocytosis can result in the degranulation of phagocytes, ultimately leading to an overreaction of the immune system and disruption of the ecological balance in chronic wounds.45 Due to the increase in bacterial species and numbers, the primary goal of nanoparticles is to inhibit bacterial proliferation and aggregation during the early colony formation phase.

Direct Inhibition of Bacterial Proliferation

Metal or metal oxide nanoparticles exhibit synergistic antimicrobial properties against planktonic bacteria through mechanical perturbations (Figure 6)46 (encapsulation47 bacterial membrane insertion48 and bacterial membrane perforation)49 and oxidative stress (Figure 7),50 These mechanisms directly inhibit bacterial proliferation and interfere with bacterial micro-colony formation in wound areas.51 For example, a novel chitosan dressing impregnated with ZnO/N-halamine hybrid nanoparticles demonstrated rapid antimicrobial properties, reducing the bacterial cell proliferation rates of S. aureus and E. coli O157 by approximately 90% within 30 min of contact with wound colonies.52 Similarly, the scaffold composite of silk fibroin protein/chitin/AgNPs, with high antimicrobial activity, successfully inhibited the proliferation and aggregation of E. coli, S. aureus, and C. albicans in the wound area, preventing the growth of bacterial colonies.53

Figure 6 (A and B) The inhibition zone diameters of EPL-g-butyl@AgNPs (16.74 mm for S. aureus, 16.6 mm for P. aeruginosa) were significantly larger than those of EPL-g-butyl (9.06 mm, 9.05 mm) and AgNPs (9.1 mm, 9.1 mm), indicating the synergistic antibacterial effect of EPL-g-butyl and AgNPs. (C–H) The scanning electron microscopy (SEM) image shows that EPL-g-butyl@AgNPs was achieved by anchoring on the bacterial cell surface, damaging the cell walls as well as membranes, inhibiting intracellular biological functions, and finally leading to the cell death. (I–N) A live/dead staining test was performed on Pseudomonas aeruginosa and Staphylococcus aureus, with fluorescent micrographs of bacteria after 1 hour of antimicrobial drug treatment. Under the fluorescence microscope, live bacterial cells with intact membranes appeared green and dead bacterial cells with damaged membranes appeared red. In contrast to the green signal emitted by live bacteria (I and L), all cells died and aggregated after 1 h of EPL-g-butyl@AgNPs treatment (J and M). Figures (K) and (N) show that a large number of bacterial cells remained alive without any aggregation after 1 h of AgNPs treatment. epl -g-butyl ligands exhibited direct multivalent interactions with bacteria, giving the nanocomposites excellent antibacterial activity against both Gram-negative and Gram-positive bacteria. The antibacterial effect of EPL-g-butyl@AgNPs was confirmed. * p < 0.05. Reproduced with permission from Dai X, Guo Q, Zhao Y, et al. Functional Silver Nanoparticle as a Benign Antimicrobial Agent That Eradicates Antibiotic-Resistant Bacteria and Promotes Wound Healing. ACS Appl Mater Interfaces. 2016;8(39):25798–25807.. Copyright 2016, American Chemical Society.46

Figure 7 Continued.

Figure 7 (A) Potent chitosan-stabilized PtAu nanoparticles (CSPA) with polymerase-like activity promote the production of large amounts of ROS by bacteria, leading to a decrease in ATP content and glutathione (GSH) consumption. In addition, the molecular mechanism of the antibacterial properties of CSPA was revealed by transcriptomic analysis of differentially expressed genes (DEGs). Finally, an in vivo wound infection model was developed to investigate the antimicrobial effect of CSPA and its ability to promote wound healing.In vitro antimicrobial activity of CSPA. (B–E) The number of colonies on the agar plates gradually decreased with the increase of CSPA concentration. No bacterial growth was observed in the agar dishes at a treatment concentration of 2.0 μg mL-1 for E. coli, and almost no colonies were found in the agar dishes at a treatment concentration of 20.0 μg mL−1 for S. aureus. However, the number of colonies after CS treatment at the same concentration was significantly higher than that after CSPA treatment, indicating that the antimicrobial activity of CSPA was superior to that of CS. (F and G) The control and CS groups exhibited predominantly green fluorescence, indicating bacterial survival. In contrast, the bacteria exhibited a distinct red fluorescence, indicating massive bacterial death. These findings confirm previous antimicrobial testing and emphasize the significant antimicrobial efficacy of CSPA. (H) Using vancomycin hydrochloride (Van) as a control, no significant resistance to CSPA was detected in S. aureus over the course of 16 treatments, and the multiplicative change in minimum bactericidal concentration (MBC) remained constant. In contrast, (S) aureus showed increased resistance to Van during the 11th passaging and this resistance escalated dramatically after the 3rd passaging. (I) The results indicate that S. aureus is not particularly susceptible to resistance to CSPA. This may be due to the fact that the antibacterial mechanism of CSPA is through electrostatic contact, which then enters the bacteria in order to stimulate intracellular ROS production. (J) This observation suggests that CSPA has a stronger ability to completely inhibit biofilm formation, making it more effective in preventing bacterial resistance. (K) This observation suggests that CSPA has a stronger ability to completely inhibit biofilm formation, thus more effectively preventing the development of drug resistance in bacteria. *p < 0.05, **p < 0.01, ***p < 0.001. Reprinted from Wen Y, Chen W, Wu R, et al. Chitosan‐stabilized PtAu nanoparticles with multienzyme‐like activity for mixed bacteria infection wound healing and insights into its antibacterial mechanism. Small Structures. 2024;5(6):2300553. © 2024 The Authors. Small Structures published by Wiley-VCH GmbH.

Although clinical strains exhibit diverse antimicrobial resistance profiles and continuously emerge with new drug resistance mechanisms, some scholars have reported that, in in vitro experiments, nanoparticles can inhibit the proliferation of laboratory standard bacteria and interfere with forming non-standard bacterial colonies isolated from clinical wounds. Amsari et al proposed that nanoparticles possess excellent anti-proliferative ability against clinical strains; their research demonstrated that ZnO-NPs inhibited the growth of colonies formed by clinical strains isolated from skin and soft tissue infected with methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and methicillin-resistant S. epidermis (MRSE) clinical strains.54 In addition, Huang55 confirmed that Ag-NPs can prevent the rapid proliferation of P. aeruginosa isolated from chronic wounds and inhibit the formation of microflora in the wound environment, reducing the microbial load. Therefore, it is of great clinical significance to further study the inhibition of bacterial proliferation and colony formation by nanoparticles in vivo to disrupt bacterial biofilms in chronic wounds.

The in vivo antibacterial activity of different nanoparticles was reported. For example, P. aeruginosa is a common pathogenic bacterium in burn wounds. In mice with non-treated chronic wounds, Ag-NPs significantly reduced the incidence of burn wound infections caused by P. aeruginosa colonies, effectively treated the chronic wounds, and led to complete wound healing after four weeks.56 Studies have demonstrated that nanoparticles can inhibit the proliferation of P. aeruginosa in a mouse model of excised wounds. One possible explanation is that when nanoparticles bind to glutathione in vivo, they form the nitroso intermediate S-nitroso glutathione (GSNO), which inhibits the high-density growth of P. aeruginosa in the wound area.57

Indirect Inhibition of Bacterial Proliferation

Monocytes and macrophages are critical immune cells involved in regulating skin wound healing. These cells play a crucial role at the onset of bacterial colony formation. Some researchers have proposed the theory of “macrophage polarization” to explain the role of nanoparticles in stimulating macrophage-mediated inhibition of bacterial proliferation.58 Huang et al used an in vivo rat model of S. aureus-infected skin wounds and demonstrated that i Cu(2+) release and material surface characteristics of Cu-containing micro/nano-topographical coating could activate distinct signaling pathways in macrophages. The activated M1 macrophages can reprogram macrophages to exhibit inhibitory activity against bacterial proliferation59 They reported that IONPs, in combination with the triggering of the Fenton reaction, significantly enhance the role of macrophages in inhibiting S. aureus colony formation in wounds by inducing the polarization of M1 macrophages to stimulate ROS production. Saleh et al supported this theory and developed hyaluronan-based nano-hydrogels to regulate wound skin tissue macrophages during the polarization of the healing process. The results showed increased anti-inflammatory Arg-1 gene expression and decreased proinflammatory markers (including TNF-α, β, and IL-1-6). This provides further evidence that nanoparticle hydrogels have strong potential to combat biofilm-related infections and accelerate healing.60 More excitingly, in order to achieve a harmonious balance of proinflammatory and anti-inflammatory properties in the infected area, Deng proposed a “combinatorial” strategy utilizing azithromycin (AZM)-hybrid nanocomposite termed GOx@FexSy/AZM for the on-demand treatment of diabetic wounds. On-demand treatment of diabetic wounds showed that these components inhibit the activity of proinflammatory transcription factors in macrophages, promoting macrophage polarization towards a reparative M2 phenotype and facilitating tissue remodeling. A rational transition from the inflammatory to the reparative phase is accelerated by meeting the requirements of different stages of wound healing.61

Some studies believe that the indirect immune effect of nanoparticles on biofilms in chronic wounds cannot be explained solely by stimulating macrophage polarization and that there can also be an influence on the gene expression of immune cells. Rahimi investigated the inhibitory effect of gold nanoparticles on Candida albicans in infected burn wounds. Their results and theoretical evidence indicate that nano-complexes can significantly reduce the gene expression level in macrophages when treating chronic infectious wounds, playing an immunomodulatory role, reducing the release of cytokines by macrophages, and thus inhibiting bacterial infection.62 Dai also confirmed that the ε-polylysine/AgNP nanocomposite (EPL-G-Butyl@AgNPs) can regulate the relative gene expression levels of CD3 T cells and CD68 macrophages, inhibiting bacterial proliferation and effectively promoting the healing of infected wounds in rats.46

Although the theories explaining the mechanisms by which nanoparticles regulate the immune response to chronically infected wounds are incomplete, the studies above indicate that mononuclear macrophages exert an indirect effect in nanoparticle-mediated bacterial proliferation inhibition and play a key role in promoting the healing of skin wound infections.

Induction of Bacterial-Specific Aggregation

The induction of specific bacterial aggregation is an essential and intriguing bacteriostatic mechanism. Many researchers have gone beyond the conventional bacteriostatic approach and demonstrated that when ethylene glycol chitosan-conjugated nanoparticles are located at the site of acidity-related focal infection, the surface charge transfer causes the pH-responsive GCS surface to become positively charged. These nanoparticles and the negatively charged MRSA cell wall induce bacterial aggregation through strong electrostatic interactions. Subsequently, near-infrared (NIR) light thermal activation, which enables direct and specific heating of bacterial colonies, can further maximize bacteriostatic activity.63 Further, Yang et al demonstrated that TS4-NP can encapsulate Staphylococcus aureus (S. aureus) cells by binding tightly to the bacterial surface through hydrophobic and electrostatic interactions and inducing TS4-S formation. S. aureus aggregation exerts membrane disruption and reactive oxygen species (ROS) production effects, leading to intracellular DNA breaks and bacterial death (Figure 8).64 Similar results were observed when GO-quaternary ammonium salt (GO-QAS) nanocomposites were applied to E. coli.65,66 Researchers have also found that nanoparticles can induce specific aggregation to eliminate infections caused by a single microbial community dominated by one bacterium and play this role in complex multi-microbial infection communities.67 These findings show that nanoparticles can be utilized to induce specific bacterial aggregation in the acidic environment of focal infections. However, when exposed to host fibroblasts in the physiological environment, the nanoparticles exhibit a net neutral charge and do not adhere to the negatively charged adjacent host cells, resulting in minimal photothermal damage to healthy tissues. This protects the surrounding skin while inhibiting bacterial growth and promoting wound healing. Therefore, nanoparticles can induce specific bacterial aggregation, competitively bind to bacteria in the wound area, and inhibit bacterial proliferation and colony formation.

Figure 8 (A)TS NPs prepared with a TEOS/Si-QAC molar ratio of 4:1 (the corresponding products are called TS4 NPs) can kill Gram-positive Staphylococcus aureus (S. aureus) bacteria by an “adhesion-aggregation-disruption” mechanism: NPs can tightly bind to the surface of S. aureus by hydrophobic and electrostatic interactions (“adhesion”) and cover the entire bacterial cell. NPs can tightly bind to the surface of S. aureus through hydrophobic and electrostatic interactions, covering the entire bacterial cell (“adhesion”). (B) After treatment with TS4 NPs, (E) coli cells were only partially adsorbed by TS4 NPs, whereas S. aureus cells could be completely surrounded by NPs, which may limit S. aureus growth, propagation and metabolism. (C and D) Only ts4-treated S. aureus cells exhibited strong green fluorescence, indicating intracellular ROS production. (E) DNA extracted from untreated Staphylococcus aureus cells showed a clear bright band, whereas a weaker band appeared in the TS4 group of Staphylococcus aureus cells, suggesting that reactive oxygen species caused severe damage to DNA. (F) TS4-mediated bacterial aggregation was observed by confocal microscopy imaging within 5 min after the addition of TS4 to S. aureus suspensions, and TS4-induced bacterial cell aggregation was also observed at other time points (30 min, 2 h, and 6 h). Bacterial aggregation was not evident in the control group not treated with TS4. (G) DLS results also confirmed the presence of micrometer-sized aggregates in the TS4 group. Used with permission of The Royal Society of Chemistry from Yang J, Zhu YX, Lu P, et al. One-step synthesis of quaternized silica nanoparticles with bacterial adhesion and aggregation properties for effective antibacterial and antibiofilm treatments. J Mater Chem B. 2022;10(16):3073–3082. Copyright 2022; permission conveyed through Copyright Clearance Center, Inc.64

Cleavage of Mature Bacterial Biofilms in the Chronic Wound

Mature biofilms can form within 10 h in chronic wounds and persist in open wounds. Microbial behavior in high-viscosity wound environments resembles biofilms, while behavior in low-viscosity wound environments resembles bacterioplankton. Many studies have shown systemic antimicrobial agents are ineffective against bacteria in chronic wounds.68,69 Previously, researchers often attributed this to insufficient blood supply. However, many studies have found that heavy exudative wounds can present with infection and inflammation, even when the soft tissue near the wound has an abundant blood supply. This is presumably related to the presence of microorganisms with biofilm phenotypes. The evaporation of moisture in heavily exudative wounds leads to increased extracellular fluid viscosity, making microorganisms in this environment more prone to exhibit biofilm phenotypes, including reduced sensitivity to antimicrobials, resulting in difficult-to-control infections. These findings can have far-reaching implications for the treatment of chronic wounds. As a result, nanoparticles must employ multiple mechanisms to disrupt the development of mature biofilms.

Buffer Weak Alkalinity of Wound Microenvironment

Normal tissues typically exhibit pH values between 5.0 and 7.0, with a mildly acidic character on the surface. The skin wound microenvironment plays a critical role in inhibiting pathogenic microorganisms. Studies have shown that the pH of chronic infection wounds increases to 6.5–8.5, with the alkaline environment likely resulting from necrotic tissue, bacteria, and bacterial byproducts such as ammonia, which create favorable conditions for bacterial growth. In addition, mature bacterial biofilms in chronic wounds can inactivate skin keratinocytes and fibroblasts, accelerating their apoptosis and reducing epithelial keratinocyte migration, impairing wound healing. Thus, adjusting the wound’s pH can alter the bacterial growth environment’s microecology and regulate the biofilm’s pathogenic effects to accelerate wound healing. Nanoparticles can adjust the biofilm’s pH to enhance the wound’s defensive buffering capacity. On the one hand, nanoparticles can counteract alkali production and reduce the pH in chronic wound regions by slowly releasing phosphate or bicarbonate, disrupting mature biofilms. For example, AgNPs exhibit significant bactericidal activity and can promote wound healing in a chronic wound model in infected rats due to their high oxidative stress effect, acidic pH, and ROS-stimulated release of silver ions (Ag) and AMPs70 (Figure 9). On the other hand, Nanoreactors can intelligently activate multienzyme cascade reactions at high sugar levels to produce acidic substances such as gluconic acid (GA) and hydrogen peroxide, leading to the release of nitric oxide (NO), which can combat bacterial infection and inflammation in chronic wounds of diabetes.71

Figure 9 (A) Preparation process of AgNPs-AMP@PSiMPs. (B) AgNPs-AMP@PSiMPs as a synergistic antibacterial platform for wound disinfection and healing. (C–H) Mechanisms of ROS- and H+-stimulated release of Ag+ and AMPs. The in vitro antibacterial activity testing results verified that AgNPs-AMP@PSiMPs displayed the highest antibacterial activity among the three types of bactericidal agents. Reproduced with permission from Jin Y, Yang Y, Duan W, et al. Synergistic and On-Demand Release of Ag-AMPs Loaded on Porous Silicon Nanocarriers for Antibacteria and Wound Healing. ACS Appl Mater Interfaces. 2021;13(14):16127–16141. Copyright 2021, American Chemical Society.70

“Digestion” of Biofilm EPS Matrix

The EPS matrix surrounding biofilms acts as a physical barrier, protecting bacteria from bacteriostatic agents and immune cells. Nanoparticles can induce the degradation of the EPS matrix through various mechanisms.72

Previous studies have shown that nanoparticles can inhibit the production of EPS components and mediate EPS degradation, causing biofilms’ “physical” collapse. In this scenario, bacteria shed from the biofilm become exposed and lose their inherent protection. Ultimately, the connections between bacteria are disrupted, rendering them susceptible to drugs. Nanoparticles further exert a bacteriostatic effect on biofilms, eventually killing the bacteria.73 Ali demonstrated that nanoparticles attached to the EPS surface of wound biofilms inhibit the generation of extracellular polysaccharides, migrate within the EPS matrix, and infiltrate bacterial cell walls, inhibiting early biofilm growth via intracellular ROS activity.74

Subsequent research has shown that metal nanoparticles can interact with the EPS composition. Hiebner et al demonstrated that both the charge and surface functional groups of silica NPs (Si-NPs) significantly influence the degree of interaction and localization between Si-NPs and EPS macromolecules (including proteins, polysaccharides, and DNA) by evaluating the affinities of different functionalized Si-NPs and EPS macromolecules in biofilms.75 Weldrick demonstrated that protease-functionalized nano-carriers can “digest” EPS substrates, cross bacterial biofilms, reach bacteria formerly protected by the biofilm, and deliver antibiotics directly to bacterial cell walls, enhancing the clearance of bacterial biofilms formed in six chronic wounds76 (Figure 10).

Figure 10 (A–F and J–M) Diagram of the mechanism of action of the Carbopol Aqua SF1−Alcalase 2.4 L FG nano-gel particles on biofilms adhered to a substrate. Fluorescent images and tapping mode atomic force microscopy of S. aureus biofilm formation show that Alcalase-nanogel Particles can digested the biofilm matrix but left the cells largely untouched. Reproduced with permission. (G) It shows that after seeding a membrane with 1×105 CFUmL−1 for 24 h at 37 °C, the growth control continued to increase in cell density after a further 1, 6, and 24 h of growth. (H) It shows that there is a correlation between the increase of the concentration of free ciprofloxacin in the presence of an equivalent amount of Alcalase−Carbopol NPs and the residual bacterial cell viability after treatment. (I) It shows the results on the concentration dependence of the cell viability on the antibiotic treatment in experiments where the ciprofloxacin was encapsulated within the Carbopol nanogel, rather than being delivered separately as a co-treatment. **p < 0.01, ***p < 0.001. Reproduced with permission from Weldrick PJ, Hardman MJ, Paunov VN. Enhanced Clearing of Wound-Related Pathogenic Bacterial Biofilms Using Protease-Functionalized Antibiotic Nanocarriers. ACS Appl Mater Interfaces. 2019;11(47):43902–43919. Copyright 2019, American Chemical Society.76

Further studies indicate that nanoparticles can adsorb and compromise the integrity of EPS through physical action. For instance, AgNPs can pierce the EPS, forming pores that destroy its structure, facilitating the action of antimicrobials in combating bacterial biofilms in wounds.77 Consistent with these findings, Dong confirmed that nanoparticles can adsorb to the surface layer of biofi