Antibiotic Nanoparticles-Loaded Wound Dressings Against Pseudomonas aeruginosa’s Skin Infection: A Systematic Review

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a human opportunistic pathogen and one of the most common nosocomial pathogens associated with life-threatening acute and chronic infections in critically ill or immunocompromised patients. These include ventilator-associated pneumonia, structural lung disease such as cystic fibrosis, bloodstream and urinary tract infections. Furthermore, it is known to cause serious infections in cutaneous wounds, chronic ulcers and burn patients with trauma.^1–4 It is a multi-drug resistant (MDR) Gram-negative bacillus that, along with Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Enterobacter species, is considered an “ESKAPE” pathogen. This classification serves to highlight the impact of this bacterium on hospital infections and its ability to evade the effect of antibacterial drugs.^4,5 Importantly, the World Health Organization (WHO) has classified this pathogen as critical, priority 1, due to the urgent necessity for the development of new antibiotics and treatment strategies.⁵

From an epidemiological perspective, it is estimated that P. aeruginosa has a prevalence of approximately 7.2% amongst all healthcare-associated infections, with notable morbidity and mortality associated with it.⁶ Individuals with frequent or prolonged exposure to aqueous environments, diabetics, and immunocompromised patients are at high risk for developing P. aeruginosa skin infections.⁷ Regarding skin infections, this bacterium is the most prevalent organism responsible for infection in burn-injured patients, associated with sepsis and mortality. MDR P. aeruginosa is becoming an increasingly common cause of death in these patients, with approximately 86% of sepsis deaths occurring in a pediatric burn intensive care unit.⁶ Together with Staphylococcus aureus, it represents one of the most common agents isolated from chronic leg ulcers, colonizing approximately 52.2% of patients. It is typically found in the deepest region of the wound bed.⁸

Biofilms, which are complex clusters of bacteria attached to a surface and embedded in a self-produced matrix, lead to the development of an organized microbial ecosystem in which P. aeruginosa develops antibiotic tolerance. The formation of biofilms is initiated by the attachment of a cell to a surface, subsequent multiplication, maturation, and the production of an extracellular polymeric matrix. This matrix serves to resist environmental impacts including mechanical forces and antibiotics.⁹ Indeed, bacteria entrapped in biofilms can exhibit up to 1000-fold greater antibiotic tolerance than free-living bacteria.^2,10 In chronic wounds, P. aeruginosa exists as a polysaccharide-coated biofilm. The bacterial-derived polysaccharides facilitate bacterial survival, protection from the immune response, desiccation and oxidizing agents increasing the antibiotic resistance.⁶

In addition to the development of antibiotic tolerance, P. aeruginosa is intrinsically resistant to a number of antimicrobials due to the action of different mechanisms.^2,4,7 Briefly, the outer membrane permeability of this bacterium is reduced, which restrict the diffusion of antibiotics such as aminoglycosides and polymyxins through the cell envelope. Furthermore, these bacteria express multidrug efflux pumps which result in the exclusion of small hydrophilic antibiotics such as β-lactams and quinolones from the outer membrane through porins.² Finally, it produces antibiotic modification enzymes, such as chromosomal AmpC cephalosporinases. In fact, the hyperproduction of the chromosomal AmpC β-lactamase represents the primary mechanism responsible for β-lactam resistance in this bacterium.¹¹ Importantly, P. aeruginosa is capable of developing antibiotic resistance through chromosomal mutations, particularly those encoding metallo-β-lactamases or extended-spectrum β-lactamases, often co-transferred with genes encoding resistance to aminoglycosides and/or fluoroquinolones³ and through horizontal gene acquisition.² Consequently, treatments for P. aeruginosa infection are extremely difficult due to its intrinsic resistance mechanisms, biofilm’s tolerance development to antibiotics, and rapid mutations.

The conventional treatments for infected skin wounds are based on application of topical or systemic antibacterial compounds including antiseptics and antibiotics with the objective of overcoming the risk of bacterial resistance and reducing the systemic exposure to drugs.^12,13 In this context, wound dressings play an important role. Conventionally, they are used only to prevent external contamination of the wound. However, advances in this field have led to the development of advanced wound dressings that not only prevent bacterial infection but also combat it and promote wound healing. Thus, wound dressings can be functionalized with many classes of antibiotics or antibacterial compounds.¹⁴ Such dressings allow the treatment of infected wounds that require high concentrations of antibiotics. However, the use of antibiotic-embedded wound dressings can result in the generation of systemic toxicity. Together with the emergence of bacterial resistance, this systemic toxicity determines the need for NPs- based advances.

NPs have demonstrated potent bactericidal effects on their own, when loaded with antibiotic or when used in combination. Thus, they can inhibit the growth of bacterial biofilms through physical damage, oxidative stress, thermal damage, and other specific mechanisms thereby enhancing the efficacy of antibacterial drugs and the overcoming of bacterial resistance caused by biofilms.^12,13,15 Due to their unique characteristics, including their small size, surface charge, and large specific surface area, NPs can gather in infection sites.¹⁶ Moreover, depending on the design strategy employed, NPs can be engineered to release the drug in a controlled manner. This targeted approach enables the delivery of higher concentrations of therapeutic agents to cells and tissues, thus allowing for the use of lower doses. Moreover, by increasing the concentration of therapeutic agents in the target location, their effectiveness is enhanced making them more tolerable in biological systems.¹⁷ Importantly, NPs should be biodegradable, non-toxic and non-immunogenic. These properties make them attractive to be included in wound dressings for the management of skin infections. This systematic review focuses on the latest advances in the wound dressings’ development containing antibiotic NPs for the treatment of infected skin wounds caused by P. aeruginosa.

Characteristics of an Ideal Wound Dressing

It is evident that a considerable amount of blood is lost following an injury. Stopping this loss is often the first step in wound repair. Therefore, the dressing employed must possess a robust hemostatic capacity. This becomes a relevant factor in assessing the biocompatibility of dressings that come into direct contact with red blood cells.¹⁸ In accordance with the American Society for Testing and Materials (ASTM F 756–00, 2000), the internationally safe hemolysis rate should be less than 5%.^19,20 In addition to being hemocompatible, the dressings must also be cytocompatible. In order for a substance to be considered non-toxic in accordance with the ISO 2009 international standard for the biological evaluation of medical devices, the viability of the tested cell types must exceed 70%.²¹

During the initial stages of wound healing, wounds produce a considerable amount of exudate, particularly those resulting from burns. This excessive exudate presents a challenge as it impairs gas exchange, increasing the risk of wound infection.²² Such infections can impede the repair and regeneration stages involved in the healing process, and even affect the restoration of anatomical and physiological integrity, leading to chronic wounds.²³ To eliminate this issue, dressings with a high absorption capacity, which can be assessed by the swelling ratio of the gel, are required to protect the wound from microbial contamination.^24,25

At this level, it is important to consider a highly porous internal structure, with porosity ranging from approximately 60 to 90%.²⁶ This structure not only absorbs excess fluid but also allows for gas diffusion, thus enabling the wound to obtain nutrients and oxygen, creating an appropriate environment for cell growth.^24,27 The interconnected porous structure facilitates dermal fibroblast growth and migration, thereby promoting wound healing.¹⁹ It also allows the loading of antibacterial drugs.²⁸

However, although it may seem contradictory, it is essential to maintain a moist wound environment. The high-water content of the human body facilitates epidermal cell migration, angiogenesis, proliferation, collagen deposition, and re-epithelialization in an optimal aqueous environment.^24,27,29 In fact, Zhiyong et al²⁷ have investigated the potential of asymmetric materials, wherein one side would exhibit hydrophilic properties to absorb exudates, while the other would display hydrophobic characteristic to protect against airborne microorganisms, thereby reducing the risk of infection. In addition, it prevents evaporative water loss from inside the wound, maintaining the moist environment that would enhance wound healing. Therefore, wound dressings should regulate moisture loss and absorption to create an optimal moist environment that facilitates drug release.^28,30

The Water Vapor Transmission Rate (WVTR) determines the capacity of wound dressings to regulate water loss during the healing process. Dressings with a WVTR value of 1800 −2200 g · m ⁻² · 24 h ⁻¹ are ideal for maintaining optimal moisture content. A higher value may result in scarring of the surface due to excessive dehydration, while lower values may lead to the formation of exudates on the wound surface, increasing the risk of bacterial contamination.³¹

The healing process can be affected by various factors, including high oxidative stress at the wound site caused by immune cells during inflammation. The release of high levels of reactive oxygen species (ROS) can trigger cell apoptosis, which can disrupt the healing process. As a result, there is a search for bandages with antioxidant properties to protect cells from damage.^23,32

Regarding the mechanical properties of the wound dressing, rheology represents the most crucial technique for defining the characteristics of gels, which are essential for the creation of a robust scaffold.³³ Biomaterials with good strength and mechanical stability are preferred to reduce susceptibility to damage during application.^22,34 Additionally, materials with the ability to revert to their original shape (memory materials) have been studied. They can be compressed into a small volume, allowing for easy transportation and application to the wound using a syringe, without any deformation.^22,35 Accordingly, a reduction in viscosity at high shear rates and during storage is preferable for this type of material.³⁶ Several factors can affect the rheological properties, including the distribution of fibers within the material.^33,37

Another important aspect is to maintain a pH within the range of the skin (6.0–7.0). Basic or acidic pHs could irritate the skin and affect the penetration of biomaterial components as well as their ability to adhere to the skin.³⁸

Wound dressings should be biodegradable to avoid painful removal procedures for patients. If the compound used can be absorbed by the body, it would be expected to improve the patient’s quality of life.²² In this context, transparent dressings are preferred in order to allow for proper wound monitoring by clinicians. To prevent the disruption of granulation tissue and causing trauma to the wound,²³ it is recommended that the dressing be left in place until the wound has healed sufficiently. Traditional dressings can damage the granulation tissue and cause bleeding when changed, which can delay wound healing.²⁸ Therefore, it is important to find a dressing that is both cosmetically acceptable and cost-effective.²⁴

Hydrogels used as wound dressings provide physical support and influence the survival of normal cells through interactions with cell membrane receptors. Consequently, the histological assessment will encompass the reduction of inflammatory cells, collagen deposition, and the appearance of new blood capillaries which facilitate the delivery of growth factors and nutrients to the damaged tissue.³⁹

Finally, dressings are designed to have antimicrobial properties by incorporating compounds that can kill microorganisms, both individually and in biofilm formation. These compounds must not be eliminated by wound secretions such as blood, albumin, or pus.^24,30,40 This review will discuss the different alternatives.

Therefore, it is essential to develop a wound dressing with optimal hemostatic capacity, absorption, and adhesion, while maintaining wound moisture, high porosity, and superior physical properties (strength, stability, biodegradability, and transparency). The dressing should also be biocompatible, possess an appropriate pH, and exhibit antimicrobial properties to facilitate wound repair, enhance wound appearance, and reduce healing time (Figure 1). ^25,35,41

Figure 1 Characteristics of an ideal wound dressing. Mechanical stability, moisture balance, high porosity, gas permeability, hemostatic and antimicrobial capacity, cytocompatibility, biodegradability and cost-effectiveness are represented. Created with Biorender.com.

Materials and Methods

Search Strategy

This systematic review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline.⁴² On December 27, 2023, a systematic bibliographic search was performed using MeSH and free-text terms at two distinct electronic databases: “Pubmed” and “Scopus”. For this, a combination of terms was used in the search algorithm: the term “ANTIBIOTIC NANOPARTICLES” was combined using the boolean operator “and” with “PSEUDOMONAS AERUGINOSA” [Mesh] and with the following terms: “WOUND DRESSING”, “ARTIFICIAL SKIN”, “SKIN SUBSTITUTE” or “HYDROGEL”. A total of 327 results were obtained.

Eligibility Criteria

The inclusion criteria were: (1) wound dressing/artificial skin/skin substitute/hydrogel for skin infections, (2) loaded with antibiotic NPs, (3) with antimicrobial activity against P. aeruginosa, (4) where cytotoxicity of NPs was evaluated, (5) original article, (6) written in English language, (7) publications dated from January 2019 to December 2023. Exclusion criteria were: (1) non-research papers (books, reviews, letters to editor, protocols, clinical trials and unpublished literature), (2) non-skin infections and (3) articles where the antimicrobial capacity against P. aeruginosa was not evaluated. Finally, a total of 49 original research studies about wound dressing containing antibiotic NPs with antimicrobial activity against P. aeruginosa were included (Figure 2).

Figure 2 Procedure to select studies for inclusion in the systematic review following PRISMA guidelines. From 327 records identified, 158 full-text articles were assessed for eligibility criteria after date restriction and removal of duplicates, unpublished literature, clinical trials, protocols, letters to editor, books and reviews. Finally, 49 results met the inclusion criteria and were included in the qualitative analysis. Created with Biorender.com.

Study Selection

After date restriction and removal of duplicates, unpublished literature, clinical trials, protocols, letters to editor, books and reviews, search results were screened by two independent authors (MI.Q.V and A.U.R) based on title and abstract. References not meeting the inclusion criteria were excluded. After that, candidate articles were full text read autonomously by the same two authors ensuring that they fulfilled the rest of inclusion criteria. Any discrepancies about inclusion or exclusion of articles were discussed and resolved by a third independent reviewer (A.F.G).

Results

Organic NPs Loaded Wound Dressings

Carbon Based NPs Loaded Wound Dressings

This systematic review identified two different approaches of wound dressings loaded with carbon-based NPs: graphene quantum dots (GQD) and carbon dots (C-dots). Table 1 presents the composition of the wound dressing, the antimicrobial agent, the properties of NPs, the properties of the wound dressing, the antimicrobial efficacy and the biocompatibility of each approach. Specifically, three studies have been found in this search.

Table 1 Carbon Based NPs-Loaded Wound Dressings Against P. aeruginosa Skin Infection

GQD-based wound dressings have demonstrated several properties of an ideal wound dressing including good wound fluid absorption, water retention and hemostatic capacity, a porous network structure and self-healing ability, among others.^43,44 Furthermore, Cheng et al demonstrated that the dressing exhibited a photothermal effect following xenon light (XL) irradiation, resulting in a pronounced antibacterial efficacy.⁴⁴ In addition to being biocompatible to human fibroblasts (HFs), these GQDs-based dressings have been demonstrated to promote angiogenesis and accelerate wound healing in infected diabetic mice.^43,44 Carbon-based NPs-loaded into wound dressings are also capable of suppressing bacterial proliferation by removing Fe³⁺ ions from the environment. This exemplified by C-dot-loaded dressings, which are also biocompatible and capable of being 3D bioprinted as wound patches.⁴⁵

With regard to antimicrobial activity, Zmejkoski et al⁴³ observed the absence of bacterial growth on plates, although no quantified data were provided. Cheng et al⁴⁴ reported almost zero growth on plates and observed pores in the bacterial membrane under microscopy. Chekini et al⁴⁵ evaluated the reduction of bacterial colonies grown on plates and obtained a value of approximately 66%.

Polymeric NPs Loaded Wound Dressings

Polymeric NPs loaded in wound dressings are composed of poly(lactic-co-glycolic) acid (PLGA), alginate, chitosan, polydopamine (PDA) and bacterial cellulose (BC). Table 2 provides a summary of the eight approaches of polymeric NPs that have been developed to date to deliver antimicrobial agents to the wound site through wound dressings.

Table 2 Polymeric NPs-Loaded Wound Dressings Against P. aeruginosa Skin Infection

Ciprofloxacin (CIP), cefepime and polymyxin B were the delivered antimicrobial agents by polymeric NPs. Particularly, CIP was encapsulated by PDA⁵⁰ and PLGA NPs.⁵² Sodium alginate NPs were used for the encapsulation of polymyxin B⁵¹ while chitosan NPs were employed for the encapsulation of cefepime.⁴⁷ Three studies evaluated wound dressings containing empty NPs made of PDA⁴⁶ and a combination of BC and chitosan.^48,49 All these studies showed antibacterial efficacy against P. aeruginosa.

Regarding the cytotoxic impact of these wound dressings, those loaded with PDA NPs exhibited biocompatibility with a range of cell lines in vitro, including human lung embryonic cells (MRC-5),⁴⁶ mouse embryonic osteoblasts (MC3T3-E1)⁴⁶ and HFs.⁵⁰ They also demonstrated complete wound healing in vivo.⁴⁶ Chitosan NP-loaded dressings were found to be biocompatible with HT1080⁴⁷ and human gingival cells (HGCs).^48,49 As PDA NPs, they showed 100% of wound closure in vivo.⁴⁷ Wound dressings incorporating alginate NPs exhibited biocompatibility in vivo promoting wound healing. However, they reduced L929 cell viability in vitro after 24 hours of exposure.⁵¹ PLGA NPs-loaded dressing showed no cytotoxicity to human mesenchymal stem cells (hMSCs) and HaCaTs in vitro but in vivo biocompatibility was not evaluated.⁵²

All these dressings presented essential properties, including adequate mechanical strength, thermal stability, water retention and absorption and flexibility. Zhe et al also reported the extrudability and self-healing capacity of their wound dressing, which incorporated chitosan NPs.⁴⁶ Similarly, Zmejkoski et al reported the use of chitosan NPs in a similar dressing type. In this case, the dressing exhibited antioxidant properties, although the porosity was relatively low (up to 2.4%).^48,49

As with other NPs groups, a variety of assay methods have been used to evaluate antimicrobial activity. Three papers assessed the agar disk diffusion assay,^47,51,52 with inhibition zones ranging from 19 mm⁵¹ to 40 mm.⁵² One paper examined optical density measurements through turbidity tests,⁵⁰ while another used the bacteria killing assay.⁴⁶ Only one paper evaluated the minimum inhibitory concentration (MIC), defined as the lowest concentration at which a material exhibits antimicrobial activity through serial dilution.⁵¹ Only two articles reported on the ability of experimental dressings to damage bacterial biofilms.^48,49

Inorganic NPs Loaded Wound Dressings

Metal NPs are a typical example of this class. They are composed exclusively of metal precursors and can be monometallic, bimetallic or polymetallic. All articles on the use of inorganic NPs found during the search are listed in Table 3.

Table 3 Inorganic NPs-Loaded Wound Dressings Against P. aeruginosa Skin Infection

Silver NPs

A total of 28 papers on the evaluation of AgNP-loaded wound dressings were identified. They were found not to be cytotoxic in vitro against various cell types, including mouse fibroblasts (L929 cells,^{18,20,22,26,27,34,38,61} 3T3-L1,²⁹ NIH 3T3^28,54), HFs (NHDF,²⁹ CCD-986sk,³⁹ HFs,^27,56 HFFs,²¹ MSTO), keratinocytes (HaCaTs^19,21,38), endothelial cells (HUVECs¹⁹), epithelial cells (Vero-E6,^33,57 Panc 1²³), macrophages (RAW 264. 7^32,38), and mesenchymal cells (HUMSCs²⁷). The commercial Acticoat^TM dressing exhibited toxicity to hKT, which disqualifies it as a viable candidate.⁵⁸ Six of the papers evaluated hemocompatibility.^{18–20,22,34,55} Gupta et al stated that their dressing was hemolytic and therefore does not meet one of the ideal characteristics of a good dressing.²³ Regarding in vivo results, two studies evaluated the absence of damage to vital organs,^24,34 while 13 studies have evaluated histological parameters such as re-epithelialization, collagen deposition, angiogenesis, and reduced presence of inflammatory cells.^{19,20,22–24,26,27,29,31,34,54,62} Finally, the antioxidant capacity has been evaluated in five studies with positive results.^{18,23,29,32,38}

In terms of the most significant ideal properties evaluated for wound dressings, 18 papers assessed moisture balance/swelling capacity,^{19–23,26–28,31,32,34,38,39,53,54,56,57} and 11 papers reported high porosity.^{19,21,22,26–28,31,32,39,54} 16 studies evaluated the biomechanical properties,^{18–22,26,29,32–34,36,38,39,54,62} while eight papers analyzed the Ag ion release pattern.^{21,27–29,31,32,34,62} Additionally, five papers evaluated biodegradability,^{20,22,26,39,62} and seven studies provided stability data.^{19,22,24,32,38,57,62} Three papers did not provide any data on these parameters.^55,58,61 Therefore, the most evaluated properties were moisture balance/swelling capacity and biomechanical properties. Regarding porosity, Singh et al did not show high values, which are desirable for a good dressing (15%),³⁸ while those showing higher porosity were 72.2%³¹ and 72.6%.²⁶ The capacity to be 3D bioprinted was also demonstrated.^54,56

Although all studies demonstrated antimicrobial activity against the microorganism of interest, they employed different techniques. In particular, 14 papers used the agar disk diffusion assay.^{18,20,21,23,24,26,27,29,31,39,54,56,57,62} Overall, the antimicrobial efficacy of the NPs was enhanced when incorporated into the hydrogel. This resulted in bacterial inhibition zones ranging from 6 mm⁵⁶ to 27 mm.²⁶ Seven papers evaluated the MIC^{18,29,32,33,53,55,61} and three papers assessed the minimum bactericidal concentration (MBC), defined as the lowest concentration of the hydrogel that does not show colony growth in the culture medium.^32,33,53 Although the importance of biofilm elimination has been recognized, only four papers^21,22,32,33 reported the ability of the hydrogel to eradicate biofilm formation. Some studies evaluated bacterial death using live/dead assays, achieving over 80% death.^19,21,28 Other methods included optical density measurements by turbidity tests,^22,34,36 observation of pores in the bacterial membrane by microscopy,^27,28,34 plate count assays³⁸ and time-kill assays.^55,56 It should be noted that bacterial growth was observed in the commercial Acticoat^TM dressing,⁵⁸ indicating that its ability to eliminate bacteria was no better than the other experimental dressings evaluated in this review.

Others Inorganic NPs

A total of 12 studies have been conducted evaluating wound dressings loaded with other metallic NPs. These inorganic NPs include iron (Fe),^59,60 zinc (Zn),^63–66 copper (Cu)^69,70 cerium (Ce)⁶⁷ and titanium (Ti)⁷¹ oxides, magnesium hydroxide (Mg[OH]₂)⁶⁸ and gold (Au) NPs.⁷² The analyzed wound dressings, like those loaded with AgNPs, were biocompatible in vitro not only with mouse fibroblasts,^59,63,66,71 HFs^60,64,69 and epithelial cells⁷⁰ but also with urothelial cells.^67,68 Furthermore, Mg(OH)₂,⁶⁸ ZnO,^65,66 TiO₂⁷¹ and Au⁷² NPs-loaded wound dressings have shown rapid and complete re-epithelialization in vivo, with collagen deposition^65,66,72 and fewer inflammatory cells.^66,72

Importantly, these wound dressings exhibited key properties. Iron oxide (IO) NPs-loaded dressings showed smooth surfaces⁵⁹ and when iron was incorporated in the form of MIL-53(Fe) NPs, they exhibited thermal stability, water retention and absorption ability and good mechanical strength.⁶⁰ These properties were also present in dressings incorporating ZnO,^64,66 Mg(OH)₂⁶⁸ and TiO₂⁷¹ NPs. ZnONPs-loaded dressings also showed flexibility and antioxidant ability.⁶⁴ However, only CuONPs-loaded dressings showed good spreadability and extrudability. For dressings incorporating AuNPs, wound dressing properties were not evaluated.⁷² Therefore, the most evaluated properties in this type of dressing were water retention and absorption ability and mechanical strength. Interestingly, only one study reported porosity in the developed dressing.⁶⁰

The microbial activity of the remaining inorganic NPs was evaluated using various techniques; six papers assessed the agar disk diffusion assay,^{59,60,63,69–71} which resulted in bacterial inhibition zones ranging from 17 mm⁵⁹ to 33 mm.⁶⁹ Additionally, six papers evaluated the MIC,^{59,60,63,64,67,69} while five papers evaluated MBC.^{59,60,63,67,69} Three papers analyzed the reduction of bacterial growth using optical density measurements by turbidity tests^65,66 or the viable bacterial count method,⁷² In addition, four papers investigated the elimination of biofilm.^59,63,68,69 Of note is the study by Zamani et al, which evaluated the expression levels of genes involved in antibiotic resistance.⁶⁷

Discussion

P. aeruginosa is a common hospital-acquired pathogen that causes life-threatening infections in critically ill patients. It is resistant to many drugs, making it challenging to treat. P. aeruginosa infections are common in healthcare settings and can cause significant morbidity and mortality.^1–3 Traditional treatments of infected wounds involve the topical application of antibiotics, but this can lead to systemic toxicity and the development of resistance.^12,13 Advanced wound dressings containing antibiotic NPs have shown promise in overcoming these challenges. To be considered an “ideal” wound dressing, they should fulfil several criteria. These criteria include hemostatic capacity, absorption, adhesion, moisture retention, porosity, mechanical properties, pH, biodegradability, transparency, biocompatibility, and antimicrobial properties.^{18,22–24,28,31,33,38,39} These properties are crucial in promoting wound repair, improving wound appearance, and reducing healing time. Both natural (such as chitosan, cellulose, collagen, HA, gelatin, alginate, dextran, pullulan, agarose and SF) and synthetic polymers (including polyvinyl alcohol, PCL, PEG, PLGA and PDA) provide these features to the dressings. This systematic review focuses on the latest advances in the development of NPs-based wound dressings for the treatment of P. aeruginosa infected skin wounds.

In this sense, different types of wound dressings loaded with NPs were identified and analyzed, including organic (carbon-based and polymeric NPs) and inorganic (metallic, magnetic, metal oxides, metal hydroxide NPs and MOF). All of them exhibited antibacterial activity against P. aeruginosa. Figure 3 shows the percentage of publications by date for different wound dressings loaded with NPs. In particular, the 61.2% of the studies evaluated metallic NPs-loaded wound dressings. The next group with a higher percentage of publications is that evaluating polymeric NPs-loaded wound dressings (14.3%) followed by metal oxides NPs-loaded wound dressings (12.2%) and carbon-based NPs-loaded wound dressings (6.1%). Magnetic, metal hydroxides NPs and MOF represent 2% of the publications analyzed. It is noteworthy that most of the recent publications belong to the group of metallic NPs-loaded wound dressings.

Figure 3 Percentage of publications by date of NPs-loaded wound dressings against P. aeruginosa. The studies reviewed focused on wound dressings loaded with different types of NPs: 61.2% with metallic NPs, 14.3% with polymeric NPs, 12.2% with metal oxides NPs, and 6.1% with carbon-based NPs. Magnetic NPs, metal hydroxides, and MOF accounted for 2% of the publications.

Antibacterial activity was determined through different methods and evaluations including MIC and MBC determinations, bacterial kill assays, agar diffusion methods, live/dead assays and biofilm reduction or inhibition evaluations. It should be highlighted that biofilms can resist antibiotics and immune system clearance, leading to delayed wound healing and chronic inflammation.^9,73 As described above, their formation is mediated by the bacterial virulence factors and regulated by quorum-sensing mechanisms⁷⁴ and their dispersal can exacerbate disease, leading to acute life-threatening conditions.⁷⁵ It is therefore crucial to evaluate how NPs- loaded wound dressings affect P. aeruginosa’s biofilm. Despite this, only nine of the 49 studies identified in this systematic search, carried out this evaluation.^{21,22,32,33,48,49,59,63,69}

Focusing on the reported wound dressings loaded with NPs, three studies regarding carbon-based NPs-loaded dressings were found in this search. These include GQDs and C-dots. GQDs are carbon-based zero-dimensional fluorescent nanomaterials with a graphene lattice inside.⁷⁶ They are very small, usually less than 20 nm and can easily penetrate through biological membranes and exhibit antimicrobial activity through the oxidative stress caused by the generation of ROS following their photoexcitation.^43,76 Besides being antimicrobial, these carbon-based dressings have shown not only biocompatibility in vitro and in vivo but also promotion of angiogenesis in vivo. This may be attributed to their ability to interact with ROS in wound tissue, leading to a reduction in ROS levels and activation of cell signaling molecules such as hypoxia-inducible factor 1α (HIF-1α) and the p38MAPK/Akt pathway, which enhances the production of angiogenic factors.⁷⁷ C-dots are generally defined as carbon nanomaterials composed of quasi-spherical carbon NPs.⁷⁸ As with GQDs, the antimicrobial mechanism of photoinduced C-dots includes the production of ROS, the disruption and penetration of the bacterial membrane and the induction of oxidative stress with damage to bacterial DNA.⁷⁸ They are also able to suppress bacterial proliferation by removing Fe³⁺ ions from the environment.⁴⁵ It is important to note that the incorporation of these NPs into wound dressings may be more costly than traditional methods that do not involve NPs.

Inorganic NPs incorporated into wound dressings include magnetic NPs (iron oxide NPs, IONPs), metallic-organic framework (MOF), metal oxides NPs (Zn, Cu, Ce and Ti oxides), metal hydroxides NPs (Mg[OH]₂) and metallic (Au, Cu and Ag) NPs. Specifically, this review has identified a total of 39 studies in which these types of dressings were developed and evaluated.

Ag is a well-known antimicrobial agent against fungi, yeasts, and bacteria, including antibiotic-resistant strains.²³ Researchers in various fields have widely used AgNPs for the treatment of infections due to their broad-spectrum antibacterial capacity.²² The traditional synthesis of these NPs involves toxic organic solvents and reducing agents. However, there is a growing interest in “green chemistry synthesis” using natural substances, such as plant extracts and microorganisms, which are fast, economical, safe, and environmentally friendly.³⁸ For example, Cur extracted from turmeric has antimicrobial, antioxidant, and anti-inflammatory effects and can be used to reduce and protect Ag during NPs synthesis, enabling combined therapy with a synergistic effect.^19,20,23 AgNPs exert their antimicrobial effect by increasing the permeability of the bacterial cell membrane, interfering with DNA replication by binding to phosphorus-containing compounds, denaturing proteins, and releasing Ag ions inside the bacteria, which enhances their bactericidal effect.²³ They bind directly to the thiol groups of different biomolecules, including peptides, DNA, cofactors, and oxidized glutathione (GSH) to form glutathione disulphide (GSSG). This leads to increased levels of ROS, which in turn causes additional damage to bacteria.²⁶

Importantly, AgNPs not only have antibacterial properties but also possess anti-inflammatory capacity. They prevent the expression of pro-inflammatory factors such as tumor necrosis factor alpha (TNF-α), interleukins 6 and 1ß, which accelerates wound healing by promoting the proliferation and migration of keratinocytes to the wound site.^24,26,39 Additionally, they induce the transformation of fibroblasts into myofibroblasts, leading to wound contraction. It is important to note that Gram-negative bacteria are more susceptible to the effects of Ag than Gram-positive bacteria. This may be because Gram-positive bacteria have a plasma membrane coated with a peptidoglycan layer, which may limit the penetration of AgNPs.^18,37

However, despite being highly antibacterial and their anti-inflammatory ability, they are not stable in water and tend to agglomerate. Incorporating them into a hydrogel could reduce this issue.³⁹ Moreover, their use is still limited because they can pass through biological mucosal surfaces and penetrate animal organs. Therefore, it is recommended to use them in the lowest possible concentration to ensure sustained release.²⁷ These characteristics would also be improved by incorporating them into a biomaterial.

Other inorganic NPs include MOFs, IONPs, ZnONPs, Ce₂O₃NPs and TiO₂NPs, Mg(OH)₂NPs, CuNPs and AuNPs. These metal-based NPs are valuable for biofilm inhibition and treatment of microbial diseases due to their ultra-small size and high surface area.^59,79 IONPs are highly promising for antimicrobial therapy because of their strong magnetic and semiconductor properties.⁵⁹ Magnetic fields cause bacterial death and biofilm eradication through vibration damage, local hyperthermia, and ROS production.⁸⁰ A MOF-based hydrogel was reported in this systematic review. Specifically, a carboxylic acid iron MOF, called MIL-53(Fe), which is made from iron ions and terephthalic acid. These frameworks are a distinct type of hybrid materials formed by combining inorganic and organic components⁶⁰ and are well-suited for biomedical applications due to their specific characteristics, such as high porosity, high surface area, customizable chemical surface, good compatibility with the physico-chemical interaction of drugs, and interconnected channels suitable for use in drug loading and release systems.⁶⁰ In addition to causing physical damage to bacterial cells through direct contact, they can also store and slowly release metal ions, leading to the generation of oxidative stress and/or photothermal effects.^81,82 ZnONPs have been reported as antimicrobial and anti-inflammatory agents and also, to play a role in fibroblast proliferation, angiogenesis, and increased re-epithelialization.^63,64 Their antibacterial mechanism is mediated by direct contact with bacterial membranes, resulting in destruction of cell integrity, release of antimicrobial ions, mainly Zn²⁺ ions, and ROS generation.⁸³ Ce₂O₃ is a natural scavenger of free radicals such as ROS and reactive nitrogen species (RNS). Ce₂O₃NPs are likely to exert their antibacterial mechanism by inducing oxidative stress and interfering with the nutrient transport functions.⁸⁴ Furthermore, unlike TiO₂NPs which required photoactivation, Ce₂O₃NPs exhibit antibacterial effect without external activation.⁸⁵ The photoactivation of TiO₂NPs is possible due to its good photocatalytic activity in the anatase phase. As a result, these NPs produce ROS that destroy the bacterial outer membrane and ultimately destroy the bacterial cell.^71,86

Mg(OH)₂NPs have been reported as an approved drugs and food additives due to their excellent biocompatibility, low toxicity, thermal stability and low cost of production.^68,87 They are effective agents against several bacteria, including P. aeruginosa, exerting their antibacterial activity through two potential mechanisms. The first mechanism involves the direct infiltration of the NPs into the cell membrane, causing membrane damage and resulting in cell death. The second mechanism involves the absorption of water moisture on the surface of the NPs, creating a thin layer of high pH water that can harm bacterial membranes.⁸⁸ Nevertheless, this antibacterial mechanism is still unclear.⁸⁷ In fact, only one study has evaluated the incorporation of these NPs into a wound dressing and analyzed its properties, biocompatibility and antibacterial capacity.⁶⁸ Cu and AuNPs were also reported in this systematic review as inorganic NPs based wound dressings. CuNPs are known to have the highest antimicrobial property among the known synthesized metal NPs, with high level of biocompatibility.⁷⁰ The antibacterial mechanism is based on the interaction of Cu with amine and carboxyl groups on the surface of microbial cells, the inhibition of amino acid synthesis, and the inactivation of microbial proteins through thiol interaction.⁶⁹ Like Cu, the main antimicrobial mechanism of AuNPs is based on the direct adhesion of AuNPs to the bacterial surface, driven by electrostatic forces.⁸⁹ These NPs are colloidal or clustered particles composed of a Au core with high synthetic versatility and non-toxic nature.^72,89

Taken together, all the studies evaluating wound dressing containing inorganic NPs showed biocompatibility with several cell lines and antibacterial activity. Nevertheless, only 18 out of 39 (46.1%) studies performed in vivo evaluations. Therefore, more research is needed to confirm the beneficial role of these dressings in vivo.

In addition to the proposed mechanisms, emerging therapies are gaining attention to enhance the antibacterial capacity of metallic NPs due to their high target selectivity, minimal invasiveness and reduction in bacterial drug resistance.⁹⁰ These include photothermal therapy (PTT), photodynamic therapy (PDT) and chemodynamic therapy (CDT).

PTT uses photoinduced heat to destroy bacterial pathogens and is generally developed by combining a near infrared (NIR) laser with highly effective NIR light-absorbing nanomaterials.⁹¹ Several studies have employed this strategy against Gram-negative bacterial infections including P. aeruginosa infections. For instance, He et al reported efficient bactericidal and anti-biofilm effects by photothermal enhancement and ROS generation of high-entropy transition metal carbides or nitrides (HE MXenes) monolayers that eradicated methicillin-resistant Staphylococcus aureus infected tissue inflammation and stimulated angiogenesis in vivo.⁹² With regard to P. aeruginosa infections, Ye et al developed CuO- and AgO-doped ZnO nanocomposites that exhibited excellent photothermal stability and intrinsic antibacterial activity.⁹³ Another strategy in this field was carried out by Lv et al who developed a nanorod based on molybdenum disulphide nanosheets coated with AuNRs to target lipopolysaccharide present on the surface of bacteria, demonstrating a strong photothermal effect as well as a more significant antibacterial effect than non-targeted PTT.⁹⁴

PDT employs a photosensitizer that absorbs light energy and transfers it to oxygen to produce ROS, resulting in bacterial cell death.⁹⁰ Cur has been used as a photosensitizer in combination with silica,^95,96 AgNPs^97,98 and GQT⁹⁹ to combat planktonic, biofilm and clinical isolates of P. aeruginosa showing promising results for the application of this therapy in vivo. AgNPs have also been photosensitized with methylene blue.¹⁰⁰ GQT,⁹⁹ AuNPs¹⁰¹ and ZnONPs¹⁰² have also been reported to play the role of photosensitizer achieving broad antibacterial efficacy and strong biofilm inhibition after light irradiation. All these studies used visible light to activate the photosensitizer, which may result in poor tissue penetration.

Garin et al¹⁰³ proposed the combination of PDT with PTT by using copper sulphide NPs together with indocyanine green activated in the NIR region of the electromagnetic spectrum for the elimination of P. aeruginosa to reach superior tissue penetration. This kind of therapy combination was also reported by Mo et al¹⁰⁴ who developed a C₇S₄ nanosheet with excellent antibacterial activity through synergistic PDT and PTT against drug-resistant P. aeruginosa under NIR light irradiation, which inhibited skin infection in mice.

CDT, also known as peroxidase-mediated chemokinetic therapy refers to the ability of metallic NPs to produce hydroxyl radicals that damage bacterial cells. These NPs with peroxidase activity are also known as nanozymes. However, the antibacterial effects of these nanozymes are limited due to the insufficient production of hydroxyl radicals and the short range of action.¹⁰⁴ PTT can improve the catalytic activity of nanozymes by stimulating them with NIR light. For example, He et al combined the photothermal potential of V₂C MXene nanosheets with the chemodynamic activity of platinum (Pt) NPs to create a Pt@V₂C platform with enhanced antibacterial properties.¹⁰⁵ Furthermore, Liu et al irradiated with a NIR laser CuFeS₂ NPs to stimulate their peroxidase activity achieving more than 99% inactivation efficiency of P. aeruginosa through the combination of PTT, PDT and also by consuming intracellular glutathione in bacteria, resulting in more ROS production.¹⁰⁶

In addition to the dual therapies, a combination of PTT, PDT and CDT was also reported against P. aeruginosa. Dai et al developed a NIR-activated multimodal agent by loading fluorescein isothiocyanate, ultrasmall copper sulfide NPs (Cu₂−xSNPs), and ε-polylysine onto mesoporous silica NPs.¹⁰⁷ This agent could detect the bacteria and biofilms by fluorescence imaging, ablate bacteria and biofilms and treat bacterial infections in vivo.

However, despite the advances that these therapies offer in terms of superior antibacterial activity, high tissue penetration and biocompatibility, only one study identified in this systematic review applied PDT by using visible light irradiation of C₃N₄ to improve the antibacterial activity of AgNPs- loaded wound dressing.³⁴ Therefore, the combination of these therapies in the field of NPs-loaded wound dressings against P. aeruginosa infection needs to be further investigated.

Finally, this systematic review reported 7 strategies of polymeric NPs-loaded wound dressings including PDA NPs,^46,50 chitosan NPs,^47–49 sodium alginate NPs⁵¹ and PLGA NPs.⁵² Polymeric NPs range in size from 1 to 1000 nm and can be loaded with active compounds or drugs that are entrapped within the polymeric core or surface-adsorbed.¹⁰⁸ This review reported that CIP,^50,52 cefepime⁴⁷ and polymyxin B⁵¹ are the main encapsulated drugs used in wound dressings against P. aeruginosa skin infections. Surprisingly, non-encapsulated polymeric NPs have also shown antibacterial activity.^46,48,49 These polymeric NPs are biocompatible, as supported by the results, and biodegradable, properties that are crucial for wound healing strategies. For PLGA NPs, the degradation rate, mechanical strength and drug loading and release kinetics can be precisely adjusted by altering the ratio of lactic acid to glycolic acid.¹⁰⁹ Chitosan is a mucoadhesive cationic linear polysaccharide and is one of the most widely used natural polymeric‐based NPs for drug delivery due to its low cost, biodegradability, and availability in a wide range of molecular weights. It also has inherent antimicrobial and antioxidant capacities.^48,49,110 Alginate is an anionic mucoadhesive polysaccharide with versatile physicochemical properties that allow chemical modification for site-specific targeting.¹¹¹ Finally, PDA NPs are produced by the oxidative self-polymerization of dopamine and are employed in the manufacture of wet adhesive hydrogels. They improve the mechanical and self-healing properties of the hydrogels by exploiting reversible non-covalent interactions such as π–π stacking and hydrogen bonding.^46,112

Table 4 summarizes the benefits and drawbacks of the different strategies of NPs-loaded wound dressings against P. aeruginosa skin infection. Their antimicrobial mechanisms are represented in Figure 4.

Table 4 Benefits and Drawbacks of NPs-Loaded Wound Dressings Against P. aeruginosa

Figure 4 Main antimicrobial mechanisms of carbon-based, inorganic and polymeric NPs. (A). Carbon-based NPs: 1) ROS production after photoexcitation, bacterial membrane disruption and DNA damage (QGDs and C-dots) and 2) sequestration of Fe3+ from the environment (C-dots). (B) Inorganic NPs: 1) increment of membrane permeability after electrostatic adherence, interference with DNA replication, denaturation of proteins and ROS production (Metallic NPs); 2) ROS production, vibration damage, local hyperthermia (IONPs); 3) ROS production and nutrient transport inhibition (Ce2O3NPs); 4) destruction of cell integrity, liberation of metal ions and ROS generation without photoexcitation (ZnONPs) and after photoexcitation (TiO2NPs); 5) bacterial damage after direct contact, drug and metal ions liberation, oxidative stress and photothermal effect (MOF) and 6) bacterial damage by direct infiltration of the NPs into the cell membrane and/or absorption of water moisture on NPs’ surfaces, creating a thin layer of high pH water (Mg[OH]2NPs). C. Polymeric NPs: 1) drugs and/or active compounds release. Created with biorender.com.

Conclusions

In the last 5 years and due to the advantages provided by the field of nanotechnology to reduce the problems of toxicity and resistance of conventional treatments, a variety of wound dressings loaded with NPs have been developed for the control of skin infections caused by P. aeruginosa, one of the most common nosocomial microorganisms in this type of infection. 79.6% (39/49) of these strategies use inorganic NPs as antibacterial agents, including metallic, metal oxide, metal hydroxide and magnetic NPs and MOFs. It is worth noting that among inorganic NPs, 71.8% (28/39) incorporate AgNPs into the generated dressings, possibly due to their properties and the diversity of antimicrobial mechanisms they possess, making them the most promising and advantageous NPs to incorporate into wound dressings. Disadvantages such as their toxicity and tendency to agglomerate require further research in this area. However, despite their advantages, only 14.2% of the studies developed dressings loaded with polymeric NPs and 6.1% with carbon-based NPs. Importantly, despite the research carried out, there are still no clinical trials evaluating the efficacy of NPs-loaded wound dressings in patients. This may be because there are no universally accepted standards for assessing the specific risks associated with NPs.⁷⁷ Emerging therapies such as PPT, PDT or CDT and their combination have shown promising results against P. aeruginosa infection. However, only study to date has used PTT together with NPs-loaded wound dressings against this bacterium. Further research is therefore needed in this field to overcome these issues, ensure the safety of these treatments and ultimately translate the results of the investigations from the bench to the bedside.